![Schematic representation of the UPS. Ub (yellow ovals) is synthesized as four precursor proteins that are further processed by speciall ized deubiquitinating enzymes -ubiquitin isopeptidases (DUBs). The UPS involves tree types of ubiquitin ligase (E1, E2, and E3; see the text); it is highly specific and selective due to its hierarchical complexity. Ub is conjugated to substrates (S) as a monomer or the polyUb chain linked through internal lysine residues. The polyUb chain is elongated by E3 ubiquitin ligases or relatively recently discovered E4 ubiquitin ligg ases. There is a dynamic equilibrium between ubiquitination and Ub removal by ubiquitin isopeptidase; the optimal polyUb chain length is about six Ub residues per substrate molecule, according to the modern concept [124]. Next, the ubiquitinated substrate binds to the proteaa some subunits Rpn10, Rpn13, and Rpn1 either directly or with the involvement of shuttle proteins of the UBLLUBA family; there may also be a variant of specific autophagy. Some Ub molecules enter the proteolytic chamber together with the substrate, where they get degraded. In most cases, resident proteasomal deubiquitinase Rpn11 successfully removes the entire polyUb chain, which is then cleaved into monomers for recyy cle.](https://www.researchgate.net/profile/Anna-Kudriaeva/publication/332525882/figure/fig4/AS:1010379719196672@1617904705843/Schematic-representation-of-the-UPS-Ub-yellow-ovals-is-synthesized-as-four-precursor_Q320.jpg)
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Intracellular concentration of polypeptides is deter
mined by the rates of their synthesis and degradation.
Cellular protein homeostasis, or proteostasis, is main
tained by a complex adaptive system that controls the
entire protein life cycle: synthesis, folding, maintenance
of correct conformation and necessary amount, subcellu
lar localization, and degradation. For example, during
translation, aberrant RNA molecules are recognized and
their polypeptide products undergo degradation [13].
Most proteins fold into the predetermined threedimen
sional structures encoded in their amino acid sequences,
which is necessary for proper functioning of these pro
teins. Some proteins retain conformational mobility,
while others are partially or even completely unordered,
which increases the risk of their misfolding or even aggre
gation [4]. In addition, misfolded proteins may appear
because of genetic mutations or ribosomal errors, or when
folded proteins lose their structure due to denaturation,
aging, or chemical modifications. Therefore, the main
problem of proteostasis is not only regulation of intracel
lular protein concentrations but also protection of cells
from the harmful effects of abnormal and potentially
toxic misfolded or damaged proteins that can seriously
disrupt cell metabolism.
ISSN 00062979, Biochemistry (Moscow), 2019, Vol. 84, Suppl. 1, pp. S159S192. © Pleiades Publishing, Ltd., 2019.
Russian Text © A. A. Kudriaeva, A. A. Belogurov, 2019, published in Uspekhi Biologicheskoi Khimii, 2019, Vol. 59, pp. 323392.
REVIEW
S159
Abbreviations: ALS, autophagic–lysosomal system; IFN, inter
feron; MBP, myelin basic protein; MHC, major histocompati
bility complex; ODC, ornithine decarboxylase; Ub, ubiquitin;
UPS, ubiquitin–proteasome system.
* To whom correspondence should be addressed.
Proteasome: a Nanomachinery of Creative Destruction
A. A. Kudriaeva1,a* and A. A. Belogurov1,2,b*
1Shemyakin
−
Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia
2Lomonosov Moscow State University, 119991 Moscow, Russia
aemail: anna.kudriaeva@gmail.com
bemail: belogurov@mx.ibch.ru
Received August 10, 2018
Revised September 7, 2018
Accepted September 7, 2018
Abstract—In the middle of the 20th century, it was postulated that degradation of intracellular proteins is a stochastic
process. More than fifty years of intense studies have finally proven that protein degradation is a very complex and tightly
regulated in time and space process that plays an incredibly important role in the vast majority of metabolic pathways.
Degradation of more than a half of intracellular proteins is controlled by a hierarchically aligned and evolutionarily perfect
system consisting of many components, the main ones being ubiquitin ligases and proteasomes, together referred to as the
ubiquitin–proteasome system (UPS). The UPS includes more than 1000 individual components, and most of them are crit
ical for the cell functioning and survival. In addition to the wellknown signaling functions of ubiquitination, such as mod
ification of substrates for proteasomal degradation and DNA repair, polyubiquitin (polyUb) chains are involved in other
important cellular processes, e.g., cell cycle regulation, immunity, protein degradation in mitochondria, and even mRNA
stability. This incredible variety of ubiquitination functions is related to the ubiquitin ability to form branching chains
through the εamino group of any of seven lysine residues in its sequence. Deubiquitination is accomplished by proteins of
the deubiquitinating enzyme family. The second main component of the UPS is proteasome, a multisubunit proteinase
complex that, in addition to the degradation of functionally exhausted and damaged proteins, regulates many important cel
lular processes through controlled degradation of substrates, for example, transcription factors and cyclins. In addition to
the ubiquitindependentmediated degradation, there is also ubiquitinindependent degradation, when the proteolytic sig
nal is either an intrinsic protein sequence or shuttle molecule. Protein hydrolysis is a critically important cellular function;
therefore, any abnormalities in this process lead to systemic impairments further transforming into serious diseases, such as
diabetes, malignant transformation, and neurodegenerative disorders (multiple sclerosis, Alzheimer’s disease, Parkinson’s
disease, Creutzfeldt–Jakob disease and Huntington’s disease). In this review, we discuss the mechanisms that orchestrate all
components of the UPS, as well as the plurality of the finetuning pathways of proteasomal degradation.
DOI: 10.1134/S0006297919140104
Keywords: proteasome, protein degradation, ubiquitindependent proteolysis, ubiquitinindependent proteolysis, ubiquitin
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Proteins that have lost their functional conformation
undergo controlled degradation (including cotransla
tional degradation); aggregated protein molecules are
destroyed via different cellular mechanisms [57]. In
many cases, these processes involve different classes of
molecular chaperones and their regulators that promote
proper protein folding and maintain proteins in a soluble
state. Besides contributing to the protein folding, chaper
ones participate in the degradation of misfolded proteins
[8]. To summarize the aforementioned, proteostasis is
essential for cell metabolism, organelle biogenesis, and
cell adaptation to stress that greatly affects cell viability
[9].
Currently, the autophagic–lysosomal system (ALS)
and the ubiquitin–proteasome system (UPS) are consid
ered as the main proteolytic pathways in the cell,
although extralysosomal proteases, primarily caspases
and calpains, are also involved in protein hydrolysis. The
studies on the crucial role of protein degradation systems
were recognized by three Nobel prizes: in 1974, bio
chemist Christian de Duve was awarded the Nobel Prize
in Medicine and Physiology for his contribution to the
discovery of the structural and functional organization of
the cell (discovery of lysosomes); in 2004, the Nobel Prize
in Chemistry was awarded jointly to Aaron Ciechanover,
Avram Hershko, and Irwin Rose for the discovery of ubiq
uitinmediated protein degradation; and in 2016,
Yoshinori Ohsumi received the Nobel Prize in Medicine
and Physiology for discovery of the autophagy mecha
nisms.
UPS is the main component in the degradation of
shortlived, misfolded, damaged, and obsolete proteins.
UPS actively participates in cell signal transduction, tran
scription, cell cycle progression, proliferation, and apop
tosis [10]. ALS recognizes and removes large and poten
tially dangerous cellular components, such as protein
aggregates and nonfunctional or superfluous organelles.
Its functioning is an important adaptive mechanism for
fighting various types of cell stress, such as nutrient
deficit, hypoxia, oxidative stress, etc. [11]. UPS and ALS
are responsible for approximately 8090 and 1020% of
cellular proteolysis, respectively, although these ratios
may vary depending on physiological conditions and cell
type [12, 13]. In the cytosol, UPS and ALS act simulta
neously; they share some components and constantly
interact with each other [14]. Ubiquitination is used as a
degradation signal for tagging substrates in both systems.
Protein degrons (protein sequences involved in the regu
lation of protein degradation) modified with ubiquitin
(Ub) are recognized and bound by proteins containing
Ubbinding domains (UBDs) [15]. UBDs decode the
polyUb code (linkage type and chain length) and deliver
ubiquitinated substrates to the proteasome (RAD23 and
UBQLN), autophagosomal vacuoles [p62 and BRCA1
(NBR1)], or targets not involved in proteolytic processes,
e.g., in the cases of DNA damage and inflammation [16].
In this review, we will discuss the mechanisms of
UPS functioning and regulation. Because protein hydro
lysis is a continuously occurring process, it has an impor
tant physiological significance. Its impairment leads to
multiple disturbances in the normal cell functioning and,
ultimately, to serious disorders, e.g., diabetes, malignant
transformation, and neurodegenerative diseases, such as
multiple sclerosis, Alzheimer’s disease, Parkinson’s dis
ease, Creutzfeldt–Jakob disease, and Huntington’s dis
ease [17]. By now, it has become obvious that degradation
of intracellular proteins is a specific process. The stability
of an intracellular protein varies widely, depending on
both the properties of the protein itself and its environ
ment. In the middle of the 20th century, protein degrada
tion was postulated as probabilistic and uncontrollable,
but now it is commonly accepted that protein breakdown
is a very complex, tightly controlled in time and space
process that plays an essential role in most metabolic
pathways.
UBIQUITIN–PROTEASOME SYSTEM
Proteasome
Proteasome is a multisubunit proteinase complex
responsible for the degradation of most intracellular pro
teins. In addition to the breakdown of obsolete and dam
aged proteins, proteasomes regulate many important cel
lular processes via controlled degradation of transcription
factors, cell cycle regulators, enzymes, etc. Eukaryotic
proteasome contains three catalytic subunits: β1, β2, and
β5, each having its own substrate specificity. Besides, var
ious cell types contain proteasome variants with different
activity and specificity profiles; the functions of these
proteasomes have not been fully understood yet. Many
inhibitors of proteasomal proteolytic activity are current
ly used as drugs in the treatment of hematologic malig
nancies; potential application of proteasome inhibitors in
the treatment of other diseases, including autoimmune
disorders, is being actively studied.
The proteasomal degradation signal represents a
chain of Ub molecules covalently bound to the target pro
tein. The signal specificity is determined by the length
and structure of the polyUb chains that are recognized by
receptors of the proteasome regulatory particles. It has
become evident that the Ubmediated modification sys
tem is far from simple, because at least seven functional
lysine residues in the Ub sequence can be involved in the
polyUb chain growth. After polypeptide substrate binds
to the proteasome via the Ub tag, its hydrolysis starts at
the unstructured initiation site, one of the elements of the
protein degradation signal.
In addition to the Ubdependent degradation, there
is also Ubindependent degradation. In this case, the
degradation signal is either a specific motif in the protein
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sequence or some auxiliary molecule. Currently, about
two dozen proteins are known to be hydrolyzed without
preliminary modification with Ub. For example, some
protooncoproteins and oncosuppressive proteins are
Ubindependent proteasomal substrates, whose degrada
tion might have a tumorigenic effect. Identification of
Ubindependent degradation mechanisms suggests the
existence of multiple protein degradation pathways.
Proteasome structure and function. Most proteins in
the cytoplasm and nucleus of eukaryotic cells are
hydrolyzed by proteasome. Proteasome is a 2.5MDa
multisubunit protein complex that controls the content of
more than a hundred regulatory proteins and destroys
misfolded and damaged proteins [18]. Proteasomes are
present in all eukaryotic cells, as well as in some bacterial
species, which confirms their high significance for nor
mal cell activity [19]. Just as protein synthesis is regulat
ed at various levels, an equally complex system exists to
control protein hydrolysis by the proteasome. That is why
proteasomes are incredibly selective. In principle, the
proteasome is most likely able to hydrolyze any cellular
protein modified by Ub; at the same time, the risk of acci
dental degradation of other components of cellular pro
teome is excluded.
The proteasome complex consists of two functional
and structural parts. The name proteasome refers to differ
ent types of particles with different molecular weights.
The 20S proteasome, which is the catalytic core of the
proteasome, is capable of binding many regulatory parti
cles, such as the 19S regulator, PA28αβ, PA28γ, PA200,
Emc29, PI31, etc., thereby forming various proteasome
types known as the 26S proteasome (19S–20S), 30S pro
teasome (19S–20S–19S), hybrid proteasome (19S–20S–
PA28), PA28 proteasome (PA28–20S–PA28), etc. [20].
Free 20S proteasome does not hydrolyze structured pro
teins, but cleaves small peptides and unfolded protein
molecules [21]. Evaluation of the relative content of all
proteasome complexes in HeLa cells using immunopre
cipitation and immunoblotting revealed that about 40%
of all 20S proteasomes were in the free form, while the
remaining fraction was associated with PA28 and 19S
regulators in various combinations [22]. Higher levels of
free 20S proteasome were found in U947 cells treated
with γinterferon (IFNγ) [23]. Electronic cryotomogra
phy provided information on the number of 26S and 30S
proteasomes in neurons lacking the PA28 regulator; it
was found that a quarter of all proteasomes were 30S pro
teasomes, while the rest were 26S proteasomes [24].
Given specific functions of many cell types in mammals
and the fact that some cell types lack certain proteasomal
regulatory particles, it is highly likely that each cell type
contains its own individual pool of proteasomal complex
es [25]. Thus, the 20S proteasomal complex associated
with the neuronal plasma membrane through GPM6 gly
coproteins was recently found [26]. This complex can
degrade intracellular proteins into biologically active
extracellular peptides that induce calcium signaling via
NMDA receptors.
20S proteasome. According to the Xray diffraction
data, the 20S proteasome consists of four heptameric
rings forming a hollow cylinder approximately 1517 nm
in length and 11 to 12 nm in diameter [27]. Each ring
contains seven subunits with molecular masses from 20 to
35 kDa; the molecular weight of the total complex is
~750 kDa. The outer rings are composed of αsubunits,
and the inner ones are composed of βsubunits. Four
stacked rings form three internal chambers with a diame
ter of ~5 nm [28]. The volume of the central proteolytic
chamber is approximately 84 nm3; it can accommodate
proteins up to 70 kDa, but the entrance to the chamber is
restricted by a small width of the entrance opening [29].
The presence of two copies of 14 different subunits is a
common feature of all eukaryotic 20S proteasomes.
Proteasomes with a simpler composition but with the
same basic architecture (one type of αsubunits and one
type of βsubunits) were found in archaea (Thermoplasma
acidophilum) and some bacteria (Rhodococcus erythropolis
and Mycobacterium tuberculosis) [30]. However, the com
position of such proteasomes may vary: one αsubunit
type and two βsubunit types, two αsubunit types and
two βsubunit types, or two αsubunit types and one β
subunit type. Among the species that synthesize two β
subunit types, one of the two βsubunits is believed to be
inactive in Sulfolobus, Pyrobaculum, and Aeropyrum.
The central chamber of the eukaryotic 20S protea
some contains six catalytic sites formed by two β1, two
β2, and two β5 subunits. Therefore, the proteasome has
three main types of proteolytic activity: trypsinlike
(cleavage after positively charged amino acid residues),
chymotrypsinlike activity (cleavage after aromatic
amino acid residues), and caspaselike (cleavage after
negatively charged residues). Since the proteasome also
possesses some additional activities, it can cleave the
polypeptide chains between almost any amino acid
residues. The catalytic mechanism of the peptide bond
hydrolysis involves an attack of the carbonyl carbon atom
by the hydroxyl of the Nterminal threonine of the pro
teasome catalytic subunit followed by hydroxyl reactiva
tion with a water molecule (Fig. 1).
A distinctive feature of large intracellular proteases,
such as the proteasome or tripeptidyl peptidase II
(TPPII), is the active site location in the internal cavity
formed by the complex subunits (compartmentalization).
Isolation of the active sites from the cellular environment
prevents undesirable degradation of cellular proteins [31].
The proteasome assembly from subunits is a complex
multistep process. In cells, βsubunits are synthesized as
inactive precursors with longer Nterminal sequences
compared to the mature βsubunits. The Nterminal pep
tides are signals for protein factors involved in the protea
some assembly and also prevent premature activation of
βsubunits. During the assembly, relatively stable inter
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mediates, preproteasomes, are formed first that consist of
one αring and a partially assembled ring composed of
the βprecursors and auxiliary factors. Then, the two
intermediates join to form the 20S proteasome. The N
terminal propeptides are autocatalytically cleaved off,
releasing the threonine residues of the catalytic sites and
simultaneously sequestering them from the cellular envi
ronment [32]. The entrance to the 20S proteasome prote
olytic chamber is blocked by the Nterminal hydrophobic
regions of αsubunits, which also prevents spontaneous
hydrolysis of proteins. It was shown by atomic force
microscopy that the gates to the catalytic chamber are in
a dynamic equilibrium between open and closed states,
with the equilibrium being shifted towards the closed state
[33]. Therefore, the spatial configuration of the 20S pro
teasome and a small size of the openings leading into the
catalytic chamber prevent degradation of folded proteins.
αSubunits interact with the 19S regulatory complex
and PA28 and PA200 activators that induce conforma
tional changes in αsubunits leading to opening of the
entrance to the catalytic chamber [34]. It was found that
the 20S proteasome can be activated by certain low
molecularweight compounds [35]. In addition, deletion
of the Nterminal portion of the α3subunit (α3ΔN)
leads to the activation of the proteasome in mammalian
cells. Because the proteasomal degradation is the main
mechanism that regulates the level of toxic aggregated
proteins [36], an increase in the proteasomal activity due
to the opening of the proteolytic chamber may prevent
the toxicity of such proteins and suppress associated
development of proteotoxic disorders (e.g., Alzheimer’s
disease) [37]. For example, cells expressing α3ΔN protea
somes have a lower level of tau proteins and their aggre
gates [38].
proteasome
proteasome
proteasome
proteasome
1. Nucleophilic attack
2. Acylenzyme
3. Hydrolysis
of acylenzyme
4. Reaction product
removal
Fig. 1. Catalytic mechanism of the peptide bond cleavage by the proteasome. The mechanism of catalysis was revealed by Xray diffraction,
and the catalytically important residue was identified by mutagenesis. During the reaction, a proton of the threonine hydroxyl group is trans
ferred to the amino group of the same amino acid, and the water molecule in the active site acts as a base. Similar mechanism underlies activ
ity of three other enzymes: penicillin acylase (Nterminal serine in the active site), glutamine amidotransferase (Nterminal cysteine in the
active site), and aspartyl glucosaminidase (Nterminal threonine in the active site).
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In addition to the proteolytic activity, the 20S pro
teasome (especially, the α5subunit) exhibits the RNase
activity in the presence of divalent cations (not required
for the proteolytic activity) [39]. The RNase activity of α5
subunit strongly correlates with the extent of phosphory
lation of α6 and α7 subunits and markedly increases dur
ing erythroid differentiation and programmed cell death
[39]. Most αsubunits (except α3) are also known to
exhibit RNase activity against p53 mRNA in vitro [40].
In mammals, five additional subunits, β1i, β2i, β5i,
β5t, and α4s, have been found [41]. The genes encoding
β1i and β5i are located in the MHC (major histocompati
bility complex) class II gene region on chromosome 6, and
their transcription is induced by the proinflammatory
cytokines, e.g., INFγ[42]. Subunits β1i, β2i, and β5i are
stably expressed in spleen and hematopoietic professional
antigenpresenting cells due to continuous activation of
the INFγinducible promoter of the β1i and β5i genes via
binding of the unphosphorylated transcription factor Stat1
dimer to the IRF1 protein [43]. These subunits replace the
constitutive subunits of the 20S proteasome and change its
proteolytic specificity. The 20S proteasome containing
β1i, β2i, and β5i subunits is called immunoproteasome; it
lacks the caspaselike activity, but displays elevated
trypsinlike and chymotrypsinlike activities compared to
the constitutive proteasome. A set of antigenic peptides
produced by the immunoproteasome differs from a set of
peptides produced by the constitutive proteasome [44, 45].
Due to the difference in the substrate specificity between
the constitutive proteasome and the immunoproteasome,
the antigenic peptides produced by the immunoprotea
some have hydrophobic Ctermini and therefore, are more
structurally suitable for loading onto the MHC class I mol
ecules. Cells containing the immunoproteasome present
antigens on their surfaces more efficiently [42].
There are cases of the socalled proteasomemediat
ed peptide splicing, when the two peptides (proteasomal
cleavage products) that are normally not adjacent in the
original protein, are ligated by the proteasome. This addi
tional proteasome activity contributes to the expansion of
the pool of epitopes presented on MHC class I. Peptide
splicing can occur by either transpeptidation or conden
sation [46]. The mechanism of peptide splicing by the
proteasome is not completely understood yet. It is only
known that splicing does not occur spontaneously, but
requires certain conditions to be met, e.g., one of the
peptides should contain lysine or arginine at the Ctermi
nus [47]. Splicing of peptides from the same protein is
more likely than splicing of peptides from different pro
teins [48]. There is evidence that peptide splicing is not a
rare event, as previously assumed, and spliced peptides
account for one third of the peptidome diversity generat
ed by the proteasome for the presentation on MHC class
I molecules [49].
In many nonimmune cells, expression of protea
some immune subunits depends on interferons, TNFα,
and liposaccharides [42]. However, their biosynthesis can
also be induced by less specific physiological triggers –
aging and environmental stress factors, such as heat
shock. Therefore, in addition to the generation of epi
topes for MHC class I, immunoproteasomes are involved
in other cell processes, rapid elimination of damaged pro
teins after oxidative stress, cell proliferation control, and
cytokine production [50]. Hence, immunoproteasomes
can either aggravate and alleviate the course of various
diseases, such as viral infections, colitis, myocarditis, and
diabetes.
Although IFNγinducible αsubunits are unknown,
there is an alternative α4 subunit that was designated as
α4s, because it was localized exclusively to spermatocytes
differentiated from male germ cells [51]. Also, the β5t
subunit expressed exclusively in the cortical thymic
epithelial cells was found. This subunit replaces the β5i
subunit, leading to the β1iβ2iβ5t configuration called
the thymoproteasome. The mechanism of the β5t expres
sion regulation is still unknown [52]. The chymotrypsin
like activity of thymoproteasomes is lower than that of
constitutive proteasomes and immunoproteasomes,
which is related to the hydrophilic nature of the substrate
binding pocket in β5t [53]; in addition, these proteasome
types differ in their susceptibility to proteasome inhibitors
[54]. It is believed that the thymoproteasome increases
the repertoire of peptides for the positive selection of T
cells during their development in the thymus [55] (Fig. 2).
26S proteasome. The 26S proteasome is the largest
and most complex member of the ancient superfamily of
ATPdependent proteases [56]. These proteases are char
acterized by the presence of the AAA ATPase (ATPase
associated with diverse cellular activities) ring responsible
for the substrate unfolding and translocation through a
narrow channel into the proteasome proteolytic chamber.
The ATPase ring converts chemical energy of ATP
hydrolysis into mechanical force for substrate unfolding.
Most often, the term 26S or 30S proteasome refers to the
20S proteasome associated with one or two, respectively,
PA700 (protein activator) or 19S regulatory complexes
containing many additional specialized subunits in addi
tion to the ATPase ring.
The regulatory particles of the proteasome are
responsible for the binding, deubiquitination, unfolding,
and transfer of the substrates to the proteasome catalytic
chamber and for opening of the channel in the αsubunit
ring. The 19S particle contains at least 19 subunits with a
total molecular mass of about 1 MDa. This regulatory
complex may be divided into two subcomplexes called the
lid and the base. The base consists of nine subunits (regu
latory particles); of these, six are homologous AAA
ATPases (Rpt16) and three are nonATPases (Rpn1,
Rpn2, and Rpn13). Rpt16 form a heterohexameric ring
that directly contacts the αsubunit ring of the 20S pro
teasome [57]. Rpn1 and Rpn2 are the two largest struc
tural proteasomal subunits. The central parts of these sub
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units consist of 11 αhelical repeats that presumably form
the frame for the accommodation of the lid subunits and
the substrates. Rpn13 and Rpn10 can bind Ub and act as
receptors of ubiquitinated substrates [58, 59]. Recent
studies have also assigned Rpn1 to the Ubbinding sub
units [60]. In addition to the resident proteasome sub
units, the 19S particle is associated with a large number of
proteins indirectly involved in the degradation process.
Some of these proteins (Rad23, Dsk2, and Ddi1 in yeast)
contain Ublike (UBL) and Ubassociated (UBA)
domains and function as alternative Ub receptors. The lid
consists of nine different Rpn subunits: Rpn3, Rpn59,
11, 12, and Sem1 (Rpn15). Rpn11 is a deubiquitinating
enzyme in the proteasome [61]. The lid is structurally
similar to the COP9 signalosome and eIF3 complexes of
translation initiation factors [62]; it plays an important
role in the stabilization of the entire 26S proteasome
complex and also integrates and coordinates the func
tioning of different proteasomal components by allosteric
regulation [63]. Binding of a ubiquitinated substrate caus
es many structural changes in the 19S regulatory com
plex. The most noticeable changes revealed by the cryo
electron microscopy include expansion of the channel in
the ATPase ring, as well as its alignment with the entrance
to the 20S proteasome complex. Similar structural
changes also occur upon binding of the nonhydrolyzed
adenosine triphosphate analogue ATPγS, that “freezes”
the enzymatic complex in a state that the proteasome
temporarily acquires after binding ATP [64]. Biochemical
and structural studies have revealed that the entrance to
the channel leading to the 20S proteasome proteolytic
chamber opens when the Cterminal HbYX motifs of
three ATPase subunits (Rpt2, Rpt3, and Rpt5) of the 19S
regulatory complex bind to the lysine residues in the
intersubunit pockets in the 20S proteasome outer ring
formed by the αsubunits. This interaction occurs after
Positive selection
Positive
selection
Golgi apparatus Golgi apparatus
thymo
proteasome
immuno
proteasome
peptides peptides
cTECs β5tdeficient cTECs
TAP TAP
MHC I MHC I
ER
Fig. 2. The unique catalytic subunit β5t. Together with β1i and β2i, β5t is forms the thymoproteasome with reduced chymotrypsinlike activ
ity. This proteasome type plays an important role in the positive selection of MHC Ispecific T cells, which significantly expands the final
repertoire of cytotoxic lymphocytes. cTEC, cortical thyroid epithelial cell; ER, endoplasmic reticulum; MHC I, major histocompatibility
complex class I; TAP, the transporter associated with antigen processing.
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BIOCHEMISTRY (Moscow) Vol. 84 Suppl. 1 2019
ATP binds to the ATPase ring subunits and triggers dis
placement of the Ntermini of the αsubunits, which
opens the entrance to the proteolytic chamber for the
substrate. The Cterminal parts of the remaining three
ATPase subunits lacking the HbYX motifs support the
association between the regulatory particle and the 20S
proteasome subunits.
During the last decade, highresolution electron
microscopy combined with Xray crystallography of indi
vidual proteasome subunits and generation of homolo
gous models have been used to determine the structures of
the 19S regulatory particle and the entire 26S proteasome
at the nearatomic resolution [65, 66] (Fig. 3).
According to the latest data, six ATPase subunits
(Rpt16) form a ring that anchors to the 20S particle.
Each ATPase consists of three domains: the extended α
helical domain, followed by the oligonucleotide and
oligosaccharidebinding domain (OB domain), and the
AAA+ domain [67] containing elements necessary for
ATP binding and hydrolysis. All six OB domains, togeth
er with the AAA+ domains, form two concentric rings.
The Cterminal tails of the AAA+ domains are located in
recesses on the 20S proteasome surface, opening the
entrance to the catalytic chamber (AAAring). The N
terminal αhelices of the AAA+ domains form three pairs
of coiled coils that constitute the outer border of the
cylindrical part of the 26S proteasome (Nring) (Fig. 4).
The ATPase ring accounts for about 2/3 of the total vol
ume of the 19S regulatory particle and provides a link
between the Ub receptors and the ATPase motor [65, 66].
The structural subunit Rpn1 is attached laterally to
the outer side of the ATPase ring, near the 20S subunit;
Rpn2 is attached to the top on the opposite side. Ub
receptors Rpn10 and Rpn13 are located at the protea
some periphery, where they can bind the substrates.
Rpn13 is associated with Rpn2 and situated at the maxi
mal distance from the proteasome center. A quarter turn
away from Rpn13, a little closer to the ATPase ring, there
is Rpn10 attached to Rpn11 deubiquitinase. Therefore,
the active sites are located along the proteasome central
Fig. 3. Proteasome structure. According to the Xray structural analysis (PDB 5GJR), the 20S proteasome is a hollow cylinder of 1517 nm
in length and 1112 nm in diameter, consisting of four rings lying on top of each other, with two identical peripheral rings formed by αsub
units (lilac), and two identical central rings formed by catalytic βsubunits (violet). Each of the rings consists of seven 2035 kDa subunits.
PA700 (also called the 19S regulatory complex) consists of 19 different protein subunits and contains two basic elements in its structure: the
lower (base) element consists of six subunits differing in structure and having ATPase activity (Rpt16, blue), Rpn2 structural subunit, and
three Ubbinding subunits, Rpn1, Rpn10 and Rpn13 (yellow; Rpn13 is not shown due to low resolution). PA700 binds directly to the αring
of the catalytic core and provides unfolding of the polypeptide chain of protein substrates. The energy of ATP hydrolysis is spent on the
polypeptide chain unfolding and its translocation into the catalytic chamber. The upper element consists of nine nonATPase subunits: Rpn3,
59, 1112 (gray). Rpn11 (orange) scans the substrate polypeptide chain and removes the polyUb chain.
26S proteasome
ββring
ααring
ATPase ring
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axis, on the way into the proteolytic chamber, according
to the order of substrate conversion. Lid structural sub
units (Rpn3, Rpn5, Rpn6, Rpn7, Rpn9, and Rpn12)
form a Ushaped frame connecting most of the proteaso
mal elements: Rpn2, Rpn11, ATPase ring, and αring of
the catalytic particle. The lid plays an important role in
the stabilization of the entire 26S proteasome complex
[68]; it also integrates and coordinates the functions of
different proteasome parts through allosteric regulation
[63, 69].
The amount of 20S proteasomes in the cell can be
increased via 26S proteasome disassembly into its compo
nents: 20S proteasome and 19S regulator. Several studies
have demonstrated that such disassembly actually takes
place under oxidative stress, when there is a need to
increase the efficiency of damaged proteins degradation
[70]. Apparently, the disassembly process may involve dif
ferent proteins. In mammalian cells, Hsp70 chaperone is
involved in the stabilization of the 19S regulator after its
dissociation from the 20S proteasome, as well as in the
reassembly of functional 26S proteasomes after oxidative
stress [71]. It was also found that low cellular levels of
Hsp90 cause almost complete disassembly of yeast 26S
proteasome and, correspondingly, an increase in the
amount of free 20S proteasomes [44]. It was shown
recently that the contents of 26S and 20S proteasomes
depend on the cell metabolic state. For example, low
NADH/NAD+ratio destabilizes the 26S proteasome
complex, leading to the emergence of free 20S protea
somes [72]. Similarly, a decrease in the ATP content
affects the 26S to 20S proteasome ratio in cells [73].
Substrate hydrolysis by the proteasome is associated
with a significant conformational reorganization of the
Rpt1Rpt6 ATPase subunits. Due to the cascade hydroly
sis of ATP in the substratebinding pockets, the Rpt16
subunits turn like a propeller and align in the horizontal
plane. This global structural reorganization leads to the
alignment of the channel formed by the ATPases and the
axis of the 20S catalytic core. Another important conse
quence of the ATPase rotation is displacement of Rpn11
deubiquitinase to a position directly above the catalytic
chamber entrance, which greatly facilitates scanning of
the polypeptide chain and subsequent cleavage of conju
gated Ub.
Posttranslational modifications of the proteasome.
Recent studies have demonstrated that posttranslational
modifications, such as phosphorylation, Nacetylation,
ubiquitination, myristoylation, glycosylation, ribosyla
ααhelical regions
Nring
AAAring
lid
anchoring Ctermini
coiledcoil
OB domain
AAA+ domain
Fig. 4. Structure of the ATPase ring of the 26S proteasome. Rpt16 subunits with ATPase activity are shown in blue, and the deubiquitinase
subunit Rpn11 is shown in orange. Each ATPase consists of three domains: the extended αhelical region, the oligonucleotide and oligosac
charidebinding domain (OB domain), and the AAA+ domain.
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tion, and proteolytic cleavage of proteasomal subunits,
regulate proteasome assembly and localization and affect
its the proteolytic function [74]. Using modern proteom
ic techniques, about 110 co and posttranslational mod
ifications of the yeast proteasome have been identified,
but only a small part of them have been functionally char
acterized [75, 76]. The number, types, and sites of modi
fications are significantly different in the yeast, human,
and mouse proteasomes despite their high amino acid
homology [76]. Currently known functional modifica
tions of the proteasome are often divided into three types.
Modifications of the first type affect proteasome assembly
and stability (e.g., ADPribosylation of the PI31 factor
facilitates proteasome assembly [77]). Modifications of
the second type directly affect the proteolytic function of
the proteasome. Thus, glycosylation of the ATPase Rpt2
inhibits proteasome by reducing the ATPase ring activity
[78]. Modifications of the third type control proteasome
localization (e.g., Nmyristoylation of Rpt2 in yeast
[79]).
Phosphorylation. Almost all 26S proteasome sub
units are phosphorylated [76]. Phosphorylation of the
20S proteasome αsubunits primarily affects proteasome
stability. For example, phosphorylation of the α7 subunit
promotes association between the 19S regulatory complex
and the 20S proteasome with the formation of the 26S
proteasome. In the presence of INFγ, α7 subunit phos
phorylation is partially suppressed, which leads to the
destabilization of the 26S proteasome complex and,
according to [80], replacement of 19S complex with the
PA28 regulatory complex, although this fact has not been
confirmed in other studies [81]. Phosphorylation of the
regulatory complex subunits predominantly affect the
access of the substrate to the proteolytic chamber. For
example, phosphorylation of the Rpt6 ATPase subunit by
protein kinase A (PKA) or Ca2+/calmodulindependent
protein kinase II (CaMKII) [8284] increases the rate of
substrate degradation. PKA also phosphorylates the non
ATPase Rpn6 subunit, resulting in the acceleration of
substrate hydrolysis in vitro [85].
Ubiquitination. Ubiquitination of proteasome sub
units can be induced by proteasome inhibitors and pro
teotoxic stress [86, 87]. Under these conditions, five E3
ligases (Ube3a/E6AP, Ube3c/Hul5, Rnf181, Huwe1, and
Ubr4) mediating ubiquitination of approximately 14 sub
units were found to associate with the proteasome.
Multiple ubiquitination leads to the elimination of inac
tive proteasomes by autophagy (the socalled pro
teaphagy). However, modification of the Ub receptor
Rpn13, a specific substrate of E3 ligase Ube3c/Hul5,
does not result in proteaphagy. Ubiquitination of lysines
K21 and K34 located in the Nterminal fragment of the
Ubbinding Pru domain of Rpn13 reduces the ability of
Rpn13 to interact with ubiquitinated proteins, thereby
preventing their degradation [87]. Interestingly, partial
inhibition of the proteasome causes specific ubiquitina
tion of Rpn13 both in vivo and in vitro, which suggests that
this modification prevents the binding of ubiquitinated
substrates when the proteasome proteolytic functions are
impaired, although the physiological significance of this
mechanism is not yet completely understood. Another Ub
receptor, Rpn10, was found to be monoubiquitinated.
Monoubiquitination of Ub receptors often blocks their
ability to bind ubiquitinated substrates due to the
intramolecular interaction between the UBD and Ub [88].
This is also true for the Rpn10 subunit [74], whose ability
to recognize proteasomal substrates markedly decreases
upon ubiquitination. While proteotoxic stress upregulates
Rpn13 ubiquitination, the amount of monoubiquitinated
Rpn10 decreases under stress conditions.
Alternative regulators. The PA28 regulator (also
known as REG or 11S) is a 180kDa heptameric ring
shaped complex. It binds to one or both sides of the 20S
proteasome in an ATPindependent manner and signifi
cantly increases its ability to hydrolyze short peptide sub
strates, but not proteins or ubiquitinconjugated proteins.
However, there have been a growing number of studies
that claim that the 20S proteasome with the PA28 regula
tors can also degrade proteins. In addition, PA28 can bind
to the free end of the asymmetric 26S proteasome
(19S–20S) to form a hybrid proteasome (19S–20S–
PA28) that hydrolyzes tri and tetrapeptides at a higher
rate compared to the 26S proteasome [89].
PA28
αβ
.In mammals, PA28 consists of two homol
ogous subunits, PA28α(REGαor PSME1) and PA28β
(REGβor PSME2), whose expression is induced by
INFγ[90]. Professional antigenpresenting cells normal
ly express high amounts of PA28αβ, which is consistent
with the potential involvement of this complex in the
presentation of antigens on the MHC class I molecules
[91]. In addition, this complex is involved in the mainte
nance of protein homeostasis during oxidative stress [92],
regulation of cell growth, and apoptosis. PA28αand
PA28αβ were found to promote presentation of some, but
not all, antigens on MHC class I. Cells lacking this regu
latory complex have a lower ability to present certain
antigens. INFγinduced coordinated expression of
PA28αand PA28β, as well as β1i, β2i, and β5i protea
some immune subunits, leads to the formation of the
PA28αβ–20S immunoproteasomes in vitro and in vivo.
However, PA28αβ was also found in cells, tissues, and
organs lacking immunoproteasomes, e.g., red blood cells
and muscles. PA28αβ also facilitates the release of pep
tides that cannot be presented on MHC class I [93],
which indicates that this complex can function as a regu
lator of autoimmune processes by reducing excessive
cytotoxic response to autoantigens. In addition, PA28αβ
stimulates the degradation by the 20S proteasome of oxi
dized and misfolded proteins, regardless of their ubiquiti
nation [92, 9496].
PA28
γ
.There is also the third subunit – PA28γ
(REGγ, PSME3, or Ki antigen) that forms homohep
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tamers in the nucleus [97]. It is not INFγinducible and
not involved in the antigen presentation on the MHC
class I molecules. It was found that the PA28γand
PA28αβ differ in their activation properties and peptide
cleavage patterns. This difference may be associated with
the tendency of these regulators to bind to different pro
teasome types. Although it is still unclear how PA28γrec
ognizes substrates, it can hydrolyze short peptides as well
as intact, unstructured, or intrinsically disordered intra
cellular proteins via the Ubindependent mechanism. A
number of substrates hydrolyzed by the PA28γprotea
some have been identified; these include SRC3 [98], hep
atitis C virus core protein 3 [99], and inhibitors of cyclin
dependent kinases p21, p16, and p14 [94, 100]. PA28γ
directly interacts with the SRC3 domain of histone acety
lase (HAT) [98] and p21 [94] in vitro. PA28γknockout
mice are characterized by retarded growth, defects in
cellspecific mitosis, and reduced body weight in adults
[101, 102]. Therefore, PA28γcan be associated with the
metabolism and pathogenesis of various diseases via pro
moting Ubindependent substrate degradation.
PA200/Blm10. Another alternative regulator, the
PA200 protein encoded by the Psme4 gene, was discov
ered in rabbit reticulocyte lysate [103]. Its ortholog
Blm10 was found in yeast [104], nematodes, and plants
[105]. The PA200 subunit, a 250kDa highly conserved
protein located in the nucleus, can attached to one or
both ends of the 20S proteasome. In vitro, the proteasome
with this regulator hydrolyzes only short peptides or
unfolded proteins and is involved in the regulation of glu
tamine/glutamate homeostasis in tumor cells [106].
PA200 is present in all mammalian tissues, but it is most
abundant in the testes. It is believed that PA200 facilitates
the maintenance of protein homeostasis in mitochondria
[107]. This hypothesis is supported by the data obtained
in experiments with PA200 knockout mice. Male mice
deficient by PA200 were found to be infertile due to the
impaired spermatogenesis [108]. Further investigation
demonstrated that this regulator also contributes to the
chromosomal stability [109] and participates in the ATP
and Ubindependent degradation of acetylated histones
in somatic cells in response to DNA damage [110]. PA200
and Blm10 contain the bromodomain (BRD)like
regions that recognize acetylation. The BRDlike regions
of PA200 and Blm10 are able to specifically bind acety
lated core histones [110]. Male mice with the
PA200/PA28γdouble knockout are completely infertile,
with pronounced defects in the sperm motility due to the
decreased proteasomal activity [111]. Based on the
impairment of normal spermatogenesis in PA200defi
cient mice, PA200mediated degradation of histones is an
important component in the formation of spermatozoa.
Yeast lacking Blm10 are hypersensitive to DNAdamag
ing agents and exhibit a decreased respiratory capacity
[107]. The lack of Blm10 also results in the elevated sen
sitivity to oxidative stress, increased mitochondrial fis
sion, and reduced degradation of the fission protein
Dnm1 [112]. Blm10 proteasomes also mediate degrada
tion of the ribosomeassociated transcription factor Sfp1
in response to nutrient depletion, thereby affecting ribo
somal biosynthesis [113]. Therefore, the PA200/Blm10
proteasomes can regulate cellular metabolism by enhanc
ing proteasomal activity in the absence of Ub.
PI31. PI31, a 30 kDa prolinerich protein, was first
described as an inhibitor of proteasomal activity. It com
petes with the 19S (or PA28) regulatory particle for the
binding to the 20S proteasome [114]. A study conducted
in Drosophila melanogaster revealed that PI31 in a com
plex with the E3 ubiquitin ligase Nutcracker regulates
proteasome functions, promotes the activity of 26S pro
teasome, and suppresses the activity of free 20S protea
some. However, PI31 overexpression in mouse embryon
ic cells does not affect proteasomemediated hydrolysis
[115]. Instead, PI31 located on the nuclear or endoplas
mic reticulum membranes can selectively inhibit matura
tion of the immunoproteasome precursor complexes,
which in turn reduces antigen presentation by the MHC
class I molecules on the surface of INFγtreated mouse
embryonic cells.
Ecm29. Ecm29 is a 205kDa large protein capable of
binding to the 20S proteasome and regulating its function
by several mechanisms. Direct inhibition of proteasome
activity partially by inhibiting ATPase activity of the 19S
regulatory particle was shown in yeast [116]. On the other
hand, activation of the yeast proteasome by Ecm29 was
also described. Ecm29 was found to promote proteasome
assembly by stabilizing the intermediate 20S–19S com
plex, in which maturation of the 20S proteasome is
delayed due to a temporary lack of some βsubunits [117].
Other studies demonstrated that Ecm29 binds to the 19S
complex in response to the oxidative stress and causes the
26S proteasome disassembly [118]. It was suggested that
the Ecm29dependent disassembly of the 26S proteasome
increases the amount of the 20S proteasome, which
allows the cells to manage large amounts of oxidized pro
teins. In mammalian cells, Ecm29 (encoded by the
KIAA0368 gene) facilitates proteasome dissociation under
oxidative stress. Ecm29 is also associated with various
molecular motors and endosomal components, which
might be related to its ability to transfer 26S proteasomes
to various cell compartments, e.g., endoplasmic reticu
lum and centrosome.
Proteaphagy. The term proteaphagy is used to refer to
proteasome utilization by autophagy [119]. In
Arabidopsis, proteaphagy can is stimulated by nitrogen
starvation (i.e., conditions promoting nonselective
autophagy), while chemical or genetic inhibition of the
proteasome cause selective removal of nonfunctional
proteasomes. Selective elimination of proteasomes is
accompanied by extensive ubiquitination of proteasome
subunits, which promotes their recognition by the
extraproteasomal Rpn10 that acts as a classic selective
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autophagosome receptor by simultaneously binding to
the Ubmodified subunits via the ubiquitininteracting
motif (UIM) and to the autophagosomal membrane pro
tein ATG8 via the ATG8interacting motif (AIM) [119].
In yeast, most proteasomes localize to the nucleus,
whereas autophagy is limited to the cytosol. Furthermore,
yeast Rpn10 does not contain the AIM. Similar to
Arabidopsis, yeast have two proteaphagy pathways that
respond either to the nitrogen starvation or inactivation of
proteasome particles, respectively [120, 121]. Similar to
Rpn10, the Cue5 protein acting as a selective proteaphagy
receptor was identified [120].
Mechanisms of Substrate Recognition by the Proteasome
To be hydrolyzed by the proteasome, a protein
should contain the degradation signal. In the most gener
al case, the degradation signal includes two components:
(i) a site for the recognition and binding by the protea
some and (ii) a site initiating protein unfolding and fur
ther translocation into the catalytic chamber of the 20S
proteasome [122]. In most substrates, the role of the
recognition signal belongs to the polyUb chain.
Ubdependent proteolysis. Ubiquitination system.
Ubiquitin (Ub) is an 8.5kDa signaling protein present in
all eukaryotic cells. Posttranslational modification of
proteins with Ub (ubiquitination) regulates numerous
cellular processes, such as protein degradation, sorting,
localization, and activation and repression of protein syn
thesis. Up to 14 different families of Ub and Ublike pro
teins are known that differ in the amino acid sequences
but have the same characteristic spatial structure.
Ub is attached to the target protein via sequential
actions of several enzymes. Covalent attachment of Ub to
the substrates is catalyzed by three enzymes: E1 (activat
ing enzyme), E2 (conjugating enzyme), and E3 ubiquitin
ligase. The E1 enzyme activates Ub in the twostep ATP
dependent reaction by forming the highenergy E1Ub
thioester complex. Next, activated Ub is transferred to
the Ubconjugating enzyme E2. E3 ubiquitin ligases
include two enzyme types. RING domainbearing E3 lig
ases bind to E2 and to the substrate and bring them to a
distance sufficient for the E2catalyzed Ub transfer to the
substrate; HECT domainbearing ubiquitin ligases inde
pendently catalyze Ub transfer to the amino group of the
target protein. In some cases, E4 ubiquitin ligases that
catalyze elongation of the Ub chain are distinguished in a
separate class.
The specificity of substrate ubiquitination is achieved
due to the hierarchy of the ubiquitination system. In
mammalian cells, there are only two types of Ubactivat
ing E1 enzymes (Uba1 and Uba6 [123]), ~30 conjugating
E2 enzymes, and ~600 E3 ubiquitin ligases. E1 enzymes
activate Ub for all types of E2 enzymes, most of which
interact with several E3 ubiquitin ligases. Usually, E3
enzymes transfer Ub to various substrates with similar or
identical recognition motifs. At the same time, specific
E3 ligases can interact with more than one E2 enzyme,
and some substrates can be recognized by more than one
E3 ligase. Therefore, the ubiquitination system hierarchy
is a complex network distinct from the pyramid (Fig. 5).
There are many ways of protein modification with
Ub, including monoubiquitination (of one or several
residues) and polyubiquitination with Ub chains different
in the linkage type and length. In the Ub molecule, seven
εamino groups of lysine residues K6, K11, K27, K29,
K33, K48, and K63 can form isopeptide bonds. The first
Ub molecule is usually attached to the substrate lysine
residue through the Cterminal glycine residue (G76).
Further chain growth occurs due to formation of isopep
tide bonds between lysines of the already attached Ub and
the Cterminal glycine residue of a new Ub molecule.
Also, Ub can be directly bound to the Nterminal
methionine residue of the substrate, followed by linear
attachment of Ub molecules. The structure of polyUb
chains is extremely diverse. PolyUb chains can be homo
geneous (with Ub molecules connected via lysine residues
in a strictly defined position) and heterogeneous (com
bine different linkage types); in turn, heterogeneous
chains can branch through ubiquitination at several sites
simultaneously [125]. In addition, serine, threonine, and
lysine residues of Ub can be phosphorylated and acetylat
ed [126]. The biological meaning of the ubiquitination
signal depends on the linkage type and polyUb chain
length. The ubiquitination signals are removed by deubiq
uitinating enzymes (DUBs) that cleave off the polyUb
chains from the substrates [127].
Ub is not a simple switch that triggers on/off the
degradation of protein substrates, but rather a finely
tuned signal that can determine the order in which pro
teins involved in the regulatory pathway will be
hydrolyzed. For example, cell advancement through the
cell cycle requires degradation of the regulatory proteins
in a correct order. The timing of protein hydrolysis can be
determined by the ubiquitination time; many E3 ubiqui
tin ligases recognize substrates only when their recogni
tion site is phosphorylated [128]. The order of degrada
tion might be also controlled by the polyUb chain type.
Thus, during the cell cycle, regulatory proteins with
longer polyUb chains are hydrolyzed faster than proteins
modified with shorter polyUb chains [129].
PolyUb chains. Thousands of proteins are ubiquiti
nated in cells, but almost half of them are not degraded by
the proteasome [130]. So it remains unclear how the cell
distinguishes between different Ub signals (Fig. 6). It is
commonly believed that the proteasome recognizes K48
linked polyUb chains containing at least four Ub mole
cules. Protein modification by a single Ub molecule,
polyUb chains linked through other lysine residues (e.g.,
K63), and linear polyubiquitin chains are not associated
with proteasomal degradation, but play a role in various
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cell processes, such as chromatin structure regulation,
membrane transport, and signal transduction. However,
this is not entirely true, as polyUb chains form by binding
via K63 [131] and monoubiquitinated proteins [132] can
be recognized by the proteasome. The binding affinity of
the purified proteasome for the K63linked polyUb
chains is almost the same as for the K48linked polyUb
chains [133]; therefore, the recognition of different
polyUb chains may involve some auxiliary proteins or
chain topology.
Recent advances in proteomic technologies have
provided a map of the Ub proteome [134]. By combining
quantitative proteomics and antidiglycine (diGly) anti
bodies recognizing ubiquitinated peptides in trypsin
hydrolysates, it was revealed that a large number of pro
teins (~5000) in cultured human cells are ubiquitinated
[130]. Several ubiquitination sites were found in ~60%
proteins with about ~4% proteins containing more than
10 modified sites; ~58% of the quantified sites increased
in abundance when the cells were treated with a protea
some inhibitor.
Using isotopelabeled Ub, it was demonstrated that
ubiquitinated substrates exist mainly in the monoubiqui
tinated form [135]. Therefore, proteomic studies have
shown that a significant number of endogenous protea
some substrates can by mono or polyubiquitinated at
several sites rather than modified with a single polyUb
chain. The chains formed through K48 quickly accumu
late in the cells treated with a proteasome inhibitor, while
chains formed through other residues (K6, K11, K27,
K29, K33, and to a lesser extent K63) also became more
abundant, which indicates involvement of such chains in
proteasomal degradation [136].
In line with the in vivo observations, recently report
ed in vitro studies have shown that purified proteasomes
can recognize a very wide range of polyUb chain topolo
gies in different substrates. A singlemolecule approach
was used to demonstrate that the proteasome efficiently
hydrolyzes cyclin B1 modified with several short polyUb
chains [137, 138]. It was found using a reconstituted sys
tem that the large multiprotein E3 ligase APC/C rapidly
modifies substrates with mono, di, and triUb at sever
signaling ubiquitination
ubiquitin precursors
Ub ligase cascade
free ubiquitin pool
proteasome
autophagy
Fig. 5. Schematic representation of the UPS. Ub (yellow ovals) is synthesized as four precursor proteins that are further processed by special
ized deubiquitinating enzymes – ubiquitin isopeptidases (DUBs). The UPS involves tree types of ubiquitin ligase (E1, E2, and E3; see the
text); it is highly specific and selective due to its hierarchical complexity. Ub is conjugated to substrates (S) as a monomer or the polyUb chain
linked through internal lysine residues. The polyUb chain is elongated by E3 ubiquitin ligases or relatively recently discovered E4 ubiquitin lig
ases. There is a dynamic equilibrium between ubiquitination and Ub removal by ubiquitin isopeptidase; the optimal polyUb chain length is
about six Ub residues per substrate molecule, according to the modern concept [124]. Next, the ubiquitinated substrate binds to the protea
some subunits Rpn10, Rpn13, and Rpn1 either directly or with the involvement of shuttle proteins of the UBLUBA family; there may also be
a variant of specific autophagy. Some Ub molecules enter the proteolytic chamber together with the substrate, where they get degraded. In most
cases, resident proteasomal deubiquitinase Rpn11 successfully removes the entire polyUb chain, which is then cleaved into monomers for recy
cle.
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al sites [139]. In addition, cyclin B1 ubiquitinated with
chains of a certain length was produced and then used to
monitor the average dwell time on the proteasome by the
total internal reflection fluorescent microscopy (TIRF)
of individual molecules. The substrate binding increased
exponentially for the first three Ub units in any configu
ration (several monoUbs or short polyUb chains with four
to nine Ub molecules arranged linearly). Among poten
tial configurations with four Ub molecules, substrates
modified with two K48linked diUb chains were degrad
ed more efficiently than those modified with one K48
linked tetraUb chain. These findings were confirmed in a
later study that used a model GFP substrate fused to an
unstructured region [140]. At the same time, K48linked
polyUb chains are not always associated with substrate
degradation. In yeast, transcription activators Met4 [141]
and Cdc34 [142] can be ubiquitinated with long K48
linked polyUb chains, but they are not hydrolyzed by the
proteasome. Several short chains linked through K11,
K27, and K63 also contribute to the substrate binding to
the proteasome, which suggests that the proteasome does
not distinguish among the chain types in the case of sub
strate multiple ubiquitination. It is likely that the local
concentration of Ub units, but not the type of linkage, is
important for the substrate recognition by the protea
some.
In cells, APC/C with two different E2 ligases,
UBE2C and UBE2S, can synthesize branched ubiquitin
chains with K11 and K48linkages [143]. APC/C sub
strates modified with branched K11/K48linked chains
are degraded more efficiently than those containing
homotypic chains formed through either K48 or K11
linkages [143, 144]. In addition, K11linked chains are
involved in the processes unrelated to proteasomal degra
dation. Thus, branched K11/K63linked chains promote
internalization of MHC class I molecules by endocytosis
[145]. There is evidence that in yeast, K11linked polyUb
chains are as abundant (28% of all ubiquitin chains) [136]
as the K48linked chains; however, they are much less
common in unsynchronized mammalian cells (25% of
all ubiquitin chains) [135, 146]. As found recently, cells
contain a significant amount of branched K48/K63
linked polyUb chains [147]. The number of these chains
increases after cell treatment with a proteasome inhibitor,
thereby indicating that branched chains of this type are
proteasome degradation signals. It was also demonstrated
that the amount of K11/K48linked chains increases dur
ing proteotoxic stress caused by the inhibition of the pro
teasome, HSP70, or HSP90. In some cell types, includ
ing differentiated neurons, aggregates of newly synthe
sized and misfolded proteins are modified with the
K11/K48linked polyUb chains, which suggests the func
tion of these chains as markers for substrate degradation
by the proteasome. In addition, it was shown that patho
logical 73Qhuntingtin (HTT), but not benign 23QHTT,
is modified with the K11/K48linked chains and under
goes rapid proteasomal degradation. However, K11/K48
linked chains are redistributed during the proteotoxic
stress leading to the emergence of a large amount of
unfolded proteins; they modify newly synthetized
unstructured proteins, which in turn results in the accu
mulation of unmodified 73QHTT [148].
In yeast cells, K63linked polyUb chains account for
~16% of all linkages in yeast cells and, thus, rank third in
abundance after K48 and K11linked chains. Earlier, a
simple ubiquitination system was developed in [149] that
uses the HECTtype E3 ligase Rsp5 and a degron with the
PY motif. The Sic1 protein containing the PY motif
(Sic1PY) was overubiquitinated with long K63linked
chains under the action of Rsp5 ligase; the ubiquitinated
Sic1PY was rapidly degraded by the proteasome. This sys
tem has been widely used to analyze the proteasomal
function [60, 87]; however, as mentioned above, K63
linked chains do not participate in the proteasomal degra
dation in cells. One of the explanations for this phenom
enon may be the effect of UBDcontaining proteins that
Fig. 6. Different types of polyUb chains and their modifications. Ubiquitinconjugated substrates may be divided into three main types:
monoubiquitinated, polymonoubiquitinated, and polyubiquitinated. In addition, there is homotypic ubiquitination, when each polyUb
chain contains only one linkage type, and heterotypic ubiquitination, when Ub molecules can be connected through two or more different
types of linkages, thereby creating a mixed chain. Also, Ub can be modified at two or more sites, forming branched chains. In addition, Ub
can be conjugated with ubiquitinlike modifiers, such as SUMO, NEDD8, and ISG15; it can also undergo posttranslational modifications
(PTM), such as phosphorylation and acetylation.
Substrate
monoUb polymonoUb Homotypic chains Heterotypic chains Additional modifications
mixed branched Ublike proteins PTM
Signal complexity
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specifically recognize the K63links in vivo. In addition,
polyUb chains formed via K63 residues on endogenous
substrates are too short to be recognized by proteasomal
subunits or adaptor proteins.
Linear polyUb chains bind to the proteasome less
efficiently than chains with the K48links [150]. The only
known E3 ligase capable of assembling linear polyUb
chains is the linear Ub chain assembly complex (LUBAC)
that consists of three proteins: the catalytic RNF31 sub
unit (HOIP) [151], RBCK1 (HOIL1L), and SIPL1
(SHARPIN) [152154]. OTULIN (FAM105B or
GUMBY) is the main deubiquitinating enzyme (DUB)
with a high specificity for linear polyUb chains [155].
Linear polyUb chains are not sufficient for protein degra
dation by the proteasome in vitro [140]; however, they
were found to act as degradation signals in yeast and mam
malian cells [156, 157] (target proteins were either artifi
cially modified with such chains, or natural proteasomal
targets were used, such as protein kinase C and TRIM25
[158]). Recently, ATR, BRAP, LGALS7, PLAA, SEPT2,
HDAC6, VDAC1, and TRAF6 proteins were found to be
modified by linear polyUb chains. For example, the mod
ification of TRAF6 is necessary for proper functioning of
the IL1βdependent NFκB signaling pathway [159].
Interestingly, the type of ubiquitination required for
proteasomal degradation correlates with the size and
structural features of the substrate. Thus, monoubiquiti
nation is sufficient to induce proteasomal degradation of
small (<150 amino acids) structurally disordered proteins
in reticulocyte lysates [132, 160]. Recent proteomic study
using yeast and mammalian cells in which all Ub was
replaced by modified Ub lacking lysine residues and inca
pable of forming polyUb chains [161] showed that many
proteins (~25% in yeast and ~50% in human cells) were
degraded due to single or multiple monoubiquitination.
Furthermore, the degraded substrates shared some com
mon features; in particular, most of them were small in
comparison with polyubiquitinated proteins. Monoubi
quitinated substrates are more common among the pro
teins involved in carbohydrate transport and oxidative
stress, which may indicate that they are monoubiquitinat
ed by specific E3. In another study [162], 1392 ubiquitina
tion sites were identified in 794 proteins in HEK293T
cells. The ubiquitination sites were evenly distributed
throughout all identified proteins without significant pref
erence to the N or Cterminus. No obvious correlation
was found between the number of ubiquitination sites and
protein length. Also, 506 of the 794 identified proteins
were modified by a single lysine residue, while the remain
ing 288 proteins contained several Ubconjugated lysine
residues. Chaperones HSP701 and HSC71 had 22 and 15
ubiquitinated lysine residues, respectively; however, the
biological significance of this extensive ubiquitination
remains a mystery.
While physiological functions of homotypic polyUb
chains have been characterized, heterotypic chains
remain poorly understood [163]. In addition to the
K11/K48linked chains, four other heterotypic chain
types have been described: K29/K48, K11/K63,
K48/K63, and M1/K63. The existence and physiological
role of other heterotypic combinations still remain an
open question. Previous studies demonstrated that the
presence of both linkage types in the branched K11/K48
linked chains facilitates their recognition by proteasomal
UBDs and, thereby, ensures priority access of the modi
fied substrates to the proteasome. However, signal ampli
fication due to the polyUb chain elongation is rather
unlikely; to the contrary, the binding of a long chain over
a large area might be perceived by the UBDcontaining
receptor as a decrease in the overall signal level.
Deubiquitination system. PolyUb chains can be elon
gated or shortened even in the proteasomebound sub
strates due to the activity of E3 and E4 ubiquitin ligases
and deubiquitinating enzymes (DUBs). All currently
known DUBs are cysteine proteases or metalloproteases
that specifically hydrolyze the isopeptide bond immedi
ately after the Cterminal Ub residue (Gly76). About 100
DUBs have been found in mammalian cells; at least four
of them are associated with the proteasome (Rpn11,
Ubp6, UCH37, Doa4). The isopeptidase activity of free
DUB is rather weak, but it increases when the enzyme
binds to the proteasome. DUBs associated with the pro
teasome can allosterically affect certain proteasomal
functions, such as ATPase activity, opening of the prote
olytic chamber entrance, and substrate degradation.
These enzymes remove or gradually shorten polyUb
chains on the substrate, thereby limiting its dwell time on
the proteasome. Consequently, a protein that is difficult
to hydrolyze due to its compact folding or poorly accessi
ble initiation sites will be uncoupled from the proteasome
within a certain period of time after failed attempts to
degrade it, thereby freeing up the space for the next sub
strate molecule. In this regard, inhibitors of proteasome
associated DUBs, e.g., Ubp6/Usp14, are very promising
as potential agents for treatment of neurodegenerative
diseases because they improve the ability of proteasome to
hydrolyze degradationresistant substrates presumably
due to a longer time of their association with the protea
some [164]. Interestingly, inhibition of Ubp6/Usp14 also
accelerates proteolysis of oxidized proteins and enhances
cell resistance to oxidative stress. On the contrary,
inhibitors of other DUBs can cause accumulation of
ubiquitinated proteins [165], which makes them potential
drugs for the treatment of malignant tumors. In this case,
the biological effect of such inhibitors is generally similar
to that of proteasome inhibitors that have already been
successfully used in the treatment of multiple myeloma
[166].
Determining the detailed structure of the 19S regu
latory particle by the cryoelectron microscopy allowed to
suggest the mechanism of DUB activation upon binding
to the proteasome, effect of DUBs on the 19S complex
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conformation, and their role in the substrate degrada
tion.
Rpn11. The most important DUB is the proteasomal
regulatory particle lid subunit RPN11 belonging to the
JAMM family of Zn2+ metalloproteinases. [61]. Rpn11
contains the catalytic JAMM motif that has also been
found in seven other human proteins, such as the Csn5
subunit of the COP9 signalosome, AMSH, AMSHLP,
the BRCC36 subunit of BRISC, MPND, and MYSM1
[167169]. The conservative JAMM domain contains the
EXnHS/THX7SXXD sequence, in which His and Asp
residues are fixed by coordination with the Zn2+ ion,
while the fourth coordination position is occupied by a
water molecule that forms the hydrogen bond with the
conserved Glu residue. Zn2+ ion acts as a Lewis acid and
enhances the nucleophilicity of the bound water, enabling
the nucleophilic attack on the isopeptide bond [168, 170].
Rpn11 is essential for proteasome activity and cell viabil
ity [171]. Mutations His109Ala and His111Ala in the cat
alytic site do not disrupt the assembly or conformation of
the proteasome, but largely inhibit the proteasomal func
tions and are lethal in yeast [61]. Rpn11 is located just
above the entrance to the ATPase ring [65]; therefore,
substrate deubiquitination occurs before Ub loses its con
formation. It is believed that due to this location, Rpn11
is uncapable of cleaving bonds between internal Ub mol
ecules in the polyUb chain because of steric hindrances
[172]. Deubiquitinase Ubp6 is nonstoichiometrically
associated with the proteasome and cleaves the bonds
between the Ub molecules in the polyUb chains [173].
Due to the presence of MPN (Mpr1/Pad1 Nterminal)
domain, Rpn11 forms a heterodimeric complex with
Rpn8 [174, 175]. The crystal structure of the
Rpn11–Rpn8 heterodimer revealed that the Insert1 loop
in Rpn11 is capable of blocking the access to the deubiq
uitinase active site, but the same loop also participates in
the isopeptide bond cleavage. Rpn5 stabilizes the inactive
state of Rpn11, thereby preventing its deubiquitinating
activity until Rpn11 is incorporated in the proteasome
[176]. Ub binding induces conformational rearrange
ments in Rpn11, and the Insert1 loop transits from the
closed inactive state to the βhairpin state, thereby open
ing the access to the active site. This transition is a rate
limiting step in deubiquitination and significantly accel
erates mechanical translocation of the substrate to the
ATPase ring. Therefore, rapid removal of Ub from the
captured substrates reduces the risk for Ub to be
hydrolyzed together with the substrate [177].
Ubp6/Usp14. Yeast deubiquitinase Ubp6 and its
highly conserved mammalian homologue Usp14 are asso
ciated with the 19S regulatory complex lid [178]. Ubp6
was initially identified as proteasomal DUB by using the
proteasome inhibitor ubiquitin vinyl sulfone (UbVS, Ub
derivative modified at the Cterminus) that binds to the
catalytic cysteine residue of this deubiquitinase [179].
Removal of Ubp6 from the proteasome accelerates degra
dation of model substrates in vitro [180] and is not lethal
in Saccharomyces cerevisiae [181]. However, this removal
slows down cell growth due to the elevated degradation of
proteasomal substrates and depletion of free Ub [182].
Ubp6knockout embryonic fibroblasts display an upregu
lated deubiquitinating activity of Rpn11, as well as an
increased number of Rpn13 and Uch37 deubiquitinase
subunits in the proteasome. In addition, proteasomes
lacking Ubp6 hydrolyze nonubiquitinated proteins more
actively [183]. In the absence of ubiquitinated substrates,
Ubp6 suppresses basal hydrolysis of ATP and nonubiqui
tinated substrates. Inactive proteasomes do not hydrolyze
ATP; the probability of the nonspecific proteolysis
decreases, while the specificity of hydrolysis of ubiquiti
nated substrates increases. Interaction with the protea
some activates Ubp6 about 300fold [182]. In the pres
ence of the nonhydrolyzed analogue ATPγS, Ubp6 shifts
the conformation of the 19S regulatory particle to that
similar to the substratebound state (s3), which leads to a
2fold increase in the efficiency of cleavage of the sub
strate UbAMC (Ub7amino4methylcoumarin)
[184]. Before the substrate is captured by the proteasome,
the Nterminal UBL domain of Ubp6 binds to Rpn1,
while its catalytic USP domain can take differently posi
tions. Ubp6 interaction with the ATPase ring stimulates
its deubiquitinating activity, probably by changing the
conformation of two surface loops (BL1 and BL223). The
activity of Ubp6 further increases upon the capture of the
ubiquitinated substrate [184, 185]. Ubbound Ubp6
maintains the coaxially aligned state of the Nring,
AAA+ ring, and 20S proteasome [184]. Therefore, ubiq
uitinbound Ubp6 stimulates the ATPase activity, pro
motes the opening of the entrance to the 20S proteasome,
and facilitates inhibition of the substrate capture by desta
bilizing the s1 (substrateacceptor) state or preventing the
proteasome from returning to this conformational state
[182, 184, 185]. Taking these facts together, Ubp6 plays a
key role in the allosteric regulation of the proteasome,
partly through its binding to Ub. It was also was shown
that Ubp6containing proteasomes are able to recognize
substrates with multiple ubiquitination, while protea
somes lacking Ubp6 do not exhibit this activity. Ubp6 and
Rpn11 cleave K1 and K63linked chains with equal effi
ciency that increases with the chain elongation. In the
case of K48linked chains, an inverse dependence is
observed. Ubp6 removes K48linked chains more effi
ciently than Rpn11 [186]; the cleavage occurs only when
more than one chain is attached to the substrate [173].
Ubp6 cannot remove the closest to the substrate Ub mol
ecule regardless of the substrate positioning probably due
to steric hinderances. If the chain bypassed by Ubp6 is
short, then the substrate hydrolysis is suppressed; if the
chain length is sufficient, then the proteasomesubstrate
interaction will be preserved and the chain will be
removed by Rpn11 after initiation of the substrate
translocation [173]. Therefore, Ubp6 is most likely
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involved in the removal of the excessive Ub molecules
from the substrate [173].
Uch37. Cysteinedependent deubiquitinase Uch37
was identified as a subunit of the 19S regulatory complex
in Homo sapiens and D. melanogaster. This enzyme was
also found in Schizosaccharomyces pombe (Uch2), but not
in S. cerevisiae. Like Ubp6, Uch37 is activated by binding
to the proteasome. The crossover loop of the Uch37
active site blocks the catalytic cysteine [187], but interac
tion with the Cterminal deubiquitinase adaptor domain
(DEUBAD) of Rpn13 stabilizes the loop in one state,
which increases protein affinity for Ub [188, 189]. In the
extraproteasomal Rpn13, the DEUBAD interacts
intramolecularly with the Pru domain and decreases the
affinity of this receptor subunit for Ub. This interaction is
disrupted by Rpn13 binding to the proteasome, which
makes the above domains accessible for Ub binding and
facilitates Uch37 activation [190]. These facts suggest
that Rpn13 activates Uch37 only in the proteasome com
plex [188, 189]. In the proteasome, Uch37 is able to
cleave distal K48, K6, and K11linked polyUb chains.
It was suggested that Uch37 can edit ubiquitinated sub
strates and facilitate removal of insufficiently ubiquitinat
ed and very slowly hydrolyzed ubiquitinated proteins
from the proteasome [191]. In addition, Uch37 can
remove regulatory Ub from proteasome subunits [86],
cleave polyUb chains, and vacate Ubbinding proteasome
receptors, thus allowing continuous substrate loading
[192]. Despite a large distance between this deubiquiti
nase and the ATPase ring [193], Uch37 also stimulates
the gate opening and the activity of proteasomal ATPases,
which suggests that Uch37, like Ubp6, is able to affect the
proteasome conformational state [194]. These data
demonstrate close allosteric relationships between all
proteasome subunits. The yeast protein Doa4 is another
DUB associated with the 19S regulator, but this associa
tion is less strong than proteasome interactions with
Ubp6 and Uch37 [195].
E3 ubiquitin ligases were shown to be capable of
binding to the proteasome [196]. In particular, E3 ubiqui
tin ligase Hul5 is associated with Ubp6 in the 19S regula
tory complex and regulates the protein degradation rate
by counteracting the activity of Ubp6, i.e., by catalyzing
elongation of polyUb chains [197]. It is important to note
that Ub attachment to the substrate directly in the pro
teasome makes substrate degradation more processive
because it allows to avoid formation of partially degraded
protein fragments [198] through reubiquitination as the
proteasome moves along the polypeptide chain of extend
ed proteins [199].
Ublike proteins. Some Ublike (UBL) proteins,
sometimes called Ublike modifiers (ULMs), are struc
turally similar to Ub and can be conjugated to other pro
teins in the reactions catalyzed by enzymes that are simi
lar (but not identical) to the E1E3 enzymes of the ubiq
uitination system. ULM functions include a wide variety
of processes, such as autophagy, nuclear protein transfer,
alignment/segregation of replicated chromosomes, DNA
repair, and various signaling pathways. Each member of
this family has a βgrasp fold consisting of a fivestranded
βsheet that is partially wrapped around the central α
helix [200]. UBL proteins are divided into two classes:
type I proteins modify the substrates (SUMO, NEDD8,
ATG8, ATG12, URM1, UFM1, FAT10, and ISG15 fam
ily proteins) [201], while type II UBLs are usually com
ponents of multidomain proteins – many of them are
found in some E1 activating enzymes, E3 ligases, and
Ub/Ubl proteases. For example, Hub1 and Esc2 proteins
comprising the autonomous Ubl domain may also be
considered as type II UBLs because they do not form
conjugates with the substrates [202, 203].
SUMO. Modification of proteins through attach
ment of the small ULM SUMO (sumoylation) plays an
essential role in the regulation of various cellular process
es, such as nuclear transport, transcription, DNA repair
and replication, apoptosis, stabilization of protein mole
cules, and cell advancement through the cell cycle [204].
Like ubiquitination, SUMO conjugation to the substrate
occurs through the isopeptide bond formation between
the Cterminal glycine residue in the SUMO molecule
and εamino group of a lysine residue in the substrate
molecule. Pathogens and stress (e.g., heat, oxidative
stress, insufficient blood supply) lead to the global
changes in the sumoylation pattern. Impaired sumoyla
tion and disorders in the sumoylation regulation con
tribute to the development of serious disorders, such as
malignant neoplasms and heart failure [205].
FAT10. Covalent modification by the Ublike FAT10
protein (human leukocyte antigenF adjacent transcript
10) facilitates Ubindependent proteasomal degradation
of proteins [206]. Like other ULMs, FAT10 is bound to
the substrates through the isopeptide bond formation by a
cascade of E1 and E2 enzymes, in which UBA6 (also
called UBE1L2, E1L2, or MOP4) activates FAT10 and
USE1 (UBA6specific E2) conjugates it to the substrates
[207, 208]. FAT10 is involved in several important cellu
lar processes, including apoptosis and NFκB activation.
Basal expression of FAT10 is most pronounced in the
immune system organs, such as thymus, liver, lymph
nodes, and spleen. FAT10 expression can be induced by
proinflammatory cytokines IFNγand TNF; its expres
sion is increased in dendritic cells during their maturation
[206]. Mice lacking FAT10 are viable and fertile, which
indicates a weak dependence of constitutive functions on
FAT10 [209]. However, lymphocytes of FAT10deficient
mice are more prone to spontaneous apoptotic death;
furthermore, FAT10knockout mice demonstrate a high
sensitivity to endotoxins [210]. According to some data,
FAT10 itself can undergo Ubindependent hydrolysis by
the proteasome due to its ability to interact with the VWA
domain of the Ubbinding subunit Rpn10 [206].
However, an alternative hypothesis exists that FAT10 is
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degraded by the proteasome with the involvement of Ub
[211].
Ub receptors. Ubiquitinated substrates are recog
nized by Ub receptors before undergoing proteasomal
degradation. The Ub receptors can be classified accord
ing to their association with the proteasome: components
of the 19S regulatory complex and nonproteasomal pro
teins that bind ubiquitinated substrates and deliver them
to the proteasome.
A) Proteasomal Ub receptors. Three subunits of the
19S regulatory complex were shown to bind ubiquitinated
substrates: Rpn13 [59], Rpn1 [60], and Rpn10 [212].
Rpt5 [213] and Rpn15 [214] are also considered as poten
tial Ub receptors because they have been demonstrated to
bind Ub; however, whether these proteins recognize ubiq
uitinated substrates targeted to proteasomal degradation
still remains unclear.
Ub receptors are located at a distance of 7080 Å
from the entrance to the Nring of ATPases, which
approximately corresponds to four Ub molecules in the
polyUb chain. The process of substrate translocation into
the proteolytic chamber starts through the Nring. The
arrangement of Ub receptors at the proteasome complex
periphery provides the proteasome with the ability to cap
ture substrates with diverse geometry of polyUb chains
and folded domains for their more efficient binding,
unfolding, and deubiquitination.
Rpn10. Rpn10 (S5a/PSMD4 in mammals) was the
first identified Ub receptor. It binds polyUb chains and
UBL domains of substrates through the UIMs [215, 216],
while its affinity for free Ub is much lower. Rpn10 is a
unique Ub receptor because it is able to function both in
the proteasomeassociated and free state, as it has been
shown for D. melanogaster, S. cerevisiae, and Arabidopsis
thaliana [217]. Rsp5dependent monoubiquitination
mediates Rpn10 dissociation from the proteasome and
leads to its accumulation in the cytoplasm [74]. Free
Rpn10 has been suggested to act as an adaptor (shuttle)
protein that binds ubiquitinated substrates, thereby
increasing proteasomal processivity and presumably
compensating for a low diffusion capacity of this large
proteolytic complex [218]. Proteasomebound Rpn10
captures polyubiquitinated substrates, while its free form
exhibits high affinity for the UBL domains of Dsk2 [217]
and Rad23/hHR23 proteins [219]. Monoubiquitination
of Rpn10 regulates its ability to bind substrates, because it
promotes intramolecular interactions that reduce the
ability of the Rpn10 UIM to bind ubiquitinated proteins
[220]. Rpn10 contains the Nterminal von Willebrand A
(VWA) domain that facilitates Rpn10 binding to the pro
teasome and promotes degradation of some ubiquitinated
substrates [221]. Rpn10 also has two UIM domains
(UIM1 and UIM2) that enable Rpn10 to bind K48 and
K63branched polyUb chains [222]. The yeast ortholog
of Rpn10 has only one UIM that recognizes K48linked
polyUb chains [223]. Disruption of Ub binding to the Ub
receptor does not cause the death of S. cerevisiae cells,
but results in the accumulation of polyubiquitinated pro
teins [59, 60]. In higher eukaryotes, deletion of the Rpn10
UIM was found to be lethal in Mus musculus. Rpn13
deficient mice are viable, but have tissuespecific defects
in proteasome functions [224, 225], which probably indi
cates the existence of specific substrates of some Ub
receptors.
Rpn13. Rpn13 was initially identified as a subunit of
the 19S regulatory complex in yeast. In mammals, it was
first discovered as a 46kDa membrane glycoprotein
(called ADRM1/GP110), whose expression was induced
by IFNγduring cell adhesion [226]. ADRM1/GP110 was
later identified as a subunit of the 19S regulatory particle
and its homology to the yeast Rpn13 was demonstrated
[227]. Rpn13 binds ubiquitinated substrates through its
Nterminal pleckstrinlike domain [58]. Another impor
tant region of Rpn13 is the KEKE motif of the Ctermi
nal DEUBAD that binds and activates Uch37 deubiquiti
nase. Together, they function as an editing complex that
removes extra Ub monomers and thereby limits the length
of polyUb chains in ubiquitinated substrates [228] to the
length optimal for substrate association with the protea
some in order to ensure efficient substrate degradation.
Rpn13 and Uch37 exert a synergistic effect on the degra
dation of ubiquitinated substrates, such as the inhibitor of
NFκBα(IκBα) and inducible nitric oxide synthase
(iNOS) [229]. Mice deficient by Rpn13 are viable, but
they are sterile and display decreased proteasomal activi
ty in the testes and brain. In addition, Rpn13 knockout
leads to an increase in the body fat and abnormal levels of
the growth hormone and follicular stimulating hormone
[225]. Rpn13 is associated with the degradation of liver
kinase B1 (LKB1), a key regulator of autophagy [230],
which may explain how Rpn13 is involved in the regula
tion of fat deposits degraded by autophagy [231]. The lack
of Rpn13 reduces recruitment of Parkin to the 26S pro
teasome and delays the autophagosomal clearance of
mitochondrial proteins [232], thereby indicating that
Rpn13 might be involved in the regulation of mitophagy.
Overexpression of Rpn13 promotes the growth of cancer
cells and their migration, whereas inhibition of Rpn13
induces apoptosis and suppresses tumor progression
[233]. Elevated Rpn13 levels are associated with multiple
myeloma [234] and other types of malignant tumors,
including ovarian [235], colorectal [236], and gastric
[237] cancers. Two lowmolecularweight Rpn13
inhibitors, RA190 [238] and KDT11 [239], were devel
oped for the treatment of malignant neoplasms. Unlike
proteasome inhibitors that target proteasome catalytic
subunits, bisbenzylidene piperidone RA190 inhibits
Rpn13dependent functions. It covalently binds to cys
teine 88 of the Pru domain in Rpn13 and disrupts inter
domain interactions in the protein [238]. RA190 can
cause apoptosis associated with the endoplasmic reticu
lum stress in multiple myeloma cells. It is important to
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note that application of this inhibitor may help to avoid
serious clinical complications that accompany the use of
Bortezomib and Carfilzomib [240]. KDT11 is struc
turally different from RA190 and interacts with a specific
Rpn13 region with medium affinity (KD ~2 μM). This
inhibitor exhibits a synergistic effect with Bortezomib in
the combination therapy of multiple myeloma [239].
Rpn1. Rpn1 was identified as a Ub receptor only
recently [60]. It contains two proteins binding sites, T1
and T2. UBLUBA proteins and polyUb chains bind to
T1, while the UBL domain of Ubp6 binds to the T2 site
[60, 241]. This binding facilitates Rpn1 association with
its substrates, adaptor proteins, and DUBs followed by
substrate binding. Rpn1 is a part of the regulatory particle
and interacts with the 20S proteasome [65]. Rpn1 and
Rpn2 form αhelical toroidal solenoids that serving as
substraterecruiting sites. Rpn1 contains nine segments
known as leucinerich repeats (LRRs) [242]. Rpn1, like
other Ub receptors, is able to interact with the adaptor
proteins, including Ddi1 and Ubp6. For example, bind
ing of Ddi1 to the proteasome depends directly on the
modification of D517 residue in the LRR1 domain of
Rpn1 [243].
B) Nonproteasomal Ub receptors. In addition to the
stoichiometric proteasomal Ub receptors, the nonpro
teasomal adaptor proteins UBLUBA can act as Ub
receptors. At present, there are three known families of
yeast mediator proteins that have homologues in higher
eukaryotes and act through a similar mechanism: Rad23,
Dsk2, and Ddi1. Each of these proteins contains the UBL
domain interacting with the proteasome, as well as one or
two ubiquitinassociated (UBA) domains that bind
polyUb chains. This UBLmediated interaction occurs
by binding to the Rpn1, Rpn13, or Rpn10 subunits. The
role of shuttle proteins in proteasomal degradation is con
troversial: depending on the concentration, they can
either accelerate and inhibit substrate hydrolysis.
Overexpression of Dsk2 inhibits proteolysis and has a
cytotoxic effect [217] that is reduced by binding of the
UIM domain of nonproteasomal Rpn10 to the UBL
domain of Dsk2.
Ubiquilins, eukaryotic orthologs of yeast Dsk2, are a
family of four UBL proteins that function as adaptors.
Ubiquilins facilitate degradation of damaged proteins
after oxidative stress. Mutations in ubiquilin impairing its
ability to bind to Rpn10 increase the amount of ubiquiti
nated proteins in the cell, which in turn leads to the pro
tein aggregate formation that may be associated with the
pathogenesis of some neurodegenerative diseases (e.g.,
amyotrophic lateral sclerosis, Huntington’s disease, and
Alzheimer’s disease [244]).
Rad23 contains two UBA domains: the central
UBA1 domain and the Cterminal UBA2 domain that
bind mono and polyubiquitinated substrates with differ
ent affinities. UBA1 binds K63linked polyUb chains
with higher affinity than K48linked chains, while the
UBA2 domain preferably binds K48linked chains [245].
In addition, ubiquitinated proteins associated with Rad23
are protected from subsequent modifications (e.g., elon
gation) of their polyUb ubiquitin chains, as well as from
deubiquitination. This stabilizing effect is supposed to
provide an efficient substrate delivery to the proteasome
[246]. Rad23 also participates in the endoplasmic reticu
lumassociated degradation (ERAD) through association
between its Rad4binding domain and deglycosylase
Png1 with the formation of a complex mediating protea
somal degradation of specific endoplasmic reticulum pro
teins [247].
Despite the fact that UBLUBA proteins directly
interact with the proteasome, they themselves are not
hydrolyzed by it. The stability of Dsk2 and Rad23 is
explained by the presence of the Cterminal UBA
domains protecting unstructured initiation sites in these
proteins [248, 249]. According to some data, the unstruc
tured initiation site of Rad23 cannot initiate hydrolysis,
regardless of the presence of the UBA domain, which may
indicate that proteasome has certain preferences for the
amino acid composition of the unstructured sites [142].
The p62 protein (sequestosome 1) is an adaptor pro
tein [250] that binds ubiquitinated substrates with its C
terminal UBA domain and attaches to the proteasomal
Rpt1 and Rpn10 subunits with its Nterminal PB1136
domain, thereby facilitating the delivery of protein sub
strates (e.g., tau protein) for proteasomal degradation
[251]. p62 also acts as a Ub receptor in autophagy by
directly binding to the LC3 protein, a known mediator of
autophagosome formation [252]. The role of p62 as a Ub
receptor in both proteasomal and autophagymediated
degradation of ubiquitinated proteins is also confirmed by
the fact that downregulation of endogenous p62 leads to
the accumulation of ubiquitinated proteins.
p97/VCP/Cdc48p. In addition to proteasomal Ub
receptors and UBAUBL adaptor proteins, there are
other proteins that can bind ubiquitinated substrates and
recruit them for proteasomal degradation. The p97 pro
tein [also known as valosincontaining protein (VCP) in
mammals and Cdc48p in S. cerevisiae] is an evolutionar
ily conserved ATPase present in all eukaryotes and
archaebacteria. p97/VCP/Cdc48p belongs to a large
ATPase family called AAA+ ATPases. Enzymes of this
family often act as chaperones promoting protein fold
ing/unfolding. p97 is also involved in DNA synthesis and
repair, membrane fusion, mitotic spindle disassembly,
autophagy, and proteasomal degradation. Given an
important role of this enzyme in protein quality control,
p97 mutations can cause some neurodegenerative dis
eases [253].
p97/Cdc48p has two AAA ATPase domains (D1 and
D2, respectively). A short linker (D1D2 linker) connects
the two ATPase domains, and another linker (ND1 link
er) connects D1 to a large Nterminal domain. A short
(~40amino acid) region is attached to the Cend of the
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D2 domain. Interactions of p97/Cdc48p with partner
proteins are mainly mediated by the Nterminal domain,
although some proteins bind to p97/Cdc48p at its Cter
minus. Six Cdc48 monomers form a double ring sur
rounding the central pore. Although D1 and D2 domains
are homologous in both their sequence and structure,
they have different functions. For example, only the D1
domain, but not the D2 domain, is required for the p97
hexamer assembly.
In mammalian cells, p97 is located mainly in the
cytoplasm in fractions associated with the membranes of
subcellular organelles, such as the endoplasmic reticu
lum, Golgi apparatus, mitochondria, and endosomes.
Apparently, its perimembrane localization is mediated by
some membrane receptors that have not been identified
yet. p97/Cdc48p is also present in the nucleus, where it is
involved in quality control of nuclear proteins [254]. p97
is one of the most abundant proteins in eukaryotic cells.
In humans, expression of the p97 mRNA is moderately
upregulated in certain cancers; the expression level of p97
mRNA to some extent correlates with the sensitivity of
cancer cells to the p97 inhibitor currently considered as a
potential agent for treatment of malignant transforma
tions [255].
The mechanism of Cdc48/p97 action is poorly
understood, despite its crucial role in many cellular
processes. The best known p97/Cdc48 substrates are con
jugated to polyUb chains and degraded by the 26S pro
teasome. Accordingly, many cofactors and adaptors of
p97/Cdc48 are capable of recognizing Ub conjugates
[256]. The association of Cdc48 with ubiquitinated pro
teins is believed to be achieved by intermediaries (e.g.,
ubiquitin ligase) able to recognize both p97 and Ub. p97
can bind to Ub ligases and deubiquitinases that edit the
polyUb chains by making them suitable for the substrate
to be recognized by the proteasome or, on the contrary, to
avoid hydrolysis. Finally, the p97associated Ub ligase
recruits an adaptor protein that delivers the substrates to
the proteasome. Therefore, p97 dictates the fate of pro
teins and plays the key role in Ubdependent degradation
[257].
Initiation of substrate degradation. The proteasome
recognizes and binds substrates based on the presence of
the Ub tag, but the degradation itself is initiated at the
unstructured initiation site in the substrate [122]. After
substrate binding to the proteasome, the latter initiates
the unfolding of the polypeptide chain starting from the
initiation site and then translocates the substrate into the
proteolytic chamber, where protein degradation occurs.
Bacterial proteases of the AAA+ family recognize
degrons in their substrates due to the loops located in the
center of the ring of ATPase subunits. It is likely that the
proteasome recognizes the initiation sites in a similar way
[258]. In the proteasome, equivalent loops surround the
degradation channel, being at a distance of 3060 Åfrom
the entrance to the proteolytic channel. The channel
entrance is too narrow for the folded proteins to pass
through it; therefore, the unstructured initiation site
should be at least 2030 amino acid residues in length to
be within the reach of the ATPase loops. This length of
the unstructured site is consistent with the results of in
vitro degradation experiments using model proteasome
substrates. These experiments demonstrated that protein
hydrolysis by purified yeast proteasomes occurred faster
when the protein substrate contained an unstructured tail
of approximately 30 amino acids [259]. Therefore, the
loops of the AAA+ domains of the ATPase ring most like
ly function as “lobes”, initiating the substrate unfolding
and pushing it into the proteolytic chamber.
The need for unstructured initiation sites is reflected
in the global protein stability profile. At least 30% eukary
otic proteins contain unstructured regions that are neces
sary for these proteins to perform their functions in cells
[260]. There is bioinformatic evidence that proteins con
taining unstructured regions have on average shorter half
lives than proteins lacking these regions [261, 262]; how
ever, this correlation should be confirmed by more exper
imental data, since a number of other studies failed to
find it [263265]. Furthermore, it was found that the sites
of substrate ubiquitination are predominantly located in
unstructured protein regions [266, 267]. Even when the
protein lacks an unstructured region, ubiquitination itself
can cause local protein unfolding near the ubiquitinated
residue, which can create the initiation site for the pro
teasome [268].
The Ub tag and the initiation site do not have to be
in the same polypeptide chain. They can function sym
batically while being located in different polypeptide
chains of the protein complex, so that the ubiquitinated
subunit can facilitate degradation of its unubiquitinated
partner [269]. In this case, the ubiquitinated subunit acts
as an adaptor that binds to the proteasome and targets the
associated protein to proteolysis. Presumably, UBLUBA
proteins function in a similar way, acting as nonstoichio
metric Ub receptors for the proteasome [178].
On the other hand, the proteasome is able to
rearrange protein complexes by hydrolyzing only ubiqui
tinated subunit and leaving other proteins in the complex
intact [270, 271]. This rearrangement is important for
many regulatory processes. For example, during the cell
cycle in yeast, the proteasome removes the cyclin
dependent kinase inhibitor Sic1 from its complex with
cyclin and cyclindependent kinase by hydrolyzing Sic1
exclusively [272]. Shortly thereafter, cyclin is ubiquitinat
ed and then hydrolyzed, leaving the intact but inactive
kinase [273]. The most likely site for the degradation ini
tiation is the unstructured substrate region located closer
to the entrance to the proteasome proteolytic chamber.
Indeed, it was experimentally demonstrated that the ini
tiation site should be located at some distance from the
Ub tag to enable protein hydrolysis by the proteasome,
since substrate degradation requires simultaneous binding
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BIOCHEMISTRY (Moscow) Vol. 84 Suppl. 1 2019
of the Ub tag and the substrate unstructured site by the
proteasomal ATPase ring [259].
Processing. Although most substrates are completely
digested by the proteasome, some of them undergo limit
ed proteolysis. Proteasomes are able to recognize the so
called stop signals encoded in the amino acid sequences
of proteins, such as ~60300amino acid glycinealanine
repeats (GArs) found in the EBNA1 protein of the
Epstein–Barr virus. EBNA1 is recognized by the protea
some with subsequent initiation of degradation; however,
its translocation through the proteolytic channel stops
when the GAr region reaches the ATPase ring. Aromatic
hydrophobic loops of the ATPase subunits are incapable
to properly interact with glycine and alanine residues due
to their small size, which causes polypeptide chain slip
ping followed by interruption of substrate translocation
and formation of partially processed products [274]. The
blockade of proteolysis helps the Epstein–Barr virus to
avoid presentation of EBNA1 antigenic peptides to MHC
class I and, therefore, to prevent immune response.
Another wellstudied example of limited proteolysis
is processing of the NFκB transcription factor p50 sub
unit from its p105 precursor. NFκB can exist as a homo
or heterodimer, with the p50–p65 complex being its most
common form. The p105 precursor can inhibit the NF
κB activity by forming dimers with proteins of the NFκB
family. The precursor blocks the Nterminal nuclear
localization signal of NFκB, thereby keeping the protein
in the cytoplasm. Activation of the NFκB signaling
pathway triggers the Ubdependent processing of p105 by
the proteasome, which leads to the hydrolysis of the C
terminal fragment of p105 and release of the nuclear
localization signal. After hydrolysis, p50 dimerizes with
p65 to form active NFκB complex and translocates to
the nucleus, where it regulates transcription. The block
ade of the proteasome during p105 processing into p50
occurs with the involvement of the stop signal consisting
of a glycinerich sequence followed by the tightly folded
Rel homology domain [198]. Similar mechanisms of lim
ited degradation were described for other transcription
factors, such as Spt23 and Mga2 in yeast and Cubitus
interruptus (Ci) in D. melanogaster. However, p105 can
also undergo complete hydrolysis, which is difficult to
explain in terms of the above concept alone. Recently, it
was shown that in addition to the previously known ubiq
uitinligase βTrCP, p105 can be modified by the alterna
tive KPC1 ubiquitin ligase [275]. The fate of p105 was
found to be determined by the type of ubiquitin ligase
involved in its modification, which suggests that these
enzymes perform different types of ubiquitination, and,
as a consequence, different signals are generated.
Ubindependent proteolysis. Most cellular proteins
are degraded by the proteasome with the involvement of
Ub; however, a number of proteins can be degraded with
out ubiquitination [276]. In the latter case, the substrate
should have alternative ways for association with the pro
teasome, such as auxiliary molecules or specific protein
regions (degrons). Degrons are minimal structural ele
ments that mediate interaction between protein substrates
and the proteasome resulting in protein degradation.
Despite the importance of these elements in proteostasis,
the number of known Ubindependent degrons is very
small.
The first discovered and most studied example of
Ubindependent proteasomal substrate is ornithine
decarboxylase (ODC), a protein involved in the biogene
sis of polyamines. Regulation of the polyamine levels is
important for proper cell proliferation, and increased
polyamine concentrations are associated with various
pathological processes, including carcinogenesis.
Therefore, the intracellular levels of ODC should be
strictly controlled. The optimal content of polyamines in
eukaryotic cells is maintained by a negative feedback
mechanism. High spermine and spermidine concentra
tions lead to the increased expression of the antizyme
(AZ) that replaces one of the ODC molecule in the cat
alytically active homodimeric complex, thereby inhibit
ing the enzymatic activity of ODC. In addition, AZ bind
ing changes the conformation of ODC, which results in
the exposure of its unstructured Cterminal fragment and
significantly acceleration of ODC hydrolysis [277, 278].
ODC degradation is carried out by the 26S proteasome,
but does not require Ub involvement. The 37amino acid
Cterminal fragment of ODC contains the proteasome
binding site and the unstructured hydrolysis initiation site
[277, 278]. The attachment of this ODC fragment to pro
teins that are not proteasomal substrates facilitates their
degradation via the Ubindependent pathway.
One possible explanation for the Ubindependent
degradation is that unstructured protein regions bind
closely enough to the ATPase ring loops, so that Ub is not
required for the association between the substrate and the
proteasome. This mechanism can be considered as a vari
ant of a common proteasomal degron that lacks one of
the components, namely, the ubiquitin tag, and resembles
degrons in archaea and bacteria [281].
The mechanisms of Ubindependent hydrolysis are
poorly understood. It is possible that proteins cleaved by
the isolated 20S particles in the absence of ATP in vitro
[282] are hydrolyzed by either the 20S proteasome acti
vated by alternative regulatory complexes [100] or even
the 26S proteasome under natural conditions [283].
Proteins belonging to Ubindependent proteasome sub
strates have long unstructured regions. Moreover, two
alternative proteasomal degradation mechanisms, Ub
dependent and Ubindependent, are not mutually exclu
sive; different fractions of the same protein can be
degraded via either of these two pathways.
Most substrates of the 20S proteasome are proteins
whose structure is partially or completely disordered due
to aging, mutations, or oxidation [284]. Native proteins
that contain large unstructured regions (>30 amino acids)
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BIOCHEMISTRY (Moscow) Vol. 84 Suppl. 1 2019
or intrinsically disordered regions (IDRs), as well as pro
teins with a completely unstructured sequence called
intrinsically disordered proteins (IDPs) [285], are also
degraded by the 20S proteasome. The latter group of sub
strates mostly includes key regulatory and signaling pro
teins that promote cell advancement through the cell
cycle and participate in the cell growth regulation and
carcinogenesis. Obviously, the content of these proteins
in the cells should be strictly controlled, because changes
in their concentration may lead to the development of
various diseases [286].
Currently, several proteins are known that are
hydrolyzed by the 20S proteasome due to their binding
directly to it. The IκBαprotein was demonstrated to
interact with the PSMA2/α2 subunit of the 20S protea
some due to the presence of certain repeated fragments in
the IκBαsequence [287], which presumably leads to the
Ubindependent hydrolysis of this protein. Recently, cal
cineurin was demonstrated to interact with PSMA2/α2
and to promote degradation of IκBαvia the Ubdepend
ent pathway [288]. The PSMA4/α3 subunit interacts with
the F protein of the hepatitis C virus, thereby facilitating
Ubindependent hydrolysis of this protein [289]. The
PSMA7/α4 subunit interacts with the REGα/βregulato
ry subunits (PA28α/β), as demonstrated by using a two
hybrid yeast system, as well as by inhibiting proteasome
activation by the hepatitis B virus polypeptide X that
binds directly to the PSMA7/α4 subunit [290]. The C
terminal part of PSMA7/α4 specifically interacts with the
Nterminal region of Rab7 and participates in the cargo
transport at the late stages of endocytosis; however, this
interaction does not facilitate Rab7 degradation [291].
Parkin (E3 ligase involved in the pathogenesis of
Parkinson’s disease) interacts with the Cterminal region
of PSMA7/α4 via its Cterminal IBRRING domain and
can function as an auxiliary protein in proteasomal
hydrolysis of substrates [292]. Also, PSMA7/α4 interacts
with the nucleotidebinding oligomerization domain
containing protein 1 (NOD1), thus promoting NOD1
degradation by the proteasome [293]. PSMA3/α7 also
interacts with the REGα/β(PA28α/β) particles that
mediate proteasome activation together with PSMA1/α1
and PSMA7/α4. Interaction of the p21WAF1/CIP1 C
terminus with PSMA3/α7 facilitates p21 degradation by
the Ubindependent mechanism [294]. Several proteins
were also shown to act as mediators to promote p21
hydrolysis by the proteasome. For example, MDM2 (E3
ubiquitin ligase) does not ubiquitinate p21, but rather
binds to this protein, thereby promoting its binding to the
PSMA3/α7 proteasome subunit [295]. The binding of
p21 to the REGγ(PA28γ) regulatory subunit also medi
ates p21 degradation by the proteasome [94]. The SRC3/
AIB1 protein is a steroid receptor coactivator that inter
acts directly with the PSMA3/α7 subunit [296] or binds
to REGγ(PA28γ) for the proteasomal degradation [98].
MDM2 binds to PSMA3/α7 and promotes interaction
between the retinoblastoma (Rb) protein and this sub
unit, which leads to the Ubindependent degradation of
Rb [297]. The Nterminal region (amino acids 160) of
αsynuclein, a protein involved in Parkinson’s disease,
interacts with the Cterminal region of PSMA3/α7,
thereby ensuring αsynuclein degradation [298].
PSMB6/β1 binds directly to the p27Kip1 protein and
promotes its proteasomal degradation [299]. The Smad1
protein undergoes hydrolysis by the proteasome, both by
the Ubdependent mechanism and without preliminary
modification with Ub [300] via binding PSMB4/β7 and
antizyme (AZ).
The list of cellular proteins whose degradation does
not require preliminary ubiquitination is constantly
expanding. Proteins that have already been described in
detail [283, 301] include ornithine decarboxylase (ODC),
p21, p53 whose degradation is inhibited by NAD(P)H
quinone oxidoreductase 1 (NQO1), cFos whose hydrol
ysis is also inhibited by NQO1 [302], Fra1 that directly
interacts with the 19S proteasome regulatory particle,
TBP1 containing a Cterminal Ubindependent degron
[303], Rb protein, αsynuclein, HIF1α, SRC3/AIB1
transcriptional coactivator, IκBα, Ybinding protein 1
(YB1), thymidylate synthase (TS), and tau protein
involved in the pathogenesis of Alzheimer’s disease.
Recently, myelin basic protein (MBP), which is one
of the main autoantigens in multiple sclerosis, was found
to undergo proteasomal hydrolysis without preliminary
modification with Ub [304, 305]. Furthermore, the num
ber of proteasome immune subunits was shown to
increase in the central nervous system in mice developing
experimental autoimmune encephalomyelitis (EAE). In
this case, the β1i subunit was localized mainly to oligo
dendrocytes (MBPexpressing cells), and β5i occurred
mainly in cytotoxic lymphocytes penetrating the central
nervous system through the damaged bloodbrain barrier
[306] (Fig. 7). Given the wellknown facts about the
mechanism of proteasome functioning, it may be suggest
ed that MBP binds either to the 19S regulatory particle or
to one of the alternative regulators due to its high positive
charge [307], because the 19S complex subunits are most
ly neutral or negatively charged. In addition, recent stud
ies have demonstrated that the immunoproteasome
hydrolyzes unstructured proteins, such as MBP and his
tones [308], at a higher rate compared to the constitutive
proteasome.
The list of Ubindependent proteasomal substrates
includes proteins that undergo Ubindependent protea
somal hydrolysis due to the interaction with alternative
proteasome activators, mainly REGγ(PA28γ) and
PA200/Blm10. In addition to p21, REGγ(PA28γ) is also
involved in the proteasomal degradation of other cell
cycle regulators, such as p16 (INK4A) and p19 (Arf) [94].
Activationinduced cytidine deaminase (AID), which is
responsible for the initiation of immunoglobulin gene
diversification in activated B lymphocytes, undergoes
S180 KUDRIAEVA, BELOGUROV
BIOCHEMISTRY (Moscow) Vol. 84 Suppl. 1 2019
Ubindependent proteolysis also due to the interaction
with REGγ(PA28γ). The PA200/Blm10 activator binds
through its Cterminal YYX motif to the 20S proteasome
and activates degradation of the tau protein in vitro [309].
More recently, PA200/Blm10 was shown to facilitate Ub
independent degradation of acetylated histones [110].
Some attempts have been made to define the mini
mum requirements for a protein substrate hydrolyzed via
the Ubindependent mechanism [310312]; however, no
generalized mechanism for this process exists so far. To
elucidate this mechanism, it is necessary to identify spe
cific and nonspecific interactions with proteasomal sub
units that mediate Ubindependent substrate degrada
tion. One of the critical problems is identification of pro
teasome subunits in the 20S, 19S, or alternative regulato
ry particles that specifically interact with proteins
hydrolyzed by the Ubindependent mechanism. At the
same time, there is a hypothesis that substrates can enter
the proteolytic chamber through the lateral surface of the
20S subunit via the free space between the α and βsub
units, thereby bypassing the end channels blocked by N
terminal peptides of the αsubunit [27].
Several mechanisms that prevent partially or com
pletely the degradation of partially or completely unstruc
tured substrates have been discovered, e.g., interaction
with the socalled nanny proteins that mask the unstruc
Fig. 7. The role of immunoproteasomes in the development of autoimmune neurodegeneration and promising approaches in targeted ther
apy of this disease. The proteasome is a major proteolytic complex that generates peptides presented on the cell surface by the MHC class
I. The lack of the ubiquitination system control in hydrolysis of the myelin basic protein (MBP) means that the qualitative and quantitative
spectrum of MBP peptides presented on the oligodendrocyte surface is almost completely determined by the proteasome catalytic subunits.
We found that in the brain of immunized mice with experimental autoimmune encephalomyelitis (EAE; an animal model of multiple scle
rosis), the constitutive proteasome was largely replaced with the PA28αβ immunoproteasome under action of IFNγ; the β1i immune sub
unit was preferably localized to oligodendrocytes. An increased content of the immunoproteasome in the brain of EAE mice elevated the
amount of some pathogenic MBP peptides, including the ENPVVHFF peptide that is a fragment of the encephalitogenic MBP region.
Activated CD8+T cells specific for this peptide efficiently lysed oligodendrocytes treated with IFNγ. The specific inhibitor of the β1i
immune subunit (IPSI001) selectively affected the immunoproteasome in vitro and suppressed the of EAE development in experimental
animals. These findings suggest a relationship between the Ubindependent proteolytic cleavage of MBP by the immunoproteasome and
development of multiple sclerosis, as well as indicate the potential of specific immunoproteasome inhibitors as drugs. ER, endoplasmic
reticulum; MHC I, major histocompatibility complex class I; TAP, the transporter associated with antigen processing; TCR, Tcell recep
tor; PAD, peptidylarginine deiminase; CTL, cytotoxic T lymphocyte; β2M, beta2 microglobulin.
immunoproteasome
Oligodendrocyte ACTIVATION
cytotoxicity
Golgi apparatus
TCR
MHC I
myelin
constitutive
proteasome
nucleus
cytoplasm ER
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BIOCHEMISTRY (Moscow) Vol. 84 Suppl. 1 2019
tured regions [313] or oxidationmediated structure sta
bilization [298]. However, the extent of occurrence of
these mechanisms is the subject of further research.
Inhibition of proteasome activity or large proteaso
mal load lead to the activation of autophagy in most cell
types, which clearly indicates a functional relationship
between the two degradation systems. By now, the shift
from proteasomal degradation to autophagy has been well
studied. Such shift provides a compensation for a reduced
proteolytic capacity of the proteasome and eliminates the
risk of accumulation of potentially toxic protein aggre
gates. So far, there is no reliable evidence for the opposite
shift from autophagy to the proteasome that would be
activated by the autophagy impairment. Autophagy inhi
bition caused by the nutrient deficiency in malignant
colon cells increases proteasome proteolytic activity and
expression of proteasomal subunits [314]. However, other
studies revealed no increase in the proteasome activity
upon inhibition of lysosomal functions [315]. Autophagy
inhibition in HeLa cells is accompanied by reduction in
the protein degradation by the UPS due to stabilization of
the p62 protein that, in its basic state, is hydrolyzed
together with the autophagosomal load. Accumulated
p62 sequesters ubiquitinated proteins thus delaying their
translocation to the proteasome; however, this protein
does not affect the proteasome activity [316]. In addition,
the compensatory function of the proteasome in the case
of impaired autophagy is unlikely due to a significant size
of most autophagosome substrates [317].
Apart from the compensatory function of autophagy
in the case of impaired proteasome activity, both systems
share several pathways involved in the coordination
between their activities in proteostasis and organelle
homeostasis. Both UPS and autophagy can affect each
other due to the mutual control of expression levels of
their key components. For example, during the oxidative
stress, the autophagy receptor p62 mediates autophagoso
mal degradation of the E3 ligase Keap1 and degradation of
whole proteasomes by proteaphagy. LC3 can undergo pro
teasomal degradation by the 20S proteasome, but can also
avoid proteolysis using the autophagy receptor p62 [14].
Depending on the cell state, autophagy and proteasomes
can have common substrates and common regulatory fac
tors. Several main regulators of both pathways were shown
to physically interact with each other, thereby providing
mutual control and coordination of the activities [14].
The most significant common feature of both prote
olytic systems is the use of Ub as a degradation signal.
Interestingly, even E3 ligases that have pronounced sub
strate specificity do not always modify substrates exclu
sively for one degradation system. For example, Parkin
E3 ligase, which plays a central role in mitophagy, medi
ates proteasomal degradation of some of its mitochondri
al substrates, while another subset of its substrates under
goes autophagosomal degradation.
Therefore, UPS and ALS together form a machinery
able to monitor the critical states of the cells, to prevent
toxicity resulting from protein misfolding, and to clean
the cells from superfluous proteins and organelles. These
two systems synergistically maintain cellular proteostasis;
moreover, the ALS is able to compensate for the UPS
dysfunction. During the past decades, considerable
efforts have been made to understand the molecular
mechanisms underlying the UPS and ALS. Investigation
of biochemical pathways connecting these two systems
may help to develop new drugs that would enhance cell
sensitivity to intracellular degradation inhibitors in the
anticancer therapy and increase the effectiveness of mis
folded protein degradation in the treatment of neurode
generative diseases. Further investigations of the UPS and
ALS molecular mechanisms and relationships between
them will not only ensure the progress in basic science but
also promote active development of translational medi
cine.
Funding
The work was supported by the Russian Science
Foundation (project 141400585P).
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