Figure - available via license: Creative Commons Attribution 3.0 Unported
Content may be subject to copyright.
The N-terminal region of Ump1 is highly disordered. Far-UV CD spectra of Ump1-C115S and its isolated N-terminal fragment. The difference spectrum for the C-terminal peptide was obtained by subtracting the N-terminal Ump1 spectrum from that of full-length Ump1-C115S. Upon deconvolution the secondary structure of Ump1 N-terminal is 1% α-helix, 25% β-strand, 22% turns and 50% coil. The C-terminal peptide secondary structure corresponds to 18% α-helix, 37% β-strand, 19% turns and 27% coil.
Source publication
Protein degradation is essential for maintaining cellular homeostasis. The proteasome is the central enzyme responsible for non-lysosomal protein degradation in eukaryotic cells. Although proteasome assembly is not yet completely understood, a number of cofactors required for proper assembly and maturation have been identified. Ump is a short-lived...
Similar publications
Production of export-competent mRNAs involves transcription and a series of dynamic processing and modification events of pre-messenger RNAs in the nucleus. Mutations in the genes encoding the transcription and mRNP processing machinery and the complexities involved in the biogenesis events lead to the formation of aberrant messages. These faulty t...
Iron-sulfur (Fe-S) clusters are ancient cofactors present in all kingdoms of life. Both the Fe-S cluster assembly machineries and target apoproteins are distributed across different subcellular compartments. The essential function of Fe-S clusters in nuclear enzymes is particularly difficult to study. The base excision repair (BER) pathway guards t...
GoxA is a glycine oxidase which possesses a cysteine tryptophylquinone (CTQ) cofactor that is formed by posttranslational modifications that are catalyzed by a modifying enzyme GoxB. It is the second known tryptophylquinone enzyme to function as an oxidase; the other being the lysine epsilon-oxidase, LodA. All other enzymes containing CTQ or trypto...
Citations
... Given the abundance and complexity of CPs, it stands to reason that proteasome biogenesis depends on dedicated chaperones that facilitate assembly but are not subunits of mature proteasomes [24][25][26][27][28] . Previous work identified five CP chaperones: proteasome maturation protein (POMP, also called hUmp1) [29][30][31][32][33][34] and proteasome assembly chaperones 1-4 (PAC1-PAC4) [35][36][37][38] . Moreover, five of the seven β subunits (β1, β2 and β5-β7) are synthesized as inactive precursors with N-terminal propeptides, some of which contribute to CP biogenesis [39][40][41][42] . ...
Dedicated assembly factors orchestrate the stepwise production of many molecular machines, including the 28-subunit proteasome core particle (CP) that mediates protein degradation. Here we report cryo-electron microscopy reconstructions of seven recombinant human subcomplexes that visualize all five chaperones and the three active site propeptides across a wide swath of the assembly pathway. Comparison of these chaperone-bound intermediates and a matching mature CP reveals molecular mechanisms determining the order of successive subunit additions, as well as how proteasome subcomplexes and assembly factors structurally adapt upon progressive subunit incorporation to stabilize intermediates, facilitate the formation of subsequent intermediates and ultimately rearrange to coordinate proteolytic activation with gated access to active sites. This work establishes a methodologic approach for structural analysis of multiprotein complex assembly intermediates, illuminates specific functions of assembly factors and reveals conceptual principles underlying human proteasome biogenesis, thus providing an explanation for many previous biochemical and genetic observations.
... The Grand Average hydropathy (GRAVY) indices was identified as 0.532. The molecular weight of a protein has a key role in its biochemical characterization (Sá- Moura et al., 2013) and is an analytical parameter in biochemistry (Jardine, 1990). The aliphatic index denotes the relative volume occupied by aliphatic side chains, which are comprised of alanine, valine, isoleucine and leucine, which contribute to the thermostability of protein (Ikai, 1980). ...
Bromodomains (BRDs) are readers that bind to acetylated lysine residues in chromatin and regulate gene expression. The crystal structure of BRD8 is not elucidated yet. As BRDs play a significant role in cancer, understanding their functional implications is important for drug discovery. The theoretical model is the only reliable technique when there is no crystal structure available. We have investigated the primary and secondary structures of BRD8 (UniProt Id: Q9H0E9|) using the ExPASy (the Expert Protein Analysis System), ProtParam and SOPMA. A model of BRD8 was determined using the template (PDB Id: 3S91) and BLASTP. The stereochemical quality of the model was validated using Ramachandran Plot (97.1% residues in the most favorable region) and ProSA-web (Z score of-5.79). PTM studies show that there are 10 functional sites present in BRD8, of which Protein kinase C and Casein Kinase II sites were abundant. Protein kinase C controls the signaling pathways in proliferation, tumorigenesis and metastasis, whereas Casein Kinase II regulates apoptosis and cell cycle in cancers. We identified three binding pockets in the model. The results from this study show the relevance of BRD8 in cancer research. Functional and structural research can help provide the basis for further studies, which is significant for the pharmaceutical industry as a whole.
... This example corresponds to the yeast proteasome maturation factor Ump1, which is disordered when free in solution, as observed using various experimental techniques. 63,64 It however forms well-ordered secondary structures in complex with the 20 S core particle, playing a key role in the dynamical assembly of proteasome. 65 An additional example of stabilization of 3D structures in complexes is provided in Figure S2b. ...
Order and disorder govern protein functions, but there is a great diversity in disorder, from regions that are – and stay – fully disordered to conditional order. This diversity is still difficult to decipher even though it is encoded in the amino acid sequences. Here, we developed an analytic Python package, named pyHCA, to estimate the foldability of a protein segment from the only information of its amino acid sequence and based on a measure of its density in regular secondary structures associated with hydrophobic clusters, as defined by the Hydrophobic Cluster Analysis (HCA) approach. The tool was designed by optimizing the separation between foldable segments from databases of disorder (DisProt) and order (SCOPe (soluble domains) and OPM (transmembrane domains)). It allows to specify the ratio between order, embodied by regular secondary structures (either participating in the hydrophobic core of well‐folded 3D structures or conditionally formed in intrinsically disordered regions) and disorder. We illustrated the relevance of pyHCA with several examples and applied it to the sequences of the proteomes of 21 species ranging from prokaryotes and archaea to unicellular and multicellular eukaryotes, for which structure models are provided in the AlphaFold2 databases. Cases of low‐confidence scores related to disorder were distinguished from those of sequences that we identified as foldable but are still excluded from accurate modeling by AlphaFold2 due to a lack of sequence homologs or to compositional biases. Overall, our approach is complementary to AlphaFold2, providing guides to map structural innovations through evolutionary processes, at proteome and gene scales. This article is protected by copyright. All rights reserved.
... The CP is assembled from two inactive precursor complexes containing Ump1 in yeast/ POMP in mammals (14)(15)(16). Ump1 is intrinsically disordered and becomes the first substrate during CP maturation (15,17,18). As the second most abundant protein complex in the cell, the proteasome is continuously synthesized during proliferation (19). ...
The ubiquitin-proteasome-system (UPS) fulfills an essential role in regulating protein homeostasis by spatially and temporally controlling proteolysis in an ATP- and ubiquitin-dependent manner. However, the localization of proteasomes is highly variable under diverse cellular conditions. In yeast, newly synthesized proteasomes are primarily localized to the nucleus during cell proliferation. Yeast proteasomes are transported into the nucleus through the nuclear pore either as immature subcomplexes or as mature enzymes via adaptor proteins Sts1 and Blm10, while in mammalian cells, post-mitotic uptake of proteasomes into the nucleus is mediated by AKIRIN2, an adaptor protein essentially required for nuclear protein degradation. Stressful growth conditions and the reversible halt of proliferation, i.e. quiescence, are associated with a decline in ATP and the re-organization of proteasome localization. Cellular stress leads to proteasome accumulation in membraneless granules either in the nucleus or in the cytoplasm. In quiescence, yeast proteasomes are sequestered in a ubiquitin-dependent manner into motile and reversible proteasome storage granules (PSGs) in the cytoplasm. In cancer cells upon amino acid deprivation, heat shock, osmotic stress, oxidative stress, or the inhibition of either proteasome activity or nuclear export, reversible proteasome foci containing poly-ubiquitinated substrates are formed by liquid-liquid phase separation in the nucleus. In this review, we summarize recent literature revealing new links between nuclear transport, ubiquitin signaling and the intracellular organization of proteasomes during cellular stress conditions.
... Human UMP1/POMP is essential for cell viability, and several studies have revealed that point mutations in the coding region or flanking regions are involved in distinct diseases [7][8][9][10][11][12][13]. On the other hand, the UMP1 gene has been considered as a drug target to overcome tumor cell resistance to proteasome inhibitors [14]. Structural studies of recombinant yeast Ump1 protein showed that it behaves similar to a natively unfolded protein with little secondary structure elements [15,16]. Structural studies based on electron microscopy (EM) and crosslinking revealed that, in the 15S PC, Ump1 loops around the inner cavity in a stretched-out conformation at the interface between αand β-rings [6,17]. ...
Biogenesis of the eukaryotic 20S proteasome core particle (PC) is a complex process assisted by specific chaperones absent from the active complex. The first identified chaperone, Ump1, was found in a precursor complex (PC) called 15S PC. Yeast cells lacking Ump1 display strong defects in the autocatalytic processing of β subunits, and consequently have lower proteolytic activity. Here, we dissect an important interaction of Ump1 with the β7 subunit that is critical for proteasome biogenesis. Functional domains of Ump1 and the interacting proteasome subunit β7 were mapped, and the functional consequences of their deletion or mutation were analyzed. Cells in which the first sixteen Ump1 residues were deleted display growth phenotypes similar to ump1∆, but massively accumulate 15S PC and distinct proteasome intermediate complexes containing the truncated protein. The viability of these cells depends on the transcription factor Rpn4. Remarkably, β7 subunit overexpression re-established viability in the absence of Rpn4. We show that an N-terminal domain of Ump1 and the propeptide of β7 promote direct interaction of the two polypeptides in vitro. This interaction is of critical importance for the recruitment of β7 precursor during proteasome assembly, a step that drives dimerization of 15S PCs and the formation of 20S CPs.
... According to the results, RGG1 and AGG1 proteins exhibited extra disordered structure with dominant α-helix content. RGG1 and AGG1 proteins had partially unfolded structures, which may be the reason for providing the functional selectivity to Gβγ dimer [48]. RGG2 protein has a more compact structure, β-strands, and turns. ...
In plants, heterotrimeric G-protein (Gγ) subunits are diverse, and they have structural plasticity to provide functional selectivity to the heterotrimer. Although the Gβ and Gγ subunits dimerize to function in the signaling pathway, the interaction mechanism of various Gγ subunits with the Gβ subunit partners is still elusive.
To better understand the interaction mechanism, one approach is to separate the subunits for the re-assembly in vitro. Hence, developing a reliable method for achieving the efficient production and purification of these proteins has become a need.
In this study, Gγ1 and Gγ2 proteins from Oryza sativa and Arabidopsis thaliana were successfully identified, cloned, expressed in bacteria, and purified as recombinant proteins with the fusion tags. Highly expressed recombinant Gγ subunits in E.coli were digested by proteases, which were also produced in the presented study.
Preliminary structural characterization studies without the Gβ partners showed that Gγ1 proteins have disordered structures with coiled-coil, α-helix extensions, and loops, whereas the Gγ2 protein has a more dominant β-sheet and turns structure. Finally, computational analyses performed on Gγ genes have laid the foundation of new targets for biotechnological purposes.
The proposed optimized expression and purification protocol can contribute to investigations on the Gβγ binding mechanism in plant G-protein signaling. The investigations on selective binding are critical for us to shed light on the role(s) of different plant Gγ subunit types in biological processes.
... This example corresponds to the yeast proteasome maturation factor Ump1, which is disordered when free in solution, as observed using various experimental techniques. 63,64 It however forms well-ordered secondary structures in complex with the 20 S core particle, playing a key role in the dynamical assembly of proteasome. 65 An additional example of stabilization of 3D structures in complexes is provided in Figure S2b. ...
... In solution, free Ump1 is largely disordered 15 . In contrast, Ump1 formed a well-defined density within the 13S and pre-15S with similar local resolution to the rest of the structures (Fig. 2c,d and Extended Data Fig. 4). ...
... Ump1 was identified over 20 years ago 24 , but little was known about its structure. It is intrinsically disordered outside the CP 15 and two low-resolution (~20 Å) structures of CP assembly intermediates could not resolve its structure 14 . Furthermore, most models assumed that Ump1 projected out of the β-ring, an orientation that could have explained its role in promoting CP half-mer dimerization/fusion 24 . ...
The proteasome mediates most selective protein degradation. Proteolysis occurs within the 20S core particle (CP), a barrel-shaped chamber with an α7β7β7α7 configuration. CP biogenesis proceeds through an ordered multistep pathway requiring five chaperones, Pba1–4 and Ump1. Using Saccharomycescerevisiae, we report high-resolution structures of CP assembly intermediates by cryogenic-electron microscopy. The first structure corresponds to the 13S particle, which consists of a complete α-ring, partial β-ring (β2–4), Ump1 and Pba1/2. The second structure contains two additional subunits (β5–6) and represents a later pre-15S intermediate. These structures reveal the architecture and positions of Ump1 and β2/β5 propeptides, with important implications for their functions. Unexpectedly, Pba1’s N terminus extends through an open CP pore, accessing the CP interior to contact Ump1 and the β5 propeptide. These results reveal how the coordinated activity of Ump1, Pba1 and the active site propeptides orchestrate key aspects of CP assembly. Cryo-EM structures of assembly intermediates of the proteasome 20S core particle show how the coordinated activity of chaperones orchestrates early steps in proteasome biogenesis.
... Construction of the β-ring is also aided by binding of the Ump1 chaperone at the center of the α-ring prior to or concomitant with β 2 binding (Figure 3; Ramos et al., 1998;Sá-Moura et al., 2013). Yeast lacking the intrinsically disordered Ump1 accumulate CP precursors, arguing that it plays a positive role in assembly (Ramos et al., 1998). ...
All eukaryotes rely on selective proteolysis to control the abundance of key regulatory proteins and maintain a healthy and properly functioning proteome. Most of this turnover is catalyzed by the 26S proteasome, an intricate, multi-subunit proteolytic machine. Proteasomes recognize and degrade proteins first marked with one or more chains of poly-ubiquitin, the addition of which is actuated by hundreds of ligases that individually identify appropriate substrates for ubiquitylation. Subsequent proteasomal digestion is essential and influences a myriad of cellular processes in species as diverse as plants, fungi and humans. Importantly, dysfunction of 26S proteasomes is associated with numerous human pathologies and profoundly impacts crop performance, thus making an understanding of proteasome dynamics critically relevant to almost all facets of human health and nutrition. Given this widespread significance, it is not surprising that sophisticated mechanisms have evolved to tightly regulate 26S proteasome assembly, abundance and activity in response to demand, organismal development and stress. These include controls on transcription and chaperone-mediated assembly, influences on proteasome localization and activity by an assortment of binding proteins and post-translational modifications, and ultimately the removal of excess or damaged particles via autophagy. Intriguingly, the autophagic clearance of damaged 26S proteasomes first involves their modification with ubiquitin, thus connecting ubiquitylation and autophagy as key regulatory events in proteasome quality control. This turnover is also influenced by two distinct biomolecular condensates that coalesce in the cytoplasm, one attracting damaged proteasomes for autophagy, and the other reversibly storing proteasomes during carbon starvation to protect them from autophagic clearance. In this review, we describe the current state of knowledge regarding the dynamic regulation of 26S proteasomes at all stages of their life cycle, illustrating how protein degradation through this proteolytic machine is tightly controlled to ensure optimal growth, development and longevity.
... Interestingly, lysine 48-linkage between the ubiquitin molecules is used for nuclear substrates suggesting that nuclear proteasomes prefer the conventional poly-ubiquitin chain with lysine-48 linked ubiquitin molecules. Cytoplasmic substrates have mixed lysine linkages which seem to be conferred by ubiquitin ligases associated with the ER (Samant et al., 2018). Whether proteasomes in the nucleo-and cytoplasm are differently configured to achieve ubiquitin-linkage specific degradation of proteasomal substrates needs to be investigated. ...
... Whether proteasomes in the nucleo-and cytoplasm are differently configured to achieve ubiquitin-linkage specific degradation of proteasomal substrates needs to be investigated. There is an intriguing coincidence that proteasomes are primarily nuclear in dividing yeast and mammalian cancer cells (Enenkel, 2014b) suggesting that substrates with conventional lysine 48linked poly-ubiquitin chains arise in the nucleus for proteasomal degradation (Samant et al., 2018). ...
Proteasomes are key proteases in regulating protein homeostasis. Their holo-enzymes are composed of 40 different subunits which are arranged in a proteolytic core (CP) flanked by one to two regulatory particles (RP). Proteasomal proteolysis is essential for the degradation of proteins which control time-sensitive processes like cell cycle progression and stress response. In dividing yeast and human cells, proteasomes are primarily nuclear suggesting that proteasomal proteolysis is mainly required in the nucleus during cell proliferation. In yeast, which have a closed mitosis, proteasomes are imported into the nucleus as immature precursors via the classical import pathway. During quiescence, the reversible absence of proliferation induced by nutrient depletion or growth factor deprivation, proteasomes move from the nucleus into the cytoplasm. In the cytoplasm of quiescent yeast, proteasomes are dissociated into CP and RP and stored in membrane-less cytoplasmic foci, named proteasome storage granules (PSGs). With the resumption of growth, PSGs clear and mature proteasomes are transported into the nucleus by Blm10, a conserved 240 kDa protein and proteasome-intrinsic import receptor. How proteasomes are exported from the nucleus into the cytoplasm is unknown.
