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To Live or Die by the Sword
Abstract
The proteasome has been implicated in the control of apoptosis by modulating the levels of both pro- and antiapoptotic molecules. A recent study published in the April 9th issue of Molecular Cell reveals that caspase-dependent inactivation of the proteasome can amplify the activation of apoptosis.
... Protein ubiquitination, the covalent modification of proteins by the addition of ubiquitin, is a mechanism by which damaged proteins are marked for subsequent degradation by the 26S proteasome [18,19]. In the ubiquitin-proteasome pathway, the sequential actions of E1, the ubiquitin-activating enzyme, E2, the ubiquitin conjugating enzyme (UBC), and E3, the ubiquitin-protein ligase, serve to attach the ubiquitin moiety (most often polyubiquitin chains) to acceptor lysine residues of protein substrates, which are in turn targeted for degradation by the 26S proteasome [20,21]. ...
... In the ubiquitin-proteasome pathway, the sequential actions of E1, the ubiquitin-activating enzyme, E2, the ubiquitin conjugating enzyme (UBC), and E3, the ubiquitin-protein ligase, serve to attach the ubiquitin moiety (most often polyubiquitin chains) to acceptor lysine residues of protein substrates, which are in turn targeted for degradation by the 26S proteasome [20,21]. The ability to degrade and regulate levels of intracellular proteins has made the proteasome an important molecule in the regulation of diverse biological processes including cell cycle progression, cell signaling, and apoptosis [18,22]. ...
... Various extracellular and intracellular stimuli can trigger this process, and once initiated the signal is tranduced through a series of protein-protein interactions. There is accumulating evidence that supports a close relationship between the ubiquitin-proteasome pathway and the apoptotic machinery [18,22]. Protein degradation mediated by polyubiquitination regulates the steady-state expression levels of many of the apoptosis regulators [22]. ...
To understand the role of glutathione (GSH) in the protection of cells from arsenite toxicity, we studied the mechanism of apoptotic cell death in cells genetically unable to synthesize GSH (GCS-2 cells). Arsenite stimulated an increase in protein ubiquitination in GCS-2 cells while the wild-type cells were unaffected. Arsenite treatment increased lipid peroxidation and induced ubiquitination of molecular chaperone Hsp90 and impaired its ability to bind cochaperone p50(Cdc-37) and client proteins Plk-1 and Cdk-4 in GCS-2 cells. Treatment with arsenite also partially inhibited proteasome activity in GCS-2 cells. In these cells stably transfected with GFP(u) (a reporter consisting of a short degron fused to the COOH-terminus of GFP), intracellular fluorescence increased, suggesting the accumulation of GFP aggregates. GCS-2 cells underwent apoptosis accompanied by release of cytochrome c into the cytoplasm. Taken together, these data suggest that a possible mechanism of arsenite-induced apoptosis is the accumulation of ubiquitinated proteins and impairment of the protein degradative pathway. Further, protection from arsenite-induced ubiquitination is mediated by GSH and to a lesser extent by available reducing equivalents in the cells.
... As a result, this proteolytic system regulates several cell functions that play significant role in carcinogenesis such as proliferation, apoptosis, response to stress, and metastasis [4,5]. Inhibition of the proteasome happens during apoptosis and is a pre-requisite for apoptosis progression [6][7][8]. Thus, proteasome inhibition appears to be promising as a cancer therapeutic strategy and indeed a small molecule proteasome inhibitor, bortezomib, has been developed and entered the clinical arena as treatment for multiple myeloma [9], while it has also been investigated in solid tumors [10][11][12]. Nevertheless proteasome inhibition has the "molecular side effect" of increasing prostaglandin E2 (PGE2) production by stimulating arachidonic acid release and cox-2 up-regulation [13,14] and by direct Cox-2 stabilization [15]. ...
... Western blotting of lysates from HCT116 (lanes 1-4), HT-29 (lanes[5][6][7][8], and CaCo2 cells (lanes 9-12) for p27 (a), p21 (b). Cell pellets were lysed in protein lysis buffer and 50 μg of protein lysates were electrophoresed in polyacrylamide gels. ...
To investigate the effect of cyclooxygenase-2 (Cox-2) inhibitor aspirin (acetylsalicylic acid, ASA) and proteasome inhibitor bortezomib in the proliferation and apoptosis of colorectal cancer cell lines.
MTT assay, trypan blue exclusion and DNA fragmentation have been used to investigate cell proliferation and apoptosis in the presence of drugs. For the determination of Cox activity a colorimetric method was used. Western blotting was used for the measurement of the effect of the drugs in different proteins expression.
Bortezomib together with aspirin inhibit the growth of colorectal cancer cell lines HCT116, HT-29, and CaCo2 more than each drug alone. In the first two cell lines ASA inhibitory effects are Cox-2 independent because HCT116 cells do not express the enzyme while in HT-29 cells, Cox-2 has no activity as shown by a Cox activity assay. In CaCo2 cells that express enzymatically active Cox-2 this activity is inhibited by ASA. ASA is also able to suppress the increase in Cox-2 activity induced by bortezomib in these cells. Cell cycle inhibitors p21 and p27 are induced in the three cell lines by bortezomib and the combination treatment. Akt1 kinase is down-regulated in all three lines by the same treatments. Transcription factor NF-kappaB is retained in the cytoplasm by drug treatment in cell lines HCT116 and HT-29, a fact that may play a role in their pro-apoptotic activity. Pro-apoptotic bcl-2 family member, bad is down-regulated in cell lines HCT116 and CaCo2 by bortezomib treatment, a neoplasia-promoting event that is reversed by combination treatment.
The combination of bortezomib and ASA cooperates to decrease proliferation and induce apoptosis in three human colorectal cell lines with different genetic lesions. These effects are at least in some cases Cox-2 independent and involve common and diverse mechanisms in the three lines.
... Expression of A53T mutant alpha-synuclein decreases proteasome activity and increases intracellular ROS in PC12 cells Proteasome inhibition has been widely reported to cause apoptosis (26,27) and has been implicated in PD (2,28 -31). Previously, we showed that proteasome activity in cells expressing A30P alpha-synuclein was lower than that in non-induced cells or cells expressing wild-type alphasynuclein (23). ...
... Oxidative stress and proteasome inhibition have been implicated in PD and other neurodegenerative disorders (3,4,28,41,42). Proteasome inhibition can cause apoptosis (26,27) and increased intracellular ROS levels can cause DNA and protein damage leading to cell death (43,44). A number of reports have shown that both proteasome inhibition and ROS can trigger mitochondrial and ER stress cell death pathways (45 -48). ...
Parkinson's disease (PD) is a neurodegenerative movement disorder characterized by selective loss of dopaminergic neurons and the presence of Lewy bodies. Alpha-synuclein is a major component of Lewy bodies in sporadic PD, and mutations in alpha-synuclein cause autosomal-dominant hereditary PD. Here, we generated A53T mutant alpha-synuclein-inducible PC12 cell lines using the Tet-off regulatory system. Inducing expression of A53T alpha-synuclein in differentiated PC12 cells decreased proteasome activity, increased the intracellular ROS level and caused up to approximately 40% cell death, which was accompanied by mitochondrial cytochrome C release and elevation of caspase-9 and -3 activities. Cell death was partially blocked by cyclosporine A [an inhibitor of the mitochondrial permeability transition (MPT) process], z-VAD (a pan-caspase inhibitor) and inhibitors of caspase-9 and -3 but not by a caspase-8 inhibitor. Furthermore, induction of A53T alpha-synuclein increased endoplasmic reticulum (ER) stress and elevated caspase-12 activity. RNA interference to knock down caspase-12 levels or salubrinal (an ER stress inhibitor) partially protected against cell death and further reduced A53T toxicity after treatment with z-VAD. Our results indicate that both ER stress and mitochondrial dysfunction contribute to A53T alpha-synuclein-induced cell death. This study sheds light into the pathogenesis of alpha-synuclein cellular toxicity in PD and provides a cell model for screening PD therapeutic agents.
... Proteasomes turn over specific stage-specific cyclins and, therefore, proteasome activity is intimately associated with the cell cycle. Proteasome activity is also associated with cell survival by recycling a huge majority of mistranslated, damaged, or redundant proteins [32,33] and ultimately with the regulation of cell death by modulating the levels of anti-and proapoptotic molecules [77]. The 20S proteasome has three active sites with caspase-like, trypsin-like and chymotrypsin-like activities that collectively digest most proteins to short peptides [78,79]. ...
Schistosomiasis ranks among the most important infectious diseases, with over 200 million people currently being infected and > 280,000 deaths reported annually. Chemotherapeutic treatment has relied on one drug, praziquantel, for four decades, while other drugs, such as oxamniquine and metrifonate, are no longer preferred for clinical use due to their narrow spectrum of activity - these are only active against S. mansoni and S. haematobium, respectively. Despite being cheap, safe, and effective against all schistosome species, praziquantel is ineffective against immature worms, which may lead to reinfections and treatment failure in endemic areas; a situation that necessitates repeated administration besides other limitations. Therefore, novel drugs are urgently needed to overcome this situation. In this paper, an up to date review of drug targets identified and validated against schistosomiasis while also encompassing promising clinical and preclinical candidate drugs is presented. While there are considerable efforts aimed at identifying and validating drug targets, the pipeline for new antischistosomals is dry. Moreover, the majority of compounds evaluated preclinically are not really advanced because most of them were evaluated in very small preclinical species such as mice alone. Overall, it appears that although a lot of research is going on at discovery phases, unfortunately, it does not translate to advanced preclinical and clinical evaluation.
... The proto-oncogene c-Casitas b-lineage lymphoma (c-Cbl) is an E3 ubiquitin ligase containing the RING Finger (RNF) domain, which can either upregulate or downregulate signal transduction 12,13 . E3 ubiquitin ligase, which presents with the greatest significant tissue and substrate specificity, acts on the last step of the formation of ubiquitin-protein conjugates 14,15 . The protein first labeled by ubiquitin (polypeptide) and then recognized and degraded by the 26 S proteasome complex 16 . ...
The chronic wound induced by diabetes has poor efficacy and could lead to amputation. The repair function of mesenchymal stem cells (MSCs) impaired after long-term culture in vitro. Studies have shown that the proto-oncogene c-Casitas b-lineage lymphoma (c-Cbl) can regulate receptor- and non-receptor tyrosine kinase, which was also involved in the angiogenesis process. This study aimed to explore the regulative effect of c-Cbl on the proangiogenic functions of long-term cultured MSCs and evaluate its pro-healing effect on diabetic wounds. In this study, the c-Cbl level was downregulated by locked nucleic acid-modified antisense oligonucleotide gapmers (LNA Gapmers). We detected the effect of c-Cbl downregulation on long-term cultured MSCs in terms of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signal, cellular proliferation, senescence, migration, and angiogenic factors paracrine activity in vitro. In vivo, we observed the pro-healing effect of long-term cultured MSCs, with or without c-Cbl downregulation, on the diabetic wound. We found that the phosphorylation level of c-Cbl increased and that of Akt decreased in passage 10 (P10) MSCs compared with passage 3 (P3) MSCs (P < 0.05). Additionally, the proliferation, paracrine, and migration capacity of P10 MSCs decreased significantly, accompanied by the increase of cellular senescence (P < 0.05). However, these functions, including PI3K/Akt activity of P10 MSCs, have been improved by c-Cbl downregulation (P < 0.05). Compared with P10 MSCs treatment, treatment with c-Cbl downregulated P10 MSCs accelerated diabetic wound healing, as defined by a more rapid wound closure (P < 0.05), more neovascularization (P < 0.05), and higher scores of wound histological assessment (P < 0.05) in a diabetic rat model. Our findings suggested that c-Cbl downregulation could attenuate the impairment of proangiogenic functions in MSCs induced by long-term culture in vitro and improve the effect of long-term cultured MSCs in promoting diabetic wound healing.
... For cell growth and survival, UPS is crucial, whereas for apoptosis to occur, there needs to be an inhibition of UPS. For example, p53 activity is tightly regulated by UPS and it plays a crucial role in the suppression of tumors (Adrain et al., 2004;Friedman and Xue, 2004;Gupta et al., 2018;Sun et al., 2004). Another example is that PIs can induce endoplasmic reticulum (ER) stress and produce apoptosis in many cancers (Best et al., 2019). ...
Drug resistance is a major obstacle in the field of pre-clinical and clinical therapeutics. The development of novel technologies and targeted therapies have yielded new modalities to overcome drug resistance, but multidrug resistance (MDR) remains one of the major challenges in the treatment of cancer. The ubiquitin-proteasome system (UPS) has a central role in regulating the levels and activities of a multitude of proteins as well as regulation of cell cycle, gene expression, response to oxidative stress, cell survival, cell proliferation and apoptosis. Therefore, inhibition of the UPS could represent a novel strategy for the treatment and overcoming of drug resistance in chemoresistant malignancies. In 2003, bortezomib was approved by the FDA for the treatment of multiple myeloma (MM). However, due to its limitations, second generation proteasome inhibitors (PIs) like carfilzomib, ixazomib, oprozomib, delanzomib and marizomib were introduced which displayed clinical activity in bortezomib-resistant tumors. Past studies have demonstrated that proteasome inhibition potentiates the anti-cancer efficacy of other chemotherapeutic drugs by: i) decreasing the expression of anti-apoptotic proteins such as TNF-α and NF-kB, ii) increasing the levels of Noxa, a pro-apoptotic protein, iii) activating caspases and inducing apoptosis, iv) degrading the pro-survival protein, induced myeloid leukemia cell differentiation protein (MCL1), and v) inhibiting drug efflux transporters. In addition, the mechanism of action of the immunoproteasome inhibitors, ONX-0914 and LU-102, suggested their therapeutic role in the combination treatment with PIs. In the current review, we discuss various PIs and their underlying mechanisms in surmounting anti-tumor drug resistance when used in combination with conventional chemotherapeutic agents.
... Interferon-stimulated gene 15 (ISG15) is a small protein with a low molecular weight induced by the expression of type I interferon, which is an upstream regulator in the UPP pathway. A previous study revealed that UPP participates widely in cell cycle regulation and apoptosis (12). Desai et al (13) demonstrated that the overexpression of ISG15 in cancer cells is able to negatively regulate ubiquitin-26S UPP via inhibiting the poly-ubiquitination of target proteins. ...
The aim of the present study was to investigate the roles of microRNA (miR)-138 and interferon-stimulated gene 15 (ISG15) in patients with oral squamous cell carcinoma (OSCC). miR-138 and ISG15 expression in cancer tissues was detected, and the influence on proliferation, migration and invasion of OSCC cell lines was assessed. Reverse transcription-quantitative polymerase chain reaction was performed to analyze the expression of miR-138 and ISG15 in resected cancer tissues and pericancerous tissues harvested from patients with OSCC. The protein level of ISG15 was determined via western blot analysis. The constructed pGCMV/EGFP/miR-138 plasmid was transfected into CAL27 and SCC-15 OSCC cell lines via a liposome method to upregulate miR-138 expression. The transfection efficiency was determined based on miR-138 expression levels, and changes in proliferation, migration and invasion were subsequently compared with those in untransfected cells. The expression of ISG15 mRNA and protein was also detected in OSCC cells. miR-138 was significantly downregulated (P<0.05) in cancer tissues compared with adjacent normal tissues in patients with OSCC, whereas ISG15 mRNA expression levels were significantly higher in pericancerous tissues (P<0.05). ISG15 protein levels were also significantly higher in pericancerous tissues (P<0.05). ISG15 protein and mRNA levels were significantly decreased in the transfected cells compared with the untransfected cells, which indicated that miR-138 overexpression inhibited ISG15 expression. Additionally, the invasion, migration and proliferation abilities of successfully transfected CAL27 and SCC-15 cells were significantly decreased compared with the untransfected cells (P<0.05). The results of the present study suggest that miR-138 functions as a tumor-suppressive miR and serves an important role in OSCC via regulating ISG15 expression. These findings suggest that miR-138 is able to inhibit the proliferation, migration and invasion of OSCC cell lines.
... As a pathway of protein degradation and posttranslational modification [1], ubiquitin-proteasome pathway (UPP) is responsible for the degradation of 80% -90% bulk protein in eukaryotic cell [2]. UPP is involved in a complex series of intracellular processes, such as gene expression regulation [3], cell cycle regulation, DNA damage repair, immune response [4], cell apoptosis [5] and so forth. To prevent cells from undergoing proteotoxic stress, UPP controls the levels of cellular proteome by regulating the complex series of intracellular processes [6]. ...
Ubiquitin-proteasome
pathway (UPP) is a significant
way of protein degradation and modification in eukaryotic cell and involved in
a complex series of intracellular processes. As a key component in UPP, ubiquitin-conjugating enzyme (E2) plays an extremely
important role in ubiquitin (Ub) transferring and substrate specific
recognition. Abundant evidences have proved that UPP is involved in cells
immune reaction caused by pathogens and the attendance of E2 has a significant
effect on host cells and pathogen. This article presents an overview of the
current research on E2s that is involved in immune response caused by viruses
and bacteria.
... One question to be asked is how Drosophila SOD3 went unnoticed despite intense work to understand how the SODs function in Drosophila ageing [19,30,36,37,[62][63][64][65]. To address this we analysed expression and activity of SOD3 in two classic SOD1 mutants: sod1 x39 and sod1 n108 [19,30,62]. ...
In this study we cloned and sequenced the mRNAs of the extracellular Cu Zn superoxide dismutase (Sod3) gene in Drosophila and identified two mRNA products formed by alternative splicing. These products code for a long and short protein derived from the four transcripts found in global expression studies (Flybase numbers Dmel\CG9027, FBgn0033631). Both mRNA process variants contain an extracellular signalling sequence, a region of high homology to the cytoplasmic Cu Zn superoxide dismutase (Sod1) including a conserved AUG start, with the longer form also containing a hydrophobic tail. The two fully processed transcripts are homologous to Caenorhabditis elegans extracellular Cu Zn SOD mRNA showing the same processing pattern. Using an established KG p-element+ insertion line (KG06029), we demonstrate that the Sod3 codes for an active Cu Zn SOD. We found differing expression patterns across sex with higher levels of expression of Sod3 in females. There is a correlation of Sod1 and Sod3 gene expression and activity that can explain why Sod3 was not seen in earlier studies of Sod1. Finally, we found no effect on lifespan with the Sod3 hypomorph mutation (Sod3KG06029) but did observe a significant increase in resistance to paraquat and hydrogen peroxide (H2O2). Open access at: http://www.bioscirep.org/bsr/imps/abs/BSR20140133.htm
... Previous studies have reported that the proteasome is inhibited through caspase-mediated cleavage following the activation of apoptosis in response to various apoptotic stimuli, including DNA damage (Sun et al., 2004). The inactivation of the proteasome after the initiation of apoptosis was suggested to facilitate and amplify the apoptotic cascade (Friedman and Xue, 2004;Sun et al., 2004). Therefore, aggresome formation may be a consequence of the loss of proteasome function in the early stages of apoptosis. ...
In conditions of proteasomal impairment, the build-up of damaged or misfolded proteins activates a cellular response leading to the recruitment of damaged proteins into perinuclear aggregates called aggresomes. Aggresome formation involves the retrograde transport of cargo proteins along the microtubule network and is dependent on the histone deacetylase HDAC6. Here we show that ionizing radiation (IR) promotes Ran-Binding Protein M (RanBPM) relocalization into discrete perinuclear foci where it co-localizes with aggresome components ubiquitin, dynein and HDAC6, suggesting that the RanBPM perinuclear clusters correspond to aggresomes. RanBPM was also recruited to aggresomes following treatment with the proteasome inhibitor MG132 and the DNA-damaging agent etoposide. Strikingly, aggresome formation by HDAC6 was markedly impaired in RanBPM shRNA cells, but was restored by re-expression of RanBPM. RanBPM was found to interact with HDAC6 and to inhibit its deacetylase activity. This interaction was abrogated by a RanBPM deletion of its LisH/CTLH domain, which also prevented aggresome formation, suggesting that RanBPM promotes aggresome formation through an association with HDAC6. Our results suggest that RanBPM regulates HDAC6 activity and is a central regulator of aggresome formation.
... Thus, individual subunits contribute to substrate recognition, unfolding and peptide bond hydrolysis in a highly processive manner [2]. Proteasome activity is intimately associated with the cell cycle by turning over stage-specific cyclins [3] and with cell survival by recycling a vast majority of either mistranslated, damaged or redundant proteins [4,5] and ultimately with the regulation of cell death by modulating the levels of pro-and antiapoptoptic molecules [6]. The 20S proteasome that comprises up to 1% of cellular proteins [7] is composed of four stacked rings. ...
Proteasomes are molecular machines found in virtually all cells that provide one of the mechanisms for protein turnover. We have analysed the 20S proteasome of Schistosoma mansoni, the first multimeric complex isolated from this helminth parasite. Three chromatographic steps were employed to yield a highly homogeneous preparation. 2-DE of the purified complex revealed 58 spots, of which 46 could be assigned either an alpha or a beta proteasome signature by MS. Most of the 14 transcripts (7alpha and 7beta) encoded by the parasite genome were represented by multiple spots and we suggest that this diversity is due to PTMs of subunits. For most of the isoforms, variations in pI predominated although alterations in mass were also observed. 2-DE separations of extracts from infective cercariae and blood-dwelling adult worms probed by Western blotting, using a human anti-alpha subunit antibody, revealed different patterns of reactivity, most probably in alpha3 and alpha6 subunits, on the basis of sequence conservation. This difference was rapidly lost following transformation of the cercaria to the skin schistosomulum stage, suggesting that changes in the proteasome structure, likely caused by the introduction of a new set of PTMs, precede remodelling of the parasite body prior to intravascular migration.
... The ubiquitin-proteasome system plays an important role in regulating apoptosis pathways on multiple levels [85,86]. (i) Many proapoptotic proteins, such as p53, FOXO1, and Bax are direct targets of the proteasome [52,87,88]. ...
Proteasomes are large multicatalytic proteinase complexes located in the cytosol and the nucleus of eukaryotic cells. The ubiquitin-proteasome system is responsible for the degradation of most intracellular proteins and therefore plays an essential regulatory role in critical cellular processes including cell cycle progression, proliferation, differentiation, angiogenesis and apoptosis. Besides involving in normal cellular functions and homeostasis, the alteration of proteasomal activity contributes to the pathological states of several clinical disorders including inflammation, neurodegeneration and cancer. It has been reported that human cancer cells possess elevated level of proteasome activity and are more sensitive to proteasome inhibitors than normal cells, indicating that the inhibition of the ubiquitin-proteasome system could be used as a novel approach for cancer therapy. In this review we summarize several specific aspects of research for the proteasome complex, including the structure and catalytic activities of the proteasome, properties and mechanisms of action of various proteasome inhibitors, and finally the clinical development of proteasome inhibitors as novel anticancer agents.
... The proteasome is a highly conserved, multisubunit complex that consists of several multicatalytic proteases responsible for most of the cytosolic and nuclear protein degradation (Smalle and Vierstra 2004). In animals, protesomes affect apoptosis by controlling the stability of both pro-and anti-apoptotic molecules (Jesenberger and Jentsch 2002; Friedman and Xue 2004). To examine the role of proteasomes in the R protein-mediated PCD in plants, Hatsugai et al. (2009) used proteasome inhibitors in their study. ...
Eukaryotes have evolved various means for controlled and organized cellular destruction, known as programmed cell death (PCD). In plants, PCD is a crucial regulatory mechanism in multiple physiological processes, including terminal differentiation, senescence, and disease resistance. In this issue of Genes & Development, Hatsugai and colleagues (pp. 2496-2506) demonstrate a novel plant defense strategy to trigger bacteria-induced PCD, involving proteasome-dependent tonoplast and plasma membrane fusion followed by discharge of vacuolar antimicrobial and death-inducing contents into the apoplast.
... Proteasome function is essential for normal ERAD, and its inhibition causes accumulation of misfolded proteins in the ER and activates apoptotic pathways leading to cell death (39)(40)(41)(42). Our study demonstrated that treatment with the proteasome inhibitor MG132 promoted cell death in cells expressing mutant Notch3, although cell death in cultures was not directly induced by expression of wild-type and mutant Notch3. ...
Mutations in the human NOTCH3 gene cause cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), but the pathogenic mechanisms of the disorder remain unclear. We investigated the cytotoxic properties of mutant Notch3 using stable cell lines with inducible expression of either wild-type or two mutants p.R133C and p.C185R. We found that both mutants of Notch3 were prone to aggregation and retained in the endoplasmic reticulum (ER). The turnover rates of the mutated Notch3 proteins were strikingly slow, with half-lives greater than 6 days, whereas wild-type Notch3 was rapidly degraded, with a half-life of 0.7 days. The expression of mutant Notch3 also impaired cell proliferation compared with wild-type Notch3. In addition, cell lines expressing mutant Notch3 were more sensitive to proteasome inhibition resulting in cell death. These findings suggest that prolonged retention of mutant Notch3 aggregates in the ER decreases cell growth and increases sensitivity to other stresses. It is also possible that the aggregate-prone property of mutant Notch3 contributes to a pathogenic mechanism underlying CADASIL. © The Author 2009. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
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... Polyubiquitination has the role of labelling proteins for degradation by the 26S proteasome or by lysosomes (Hochstrasser, 1996). Ubiquitin-mediated protein degradation is involved in the control of many events, including cell cycle progression (Hershko and Ciechanover, 1998), membrane receptor endocytosis (Haglund et al., 2003b) and apoptosis (Friedman and Xue, 2004). ...
Global ubiquitination in human semen has been found to negatively correlate with standard semen parameters, indicating that ubiquitination can be considered a marker of poor semen quality. However, the inclusion of all semen components in the analysis may be misleading on the biological significance of ubiquitination of sperm cells. We have recently demonstrated the variable presence of bodies of different size, with the highest concentration in oligoasthenoteratozoospermia. The purpose of the present study was to evaluate the relationship between ubiquitination and standard semen parameters, after distinguishing between ubiquitinated sperm and bodies in each sample.
Ubiquitination was evaluated by flow cytometric sperm ubiquitin tag immunoassay (SUTI) in sperm samples from 45 subjects. Semen analysis was performed according to WHO (1999) guidelines.
When only ubiquitinated sperm were considered, a positive correlation with number, motility and normal morphology was found. When correlation was evaluated considering the percentage of ubiquitinated bodies, a negative correlation was found with good semen quality.
Results indicate that the negative correlations previously found between global semen ubiquitination and parameters of semen analysis are mainly driven by components other than sperm. The positive correlation between sperm ubiquitination and good quality parameters suggests a previously unrecognized role for sperm ubiquitination.
... The initiation and execution of apoptosis is dependent upon caspases and, in many cell types, mitochondrial events (27). Other cellular proteases play regulatory roles in this process (22,31,34,61). Caspases are triggered in a hierarchical order: initiator caspases 8 and 9 mediate the cleavage-activation of effector caspase 3, either directly or following release of mitochondrial factors that promote this cascade. ...
Human cytomegalovirus carries a mitochondria-localized inhibitor of apoptosis (vMIA) that is conserved in primate cytomegaloviruses.
We find that inactivating mutations within UL37x1, which encodes vMIA, do not substantially affect replication in TownevarATCC (Towne-BAC), a virus that carries a functional copy of the betaherpesvirus-conserved viral inhibitor of caspase 8 activation,
the UL36 gene product. In Towne-BAC infection, vMIA reduces susceptibility of infected cells to intrinsic death induced by
proteasome inhibition. vMIA is sufficient to confer resistance to proteasome inhibition when expressed independent of viral
infection. Murine cytomegalovirus m38.5, whose position in the viral genome is analogous to UL37x1, exhibits mitochondrial
association and functions in much the same manner as vMIA in inhibiting intrinsic cell death. This work suggests a common
role for vMIA in rodent and primate cytomegaloviruses, modulating the threshold of virus-infected cells to intrinsic cell
death.
... Numerous studies have also demonstrated the role of calpains, cytoplasmic Ca (2+) -activated neutral cysteine proteases [85], in the apoptotic process induced by different stimuli in various cell types, but the precise underlying mechanisms are still unclear [86]. By controlling the levels of various cellular proteins, the proteasome degradation system can regulate the levels of many molecules of the cell death machinery [87][88][89]. In addition to these proteases, cytotoxic granules, which are specialized lysosome-related organelles of T lymphocytes used to kill virus-infected cells and tumor cells, contain neutral serine proteases called granzymes. ...
Death of cancer cells influences tumor development and progression, as well as the response to anticancer therapies. This can occur through different cell death programmes which have recently been shown to implicate components of the acidic organelles, lysosomes. The role of lysosomes and lysosomal enzymes, including cathepsins and some lipid hydrolases, in programmed cell death associated with apoptotic or autophagic phenotypes is presented, as evidenced from observations on cultured cells and living animals. The possible molecular mechanisms that underlie the action of lysosomes during cell death are also described. Finally, the contribution of lysosomal proteins and lysosomes to tumor initiation and progression is discussed. Elucidation of this role and the underlying mechanisms will shed a new light on these 'old' organelles and hopefully pave the way for the development of novel anticancer strategies.
... By controlling the rapid and irreversible turnover of key regulatory proteins, the ubiquitin–proteasome pathway plays important roles in a variety of biological processes, including cell cycle progression (Reed 2003), transcriptional regulation and chromatin remodeling (Kinyamu et al. 2005; Hegde and Upadhya 2006), memory and synaptic plasticity (DiAntonio and Hicke 2004), circadian rhythms (Naidoo et al. 1999), signal transduction (Ye and Fortini 2000), metabolic regulation (Hampton and Bhakta 1997), antigen processing (Kloetzel 2004), and programmed cell death (Friedman and Xue 2004). This pathway also carries out an important ''housekeeping'' function, by ridding cells of potentially harmful abnormal proteins that arise as the result of mutation, misfolding, or postsynthetic damage (Kostova and Wolf 2003). ...
Two dominant temperature-sensitive (DTS) lethal mutants of Drosophila melanogaster are Pros26(1) and Prosbeta2(1), previously known as DTS5 and DTS7. Heterozygotes for either mutant die as pupae when raised at 29 degrees , but are normally viable and fertile at 25 degrees . Previous studies have identified these as missense mutations in the genes encoding the beta6 and beta2 subunits of the 20S proteasome, respectively. In an effort to isolate additional proteasome-related mutants a screen for dominant suppressors of Pros26(1) was carried out, resulting in the identification of Pros25(SuDTS) [originally called Su(DTS)], a missense mutation in the gene encoding the 20S proteasome alpha2 subunit. Pros25(SuDTS) acts in a dominant manner to rescue both Pros26(1) and Prosbeta2(1) from their DTS lethal phenotypes. Using an in vivo protein degradation assay it was shown that this suppression occurs by counteracting the dominant-negative effect of the DTS mutant on proteasome activity. Pros25(SuDTS) is a recessive polyphasic lethal at ambient temperatures. The effects of these mutants on larval neuroblast mitosis were also examined. While Prosbeta2(1) shows a modest increase in the number of defective mitotic figures, there were no defects seen with the other two mutants, other than slightly reduced mitotic indexes.
... Specifically the chymotrypsin-like and peptidyl-gloutamyl proteasomal activities are affected due to degradation of the 19S subunits Rpt5 and Rpn10 involved in substrate recognition and Rpn2 involved in holding together the lid and base components of the 19S proteasome [160,161]. Indeed the fact that activated executioner caspases degrade proteasome components leading to proteasome inhibition points to a crucial role of proteasome in cell survival that needs to be circumvented in order for apoptosis to proceed [162]. This happens despite the role that the proteasome plays in degrading IAP member XIAP and cIAP1 after a RING domain reciprocal interaction [163]. ...
Introduction
APC and the Wnt pathway
K‐ras initiated pathways
The TGF‐β pathway
P53 and its functional regulation and dysregulation in colorectal cancer
The ubiquitin‐proteasome system (UPS)
The ubiquitin‐proteasome system in apoptosis and the cell cycle
Proteasome inhibition in colorectal carcinoma
Cox‐2 in colorectal cancer
Cox‐2 and lipid metabolism in colorectal cancer
Cox‐independent anti‐carcinogenic effects of Cox inhibitors
Combined Cox and proteasome inhibition in colorectal cancer
Abstract
Pathways of the molecular pathogenesis of colorectal carcinoma have been extensively studied and molecular lesions during the development of the disease have been revealed. High up in the list of colorectal cancer lesions are APC (adenomatous polyposis coli), K‐ras, Smad4 (or DPC4‐deleted in pancreatic cancer 4) and p53 genes. All these molecules are part of important pathways for the regulation of cell proliferation and apoptosis and as a result perturbation of these processes lead to carcinogenesis. The ubiquitin–proteasome system (UPS) is comprised of a multi‐unit cellular protease system that regulates several dozens of cell proteins after their ligation with the protein ubiquitin. Given that among these proteins are regulators of the cell cycle, apoptosis, angiogenesis, adhesion and cell signalling, this system plays a significant role in cell fate and carcinogenesis. UPS inhibition has been found to be a pre‐requisite for apoptosis and is already clinically exploited with the proteasome inhibitor bortezomib in multiple myeloma. Cyclooxygenase‐2 (Cox‐2) is the inducible form of the enzyme that metabolizes the lipid arachidonic acid to prostaglandin H 2 , the first step of prostaglandins production. This enzyme is up‐regulated in colorectal cancer and in several other cancers. Inhibition of Cox‐2 by aspirin and other non‐steroidal anti‐inflammatory drugs (NSAIDs) has been found to inhibit proliferation of colorectal cancer cells and in epidemiologic studies has been shown to reduce colon polyp formation in genetically predisposed populations and in the general population. NSAIDs have also Cox‐independent anti‐proliferative effects. Targeted therapies, the result of increasingly understanding carcinogenesis in the molecular level, have entered the field of anti‐neoplastic treatment and are used by themselves and in combination with chemotherapy drugs. Combinations of targeted drugs have started also to be investigated. This article reviews the molecular pathogenesis of colorectal cancer, the roles of UPS and Cox‐2 in it and puts forward a rational for their combined inhibition in colorectal cancer treatment.
... In eukaryotes, the ubiquitin-proteasome pathway plays a critical and specific role in promoting protein degradation, thus regulating key biological processes as general as cell-cycle progression and transcriptional regulation and as specific as memory, synaptic plasticity, and circadian rhythms (Glickman and Ciechanover, 2002). The ubiquitin-proteasome pathway is also involved in antigen processing (Kloetzel and Ossendorp, 2004) and programmed cell death (Friedman and Xue, 2004) and plays a role in eliminating cells expressing potentially harmful a similarly staged embryo expressing two copies of dominant-negative forms of the two proteasome subunits specifically in macrophages (under the control of two copies of a crq-Gal4 driver), and a (D) slmb mutant embryo. All embryos were stained with the Crq antibody (green) and 7-AAD (red). ...
Many cells die by apoptosis during animal development. Apoptotic cells are rapidly removed through phagocytosis by their neighbors or by macrophages. To genetically dissect this process, we performed an in vivo screen for genes required for efficient phagocytosis of apoptotic cells by Drosophila macrophages and identified pallbearer (pall), which encodes an F box protein. F box proteins generally provide substrate specificity to Skp Cullin F box (SCF) complexes, acting as E3 ligases that target phosphorylated proteins to ubiquitylation and degradation via the 26S proteasome. We showed that Pallbearer functions in an SCF-dependent manner and provided direct evidence of a role for ubiquitylation and proteasomal degradation in phagocytosis of apoptotic corpses in vivo. This work might further our understanding of the regulation of apoptotic cell engulfment and thus our understanding of innate immunity as a whole.
After Alzheimer, Parkinson's disease (PD) is the second most common neurodegenerative disorder. Alpha synuclein (SNCA) is deemed as a major component of Lewy bodies, a neuropathological feature of PD. Five point mutations in SNCA have been reported so far, responsible for autosomal dominant PD. This study aims to decipher evolutionary and structural insights of SNCA by revealing its sequence and structural evolutionary patterns among sarcopterygians and its paralogous counterparts (SNCB and SNCG). Rate analysis detected strong purifying selection on entire synuclein family. Structural dynamics divulges that during the course of sarcopterygian evolutionary history, the region encompassed 32 to 58 of N-terminal domain of SNCA has acquired its critical functional significance through the epistatic influence of the lineage specific substitutions. In sum, these findings provide an evidence that the region from 32 to 58 of N-terminal lipid binding alpha helix domain of SNCA is the most critical region, not only from the evolutionary perspective but also for the stability and the proper conformation of the protein as well as crucial for the disease pathogenesis, harboring critical interaction sites.
The proto-oncogene Casitas b-lineage lymphoma (c-Cbl) is an adaptor protein with an intrinsic E3 ubiquitin ligase activity that targets receptor and non-receptor tyrosine kinases, resulting in their ubiquitination and down-regulation. However, the function of c-Cbl in the control of cardiac function is currently unknown. In this study, we examined the role of c-Cbl in myocyte death and cardiac function after myocardial ischemia.
We show increased c-Cbl expression in human ischemic and dilated cardiomyopathy hearts and in response to pathological stress stimuli in mice. c-Cbl deficient mice demonstrated a more robust functional recovery after myocardial ischemia reperfusion injury, as well as significantly reduced myocyte apoptosis and improved cardiac function. Ubiquitination and downregulation of key survival c-Cbl targets, epidermal growth factor receptors and focal adhesion kinase, were significantly reduced in c-Cbl knockout mice. Inhibition of c-Cbl expression or its ubiquitin ligase activity in cardiac myocytes offered protection against H2O2 stress. Interestingly, c-Cbl deletion reduced the risk of death and increased cardiac functional recovery after chronic myocardial ischemia. This beneficial effect of c-Cbl deletion was associated with enhanced neoangiogenesis and increased expression of vascular endothelial growth factor (VEGF)-a and VEGF receptor type 2 in the infarcted region.
c-Cbl activation promotes myocyte apoptosis, inhibits angiogenesis and causes adverse cardiac remodeling after myocardial infarction. These findings point to c-Cbl as a potential therapeutic target for the maintenance of cardiac function and remodeling after myocardial ischemia.
A set of 18 new C(4) and C(1) derivatives of nor-cerpegin (1,1-dimethyl furo[3,4-c]pyridine-3-one), 6 model compounds (γ- and δ-lactones) and 20 furo- or thieno[2,3-d]-pyrimidine-4-one related compounds were designed and synthesized. Each compound was assayed for inhibition of CT-L, T-L and PA proteolytic activities of 20S constitutive proteasome (c20S). Most performant compounds were also assayed on 20S immunoproteasome (i20S). Compound 10 with a benzylamino group at C(4) and dimethylated at C(1) of the furopyridine ring was the most efficient PA site-specific inhibitor of the c20S (IC50(cPA) of 600nM) without noticeable inhibition of the i20S PA site (iPA). In silico docking assays for 10 at the iPA catalytic site revealed the absence of poses normally observed for this compound and related ones at the constitutive PA site (cPA). The thieno[2,3-d]pyrimidine-4-one 40 was T-L site-specific with a mild inhibition of both c20S and i20S in vitro (IC50(cT-L) of 9.9μM and IC50(iT-L) of 6.7μM). In silico docking assays of 40 at T-L sites of c20S and i20S revealed almost identical first rank poses in the two types of sites with no possibility left for nucleophilic attack by Thr1 as observed for the fused furopyridine-3-one 10.
Ecm29 is an ˜200-K HEAT-repeat protein that associates with the 26S proteasome in yeast and HeLa cells. Whereas in most mouse organs Ecm29 is present as a single 200-K protein, in the brain there are eleven species, ranging from 55 K to greater than 250 K, that react with anti-Ecm29 antibodies. Since the largest brain-specific Ecm29 variant reacts with antibodies to sequences within the N-terminal and C-terminal regions of the protein, but not to the antibody directed to its central region, this putative isoform likely arises by alternative splicing. Some of the smaller Ecm29-positive forms appear to arise by proteolytic processing, but extraction of minced brain fragments in boiling SDS-PAGE sample buffer reveals that all forms are present prior to homogenization. Only the 220-K Ecm29 species is clearly associated with the 26S proteasome; with one exception, the other variants sediment slower than 20S. Direct dissection and immunohistochemistry reveal that these putative Ecm29 isoforms are present throughout the central nervous system. However, anti-Ecm29 immunoreactivity is differentially distributed within subregions of the mouse brain and among cell types, suggesting that Ecm29 isoform distribution contributes to the complexity and regional specificity of brain function.
The neutrophil-derived serine protease, cathepsin G (Cat.G), has been shown to induce myocyte detachment and apoptosis by anoikis through down-regulation of focal adhesion (FA) signaling. However, the mechanisms that control FA protein stability and turnover in myocytes are not well understood. Here, we have shown that the Casitas b-lineage lymphoma (c-Cbl), adaptor protein with an intrinsic E3 ubiquitin ligase activity, is involved in FA and myofibrillar protein stability and turnover in myocytes. Cat.G treatment induced c-Cbl activation and its interaction with FA proteins. Deletion of c-Cbl using c-Cbl knock-out derived myocytes or inhibition of c-Cbl ligase activity significantly reduced FA protein degradation, myofibrillar degeneration, and myocyte apoptosis induced by Cat.G. We also found that inhibition of the proteasome activity, but not the lysosome or the calpain activity, markedly attenuated FA and myofibrillar protein degradation induced by Cat.G. Interestingly, c-Cbl activation induced by Cat.G was mediated through epidermal growth factor receptor (EGFR) transactivation as inhibition of EGFR kinase activity markedly attenuated c-Cbl phosphorylation and FA protein degradation induced by Cat.G. These findings support a model in which neutrophil protease Cat.G promotes c-Cbl interaction with FA proteins, resulting in enhanced c-Cbl-mediated FA protein ubiquitination and degradation, myofibril degradation, and subsequent down-regulation of myocyte survival signaling.
Schistosoma mansoni eggs, consisting of an ovum surrounded by nutritive vitelline cells packaged in a tanned protein shell, are produced by paired worms residing in the mesenteric veins of the human host. The vitelline cells are degraded as the larval miracidium matures, the fully developed egg either crossing the gut wall to escape the host or becoming lodged in the host's tissues where it dies and disintegrates, inducing a potentially pathological immune response. Thus, the egg is central to both the transmission of the parasite and the aetiology of the disease. Here we present the first study investigating protein turnover in the egg. We establish that the ubiquitin-proteasome pathway (UPP) changes with egg development and furthermore, that the morphological components of the fully developed egg (the miracidium and the subshell envelope) also exhibit different proteasome subunit expression profiles. We conclude that the UPP is responsible not only for degrading the vitelline cells but is also more highly developed in the envelope than in the miracidium. The envelope is involved in the defence of the miracidium and produces the proteins that the egg secretes, presumably to facilitate its escape from the host, so the UPP probably has a multi-faceted role in the egg's biology.
Prostate cancer is the most common carcinoma in the male population. In its initial stage, the disease is androgen-dependent and responds therapeutically to androgen deprivation treatment but it usually progresses after a few years to an androgen-independent phase that is refractory to hormonal manipulations. The proteasome is a multi-unit protease system that regulates the abundance and function of a significant number of cell proteins, and its inhibition results in cancer cell growth inhibition and apoptosis and is already exploited in the clinic with the use of proteasome inhibitor bortezomib in multiple myeloma. In order to be recognized by the proteasome, a target protein needs to be linked to a chain of the small protein ubiquitin. In this paper, we review the role of ubiquitin-proteasome system (UPS) in androgen receptor-dependent transcription as well as in the castration resistant stage of the disease, and we discuss therapeutic opportunities that UPS inhibition offers in prostate cancer.
Plants have developed their own defense strategies because they have no immune cells. A common plant defense strategy involves programmed cell death (PCD) at the infection site, but how the PCD-associated cell-autonomous immunity is executed in plants is not fully understood. Here we provide a novel mechanism underlying cell-autonomous immunity, which involves the fusion of membranes of a large central vacuole with the plasma membrane, resulting in the discharge of vacuolar antibacterial proteins to the outside of the cells, where bacteria proliferate. The extracellular fluid that was discharged from the vacuoles of infected leaves had both antibacterial activity and cell death-inducing activity. We found that a defect in proteasome function abolished the membrane fusion associated with both disease resistance and PCD in response to avirulent bacterial strains but not to a virulent strain. Furthermore, RNAi plants with a defective proteasome subunit PBA1 have reduced DEVDase activity, which is an activity associated with caspase-3, one of the executors of animal apoptosis. The plant counterpart of caspase-3 has not yet been identified. Our results suggest that PBA1 acts as a plant caspase-3-like enzyme. Thus, this novel defense strategy through proteasome-regulating membrane fusion of the vacuolar and plasma membranes provides plants with a mechanism for attacking intercellular bacterial pathogens.
Die Therapie des duktalen Pankreasadenokarzinoms zeigt nach wie vor kaum Aussichten auf Erfolg, resultierend in einer ausgesprochen niedrigen Fünf-Jahres-Überlebensrate von ca. zwei Prozent. Eine wesentliche Ursache für die geringe Therapieeffizienz liegt in der hohen Chemoresistenz von Pankreasadenokarzinomen. Diese Chemoresistenz basiert hauptsächlich auf der geringen Sensitivität gegenüber Zytostatika-induzierter Apoptose, die Pankreasadenokarzinome entweder schon zu Therapiebeginn aufweisen (intrinsische Resistenz) oder aber erst im Laufe der Therapie entwickeln (extrinsische Resistenz). Das duktale Pankreasadenokarzinom ist weiterhin vor allem durch eine massive desmoplastische Stromabildung gekennzeichnet, deren Folge ist, dass das Tumorstroma einen großen Einfluss auf die maligne Transformation des Gangepithels und dessen biologische Eigenschaften hat.
Im Rahmen der vorliegenden Arbeit sollte die Bedeutung von Tumor-Stroma-Interaktionen für die Entstehung und Manifestation von Chemoresistenz in humanen Pankreas-karzinomzellen untersucht werden. Es konnte gezeigt werden, dass humane Pankreaskarzinomzellen, die durch Co-Kultivierung mit murinen Pankreasfibroblasten dem Einfluss von Tumor-Stroma-Interaktionen über mehrere Wochen ausgesetzt waren, sowohl in vitro als auch in vivo effektiv vor Zytostatika-induzierter Apoptose geschützt wurden. Als grundlegender Mechanismus dieser Apoptoseresistenz konnte eine erniedrigte Expression der Initiator-Caspasen 8 und 9 sowie der Effektor-Caspasen 3 und 7 in den cokultivierten Pankreaskarzinomzellen identifiziert werden. Die verminderte Expression der Procaspasen auf Proteinebene war dabei nicht bedingt durch eine veränderte proteasomale Degradation, sondern durch erniedrigte Level an Caspase-mRNA, resultierend aus einer verringerten Transkription der Caspase-Gene.
Die Resistenz sowie die herunterregulierte Expression der Procaspasen in den cokultivierten Pankreaskarzinomzellen konnte dabei mit dem DNA-Methylierungsinhibitor 5-Azacytidin aufgehoben werden, so dass ein erhöhter DNA-Methylierungsgrad der Caspase-Gene selbst oder von Caspase-regulierenden Genen als Ursache der erniedrigten Caspase-Expression angesehen werden kann.
Diese Arbeit unterstreicht die Bedeutung von Tumor-Stroma-Interaktionen bei der Entstehung und Manifestation von Chemoresistenz in Pankreasadenokarzinomzellen in vitro und in vivo und trägt durch Aufdeckung molekularer Mechanismen der Caspaseregulation zum Verständnis der Chemoresistenz des duktalen Pankreasadenokarzinoms bei. Pancreatic carcinoma is the fourth leading cause of cancer death in women and the fifth leading cause of cancer death in men, being one of the most lethal human cancers. With conventional cancer treatments having only little impact on disease course the overall five-year survival rate is approximately two percent. Poor therapy outcome is due to a high incidence of metastatic disease at the time of diagnosis and a result of resistance to chemotherapy. One major mechanism by which tumor cells acquire a chemoresistant phenotype is the protection from drug-induced apoptosis. Resistance towards cytotoxic drugs can be present before the onset of chemotherapy (intrinsic) or develops during treatment course (extrinsic).
Pancreatic ductal adenocarcinoma is also characterized by a prominent desmoplastic reaction. Interactions of tumor cells and surrounding stroma (notably fibroblasts) are known to play a critical role in tumorgenesis and the induction of a malignant phenotype. The role of tumor-stroma-interactions in development and manifestation of chemoresistance in human ductal pancreatic adenocarcinoma cells has been the subject of this thesis.
Human pancreatic carcinoma cells, which were under the influence of tumor-stroma-interactions by long term coculture with murine pancreatic fibroblasts, were effectively protected from drug-induced apoptosis in vitro and in vivo. Downregulation of the initiator caspases 8 and 9 as well as the effector caspases 3 and 7 in cocultured pancreatic carcinoma cells could be identified as the main mechanism leading to chemoresistance. The downregulated expression of procaspases was not due to enhanced protein turnover, but was caused by decreased transcription of caspase genes, resulting in low levels of mRNA.
Resistance to drug-induced apoptosis and downregulated expression of procaspases could be reversed by the methylation inhibitor 5-Azacytidine, assuming that an increased methylation of caspase genes or caspase-regulating genes are key events in downregulation of caspases and subsequent chemoresistance in pancreatic carcinoma cells.
This study underlines the importance of tumor-stroma-interactions in development and manifestation of chemoresistance in pancreatic carcinoma cells in vitro and in vivo. Additionally, the detection of molecular mechanisms regulating caspases essentially contributes to the understanding of chemoresistance in pancreatic ductal adenocarcinoma.
Human retinal dystrophies and degenerations and light-induced retinal degenerations in animal models are sharing an important feature: visual cell death by apoptosis. Studying apoptosis may thus provide an important handle to understand mechanisms of cell death and to develop potential rescue strategies for blinding retinal diseases. Apoptosis is the regulated elimination of individual cells and constitutes an almost universal principle in developmental histogenesis and organogenesis and in the maintenance of tissue homeostasis in mature organs.
Parkinson's disease (PD) is a progressive neurodegenerative movement disorder characterized by selective loss of dopaminergic neurons and the presence of Lewy bodies. Alpha-synuclein is a major component of Lewy bodies in sporadic PD, and genetic alterations in alpha-synuclein cause autosomal-dominant hereditary PD. The pathogenesis of PD remains incompletely understood, but it appears to involve both genetic susceptibility and environmental factors. Here we investigated the effect of alpha-synuclein expression on cell susceptibility to proteasome inhibition, oxidative and nitrative stresses by using a PC 12-Tet-off regulatory system. We found that inducible expression of A30P or A53T mutant alpha-synuclein decreased the proteasome activity, increased intracellular ROS levels, and enhanced lactacystin- and H2O2-induced cell death. Furthermore, 3-nitrotyrosine levels increased in cells expressing alpha-synuclein, and further increased after Sin-1 (a NO donor) treatment compared with untreated or treated non-induced cells. Expression of alpha-synuclein (mutant more than wild type) significantly enhances Sin-1 toxicity. These results indicate that genetic mutations in alpha-synuclein may increase neuronal vulnerability to cellular stress in aging and PD pathogenesis.
Infections with RNA viruses are sensed by the innate immune system through membrane-bound Toll-like receptors or the cytoplasmic
RNA helicases RIG-I and MDA-5. It is believed that MDA-5 is crucial for sensing infections by picornaviruses, but there have
been no studies on the role of this protein during infection with poliovirus, the prototypic picornavirus. Beginning at 4
h postinfection, MDA-5 protein is degraded in poliovirus-infected cells. Levels of MDA-5 declined beginning at 6 h after infection
with rhinovirus type 1a or encephalomyocarditis virus, but the protein was stable in cells infected with rhinovirus type 16
or echovirus type 1. Cleavage of MDA-5 is not carried out by either poliovirus proteinase 2Apro or 3Cpro. Instead, degradation of MDA-5 in poliovirus-infected cells occurs in a proteasome- and caspase-dependent manner. Degradation
of MDA-5 during poliovirus infection correlates with cleavage of poly(ADP) ribose polymerase (PARP), a hallmark of apoptosis.
Induction of apoptosis by puromycin leads to cleavage of both PARP and MDA-5. The MDA-5 cleavage product observed in cells
treated with puromycin is ∼90 kDa, similar in size to the putative cleavage product observed in poliovirus-infected cells.
Poliovirus-induced cleavage of MDA-5 may be a mechanism to antagonize production of type I interferon in response to viral
infection.
Ubiquitin-mediated proteolysis plays a major role in a variety of cellular functions, including cell metabolism, cell cycle progression, cellular response to DNA damage, and programmed cell death. In most cases, the crucial regulators involved in the control of these diverse cellular functions are modified by specific E3 ubiquitin ligases through the attachment of multiple ubiquitin molecules, a signal that triggers the subsequent destruction by the 26S proteasome complex. Recent studies revealed that the proteasomal degradation pathway regulates the cellular apoptosis process on multiple levels. Thus, a better understanding of the molecular mechanisms that underlie the ubiquitination and destruction of these specific regulators of apoptosis will provide us with insight on how apoptosis is properly controlled in normal cells and how tumor cells evade the apoptosis pathways. This chapter provides an overview of the common methods used to examine whether a target protein is ubiquitinated, as well as the protocols to examine how a putative E3 ligase controls the destruction of the target protein.
To determine why proteasome inhibitors prevent thymocyte death, we examined whether proteasomes degrade anti-apoptotic molecules in cells induced to undergo apoptosis. The c-IAP1 and XIAP inhibitors of apoptosis were selectively lost in glucocorticoid- or etoposide-treated thymocytes in a proteasome-dependent manner before death. IAPs catalyzed their own ubiquitination in vitro, an activity requiring the RING domain. Overexpressed wild-type c-IAP1, but not a RING domain mutant, was spontaneously ubiquitinated and degraded, and stably expressed XIAP lacking the RING domain was relatively resistant to apoptosis-induced degradation and, correspondingly, more effective at preventing apoptosis than wild-type XIAP. Autoubiquitination and degradation of IAPs may be a key event in the apoptotic program.
Ultraviolet (UV) irradiation of HeLa cells triggers an apoptotic response mediated by mitochondria. Biochemical analysis of this response revealed that the elimination of cytosolic inhibitors is required for mitochondrial release of cytochrome c and subsequent caspase activation. These inhibitors were found to be Mcl-1 and Bcl-xL, two antiapoptotic members of the Bcl-2 family. Following UV treatment, Mcl-1 protein synthesis is blocked, the existing pool of Mcl-1 protein is rapidly degraded by the proteasome, and cytosolic Bcl-xL translocates to the mitochondria. These events are sequential; the elimination of Mcl-1 is required for the translocation of Bcl-xL. The disappearance of Mcl-1 is also required for other mitochondrial apoptotic events including Bax translocation, cytochrome c release, and caspase activation.
Mdm2 has been shown to regulate p53 stability by targeting the p53 protein for proteasomal degradation. We now report that
Mdm2 is a ubiquitin protein ligase (E3) for p53 and that its activity is dependent on its RING finger. Furthermore, we show
that Mdm2 mediates its own ubiquitination in a RING finger-dependent manner, which requires no eukaryotic proteins other than
ubiquitin-activating enzyme (E1) and an ubiquitin-conjugating enzyme (E2). It is apparent, therefore, that Mdm2 manifests
an intrinsic capacity to mediate ubiquitination. Mutation of putative zinc coordination residues abrogated this activity,
as did chelation of divalent cations. After cation chelation, the full activity could be restored by addition of zinc. We
further demonstrate that the degradation of p53 and Mdm2 in cells requires additional potential zinc-coordinating residues
beyond those required for the intrinsic activity of Mdm2 in vitro. Replacement of the Mdm2 RING with that of another protein (Praja1) reconstituted ubiquitination and proteasomal degradation
of Mdm2. However, this RING was ineffective in ubiquitination and proteasomal targeting of p53, suggesting that there may
be specificity at the level of the RING in the recognition of heterologous substrates.
To identify proteins that bind mammalian IAP homolog A (MIHA, also known as XIAP), we used coimmuno-precipitation and 2D immobilized pH gradient/SDS PAGE, followed by electrospray ionization tandem mass spectrometry. DIABLO (direct IAP binding protein with low pI) is a novel protein that can bind MIHA and can also interact with MIHB and MIHC and the baculoviral IAP, OpIAP. The N-terminally processed, IAP-interacting form of DIABLO is concentrated in membrane fractions in healthy cells but released into the MIHA-containing cytosolic fractions upon UV irradiation. As transfection of cells with DIABLO was able to counter the protection afforded by MIHA against UV irradiation, DIABLO may promote apoptosis by binding to IAPs and preventing them from inhibiting caspases.
We report here the identification of a novel protein, Smac, which promotes caspase activation in the cytochrome c/Apaf-1/caspase-9 pathway. Smac promotes caspase-9 activation by binding to inhibitor of apoptosis proteins, IAPs, and removing their inhibitory activity. Smac is normally a mitochondrial protein but is released into the cytosol when cells undergo apoptosis. Mitochondrial import and cleavage of its signal peptide are required for Smac to gain its apoptotic activity. Overexpression of Smac increases cells' sensitivity to apoptotic stimuli. Smac is the second mitochondrial protein, along with cytochrome c, that promotes apoptosis by activating caspases.
The inhibitor of apoptosis (IAP) family of anti-apoptotic proteins regulate programmed cell death and/or apoptosis. One such protein, X-linked IAP (XIAP), inhibits the activity of the cell death proteases, caspase-3, -7, and -9. In this study, using constitutively active mutants of caspase-3, we found that XIAP promotes the degradation of active-form caspase-3, but not procaspase-3, in living cells. The XIAP mutants, which cannot interact with caspase-3, had little or no activity of promoting the degradation of caspase-3. RING finger mutants of XIAP also could not promote the degradation of caspase-3. A proteasome inhibitor suppressed the degradation of caspase-3 by XIAP, suggesting the involvement of a ubiquitin-proteasome pathway in the degradation. An in vitro ubiquitination assay revealed that XIAP acts as a ubiquitin-protein ligase for caspase-3. Caspase-3 was ubiquitinated in the presence of XIAP in living cells. Both the association of XIAP with caspase-3 and the RING finger domain of XIAP were essential for ubiquitination. Finally, the RING finger mutants of XIAP were less effective than wild-type XIAP at preventing apoptosis induced by overexpression of either active-form caspase-3 or Fas. These results demonstrate that the ubiquitin-protein ligase activity of XIAP promotes the degradation of caspase-3, which enhances its anti-apoptotic effect.
The ubiquitin/proteasome pathway is the main non-lysosomal route for intracellular protein degradation in eukaryotes. It is instrumental to various cellular processes, such as cell-cycle progression, transcription and antigen processing. Recent findings also substantiate a pivotal role of the ubiquitin/proteasome pathway in the regulation of apoptosis. Regulatory molecules that are involved in programmed cell death have been identified as substrates of the proteasome. Moreover, key regulators of apoptosis themselves seem to have an active part in the proteolytic inactivation of death executors.
Between the 1960s and 1980s, most life scientists focused their attention on studies of nucleic acids and the translation of the coded information. Protein degradation was a neglected area, considered to be a nonspecific, dead-end process. Although it was known that proteins do turn over, the large extent and high specificity of the process, whereby distinct proteins have half-lives that range from a few minutes to several days, was not appreciated. The discovery of the lysosome by Christian de Duve did not significantly change this view, because it became clear that this organelle is involved mostly in the degradation of extracellular proteins, and their proteases cannot be substrate specific. The discovery of the complex cascade of the ubiquitin pathway revolutionized the field. It is clear now that degradation of cellular proteins is a highly complex, temporally controlled, and tightly regulated process that plays major roles in a variety of basic pathways during cell life and death as well as in health and disease. With the multitude of substrates targeted and the myriad processes involved, it is not surprising that aberrations in the pathway are implicated in the pathogenesis of many diseases, certain malignancies, and neurodegeneration among them. Degradation of a protein via the ubiquitin/proteasome pathway involves two successive steps: 1) conjugation of multiple ubiquitin moieties to the substrate and 2) degradation of the tagged protein by the downstream 26S proteasome complex. Despite intensive research, the unknown still exceeds what we currently know on intracellular protein degradation, and major key questions have remained unsolved. Among these are the modes of specific and timed recognition for the degradation of the many substrates and the mechanisms that underlie aberrations in the system that lead to pathogenesis of diseases.
Inhibitor of apoptosis proteins (IAPs) are a conserved class of proteins that control apoptosis in both vertebrates and invertebrates. They exert their anti-apoptotic function through inhibition of caspases, the principal executioners of apoptotic cell death. Recent advances in vertebrates and Drosophila have demonstrated that IAPs use ubiquitin conjugation to control the stability, and thus the activity, of select target proteins. The Drosophila IAP1 gene is an instructive example: it employs at least two distinct ubiquitin-dependent mechanisms of protein destruction. The apoptosis-inducing genes grim, reaper and hid modulate these mechanisms, and determine the outcome.
The ubiquitin/proteasome system regulates protein turnover by degrading polyubiquitinated proteins. To date, all studies on the relationship of apoptosis and the proteasome have emphasized the key role of the proteasome in the regulation of apoptosis, by virtue of its ability to degrade regulatory molecules involved in apoptosis. We now demonstrate how induction of apoptosis may regulate the activity of the proteasome. During apoptosis, caspase activation results in the cleavage of three specific subunits of the 19S regulatory complex of the proteasome: S6' (Rpt5) and S5a (Rpn10), whose role is to recognize polyubiquitinated substrates of the proteasome, and S1 (Rpn2), which with S5a and S2 (Rpn1) holds together the lid and base of the 19S regulatory complex. This caspase-mediated cleavage inhibits the proteasomal degradation of ubiquitin-dependent and -independent cellular substrates, including proapoptotic molecules such as Smac, so facilitating the execution of the apoptotic program by providing a feed-forward amplification loop.
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