Control Of Protein Degradation Research Articles

Signaling targets of alcoholic intoxication in human skeletal muscle.

Intracellular signaling pathways were investigated in skeletal muscle cells at the early stages of alcohol addiction manifestations. No muscle fiber atrophy was observed in m. vastus lateralis of male patients. No significant changes in the signaling mechanisms that control protein degradation were detected as well. However, the concentration of the insulin-like growth factor (IGF-1) in blood plasma as well as the content of markers of intracellular signaling pathways regulating protein synthesis were significantly reduced compared to the control group.

Transcriptional activation of muscle atrophy promotes cardiac muscle remodeling during mammalian hibernation.

Background. Mammalian hibernation in thirteen-lined ground squirrels (Ictidomys tridecemlineatus) is characterized by dramatic changes on a physiological and molecular level. During hibernation, mammalian hearts show a propensity to hypertrophy due to the need for increasing contractility to pump colder and more viscous blood. While cardiac hypertrophy is quite often a process characterized by decompensation, the ground squirrel studied is an excellent model of cardiac plasticity and cardioprotection under conditions of hypothermia and ischemia. The forkhead box O (Foxo) family of proteins and myogenin (MyoG) are transcription factors that control protein degradation and muscle atrophy by regulating the expression of the E3 ubiquitin ligases, MAFbx and MuRF1. These ligases are part of the ubiquitin proteasome system by transferring ubiquitin to proteins and targeting these proteins for degradation. Regulation of Foxo1 and 3a occurs through phosphorylation at different residues. The threonine-24 (Thr-24) and serine-319 (Ser-319) residues on Foxo1, and the Thr-32 residue on Foxo3a are phosphorylated by Akt, leading to cytoplasmic localization of Foxo. We propose that the described mechanism contributes to the changes taking place in cardiac muscle throughout hibernation.Methods. Total and phosphorylated protein levels of Foxo1 and Foxo3a, as well as total protein levels of MyoG, MAFbx, and MuRF1, were studied using immunoblotting.Results. Immunoblotting results demonstrated upregulations in Foxo1 and Foxo3a total protein levels (1.3- and 4.5-fold increases relative to euthermic control, for Foxo1 and 3a respectively) during late torpor, and protein levels remained elevated throughout the rest of torpor and at interbout arousal. We also observed decreases in inactive, phosphorylated Foxo1 and 3a proteins during throughout torpor, where levels of p-Foxo1 Ser319 and Thr24, as well as p-Foxo3a Thr32 decreased by at least 45% throughout torpor. MyoG was upregulated only during late torpor by 2.4-fold. Protein levels of MAFbx and MuRF1 increased in late torpor as well as during early arousal by as much as 2.8-fold, and MAFbx levels remained elevated during interbout arousal, whereas MuRF1 levels returned to control levels.Discussion. The present results indicate that upregulation and activation of Foxo1 and 3a, in addition to the increase in MyoG levels at late torpor, may be upregulating the expression of MAFbx and MuRF1. These findings suggest that there is activation of the ubiquitin proteasome system (UPS) as ground squirrels arouse from torpor. Therefore, the signalling pathway involving MyoG, and the E3 ligases MAFbx and MuRF1, plays a significant role in cardiac muscle remodelling during hibernation. These findings provide insights into the regulation of protein degradation and turnover in the cardiac muscle of a hibernator model.

Deubiquitinases and cancer: A snapshot.

Ubiquitination is the vital system for controlling protein degradation and regulation of basic cellular processes. Deubiquitinases (DUBs) are emerging as an important regulator of several pathways related to cancer and other diseases. Their ability to detach ubiquitin from the target substrate and regulation of signaling makes it potential target to treat cancer and other fatal diseases. In the current review, we are trying to summarize deubiquitination, and their role in cancer and potential small molecules DUBs inhibitors which can be used as drugs for cancer treatment.

Regulating a Post-Transcriptional Regulator: Protein Phosphorylation, Degradation and Translational Blockage in Control of the Trypanosome Stress-Response RNA-Binding Protein ZC3H11.

The life cycle of the mammalian pathogen Trypanosoma brucei involves commuting between two markedly different environments: the homeothermic mammalian host and the poikilothermic invertebrate vector. The ability to resist temperature and other stresses is essential for trypanosome survival. Trypanosome gene expression is mainly post-transcriptional, but must nevertheless be adjusted in response to environmental cues, including host-specific physical and chemical stresses. We investigate here the control of ZC3H11, a CCCH zinc finger protein which stabilizes stress response mRNAs. ZC3H11 protein levels increase at least 10-fold when trypanosomes are stressed by heat shock, proteasome inhibitors, ethanol, arsenite, and low doses of puromycin, but not by various other stresses. We found that increases in protein stability and translation efficiency both contribute to ZC3H11 accumulation. ZC3H11 is an in vitro substrate for casein kinase 1 isoform 2 (CK1.2), and results from CK1.2 depletion and other experiments suggest that phosphorylation of ZC3H11 can promote its instability in vivo. Results from sucrose density centrifugation indicate that under normal culture conditions translation initiation on the ZC3H11 mRNA is repressed, but after suitable stresses the ZC3H11 mRNA moves to heavy polysomes. The ZC3H11 3'-UTR is sufficient for translation suppression and a region of 71 nucleotides is required for the regulation. Since the control works in both bloodstream forms, where ZC3H11 translation is repressed at 37°C, and in procyclic forms, where ZC3H11 translation is activated at 37°C, we predict that this regulatory RNA sequence is targeted by repressive trans acting factor that is released upon stress.

Inadequate fine-tuning of protein synthesis and failure of amino acid homeostasis following inhibition of the ATPase VCP/p97.

The cellular mechanisms that control protein degradation may constitute a non-oncogenic cancer cell vulnerability and, therefore, a therapeutic target. Although this proposition is supported by the clinical success of proteasome inhibitors in some malignancies, most cancers are resistant to proteasome inhibition. The ATPase valosin-containing protein (VCP; p97) is an essential regulator of protein degradation in multiple pathways and has emerged as a target for cancer therapy. We found that pharmacological depletion of VCP enzymatic activity with mechanistically different inhibitors robustly induced proteotoxic stress in solid cancer and multiple myeloma cells, including cells that were insensitive, adapted, or clinically resistant to proteasome inhibition. VCP inhibition had an impact on two key regulators of protein synthesis, eukaryotic initiation factor 2α (eIF2α) and mechanistic target of rapamycin complex 1 (mTORC1), and attenuated global protein synthesis. However, a block on protein translation that was itself cytotoxic alleviated stress signaling and reduced cell death triggered by VCP inhibition. Some of the proteotoxic effects of VCP depletion depended on the eIF2α phosphatase, protein phosphatase 1 regulatory subunit 15A (PPP1R15A)/PP1c, but not on mTORC1, although there appeared to be cross-talk between them. Thus, cancer cell death following VCP inhibition was linked to inadequate fine-tuning of protein synthesis and activity of PPP1R15A/PP1c. VCP inhibitors also perturbed intracellular amino acid levels, activated eukaryotic translation initiation factor 2α kinase 4 (EIF2AK4), and enhanced cellular dependence on amino acid supplies, consistent with a failure of amino acid homeostasis. Many of the observed effects of VCP inhibition differed from the effects triggered by proteasome inhibition or by protein misfolding. Thus, depletion of VCP enzymatic activity triggers cancer cell death in part through inadequate regulation of protein synthesis and amino acid metabolism. The data provide novel insights into the maintenance of intracellular proteostasis by VCP and may have implications for the development of anti-cancer therapies.

Insulin sensitivity of muscle protein metabolism is altered in patients with chronic kidney disease and metabolic acidosis

An emergent hypothesis is that a resistance to the anabolic drive by insulin may contribute to loss of strength and muscle mass in patients with chronic kidney disease (CKD). We tested whether insulin resistance extends to protein metabolism using the forearm perfusion method with arterial insulin infusion in 7 patients with CKD and metabolic acidosis (bicarbonate 19 mmol/l) and 7 control individuals. Forearm glucose balance and protein turnover (2H-phenylalanine kinetics) were measured basally and in response to insulin infused at different rates for 2 h to increase local forearm plasma insulin concentration by approximately 20 and 50 μU/ml. In response to insulin, forearm glucose uptake was significantly increased to a lesser extent (−40%) in patients with CKD than controls. In addition, whereas in the controls net muscle protein balance and protein degradation were decreased by both insulin infusion rates, in patients with CKD net protein balance and protein degradation were sensitive to the high (0.035 mU/kg per min) but not the low (0.01 mU/kg per min) insulin infusion. Besides blunting muscle glucose uptake, CKD and acidosis interfere with the normal suppression of protein degradation in response to a moderate rise in plasma insulin. Thus, alteration of protein metabolism by insulin may lead to changes in body tissue composition which may become clinically evident in conditions characterized by low insulinemia.

Controlled protein degradation in bacteria

Controlled protein degradation in bacteria

Cullin 4A and 4B ubiquitin ligases interact with γ-tubulin and induce its polyubiquitination.

Regulated polyubiquitination is a key step for controlling protein degradation and maintaining proper balance between the proliferation of normal and uncontrolled cells. Addition of ubiquitin to the proteins by E3 ubiquitin ligases targets them for degradation by the 26S proteosome machinery. Discrepancies in ubiquitination and/or proteosome degradation might lead to multiple genetic disorders in humans. It is reported that CUL1 and BRCA1 ubiquitin ligases localize on centrosome region and regulate the centrosome duplication cycle for genomic stability. In the current study, we predicted the possible interaction of E3 ubiquitin ligase CUL4A complex with γ-tubulin, a centrosome-specific protein, using bioinformatic protein-protein docking analysis. We also confirmed their interaction by performing co-immunoprecipitation studies using endogenous CUL4A/B and stable cell lines that overexpress Flag-CUL4A or Flag-CUL4B. We additionally noted that the γ-tubulin was polyubiquitinated by CUL4A or 4B immune complex indicating that CUL4A or CUL4B may regulate the stability of γ-tubulin. Furthermore, the inhibition of proteosomal degradation pathway using MG132 or LLNV drugs resulted in accumulation and co-localization of CUL4A with γ-tubulin in the centrosome region. Overall, our observation has identified γ-tubulin as a novel target for E3 ubiquitin ligase CUL4 complex, and might lead to the establishment of a unique mechanism for controlling centrosome stability.

Deubiquitinase inhibition as a cancer therapeutic strategy

The ubiquitin proteasome system (UPS) is the main system for controlled protein degradation and a key regulator of fundamental cellular processes. The dependency of cancer cells on a functioning UPS has made this an attractive target for development of drugs that show selectivity for tumor cells. Deubiquitinases (DUBs, ubiquitin isopeptidases) are components of the UPS that catalyze the removal of ubiquitin moieties from target proteins or polyubiquitin chains, resulting in altered signaling or changes in protein stability. A number of DUBs regulate processes associated with cell proliferation and apoptosis, and as such represent candidate targets for cancer therapeutics. The majority of DUBs are cysteine proteases and are likely to be more "druggable" than E3 ligases. Cysteine residues in the active sites of DUBs are expected to be reactive to various electrophiles. Various compounds containing α,β-unsaturated ketones have indeed been demonstrated to inhibit cellular DUB activity. Inhibition of proteasomal cysteine DUB enzymes (i.e. USP14 and UCHL5) can be predicted to be particularly cytotoxic to cancer cells as it leads to blocking of proteasome function and accumulation of proteasomal substrates. We here provide an overall review of DUBs relevant to cancer and of various small molecules which have been demonstrated to inhibit DUB activity.

Molecular pathways: translational potential of deubiquitinases as drug targets.

The ubiquitin proteasome system (UPS) is the main system for controlled protein degradation and a key regulator of fundamental cellular processes. The dependency of cancer cells on a functioning UPS coupled with the clinical success of bortezomib for the treatment of multiple myeloma have made the UPS an obvious target for drug development. Deubiquitinases (DUB) are components of the UPS that encompass a diverse family of ubiquitin isopeptidases that catalyze the removal of ubiquitin moieties from target proteins or from polyubiquitin chains, resulting in altered signaling or changes in protein stability. Increasing evidence has implicated deregulation of DUB activity in the initiation and progression of cancer. The altered pattern of DUB expression observed in many tumors can potentially serve as a clinical marker for predicting disease outcome and therapy response. The finding of DUB overexpression in tumor cells suggests that they may serve as novel targets for the development of anticancer therapies. Several specific and broad-spectrum DUB inhibitors are shown to have antitumor activity in preclinical in vivo models with low levels of systemic toxicity. Future studies will hopefully establish the clinical potential for DUB inhibitors as a strategy to treat cancer.

Gene silencing of NOB1 by lentivirus suppresses growth and migration of human osteosarcoma cells.

NIN1/RPN12 binding protein 1 homolog (Saccharomyces cerevisiae) (NOB1) encodes a chaperone protein that joins the 20S proteasome with the 19S regulatory particle in the nucleus and facilitates the biogenesis of the 26S proteasome, which plays a role in maintaining cellular homeostasis by controlling protein degradation. In order to investigate the role of NOB1 in osteosarcoma, NOB1 protein expression in human osteosarcoma cell lines was assessed using western blot analysis. Lentivirus-mediated short hairpin RNA was employed to knock down NOB1, and the effects of NOB1 silencing on cell growth were assessed using MTT, colony formation and cell cycle assays. Cell migration was observed using the Transwell assay. In addition, the expression levels of E-cadherin and β-catenin were examined by western blot analysis. Functional analysis indicated that NOB1-knockdown markedly inhibited cell growth and caused G2/M-phase arrest in human osteosarcoma cells. Furthermore, NOB1 inhibition decreased cell migration and increased E-cadherin and β-catenin expression in U2OS cells. In conclusion, the present study suggested that NOB1 depletion may inhibit osteosarcoma development by increasing E-cadherin and β-catenin expression and, for the first time, indicated the potential of NOB1 as a target in osteosarcoma treatment.

HACE1-dependent protein degradation provides cardiac protection in response to haemodynamic stress

The HECT E3 ubiquitin ligase HACE1 is a tumour suppressor known to regulate Rac1 activity under stress conditions. HACE1 is increased in the serum of patients with heart failure. Here we show that HACE1 protects the heart under pressure stress by controlling protein degradation. Hace1 deficiency in mice results in accelerated heart failure and increased mortality under haemodynamic stress. Hearts from Hace1−/− mice display abnormal cardiac hypertrophy, left ventricular dysfunction, accumulation of LC3, p62 and ubiquitinated proteins enriched for cytoskeletal species, indicating impaired autophagy. Our data suggest that HACE1 mediates p62-dependent selective autophagic turnover of ubiquitinated proteins by its ankyrin repeat domain through protein–protein interaction, which is independent of its E3 ligase activity. This would classify HACE1 as a dual-function E3 ligase. Our finding that HACE1 has a protective function in the heart in response to haemodynamic stress suggests that HACE1 may be a potential diagnostic and therapeutic target for heart disease.

Polycystin-1 Negatively Regulates Polycystin-2 Expression via the Aggresome/Autophagosome Pathway

Mutations of the PKD1 and PKD2 genes, encoding polycystin-1 (PC1) and polycystin-2 (PC2), respectively, lead to autosomal dominant polycystic kidney disease. Interestingly, up-regulation or down-regulation of PKD1 or PKD2 leads to polycystic kidney disease in animal models, but their interrelations are not completely understood. We show here that full-length PC1 that interacts with PC2 via a C-terminal coiled-coil domain regulates PC2 expression in vivo and in vitro by down-regulating PC2 expression in a dose-dependent manner. Expression of the pathogenic mutant R4227X, which lacks the C-terminal coiled-coil domain, failed to down-regulate PC2 expression, suggesting that PC1-PC2 interaction is necessary for PC2 regulation. The proteasome and autophagy are two pathways that control protein degradation. Proteins that are not degraded by proteasomes precipitate in the cytoplasm and are transported via histone deacetylase 6 (HDAC6) toward the aggresomes. We found that HDAC6 binds to PC2 and that expression of full-length PC1 accelerates the transport of the HDAC6-PC2 complex toward aggresomes, whereas expression of the R4227X mutant fails to do so. Aggresomes are engulfed by autophagosomes, which then fuse with the lysosome for degradation; this process is also known as autophagy. We have now shown that PC1 overexpression leads to increased degradation of PC2 via autophagy. Interestingly, PC1 does not activate autophagy generally. Thus, we have now uncovered a new pathway suggesting that when PC1 is expressed, PC2 that is not bound to PC1 is directed to aggresomes and subsequently degraded via autophagy, a control mechanism that may play a role in autosomal dominant polycystic kidney disease pathogenesis.

Chapter Nine - Structure and Function of the ZTL/FKF1/LKP2 Group Proteins in Arabidopsis

The ZTL/FKF1/LKP2 group proteins are LOV-domain-based blue-light photoreceptors that control protein degradation by ubiquitination. These proteins were identified relatively recently and are known to be involved in the regulation of the circadian clock and photoperiodic flowering in Arabidopsis. In this review, we focus on two topics. First, we summarize the molecular mechanisms by which ZTL and FKF1 regulate these biological phenomena based on genetic and biochemical analyses. Next, we discuss the chemical properties of the ZTL family LOV domains obtained from structural, biophysical, and photochemical characterizations of the LOV domains. These two different levels of approach unveiled the molecular mechanisms by which plants utilize ZTL and FKF1 proteins to monitor light for daily and seasonal adaptation.

Degradation of Proteins by Ubiquitin-Proteasome Pathway

All intracellular and some extracellular proteins are continually degraded and replaced by synthesis of new proteins. Both these processes need to stay in equilibrium since their balance may lead to emergence of diseases. Cells contain many proteolytic systems that ensure highly specific and controlled degradation of proteins. One of these systems is the proteasome, a very complex molecular engine allowing degradation of proteins conjugated to ubiquitin. Since the first isolation of proteasome in 1968, many details about its function have been uncovered. In 2004, Nobel Prize for chemistry was awarded for these discoveries. In our review article, we aimed to summarize information about the mechanism of highly selective degradation of proteins by the ubiquitin proteasome pathway. Individual parts of the paper summarize current knowledge about highly selective degradation of proteins by the ubiquitin proteasome system, mechanisms of protein degradation regulation and bio-logical effects of proteasome inhibitors.

Insulin/IGF-1 Signaling Regulates Proteasome Activity through the Deubiquitinating Enzyme UBH-4

The proteasome plays an important role in proteostasis by carrying out controlled protein degradation in the cell. Impairments in proteasome function are associated with severe and often age-related diseases. Here, we have characterized a molecular mechanism linking insulin/IGF-1 signaling (IIS) to proteasome activity. We show that decreased IIS, which promotes proteostasis and longevity, increases proteasome activity through the FOXO transcription factor DAF-16 in C.elegans. Furthermore, we reveal that DAF-16 represses expression of the proteasome-associated deubiquitinating enzyme ubh-4, which we suggest functions as a tissue-specific proteasome inhibitor. Finally, we demonstrate that proteasome activation through downregulation of the ubh-4 human ortholog uchl5 increases degradation of proteotoxic proteins in mammalian cells. In conclusion, we have established a mechanism by which the evolutionarily conserved IIS contributes to the regulation of proteasome activity in a multicellular organism.

A LOV2 Domain-Based Optogenetic Tool to Control Protein Degradation and Cellular Function

Light perception is indispensable for plants to respond adequately to external cues and is linked to proteolysis of key transcriptional regulators. To provide synthetic light control of protein stability, we developed a generic photosensitive degron (psd) module combining the light-reactive LOV2 domain of Arabidopsis thaliana phot1 with the murine ornithine decarboxylase-like degradation sequence cODC1. Functionality of the psd module was demonstrated in the model organism Saccharomyces cerevisiae. Generation of conditional mutants, light regulation of cyclin-dependent kinase activity, light-based patterning of cell growth, and yeast photography exemplified its versatility. In silico modeling of psd module behavior increased understanding of its characteristics. This engineered degron module transfers the principle of light-regulated degradation to nonplant organisms. It will be highly beneficial to control protein levels in biotechnological or biomedical applications and offers the potential to render a plethora of biological processes light-switchable.

Molecular cloning, characterization and expression analysis of ubiquitin protein ligase gene (As-ubpl) from Artemia sinica

Ubiquitylation is an important protein post-translational regulation pathway, which is involved in controlling protein degradation, tumor occurrence and cell cycle regulation. E3 ubiquitin protein ligase (UBPL) plays a crucial role of the conjugation of activated ubiquitin to protein substrates and leads to targeting proteins for degradation by the proteasome. We amplified one full-length cDNA of the A. sinica UBPL (As-ubpl) gene by RACE technology. The full-length cDNA of As-ubpl is composed of 2931 bp, with a 2571 bp open reading frame (ORF) that encodes a polypeptide of 856 amino acids with a C2 domain, two domains with two conserved Trp (W) residues (WW) and a homologous to E6-AP Carboxyl Terminus (HECT) domain. The amount of As-ubpl showed from real-time PCR indicates that a high expression levels of As-ubpl at 20 h, 40 h and 3 days of embryo development, with highest expression levels appearing in the larval stage (40 h). Furthermore, As-ubpl transcripts were highly up-regulated under salinity (50‰) and low temperature stress (15 °C). These results indicate that As-ubpl is involved in protein regulation of the postdiapause development and in responses to salinity and low temperature stress.

Autophagy and mitophagy in cellular damage control

Autophagy and mitophagy are important cellular processes that are responsible for breaking down cellular contents, preserving energy and safeguarding against accumulation of damaged and aggregated biomolecules. This graphic review gives a broad summary of autophagy and discusses examples where autophagy is important in controlling protein degradation. In addition we highlight how autophagy and mitophagy are involved in the cellular responses to reactive species and mitochondrial dysfunction. The key signaling pathways for mitophagy are described in the context of bioenergetic dysfunction.

Detrimental effects of proteasome inhibition activity in Drosophila melanogaster: implication of ER stress, autophagy, and apoptosis

In eukaryotes, the ubiquitin-proteasome machinery regulates a number of fundamental cellular processes through accurate and tightly controlled protein degradation pathways. We have, herein, examined the effects of proteasome functional disruption in Dmp53 (+/+) (wild-type) and Dmp53 (-/-) Drosophila melanogaster fly strains through utilization of Bortezomib, a proteasome-specific inhibitor. We report that proteasome inhibition drastically shortens fly life-span and impairs climbing performance, while it also causes larval lethality and activates developmentally irregular cell death programs during oogenesis. Interestingly, Dmp53 gene seems to play a role in fly longevity and climbing ability. Moreover, Bortezomib proved to induce endoplasmic reticulum (ER) stress that was able to result in the engagement of unfolded protein response (UPR) signaling pathway, as respectively indicated by fly Xbp1 activation and Ref(2)P-containing protein aggregate formation. Larva salivary gland and adult brain both underwent strong ER stress in response to Bortezomib, thus underscoring the detrimental role of proteasome inhibition in larval development and brain function. We also propose that the observed upregulation of autophagy operates as a protective mechanism to "counterbalance" Bortezomib-induced systemic toxicity, which is tightly associated, besides ER stress, with activation of apoptosis, mainly mediated by functional Drice caspase and deregulated dAkt kinase. The reduced life-span of exposed to Bortezomib flies overexpressing Atg1_RNAi or Atg18_RNAi supports the protective nature of autophagy against proteasome inhibition-induced stress. Our data reveal the in vivo significance of proteasome functional integrity as a major defensive system against cellular toxicity likely occurring during critical biological processes and morphogenetic courses.