Mechanisms Of Protein Turnover Research Articles

The Function, Regulation, and Mechanism of Protein Turnover in Circadian Systems in Neurospora and Other Species.

Circadian clocks drive a large array of physiological and behavioral activities. At the molecular level, circadian clocks are composed of positive and negative elements that form core oscillators generating the basic circadian rhythms. Over the course of the circadian period, circadian negative proteins undergo progressive hyperphosphorylation and eventually degrade, and their stability is finely controlled by complex post-translational pathways, including protein modifications, genetic codon preference, protein-protein interactions, chaperon-dependent conformation maintenance, degradation, etc. The effects of phosphorylation on the stability of circadian clock proteins are crucial for precisely determining protein function and turnover, and it has been proposed that the phosphorylation of core circadian clock proteins is tightly correlated with the circadian period. Nonetheless, recent studies have challenged this view. In this review, we summarize the research progress regarding the function, regulation, and mechanism of protein stability in the circadian clock systems of multiple model organisms, with an emphasis on Neurospora crassa, in which circadian mechanisms have been extensively investigated. Elucidation of the highly complex and dynamic regulation of protein stability in circadian clock networks would greatly benefit the integrated understanding of the function, regulation, and mechanism of protein stability in a wide spectrum of other biological processes.

PUB63, A Novel Early Heat Responsive Ubiquitin E3 Ligase from Rice Implicates Heat Stress Tolerance

Heat stress has emerged as a major environmental challenge triggering reduced crop yields, compromised growth, and negatively affecting the development of crops. The cellular response to heat stress involves transcriptional and translational reprogramming and regulation of protein homeostasis which is crucial for the heat stress response and leads to the differential expression of many genes associated with protein turnover mechanisms. Plant U-box (PUB) proteins are a class of the Ubiquitin-E3 ligase involved in the Ubiquitin-proteasome pathway. In the present study, we investigated the role of a PUB protein from rice, OsPUB63, involved in heat stress responses in rice. We also identified all PUB coding genes from O. sativa indica and compared with O. sativa japonica. A quantitative real time PCR demonstrated, OsPUB63 as an early heat inducible gene in rice and the subcellular localization study confirmed its presence in the nucleus and cytosol. OsPUB63 was demonstrated as a TPR repeat containing protein with molecular weight of 39.3 KDa. Using a combination of genetic, biochemical, and physiological assays, our work establishes OsPUB63 as crucial in regulating the heat stress response in rice. Specifically, we show that OsPUB63 enhances the heat stress tolerance in transgenic Arabidopsis by promoting the accumulation of proline and reduced MDA content. Our work has identified PUB63 as the first PUB gene family E3 ligase from rice which is implicated in heat stress response and provide a crucial target for improving crop yields under high-temperature conditions in rice.

Dynamic regulation of LINC complex composition and function across tissues and contexts.

The concept of mechanotransduction to the nucleus through a direct force transmission mechanism has fascinated cell biologists for decades. Central to such a mechanism is the linker of nucleoskeleton and cytoskeleton (LINC) complex, which spans the nuclear envelope to couple the cytoplasmic cytoskeleton to the nuclear lamina. In reality, there is not one LINC complex identity, but instead, a family of protein configurations of varied composition that exert both shared and unique functions. Regulated expression of LINC complex components, splice variants, and mechanoresponsive protein turnover mechanisms together shape the complement of LINC complex forms present in a given cell type. Disrupting specific gene(s) encoding LINC complex components therefore gives rise to a range of organismal defects. Moreover, evidence suggests that the mechanical environment remodels LINC complexes, providing a feedback mechanism by which cellular context influences the integration of the nucleus into the cytoskeleton. In particular, evidence for crosstalk between the nuclear and cytoplasmic intermediate filament networks communicated through the LINC complex represents an emerging theme in this active area of ongoing investigation.

Keeping synapses in shape: degradation pathways in the healthy and aging brain.

Synapses maintain their molecular composition, plasticity and function through the concerted action of protein synthesis and removal. The complex and polarized neuronal architecture poses specific challenges to the logistics of protein and organelle turnover since protein synthesis and degradation mainly happen in the cell soma. In addition, post-mitotic neurons accumulate damage over a lifetime, challenging neuronal degradative pathways and making them particularly susceptible to the effects of aging. This review will summarize the current knowledge on neuronal protein turnover mechanisms with a particular focus on the presynapse, including the proteasome, autophagy and the endolysosomal route and their roles in regulating presynaptic proteostasis and function. In addition, the author will discuss how physiological brain aging, which entails a progressive decline in cognitive functions, affects synapses and the degradative machinery.

IDENTIFICATION AND QUANTIFICATION OF DEGRADOME COMPONENTS IN SYNOVIAL FLUID

Purpose: The regulatory mechanisms of protein turnover in osteoarthritis (OA) are still poorly understood. For instance, dysregulation of proteolytic reaction may be associated with a variety of disease events and understanding the principles that govern such dysregulation is an important part of OA biomarker analysis. Synovial fluid could reflect protease activity in surrounding joint tissues and could provide important insights about OA disease progression. Thus, our aim was to characterize the degradome of human knee synovial fluid in healthy versus late-stage knee OA.

The F-Box Protein Fbp1 Regulates Virulence of Cryptococcus neoformans Through the Putative Zinc-Binding Protein Zbp1.

The ubiquitin-proteasome system (UPS) is the major protein turnover mechanism that plays an important role in regulating various cellular functions. F-box proteins are the key proteins of the UPS, responsible for the specific recognition and ubiquitination of downstream targets. Our previous studies showed that the F-box protein Fbp1 plays an essential role in the virulence of C. neoformans. However, the molecular mechanism of Fbp1 regulating the virulence of C. neoformans is still unclear. In this study, we analyzed the potential Fbp1 substrates using an iTRAQ-based proteomic approach and identified the zinc-binding protein Zbp1 as a substrate of Fbp1. Protein interaction and stability assays showed that Zbp1 interacts with Fbp1 and is a downstream target of Fbp1. Ubiquitination analysis in vivo showed that the ubiquitination of Zbp1 is dependent on Fbp1 in C. neoformans. Subcellular localization analysis revealed that the Zbp1 protein was localized in the nucleus of C. neoformans cells. In addition, both deletion and overexpression of the ZBP1 gene led to the reduced capsule size, while overexpression has a more significant impact on capsule size reduction. Fungal virulence assays showed that although the zbp1Δ mutants are virulent, virulence was significantly attenuated in the ZBP1 overexpression strains. Fungal load assay showed that the fungal burdens recovered from the mouse lungs decreased gradually after infection, while no yeast cells were recovered from the brains and spleens of the mice infected by ZBP1 overexpression strains. Thus, our results revealed a new determinant of fungal virulence involving the post-translational regulation of a zinc-binding protein.

Nucleocytoplasmic trafficking and turnover mechanisms of BRASSINAZOLE RESISTANT1 in Arabidopsis thaliana

Regulation of the nucleocytoplasmic trafficking of signaling components, especially transcription factors, is a key step of signal transduction in response to extracellular stimuli. In the brassinosteroid (BR) signal transduction pathway, transcription factors from the BRASSINAZOLE RESISTANT1 (BZR1) family are essential in mediating BR-regulated gene expression. The subcellular localization and transcriptional activity of BZR1 are tightly regulated by reversible protein phosphorylation; however, the underlying mechanism is not well understood. Here, we provide evidence that both BZR1 phosphorylation and dephosphorylation occur in the nucleus and that BR-regulated nuclear localization of BZR1 is independent from its interaction with, or dephosphorylation by, protein phosphatase 2A. Using a photoconvertible fluorescent protein, Kaede, as a living tag to distinguish newly synthesized BZR1 from existing BZR1, we demonstrated that BR treatment recruits cytosolic BZR1 to the nucleus, which could explain the fast responses of plants to BR. Additionally, we obtained evidence for two types of protein turnover mechanisms that regulate BZR1 abundance in plant cells: a BR- and 26S proteosome-independent constitutive degradation mechanism and a BR-activated 26S proteosome-dependent proteolytic mechanism. Finally, treating plant cells with inhibitors of 26S proteosome induces the nuclear localization and dephosphorylation of BZR1, even in the absence of BR signaling. Based on these results, we propose a model to explain how BR signaling regulates the nucleocytoplasmic trafficking and reversible phosphorylation of BZR1.

CD13 is a critical regulator of cell\u2013cell fusion in osteoclastogenesis

The transmembrane aminopeptidase CD13 is highly expressed in cells of the myeloid lineage, regulates dynamin-dependent receptor endocytosis and recycling and is a necessary component of actin cytoskeletal organization. Here, we show that CD13-deficient mice present a low bone density phenotype with increased numbers of osteoclasts per bone surface, but display a normal distribution of osteoclast progenitor populations in the bone marrow and periphery. In addition, the bone formation and mineral apposition rates are similar between genotypes, indicating a defect in osteoclast-specific function in vivo. Lack of CD13 led to exaggerated in vitro osteoclastogenesis as indicated by significantly enhanced fusion of bone marrow-derived multinucleated osteoclasts in the presence of M-CSF and RANKL, resulting in abnormally large cells containing remarkably high numbers of nuclei. Mechanistically, while expression levels of the fusion-regulatory proteins dynamin and DC-STAMP1 must be downregulated for fusion to proceed, these are aberrantly sustained at high levels even in CD13-deficient mature multi-nucleated osteoclasts. Further, the stability of fusion-promoting proteins is maintained in the absence of CD13, implicating CD13 in protein turnover mechanisms. Together, we conclude that CD13 may regulate cell–cell fusion by controlling the expression and localization of key fusion regulatory proteins that are critical for osteoclast fusion.

Female mice may have exacerbated catabolic signalling response compared to male mice during development and progression of disuse atrophy.

BackgroundMuscle atrophy is a common pathology associated with disuse, such as prolonged bed rest or spaceflight, and is associated with detrimental health outcomes. There is emerging evidence that disuse atrophy may differentially affect males and females. Cellular mechanisms contributing to the development and progression of disuse remain elusive, particularly protein turnover cascades. The purpose of this study was to investigate the initial development and progression of disuse muscle atrophy in male and female mice using the well‐established model of hindlimb unloading (HU).MethodsOne hundred C57BL/6J mice (50 male and 50 female) were hindlimb suspended for 0 (control), 24, 48, 72, or 168 h to induce disuse atrophy (10 animals per group). At designated time points, animals were euthanized, and tissues (extensor digitorum longus, gastrocnemius, and soleus for mRNA analysis, gastrocnemius and extensor digitorum longus for protein synthesis rates, and tibialis anterior for histology) were collected for analysis of protein turnover mechanisms (protein anabolism and catabolism).ResultsBoth males and females lost ~30% of tibialis anterior cross‐sectional area after 168 h of disuse. Males had no statistical difference in MHCIIB fibre area, whereas unloaded females had ~33% lower MHCIIB cross‐sectional area by 168 h of unloading. Both males and females had lower fractional protein synthesis rates (FSRs) within 24–48 h of HU, and females appeared to have a greater reduction compared with males within 24 h of HU (~23% lower FSRs in males vs. 40% lower FSRs in females). Males and females exhibited differential patterns and responses in multiple markers of protein anabolism, catabolism, and myogenic capacity during the development and progression of disuse atrophy. Specifically, females had greater mRNA inductions of catabolic factors Ubc and Gadd45a (~4‐fold greater content in females compared with ~2‐fold greater content in males) and greater inductions of anabolic inhibitors Redd1 and Deptor with disuse across multiple muscle tissues exhibiting different fibre phenotypes.ConclusionsThese results suggest that the aetiology of disuse muscle atrophy is more complicated and nuanced than previously thought, with different responses based on muscle phenotypes and between males and females, with females having greater inductions of atrophic markers early in the development of disuse atrophy.

Spontaneous Cleavages of a Heterologous Protein, the CenA Endoglucanase of Cellulomonas fimi, in Escherichia coli.

CenA is an endoglucanase secreted by the Gram-positive cellulolytic bacterium, Cellulomonas fimi, to the environment as a glycosylated protein. The role of glycosylation in CenA is unclear. However, it seems not crucial for functional activity and secretion since the unglycosylated counterpart, recombinant CenA (rCenA), is both bioactive and secretable in Escherichia coli. Using a systematic screening approach, we have demonstrated that rCenA is subjected to spontaneous cleavages (SC) in both the cytoplasm and culture medium of E. coli, under the influence of different environmental factors. The cleavages were found to occur in both the cellulose-binding (CellBD) and catalytic domains, with a notably higher occurring rate detected in the former than the latter. In CellBD, the cleavages were shown to occur close to potential N-linked glycosylation sites, suggesting that these sites might serve as ‘attributive tags’ for differentiating rCenA from endogenous proteins and the points of initiation of SC. It is hypothesized that glycosylation plays a crucial role in protecting CenA from SC when interacting with cellulose in the environment. Subsequent to hydrolysis, SC would ensure the dissociation of CenA from the enzyme-substrate complex. Thus, our findings may help elucidate the mechanisms of protein turnover and enzymatic cellulolysis.

Oxidative attack during temperature fluctuation challenge compromises liver protein homeostasis of a temperate fish model.

Seasonal variations in water temperature are a natural stressor of temperate fish that affect growth performance and metabolism globally. Gilthead sea bream is one of the most economically interesting species in the Mediterranean; but its liver metabolism is affected by the cold season. However, the effects of cold on protein turnover mechanisms have hardly been studied. Here, we study the relationship between liver oxidative status and protein homeostasis pathways during a 50-day low temperature period at 14 °C, and subsequent recovery at two times: 7 days (early recovery) and 30 days (late recovery). Liver redox status was determined by measuring oxidised lipids and proteins, the glutathione redox cycle and major antioxidant enzymes activities. Protein turnover was analysed via liver protein expression of HSP70 and HSP90; proteasome 26S subunits and polyubiquitination, as markers of the ubiquitin-proteasome system (UPS); and cathepsin D, as a lysosomal protease. Low temperature exposure depressed antioxidant enzyme activities, affecting the glutathione redox cycle and reducing total glutathione levels. Both the UPS and lysosomal pathways were also depressed and consequently, oxidised protein accumulated in liver. Interestingly, both protein oxidation and polyubiquitination tagging depended on protein molecular weight. Despite all these alterations, temperature recovery reverted most consequences of the cold at different rates: with delayed recovery of total glutathione levels and oxidised protein degradation with respect to enzyme activities recovery. All these findings demonstrate that protein liver homeostasis is compromised after chronic cold exposure and could be the cause of liver affectations reported in aquaculture of temperate fish.

Overexpressed Melk Promotes the Stability of EZH2 through Phosphorylation in Natural Killer/T Cell Lymphoma (NKTL)

EZH2 oncogene is extensively involved in pathophysiology of different cancer contexts including natural killer/T cell lymphoma (NKTL). Only a few studies have dealt with regulations of EZH2 stability in specific context, and it remains un-targetable in NKTL. EZH2 is over-expressed in NKTL and the mechanism is unclear. In this study, we examined EZH2 protein turnover mechanisms in the NKTL context.The serine/threonine kinase Melk is one of the overexpressed genes in NKTL patient samples and cell lines, and the interaction between Melk and EZH2 was established by co-immunoprecipitation. Inhibition of Melk using inhibitor or siRNA both resulted in a decrease of EZH2 protein levels in NKTL cells, whereas there was no change in the mRNA level of EZH2, suggesting that Melk regulated EZH2 at the protein level. Next, we observed a change of EZH2 ubiquitination upon manipulation of Melk expression.Next, in order to confirm that Melk truly affect EZH2 ubiquitination and to identify its (de)ubiquitination site, we used a SILAC-based mass spectrometry (MS) approach. We overexpressed all-lysine-mutated ubiquitin plus EZH2 with or without Melk overexpression in 293T cells, and pulled down EZH2 for MS analysis. The MS data found a decrease of K48-linked ubiquitin peptide upon Melk overexpression, which corresponded to the impact of Melk on EZH2 protein stability, as well as two possible sites of EZH2 (de)ubiquitination. One of these two sites were confirmed in later experiments as a critical site (K222).As Melk is a kinase, it is possible that the regulation of EZH2 ubiquitination is phosphorylation-based. A re-analysis of the EZH2 post-translational modification from the MS data identified S220-phosphorylated EZH2 upon Melk overexpression. And by comparing the forward and reverse H/L ratio of S220-phospho-EZH2 and K222-ubiquitinated-EZH2 peptide, we found the phosphorylation should be an earlier event before (de)ubiquitination. Correspondingly, the enzymatic-dead mutant of Melk could rescue the deubiquitination effect of Melk wildtype on EZH2.The Melk-mediated deubiquitination of EZH2 also mediates Velcade resistance in NKTL. EZH2 overexpression led to increased resistance to Velcade treatment, which could be rescued by EZH2 S220 phospho-dead mutant transfection, and promoted by EZH2 K222 ubi-dead mutant transfection. Conversely, Melk knock-down sensitized the NK lymphoma cells to Velcade treatment. Collectively, this study uncovered a role of Melk in mediating EZH2 ubiquitination through phosphorylation and thus regulating resistance to Velcade in NKTL context. DisclosuresChng:Celgene: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Aslan: Research Funding; Merck: Research Funding; Takeda: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Amgen: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding.

HMGB1 represses the anti-cancer activity of sunitinib by governing TP53 autophagic degradation via its nucleus-to-cytoplasm transport

ABSTRACTSunitinib, a multikinase inhibitor approved for a number of cancer indications has a low response rate. Identifying mechanisms of resistance could lead to rational combination regimens that could improve clinical outcomes. Here we report that resistance to sunitinib therapy was driven by autophagic degradation of TP53/p53. Deletion of ATG7 or ATG5 suppressed TP53 degradation, as did knockdown of SQSTM1/p62. Mechanistically, the transport of TP53 from the nucleus to the cytoplasm was essential for the sunitinib-induced autophagic degradation of TP53 and did not require TP53 nuclear export signals (NESs). Moreover, TP53 degradation was achieved by the transport of its nuclear binding target, HMGB1, which shifted TP53 from the nucleus to the cytoplasm. The inhibition of HMGB1 sensitized cancer cells to sunitinib. Importantly, sunitinib induced the degradation of all TP53 proteins, except for TP53 proteins with mutations in the interaction domain of TP53 with HMGB1 (amino acids 313 to 352). In conclusion, our data identify an alternative HMGB1-mediated TP53 protein turnover mechanism that participates in the resistance of sunitinib and suggest HMGB1 as a potential therapeutic target for improving clinical outcomes of sunitinib.

Antitumor Effects of Blocking Protein Neddylation in T315I-BCR-ABL Leukemia Cells and Leukemia Stem Cells.

Imatinib revolutionized the treatment of chronic myeloid leukemia (CML), but drug resistance and disease recurrence remain a challenge. In this study, we suggest a novel strategy based on blocking protein neddylation to address BCR-ABL point mutations and leukemia stem cells (LSC) that lie at the root of imatinib-resistant recurrences. On the basis of the finding that the NEDD8-activating enzyme subunit NAE1 is overexpressed in CML cells, we hypothesized that the function of certain neddylation-dependent protein substrates might be targeted to therapeutic ends in imatinib-resistant CML cells and LSCs. In support of this hypothesis, we demonstrated that the NAE1 inhibitor MLN4924 induced G2-M-phase arrest and apoptosis in bulk CML cells with wild-type p53, regardless of their T315I mutation status in BCR-ABL. Moreover, MLN4924 inhibited the survival and self-renewal of primary human CML CD34+ cells and LSCs in CML-bearing mice via accumulation of p27kip1 in the nucleus. Notably, p27kip1 silencing attenuated the suppressive effect of MLN4924 on the maintenance of LSCs in CML-bearing mice. Taken together, our findings offer a preclinical proof of concept for targeting protein neddylation as a novel therapeutic strategy to override mutational and LSC-derived imatinib resistance in CML.Significance: These findings highlight a mediator of protein neddylation, a type of protein turnover mechanism, as a viable therapeutic target against imatinib-resistant forms of chronic myelogenous leukemia. Cancer Res; 78(6); 1522-36. ©2018 AACR.

The MTM1-UBQLN2-HSP complex mediates degradation of misfolded intermediate filaments in skeletal muscle.

The ubiquitin proteasome system and autophagy are major protein turnover mechanisms in muscle cells, which ensure stemness and muscle fibre maintenance. Muscle cells contain a high proportion of cytoskeletal proteins, which are prone to misfolding and aggregation; pathological processes that are observed in several neuromuscular diseases called proteinopathies. Despite advances in deciphering the mechanisms underlying misfolding and aggregation, little is known about how muscle cells manage cytoskeletal degradation. Here, we describe a process by which muscle cells degrade the misfolded intermediate filament proteins desmin and vimentin by the proteasome. This relies on the MTM1-UBQLN2 complex to recognize and guide these misfolded proteins to the proteasome and occurs prior to aggregate formation. Thus, our data highlight a safeguarding function of the MTM1-UBQLN2 complex that ensures cytoskeletal integrity to avoid proteotoxic aggregate formation.

Abstract 2070: HMGB1 governs p53 to autophagic degradation via its nucleus-to-cytoplasm transport

Abstract P53 is under exquisitely fine regulation and acts as a transcription factor that regulates the expression of thousands of genes that control apoptosis, necroptosis, ferrotosis, cell cycle arrest, senescence, metabolism and fertility. Regulation of p53 turnover is essential not only for the response to chemotherapeutic drugs or radiation in cancer cell but also to keep p53 activity under control in a normal cell. Moreover, .p53 is found to be overactivated in various diseases including idiopathic pulmonary fibrosis (IPF), alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD). Promoting hyperactivated-p53 degradation can combat these diseases. However, the strategies that based on known proteasome-dependent degradation only can stabilize the expression level of p53 such as nutlins (inhibit the interaction between mdm2 and tumor suppressor p53). Autophagy-lysosomal degradation is the other pathway for controlling cellular protein stability. More and more researches have established that numerous proteins can be degraded through both proteasome and autophagy-lysosome pathways. However, very little is known to date about p53 turnover through autophagy-lysosome pathway. Here we discover that the activation of p53 autophagic degradation during an oral small molecule receptor tyrosine kinase inhibitor sunitinib in p53 wt cancer cells and normal cells. Mechanistically, the nucleus-to-cytoplasm shift is essential for the autophagic degradation of p53 induced by sunitinib, however, does not require p53 nuclear export signals. Indeed, p53 degradation is achieved by the nucleus-to-cytoplasm transport of its nuclear binding target HMGB1, shifting the distribution of p53 from the nucleus to the cytoplasm. Then cytoplasmic p53 directly interacts with the autophagy cargo receptor p62 to promote degradation. Importantly, inhibition of HMGB1 sensitizes cancer cell to sunitinib. Taken together, our study identified an alternative p53 protein turnover mechanism induced by sunitinib, thus not only elucidating the underlying mechanisms that limit sunitinib efficacy in cancer therapy but also opening an avenue for expanding the clinical indications of sunitinib. Note: This abstract was not presented at the meeting. Citation Format: Qiaojun He, Peihua Luo, Xiaochun Yang, Qinjie Weng, Ling Ding, Bo Yang. HMGB1 governs p53 to autophagic degradation via its nucleus-to-cytoplasm transport [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2070. doi:10.1158/1538-7445.AM2017-2070

Evidence for the Deregulation of Protein Turnover Pathways in Atm-Deficient Mouse Cerebellum: An Organotypic Study.

Interferon-stimulated gene 15 (ISG15), an antagonist of the ubiquitin pathway, is elevated in cells and brain tissues obtained from ataxia telangiectasia (A-T) patients. Previous studies reveal that an elevated ISG15 pathway inhibits ubiquitin-dependent protein degradation, leading to activation of basal autophagy as a compensatory mechanism for protein turnover in A-T cells. Also, genotoxic stress (ultraviolet [UV] radiation) deregulates autophagy and induces aberrant degradation of ubiquitylated proteins in A-T cells. In the current study, we show that, as in A-T cells, ISG15 protein expression is elevated in cerebellums and various other tissues obtained from Atm-compromised mice in an Atm-allele-dependent manner (Atm+/+ < Atm+/- < Atm-/-). Notably, in cerebellums, the brain part primarily affected in A-T, levels of ISG15 were significantly greater (3-fold higher) than cerebrums obtained from the same set of mice. Moreover, as in A-T cell culture, UV induces aberrant degradation of ubiquitylated proteins and autophagy in Atm-deficient, but not in Atm-proficient, cerebellar brain slices grown in culture. Thus, the ex vivo organotypic A-T mouse brain culture model mimics that of an A-T human cell culture model and could be useful for studying the role of ISG15-dependent proteinopathy in cerebellar neurodegeneration, a hallmark of A-T in humans.

The Lamin B receptor is essential for cholesterol synthesis and perturbed by disease-causing mutations.

Lamin B receptor (LBR) is a polytopic membrane protein residing in the inner nuclear membrane in association with the nuclear lamina. We demonstrate that human LBR is essential for cholesterol synthesis. LBR mutant derivatives implicated in Greenberg skeletal dysplasia or Pelger-Huët anomaly fail to rescue the cholesterol auxotrophy of a LBR-deficient human cell line, consistent with a loss-of-function mechanism for these congenital disorders. These disease-causing variants fall into two classes: point mutations in the sterol reductase domain perturb enzymatic activity by reducing the affinity for the essential cofactor NADPH, while LBR truncations render the mutant protein metabolically unstable, leading to its rapid degradation at the inner nuclear membrane. Thus, metabolically unstable LBR variants may serve as long-sought-after model substrates enabling previously impossible investigations of poorly understood protein turnover mechanisms at the inner nuclear membrane of higher eukaryotes.

Autophagic degradation of the inhibitory p53 isoform Δ133p53α as a regulatory mechanism for p53-mediated senescence.

Δ133p53α, a p53 isoform that can inhibit full-length p53, is downregulated at replicative senescence in a manner independent of mRNA regulation and proteasome-mediated degradation. Here we demonstrate that, unlike full-length p53, Δ133p53α is degraded by autophagy during replicative senescence. Pharmacological inhibition of autophagy restores Δ133p53α expression levels in replicatively senescent fibroblasts, without affecting full-length p53. The siRNA-mediated knockdown of pro-autophagic proteins (ATG5, ATG7 and Beclin-1) also restores Δ133p53α expression. The chaperone-associated E3 ubiquitin ligase STUB1, which is known to regulate autophagy, interacts with Δ133p53α and is downregulated at replicative senescence. The siRNA knockdown of STUB1 in proliferating, early-passage fibroblasts induces the autophagic degradation of Δ133p53α and thereby induces senescence. Upon replicative senescence or STUB1 knockdown, Δ133p53α is recruited to autophagosomes, consistent with its autophagic degradation. This study reveals that STUB1 is an endogenous regulator of Δ133p53α degradation and senescence, and identifies a p53 isoform-specific protein turnover mechanism that orchestrates p53-mediated senescence.

Arsenic hypertolerance in the protist Euglena mutabilis is mediated by specific transporters and functional integrity maintenance mechanisms

Arsenic is a toxic metalloid known to cause multiple and severe cellular damages, including lipid peroxidation, protein misfolding, mutagenesis and double and single-stranded DNA breaks. Thus, exposure to this compound is lethal for most organisms but some species such as the photosynthetic protist Euglena mutabilis are able to cope with very high concentrations of this metalloid. Our comparative transcriptomic approaches performed on both an arsenic hypertolerant protist, i.e. E. mutabilis, and a more sensitive one, i.e. E. gracilis, revealed multiple mechanisms involved in arsenic tolerance. Indeed, E. mutabilis prevents efficiently the accumulation of arsenic in the cell through the expression of several transporters. More surprisingly, this protist induced the expression of active DNA reparation and protein turnover mechanisms, which allow E. mutabilis to maintain functional integrity of the cell under challenging conditions. Our observations suggest that this protist has acquired specific functions regarding arsenic and has developed an original metabolism to cope with acid mine drainages-related stresses.