Search

Background The 26S proteasome, canonically composed of multi-subunit 19S regulatory (RP) and 20S core (CP) particles, is crucial for cellular proteostasis. Proteasomes are re-modeled, activated, or re-localized and this regulation is critical for plants in response to environmental stresses. The proteasome holoenzyme assembly and dissociation are therefore highly dynamic in vivo. However, the stoichiometric changes of the plant proteasomes and how proteasome associated chaperones vary under common abiotic stresses have not been systematically studied. Results Here, we studied the impact of abiotic stresses on proteasome structure, activity, and interacting partners in Arabidopsis thaliana. We analyzed available RNA expression data and observed that expressions of proteasome coding genes varied substantially under stresses; however, the protein levels of a few key subunits did not change significantly within 24 h. Instead, a switch in the predominant proteasome complex, from 26S to 20S, occurs under oxidative or salt stress. Oxidative stress also reduced the cellular ATP content and the association of HSP70-family proteins to the 20S proteasome, but enhanced the activity of cellular free form CP. Salt stress, on the other hand, did not affect cellular ATP level, but caused subtle changes in proteasome subunit composition and impacted bindings of assembly chaperones. Analyses of an array of T-DNA insertional mutant lines highlighted important roles for several putative assembly chaperones in seedling establishment and stress sensitivity. We also observed that knockout of PBAC1, one of the α-ring assembly chaperones, resulted in reduced germination and tearing of the seed coat following sterilization. Conclusions Our study revealed an evolutionarily conserved mechanism of proteasome regulation during oxidative stress, involving dynamic regulation of the holoenzyme formation and associated regulatory proteins, and we also identified a novel role of the PBAC1 proteasome assembly chaperone in seed coat development.

Targeting the ubiquitin-proteasome system (UPS) - in particular, the proteasome complex - has emerged as an attractive novel cancer therapy. While several proteasome inhibitors have been successfully approved by the Food and Drug Administration for the treatment of hematological malignancies, the clinical efficacy of these inhibitors is unexpectedly lower in the treatment of solid tumors due to the functional and structural heterogeneity of proteasomes in solid tumors. There are ongoing trials to examine the effectiveness of compound and novel proteasome inhibitors that can target solid tumors either alone or in combination with conventional chemotherapeutic agents. The modest therapeutic efficacy of proteasome inhibitors such as bortezomib in solid malignancies demands further research to clarify the exact effects of these proteasome inhibitors on different proteasomes present in cancer cells. The structural, cellular localization and functional analysis of the proteasome complexes in solid tumors originated from different tissues provides new insights into the diversity of proteasomes' responses to inhibitors. In this study, we used an optimized iodixanol gradient ultracentrifugation to purify a native form of proteasome complexes with their intact associated protein partners enriched within distinct cellular compartments. It is therefore possible to isolate proteasome subcomplexes with far greater resolution than sucrose or glycerol fractionations. We have identified differences in the catalytic activities, subcellular distribution, and inhibitor sensitivity of cytoplasmic proteasomes isolated from human colon, breast, and pancreatic cancer cell lines. Our developed techniques and generated results will serve as a valuable guideline for investigators developing a new generation of proteasome inhibitors as an effective targeted therapy for solid tumors.

Aberrantly high expression of EVI1 in acute myeloid leukaemia (AML) is associated with poor prognosis. For targeted treatment of EVI1 overexpressing AML a more detailed understanding of aspects of spatiotemporal interaction dynamics of the EVI1 protein is important. EVI1 overexpressing SB1690CB AML cells were used for quantification and protein interaction studies of EVI1 and ΔEVI1. Cells were cell cycle-synchronised by mimosine and nocodazole treatment and expression of EVI1 and related proteins assessed by western blot, immunoprecipitation and immunofluorescence. EVI1 protein levels oscillate through the cell cycle, and EVI1 is degraded partly by the proteasome complex. Both EVI1 and ΔEVI1 interact with the co-repressor CtBP1 but dissociate from CtBP1 complexes during mitosis. Furthermore, a large fraction of EVI1, but not ΔEVI1 or CtBP1, resides in the nuclear matrix. In conclusion, EVI1- protein levels and EVI1-CtBP1 interaction dynamics vary though the cell cycle and differ between EVI1 and ΔEVI1. These data ad to the functional characterisation of the EVI1 protein in AML and will be important for the development of targeted therapeutic approaches for EVI1-driven AML. Electronic supplementary material The online version of this article (10.1007/s11033-020-05829-1) contains supplementary material, which is available to authorized users.

The present study investigated the seminal plasma proteome of Holstein bulls with low (LF; n = 6) and high (HF; n = 8) sperm freezability. The percentage of viable frozen-thawed sperm (%ViableSperm) determined by flow cytometry varied from -2.2 in LF to + 7.8 in HF bulls, as compared to the average %ViableSperm (54.7%) measured in an 860-sire population. Seminal proteins were analyzed by label free mass spectrometry, with the support of statistical and bioinformatics analyses. This approach identified 1,445 proteins, associated with protein folding, cell–cell adhesion, NADH dehydrogenase activity, ATP-binding, proteasome complex, among other processes. There were 338 seminal proteins differentially expressed (p 2 = 0.84 and 0.79; p

The 26S proteasome is the major degradation machinery for soluble proteins in eukaryotes. Recent evidence reveals the existence of an alternative ATP-powered protein degradation complex, the Cdc48-20S proteasome complex, and we have identified yeast Sod1, a copper-zinc superoxide dismutase, as an endogenous substrate protein. Here, we identified yeast Ths1, an essential threonyl tRNA synthetase, as another endogenous substrate protein of the Cdc48-20S proteasome. In order to analyze the degradation mechanism in more details, we established an in vitro degradation system reconstituted using purified yeast components. Recombinant Sod1 and Ths1 directly interacted with Cdc48, and were degraded in a Cdc48-20S proteasome-dependent manner. Because the substrate proteins were purified from E. coli cells, no eukaryotic modifications including ubiquitination and phosphorylation exist. Therefore, although the 26S proteasome requires ubiquitination for specific recognition of the substrate proteins, the Cdc48-20S proteasome can degrade a class of substrate proteins without any modifications.

Heterozygous microdeletions of chromosome 15q13.3 (MIM: 612001) show incomplete penetrance and are associated with a highly variable phenotype that may include intellectual disability, epilepsy, facial dysmorphism and digit anomalies. Rare patients carrying homozygous deletions show more severe phenotypes including epileptic encephalopathy, hypotonia and poor growth. For years, CHRNA7 (MIM: 118511), was considered the candidate gene that could account for this syndrome. However, recent studies in mouse models have shown that OTUD7A/CEZANNE2 (MIM: 612024), which encodes for an ovarian tumor (OTU) deubiquitinase, should be considered the critical gene responsible for brain dysfunction. In this study, a patient presenting with severe global developmental delay, language impairment and epileptic encephalopathy was referred to our genetics center. Trio exome sequencing (tES) analysis identified a homozygous OTUD7A missense variant (NM_130901.2:c.697C>T), predicted to alter an ultraconserved amino acid, p.(Leu233Phe), lying within the OTU catalytic domain. Its subsequent segregation analysis revealed that the parents, presenting with learning disability, and brother were heterozygous carriers. Biochemical assays demonstrated that proteasome complex formation and function were significantly reduced in patient-derived fibroblasts and in OTUD7A knockout HAP1 cell line. We provide evidence that biallelic pathogenic OTUD7A variation is linked to early-onset epileptic encephalopathy and proteasome dysfunction.

Much of the mammalian genome is transcribed, generating long non-coding RNAs (lncRNAs) that can undergo post-transcriptional surveillance whereby only a subset of the non-coding transcripts is allowed to attain sufficient stability to persist in the cellular milieu and control various cellular functions. Paralleling protein turnover by the proteasome complex, lncRNAs are also likely to exist in a dynamic equilibrium that is maintained through constant monitoring by the RNA surveillance machinery. In this Review, we describe the RNA surveillance factors and discuss the vital role of lncRNA surveillance in orchestrating various biological processes, including the protection of genome integrity, maintenance of pluripotency of embryonic stem cells, antibody–gene diversification, coordination of immune cell activation and regulation of heterochromatin formation. We also discuss examples of human diseases and developmental defects associated with the failure of RNA surveillance mechanisms, further highlighting the importance of lncRNA surveillance in maintaining cell and organism functions and health. Mammalian genomes generate long non-coding RNAs, which are degraded by the RNA surveillance machinery. This regulated degradation is vital for various processes, including for genome integrity, stem cell pluripotency and immune cell activation. Consequently, defects in RNA surveillance cause human diseases and developmental disorders.

Objective: We propose that sirtuin (SIRT) may induce a pro-apoptotic effect by deacetylating transcription factors in A549 cells: depletion of sirtuin-1 (SIRT1) induced cell cycle arrest in cisplatin-resistant A549 (A549/CADD) cells. Methods: Protein and mRNA levels of SIRT1 were investigated using western blot and RT-PCR. In A549 and A549/CADD cells, the cytotoxicity of cisplatin administration was evaluated by MTT assay, proliferation was measured by ECIS, and the cell cycle distribution was analyzed using FACS. Cells were transfected with pcDNA3.1-Myc-SIRT1 or pcDNA3.1-Myc-Control vectors to analyze the impact of SIRT-1 on cisplatin induced drug resistance. SIRT1 localization was studied using immunofluorescence analysis. In addition, immunoprecipitation and 20S proteasome activity assay were performed to examine the relationship of SIRT1 with the proteasome complex. Results: A549/CADD cells exhibited a mesenchymal-like cell characteristic. SIRT1 expression was markedly decreased in A549/CADD cells. We observed that cisplatin regulates p53 stability through the depletion of ubiquitination following SIRT1 downregulation. Furthermore, cisplatin treatment increased proteasomal activity and significantly decreased cytoplasmic SIRT1 protein levels in A549/CADD cells. Conclusion: In this study, we found SIRT1 to be depleted in A549/CADD cells and also determined the underlying resistance mechanism which may act as novel therapeutic targets in overcoming drug resistance.

Comparison of the structures of ClpC1–ecumicin and ClpC1–rufomycin complexes reveals unique interactions that are relevant to their modes of action., The biological processes related to protein homeostasis in Mycobacterium tuberculosis, the etiologic agent of tuberculosis, have recently been established as critical pathways for therapeutic intervention. Proteins of particular interest are ClpC1 and the ClpC1–ClpP1–ClpP2 proteasome complex. The structure of the potent antituberculosis macrocyclic depsipeptide ecumicin complexed with the N-terminal domain of ClpC1 (ClpC1-NTD) is presented here. Crystals of the ClpC1-NTD–ecumicin complex were monoclinic (unit-cell parameters a = 80.0, b = 130.0, c = 112.0 Å, β = 90.07°; space group P21; 12 complexes per asymmetric unit) and diffracted to 2.5 Å resolution. The structure was solved by molecular replacement using the self-rotation function to resolve space-group ambiguities. The new structure of the ecumicin complex showed a unique 1:2 (target:ligand) stoichiometry exploiting the intramolecular dyad in the α-helical fold of the target N-terminal domain. The structure of the ecumicin complex unveiled extensive interactions in the uniquely extended N-terminus, a critical binding site for the known cyclopeptide complexes. This structure, in comparison with the previously reported rufomycin I complex, revealed unique features that could be relevant for understanding the mechanism of action of these potential antituberculosis drug leads. Comparison of the ecumicin complex and the ClpC1-NTD-L92S/L96P double-mutant structure with the available structures of rufomycin I and cyclomarin A complexes revealed a range of conformational changes available to this small N-terminal helical domain and the minor helical alterations involved in the antibiotic-resistance mechanism. The different modes of binding and structural alterations could be related to distinct modes of action.

Made available in DSpace on 2020-12-12T01:48:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-12-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Obesity is a pandemic associated with a high incidence of cardiovascular disease; however, the mechanisms are not fully elucidated. Proteomics may provide a more in-depth understanding of the pathophysiological mechanisms and contribute to the identification of potential therapeutic targets. Thus, our study evaluated myocardial protein expression in healthy and obese rats, employing two proteomic approaches. Male Wistar rats were established in two groups (n = 13/group): control diet and Western diet fed for 41 weeks. Obesity was determined by the adipose index, and cardiac function was evaluated in vivo by echocardiogram and in vitro by isolated papillary muscle analysis. Proteomics was based on two-dimensional gel electrophoresis (2-DE) along with mass spectrometry identification, and shotgun proteomics with label-free quantification. The Western diet was efficient in triggering obesity and impaired contractile function in vitro; however, no cardiac dysfunction was observed in vivo. The combination of two proteomic approaches was able to increase the cardiac proteomic map and to identify 82 differentially expressed proteins involved in different biological processes, mainly metabolism. Furthermore, the data also indicated a cardiac alteration in fatty acids transport, antioxidant defence, cytoskeleton, and proteasome complex, which have not previously been associated with obesity. Thus, we define a robust alteration in the myocardial proteome of diet-induced obese rats, even before functional impairment could be detected in vivo by echocardiogram. Department of Internal Medicine Botucatu Medical School São Paulo State University (UNESP) Centre for Proteome Research Institute of Integrative Biology University of Liverpool Liverpool Department of Morphology Institute of Biosciences São Paulo State University (UNESP) Department of Biostatistics Institute of Biosciences São Paulo State University (UNESP) Center for the Study of Venoms and Venomous Animals (CEVAP)/Graduate Program in Tropical Diseases (FMB) São Paulo State University (UNESP) Department of Internal Medicine Botucatu Medical School São Paulo State University (UNESP) Department of Morphology Institute of Biosciences São Paulo State University (UNESP) Department of Biostatistics Institute of Biosciences São Paulo State University (UNESP) Center for the Study of Venoms and Venomous Animals (CEVAP)/Graduate Program in Tropical Diseases (FMB) São Paulo State University (UNESP) FAPESP: 2014/22152-0 FAPESP: 2015/16934-8 FAPESP: 2017/09688-6

The coordinated transcription of the genome is the fundamental mechanism in molecular biology. Transcription in eukaryotes is carried out by three main RNA polymerases: Pol I, II, and III. One basic problem is how a decrease in tRNA levels, by downregulating Pol III efficiency, influences the expression pattern of protein-coding genes. The purpose of this study was to determine the mRNA levels in the yeast mutant rpc128-1007 and its overdose suppressors, RBS1 and PRT1. The rpc128-1007 mutant prevents assembly of the Pol III complex and functionally mimics similar mutations in human Pol III, which cause hypomyelinating leukodystrophies. We applied RNAseq followed by the hierarchical clustering of our complete RNA-seq transcriptome and functional analysis of genes from the clusters. mRNA upregulation in rpc128-1007 cells was generally stronger than downregulation. The observed induction of mRNA expression was mostly indirect and resulted from the derepression of general transcription factor Gcn4, differently modulated by suppressor genes. rpc128-1007 mutation, regardless of the presence of suppressors, also resulted in a weak increase in the expression of ribosome biogenesis genes. mRNA genes that were downregulated by the reduction of Pol III assembly comprise the proteasome complex. In summary, our results provide the regulatory links affected by Pol III assembly that contribute differently to cellular fitness.

Gankyrin is a regulatory subunit of the 26‐kD proteasome complex and promotes the occurrence and progression of many malignancies. However, the role of gankyrin in osteosarcoma (OS) metastasis remains unclear. Hedgehog signalling has been shown to regulate stem cell homeostasis and cancer metastasis, but the mechanisms that activate this pathway in OS are still poorly understood. Here, a series of in vitro and in vivo assays were carried out to explore the function and mechanism of gankyrin regulating Hedgehog signalling in OS. We demonstrated that gankyrin promotes migration, invasion and regulates the expression of some stemness factors by up‐regulating Gli1 in OS. Importantly, our data showed an interaction between gankyrin and Gli1. Moreover, gankyrin suppresses the ubiquitin‐mediated degradation of Gli1 protein in OS. Gankyrin also significantly promotes the lung metastasis of OS in vivo. Our findings suggest that gankyrin drives metastasis and regulates the expression of some stemness factors in osteosarcoma by activating Hedgehog signalling, indicating that drug screening for compounds targeting gankyrin may contribute to the development of novel and effective therapies for OS.

Muscle accretion is affected by the difference between protein synthesis and its degradation. Studies on different species revealed that muscle proteolysis is mediated by different pathways including the ubiquitin-proteasome pathway in which the ubiquitin protein ligases play an important role. These muscle atrophy associated ligases were not well studied in tilapia. In this study, we characterized the ubiquitin protein ligases MuRF1/2/3, Atrogin-1 and F-box25, members of the ubiquitin-proteasome pathway in tilapia, Oreochromis niloticus, and their expressions in the muscle of starved, fed, refed, and control fish. Sequences of these genes revealed presence of Ring finger, B-box, and Cos domains in all MuRF genes, as well as F-box domain in Atrogin-1 and F-box25 genes. Real-time qPCR data analysis showed that expression of MuRF1/2/3, Atrogin-1, F-box25, and proteasome complex genes was significantly upregulated in starved fish compared to fed fish. Concurrently, the proteasome activity was 1.7-folds elevated in the starved fish compared to fed fish. These results confirm the important role of these genes in muscle degradation and suggest potential usage as markers of muscle accretion in tilapia.

Introduction: Multiple myeloma (MM) is the second most commonly diagnosed hematologic malignancy and has an increasing incidence and prevalence globally, and proteasome inhibitors (PIs) form the backbone of some of our most effective regimens for all phases of this disease in fit and frail patients. Areas covered: Strong understanding of the proteasome complex is increasingly important as the rapid development of new PIs and innovative myeloma therapies complicate the use of old and new combination regimens. We focus herein on the pharmacodynamics and pharmacokinetics of the approved PIs and others in development, including their safety and efficacy in corresponding clinical studies. Expert opinion: Advancements such as the first oral PI, ixazomib, with a more convenient route of administration and improved toxicity profile led to an improved quality of life, patient compliance, and all-oral combination regimens which are efficacious for long-term management of standard and high-risk MM. Novel pan-PIs, such as marizomib, hold the promise of superior clinical activity due to irreversible targeting of all multicatalytic proteinase complex subunits. Development of clinically validated biomarkers of PI sensitivity/resistance is required to inform utilization of the most optimal and effective, rationally targeted PI treatments for all MM patients.

Cytotoxic protein aggregation-induced impairment of cell function and homeostasis are hallmarks of age-related neurodegenerative pathologies. As proteasomal degradation represents the major clearance pathway for oxidatively damaged proteins, a detailed understanding of the molecular events underlying its stress response is critical for developing strategies to maintain cell viability and function. Although the 26S proteasome has been shown to disassemble during oxidative stress, its conformational dynamics remains unclear. To this end, we have developed a new quantitative cross-linking mass spectrometry (QXL-MS) workflow to explore the structural dynamics of proteasome complexes in response to oxidative stress. This strategy comprises SILAC-based metabolic labeling, HB tag-based affinity purification, a 2-step cross-linking reaction consisting of mild in vivo formaldehyde and on-bead DSSO cross-linking, and multi-stage tandem mass spectrometry (MSn) to identify and quantify cross-links. This integrated workflow has been successfully applied to explore the molecular events underlying oxidative stress-dependent proteasomal regulation by comparative analyses of proteasome complex topologies from treated and untreated cells. Our results show that H2O2 treatment weakens the 19S-20S interaction within the 26S proteasome, along with reorganizations within the 19S and 20S subcomplexes. Altogether, this work sheds light on the mechanistic response of the 26S to acute oxidative stress, suggesting an intermediate proteasomal state(s) before H2O2-mediated dissociation of the 26S. The QXL-MS strategy presented here can be applied to study conformational changes of other protein complexes under different physiological conditions.

28 p.-6 fig., Cisplatin (CDDP) is a widely used agent in the treatment of neuroblastoma. Unfortunately, the development of acquired chemoresistance limits its clinical use. To gain a detailed understanding of the mechanisms underlying the development of such chemoresistance, we comparatively analysed established cisplatin-resistant neuroblastoma cell line (UKF-NB-4CDDP) and its sensitive counterpart (UKF-NB-4). First, using viability screenings, we confirmed the decreased sensitivity of tested cells to cisplatin and identified a cross-resistance to carboplatin and oxaliplatin. Then, the proteomic signatures were analysed using nanoLC MS/MS. Among the proteins responsible for UKF-NB-4CDDP chemoresistance, ion channels transport family proteins, ABC superfamily proteins, SLC-mediated trans-membrane transporters, proteasome complex subunits and V-ATPases were identified. Moreover, we detected markedly higher proteasome activity in UKF-NB-4CDDP cells and a remarkable lysosomal enrichment that can be inhibited by bafilomycin A to sensitize UKF-NB-4CDDP to CDDP. Our results indicate that lysosomal sequestration and proteasome activity may be one of key mechanisms responsible for intrinsic chemoresistance of neuroblastoma to CDDP., This project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement No.759585). The authors also gratefully acknowledge financial support from AZV project 15-28334A, ERASMUS + Staff Mobility from European Commission and from the Ministry of Health of the Czech Republic for conceptual development of research organization 00064203 17(University Hospital Motol, Prague, Czech Republic).

Bortezomib, a highly selective reversible inhibitor of the proteasome complex, is used to the current standard of care in the treatment of multiple myeloma. Although its most commonly reported side effects are gastrointestinal symptoms, peripheral neuropathy, neuropathic pain, and thrombocytopenia, cutaneous adverse reactions are also frequently seen. However, severe cutaneous adverse reactions (SCAR) such as Stevens-Johnson syndrome (SJS) occur very rarely. Here we report the first case of bortezomib-induced SJS with confirmed by patch test. In this case, we performed a patch test that proved bortezomib was the offensive drug in this patient, who had been treated with multiple drugs including antibiotics, allopurinol, and anticancer drugs. Although bortezomib-induced SCARs are generally very rare, we suggest that clinicians be aware of potential adverse reactions including SJS.

Multiple myeloma (MM) is still an incurable malignancy of plasma cells. Proteasome inhibitors (PIs) work as the backbone agent and have greatly improved the outcome in majority of newly diagnosed patients with myeloma. However, drug resistance remains the major obstacle causing treatment failure in clinical practice. Indirubin-3’-monoxime (Id-3) is one of the derivatives of Indirubin, a traditional Chinese medicine that has reported to be effective in the treatment of chronic myeloid leukemia and some solid tumors. In this study, we demonstrated the anti-MM activity of Id-3 in both drug sensitive and bortezomib-resistance human MM cell lines and patient-derived cells. Moreover, combination with Id-3 could sensitize MM cells to bortezomib-induced apoptosis. Mechanistically, Id-3 acts as a multifaceted regulator of cell death, which induces DNA damage and cell cycle arrest as well as abrogates NF-κB activation in MM cells. Id-3 efficiently down-regulates USP7 expression, resulting in destabilization of NEK2 and suppression of NF-κB signaling. Importantly, proteasome complex subunits PSME3 (PA28γ) and PSME4 (PA200) were down-regulated in MM cells treated with Id-3. Knockdown of PSME3 or PSME4 by shRNA caused proteasome inhibition and paraprotein accumulation, as well as sensitized MM cells to bortezomib-mediated growth arrest. Clinical data analysis demonstrated that PSME3 and PSME4 are over-expressed in relapsed/refractory MM and associated with inferior overall survival. Taken together, our study indicate that Id-3 is a novel agent triggering proteasome inhibition and represents a promising therapeutic strategy to improve patient outcome in MM. Funding: This work was supported by the Natural Science Foundation of China (8217011925, 81570181, 81920108006), the Non-profit Central Research Institute Fund of the Chinese Academy of Medical Sciences (2018PT31006, 2018RC320012), the Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences CAMS- 2017-I2M-1-005, 2016-I2M-3-023). National Program on Key Basic Research Project 2018ZX09733003. Declaration of Interest: Dr. Kenneth. C. Anderson is the consultant: Pfizer, Amgen, Astrazeneca, Janssen, Precision Biosciences, Mana and Window. Founder/Stock Shareholder: C4 Therapeutics, Oncopep, Raqia and NextRNA. Other authors declared no competing financial interests in relation to the work described. Ethical Approval: The study was approved by the Medical Ethical Committee of the Institute of Hematology & Blood Diseases Hospital, State Key Laboratory of Experimental Hematology, Chinese Academy of Medical Science & Peking Union Medical College, Tianjin, China.

MinD is a cell division ATPase in Escherichia coli that oscillates from pole to pole and regulates the spatial position of the cell division machinery. Together with MinC and MinE, the Min system restricts assembly of the FtsZ-ring to midcell, oscillating between the opposite ends of the cell and preventing FtsZ-ring misassembly at the poles. Here, we show that the ATP-dependent bacterial proteasome complex ClpXP degrades MinD in reconstituted degradation reactions in vitro and in vivo through direct recognition of the MinD N-terminal region. MinD degradation is enhanced during stationary phase, suggesting that ClpXP regulates levels of MinD in cells that are not actively dividing. ClpXP is a major regulator of growth phase–dependent proteins, and these results suggest that MinD levels are also controlled during stationary phase. In vitro, MinC and MinD are known to coassemble into linear polymers; therefore, we monitored copolymers assembled in vitro after incubation with ClpXP and observed that ClpXP promotes rapid MinCD copolymer destabilization and direct MinD degradation by ClpXP. The N terminus of MinD, including residue Arg 3, which is near the ATP-binding site in sequence, is critical for degradation by ClpXP. Together, these results demonstrate that ClpXP degradation modifies conformational assemblies of MinD in vitro and depresses Min function in vivo during periods of reduced proliferation.

Simple Summary The present study shows the role of Drosophila USP14 under ER stress and ER stress related disease, autosomal dominant retinitis pigmentosa. Drosophila USP14 protects cell from ER stress triggered by ER stress-causing chemicals Drosophila S2 cells and suppresses the retinal degeneration in disease model for retinitis pigmentosa by regulating the stability of Rhodopsin-1. This study also indicates the dynamic reorganization of proteasome complex under ER stress. The modulation of USP14 could be a potential therapeutic strategy for treating the diseases associated with protein folding. Abstract Endoplasmic reticulum (ER) stress and its adaptive cellular response, the unfolded protein response (UPR), are involved in various diseases including neurodegenerative diseases, metabolic diseases, and even cancers. Here, we analyzed the novel function of ubiquitin-specific peptidase 14 (USP14) in ER stress. The overexpression of Drosophila USP14 protected the cells from ER stress without affecting the proteasomal activity. Null Hong Kong (NHK) and alpha-1-antitrypsin Z (ATZ) are ER-associated degradation substrates. The degradation of NHK, but not of ATZ, was delayed by USP14. USP14 restored the levels of rhodopsin-1 protein in a Drosophila model for autosomal dominant retinitis pigmentosa and suppressed the retinal degeneration in this model. In addition, we observed that proteasome complex is dynamically reorganized in response to ER stress in human 293T cells. These findings suggest that USP14 may be a therapeutic strategy in diseases associated with ER stress.

The ubiquitin-associated (UBA) domain is an important motif in the modulation of many molecular functionalities. It has been mainly associated with ubiquitin-mediated proteolysis, a multistep mechanism in which undesirable proteins are tagged with polyubiquitin chains for degradation in the proteasome complex. Comparison among UBA domains reveals a quite small structural variability, displaying an overall fold with a tightly packed three-helix bundle, and a common conserved hydrophobic patch on their surface that is important for ubiquitin binding. Mutations in the UBA domain, mainly in the highly conserved hydrophobic patch, induce conformational instabilities, which can be related to weak affinity for ubiquitin. This raises the question whether such hydrophobic patch presents conserved structural arrangement for selective recognition and protein binding. A concern that led us to investigate the stability of the p62-UBA domain as a case study regarding its structural arrangement as a function of temperature and two NaCl concentrations. Our results reveal that the temperature range and ionic strengths considered in this work produced a negligible effect on the three-helix bundle fold of p62-UBA domain.

In the recent past, many of the deubiquitinases (DUB) were found to modulate mitochondrial clearance or mitophagy and thus they are currently projected as therapeutic targets against neurodegeneration. Among these DUBs, USP14 stands at a distinctive juncture, since it can influence both proteasome complex activity and autophagy process. USP14 interference can enhance mitochondrial clearance and thus can protect Parkinsonian phenotypes in Drosophila model. However, in higher animal models of neurodegenerative disorders, evaluation of the protective role of USP14 is yet to be done. In this perspective, we pointed out a few of the major considerations that should be classified before designing experiments to evaluate the therapeutic potential of this DUB in rodent models of neurodegeneration. These are mainly: level of USP14 in the concerned brain region and how the level alters in the model system. Because USP14 mediated mitophagy is Prohibitin2 dependent, the anticipated impact of this protein in this aspect is also discussed. To illustrate our view, we show that USP14 levels increases in adult rat brain substantia nigra (SN) and cerebellum compared to the young ones. We also depict that rotenone treatment can immediately lead to increased SN specific USP14 levels. Our perception thus portrays USP14 as a therapeutic target, especially for addressing SN specific neurodegeneration in adult rat brain, but may vary with the disease model.

The COP9 signalosome (CSN) is an evolutionarily conserved multisubunit protein complex, which controls protein degradation through deneddylation and inactivation of cullin-RING ubiquitin E3 ligases (CRLs). Recently, the CSN complex has been linked to the NF-κB signaling pathway due to its association with the IKK complex. However, how the CSN complex is regulated in this signaling pathway remains unclear. Here, we have carried out biochemical experiments and confirmed the interaction between the CSN and IKK complexes. In addition, we have determined that overexpression of IKKα or IKKβ leads to enhanced phosphorylation of CSN5, the catalytic subunit for CSN deneddylase activity. Mutational analyses have revealed that phosphorylation at serine 201 and threonine 205 of CSN5 impairs CSN-mediated deneddylation activity in vitro. Interestingly, TNF-α treatment not only enhances the interaction between CSN and IKK but also induces an IKK-dependent phosphorylation of CSN5 at serine 201, linking CSN to TNF-α signaling through IKK. Moreover, TNF-α treatment affects the CSN interaction network globally, especially the associations of CSN with the proteasome complex, eukaryotic translation initiation factor complex, and CRL components. Collectively, our results provide new insights into IKK-mediated regulation of CSN associated with the NF-κB signaling pathway.

Background: We previously described a novel micronutrient blend that behaves like a putative calorie restriction mimetic. The aim of this paper was to analyze the beneficial effects of our micronutrient blend in mice and C. elegans, and compare them with calorie restriction. Methods: Whole transcriptomic analysis was performed in the brain cortex, skeletal muscle and heart in three groups of mice: old controls (30 months), old + calorie restriction and old + novel micronutrient blend. Longevity and vitality were tested in C. elegans. Results: The micronutrient blend elicited transcriptomic changes in a manner similar to those in the calorie-restricted group and different from those in the control group. Subgroup analysis revealed that nuclear hormone receptor, proteasome complex and angiotensinogen genes, all of which are known to be directly related to aging, were the most affected. Furthermore, a functional analysis in C. elegans was used. We found that feeding C. elegans the micronutrient blend increased longevity as well as vitality. Conclusions: We describe a micronutrient supplement that causes similar changes (transcriptomic and promoting longevity and vitality) as a calorie restriction in mice and C. elegans, respectively, but further studies are required to confirm these effects in humans.

MinD is a cell division ATPase inEscherichia colithat oscillates from pole to pole and regulates the spatial position of the cell division machinery. Together with MinC and MinE, the Min system restricts assembly of the FtsZ-ring to midcell, oscillating between the opposite ends of the cell and preventing FtsZ-ring misassembly at the poles. Here, we show that the ATP-dependent bacterial proteasome complex ClpXP degrades MinD in reconstituted degradation reactions in vitro, through direct recognition of the MinD N-terminal region, and in vivo. MinD degradation is enhanced during stationary phase, suggesting that ClpXP regulates levels of MinD in cells that are not actively dividing. MinC and MinD are known to co-assemble into linear polymers, therefore we monitored copolymers assembled in vitro after incubation with ClpXP and observed that ClpXP promotes rapid MinCD copolymer disassembly as a result of direct MinD degradation by ClpXP. The N-terminus of MinD, including residue Arg 3, which is near the ATP-binding site, is critical for degradation by ClpXP. Together, these results demonstrate that ClpXP degradation modifies conformational assemblies of MinD in vitro and depresses Min function in vivo during periods of reduced proliferation.

Recent advances in next generation sequencing expanded the availability of tumor mutanome data that list the mutations present in cancer cells. Mutated proteins are an interesting source of neoantigens that can be used to specifically target tumor cells in the context of immunotherapy. However, identifying new antigenic peptides from mutated proteins remains challenging. In this chapter, we present Reverse Immunology as an approach to identify potential antigens from any given polypeptide sequence. First, we explain the rationale behind the identification of candidate HLA-binding peptides through mass spectrometry or in silico approaches. Then, we describe the isolation of low-frequency T-cell precursors specific for the candidate peptides using peptide-HLA multimers. Finally, we discuss validation steps leading to the identification of a T-cell clone recognizing tumor cells that endogenously process the candidate peptide. We also present approaches to study the impact of the proteasome complex on candidate peptide processing.

The proteasome complex is mainly responsible for proteolytic degradation of cytosolic proteins, generating the C-terminus of MHC I-restricted peptide ligands and CD8 T cell epitopes. Therefore, prediction of proteasomal cleavage sites is relevant for anticipating CD8 T-cell epitopes. There are two different proteasomes, the constitutive proteasome, expressed in all types of cells, and the immunoproteasome, constitutively expressed in dendritic cells. Although both proteasome forms generate peptides for presentation by MHC I molecules, the immunoproteasome is the main form involved in providing peptide fragments for priming CD8 T cells. On the contrary, the proteasome provides peptides for presentation by MHC I molecules that can be targeted by already primed CD8 T cells. Proteasome cleavage prediction server (PCPS) is a server for predicting cleavage sites generated by both the constitutive proteasome and the immunoproteasome. Here, we illustrate the usage of PCPS to predict proteasome and immunoproteasome cleavage sites and compare the results with those provided by NetChop, a related tool available online. PCPS is implemented for free public use available online at http://imed.med.ucm.es/Tools/pcps/ .

Virotherpay is emerging as a promising strategy against cancer, and three oncolytic viruses (OVs) have gained approval in different countries for the treatment of several cancer types. Beyond the capability to selectively infect, replicate and lyse cancer cells, OVs act through a multitude of events, including modification of the tumour micro/macro-environment as well as a complex modulation of the anti-tumour immune response by activation of danger signals and immunogenic cell death pathways. Most OVs show limited effects, depending on the viral platform and the interactions with the host. OVs used as monotherapy only in a minority of patients elicited a full response. Better outcomes were obtained using OVs in combination with other treatments, such as immune therapy or chemotherapy, suggesting that the full potential of OVs can be unleashed in combination with other treatment modalities. Here, we report the main described combination of OVs with conventional chemotherapeutic agents: platinum salts, mitotic inhibitors, anthracyclines and other antibiotics, anti-metabolites, alkylating agents and topoisomerase inhibitors. Additionally, our work provides an overview of OV combination with targeted therapies: histone deacetylase inhibitors, kinase inhibitors, monoclonal antibodies, inhibitors of DNA repair, inhibitors of the proteasome complex and statins that demonstrated enhanced OV anti-neoplastic activity. Although further studies are required to assess the best combinations to translate the results in the clinic, it is clear that combined therapies, acting with complementary mechanisms of action might be useful to target cancer lesions resistant to currently available treatments.

Background Homeostasis of the proteome is critical to the development of chloroplasts and also affects the expression of certain nuclear genes. CLPC1 facilitates the translocation of chloroplast pre-proteins and mediates protein degradation. Results We found that proteins involved in photosynthesis are dramatically decreased in their abundance in the clpc1 mutant, whereas many proteins involved in chloroplast transcription and translation were increased in the mutant. Expression of the full-length CLPC1 protein, but not of the N-terminus-deleted CLPC1 (ΔN), in the clpc1 mutant background restored the normal levels of most of these proteins. Interestingly, the ΔN complementation line could also restore some proteins affected by the mutation to normal levels. We also found that that the clpc1 mutation profoundly affects transcript levels of chloroplast genes. Sense transcripts of many chloroplast genes are up-regulated in the clpc1 mutant. The level of SVR7, a PPR protein, was affected by the clpc1 mutation. We showed that SVR7 might be a target of CLPC1 as CLPC1-SVR7 interaction was detected through co-immunoprecipitation. Conclusion Our study indicates that in addition to its role in maintaining proteome homeostasis, CLPC1 and likely the CLP proteasome complex also play a role in transcriptome homeostasis through its functions in maintaining proteome homeostasis. Electronic supplementary material The online version of this article (10.1186/s12870-018-1396-0) contains supplementary material, which is available to authorized users.

Colorectal cancer (CRC) is one of the most common and mortal types of cancer. There is increasing evidence that some polyunsaturated fatty acids (PUFAs) exercise specific inhibitory actions on cancer cells through different mechanisms, as a previous study on CRC cells demonstrated for two very long-chain PUFA. These were docosahexaenoic acid (DHA, 22:6n3) and arachidonic acid (ARA, 20:4n6) in the free fatty acid (FFA) form. In this work, similar design and technology have been used to investigate the actions of both DHA and ARA as monoacylglycerol (MAG) molecules, and results have been compared with those obtained using the corresponding FFA. Cell assays revealed that ARA- and DHA-MAG exercised dose- and time-dependent antiproliferative actions, with DHA-MAG acting on cancer cells more efficiently than ARA-MAG. Sequential window acquisition of all theoretical mass spectra (SWATH) &ndash, mass spectrometry massive quantitative proteomics, validated by parallel reaction monitoring and followed by pathway analysis, revealed that DHA-MAG had a massive effect in the proteasome complex, while the ARA-MAG main effect was related to DNA replication. Prostaglandin synthesis also resulted as inhibited by DHA-MAG. Results clearly demonstrated the ability of both ARA- and DHA-MAG to induce cell death in colon cancer cells, which suggests a direct relationship between chemical structure and antitumoral actions.

Heterotopic ossification (HO), the pathological extraskeletal formation of bone, can arise from blast injuries, severe burns, orthopedic procedures and gain-of-function mutations in a component of the bone morphogenetic protein (BMP) signaling pathway, the ACVR1/ALK2 receptor serine-threonine (protein) kinase, causative of Fibrodysplasia Ossificans Progressiva (FOP). All three ALKs (−2, −3, −6) that play roles in bone morphogenesis contribute to trauma-induced HO, hence are well-validated pharmacological targets. That said, development of inhibitors, typically competitors of ATP binding, is inherently difficult due to the conserved nature of the active site of the 500+ human protein kinases. Since these enzymes are regulated via inherent plasticity, pharmacological chaperone-like drugs binding to another (allosteric) site could hypothetically modulate kinase conformation and activity. To test for such a mechanism, a surface pocket of ALK2 kinase formed largely by a key allosteric substructure was targeted by supercomputer docking of drug-like compounds from a virtual library. Subsequently, the effects of docked hits were further screened in vitro with purified recombinant kinase protein. A family of compounds with terminal hydrogen-bonding acceptor groups was identified that significantly destabilized the protein, abolishing activity. Destabilization was pH-dependent, putatively mediated by ionization of a histidine within the allosteric substructure with decreasing pH. In vivo, nonnative proteins are degraded by proteolysis in the proteasome complex, or cellular trashcan, allowing for the emergence of therapeutics that inhibit through degradation of over-active proteins implicated in the pathology of diseases and disorders. Because HO is triggered by soft-tissue trauma and ensuing hypoxia, dependency of ALK destabilization on hypoxic pH imparts selective efficacy on the allosteric inhibitors, providing potential for safe prophylactic use.

Endometrial cancer incidence increases annually. Several risk factors, including high glucose intake, are associated with endometrial cancer. We investigated whether glucose affects lysine-specific demethylase 1 (LSD1) expression and the responsible molecular mechanisms. A high concentration of glucose stimulated p62 phosphorylation and increased LSD1 protein expression. Knockdown of p62 or treatment with mammalian target of rapamycin (mTOR), transforming growth factor-β activated kinase 1 (TAK1), casein kinase 1 (CK1), and protein kinase C (PKC) inhibitors abrogated glucose-regulated LSD1 expression. Unphosphorylated p62 and LSD1 formed a complex with Kelch-like ECH-associated protein 1 (KEAP1) and were degraded by the KEAP1-dependent proteasome. Phosphorylated p62 increased LSD1 protein expression by escaping the KEAP1 proteasome complex. LSD1 and KEAP1 interaction was enhanced in the presence of the nuclear factor erythroid 2-related factor 2 (NRF2) protein. LSD1 also participated in antioxidant gene regulation with NRF2. In diabetic mice, increasing LSD1and phospho-p62 expression was observed in uterine epithelial cells. Our results indicate that glucose induces p62 phosphorylation through mTOR, TAK1, CK1, and PKC kinases. Subsequently, phospho-p62 competitively interacts with KEAP1 and releases NRF2–LSD1 from the KEAP1 proteasome complex. Our findings may have public health implications for the prevention of endometrial cancer.

Background Proteasome, a complex involved in the intracellular protein degradation, consists of multiple subunits, but only three subunits have enzymatic activity to cleave and degrade proteins, namely β1, β2 and β5. Carfilzomib (CFZ), a second-generation proteasome inhibitor (PI), can induce cell death by selective and irreversible inhibition of β5 subunit of proteasome. Preclinical data suggested that high-dose CFZ could co-inhibit predominantly β2 proteasome activity, followed by β1 inhibition (Besse et al, Cell Chem Biol. 2019). Over the past few years, CFZ has become a corner stone for multiple myeloma (MM) therapy. Currently, CFZ is approved by the FDA in different dosing schedules in combination with lenalidomide or daratumumab and dexamethasone. However, the optimal CFZ dosing is still a matter of debate, with the approved dosage ranging from 20to 70mg/m 2 in different regimens. In addition, if response can be recaptured by escalating CFZ dose in patients progressing from low-dose CFZ has yet to be determined. The aim of our current study was to analyse the profile of proteasome inhibition in the respective dose cohorts and to elucidate if high-dose CFZ could recapture response in patients resistant to low-dose CFZ. Methods We prospectively collected clinical data and peripheral blood mononuclear cells (PBMC) of 32 patients with relapsed/refractory (RR) MM before and 1-8 hours after CFZ administration. PBMC were lysed and labelled for the activity of individual proteasome subunits using activity based proteasome probes and the proteasome subunits were separated using SDS-PAGE. The activity of constitutive and immunoproteasome β1, β2 and β5 subunits was evaluated by densitometry analysis and combination of the activity of constitutive and immunoproteasome individual subunit was used for further analysis. Results Overall, six, nine, twelve and five patients received CFZ at a dose of 20, 27, 36 and 56 mg/m 2, respectively. As expected, the total activity of proteasome decreased with higher doses of CFZ. Significant inhibition (median inhibition > 50%) of β5 subunit was observed already at 20 mg/m 2 dose, while β2 subunit started to be co-inhibited only at a dose of ≥27 mg/m 2. Significant co-inhibition of β2 activity was seen at 36 mg/m 2 dose, at which also β1 subunit started to be co-inhibited. Finally, at 56 mg/m 2, the activity of all active subunits was inhibited with a median inhibition of > 50%, with the strongest inhibition of the β5 subunit, followed by β2 and then β1. When we compared the patient groups low-dose CFZ (20 or 27 mg/m 2) versus high-dose CFZ (36 or 56 mg/m 2), we observed a significant difference in β2 (P=0.002) and β5 (P=0.02) subunit inhibition between the both groups. In terms of total proteasome activity, high-dose CFZ demonstrated a significantly higher proteasome inhibition in comparison with patients receiving low-dose CFZ (P=0.01). In brief, our results suggested that high-dose CFZ, in contrast to low-dose CFZ, could obtain superior proteasome inhibition by co-inhibiting β2 subunit of proteasome complex. In light of this finding, we successfully treated six RRMM patients who were resistant to low-dose CFZ with CFZ dose escalation. All six patients were heavily pretreated with 3-12 lines of therapy including daratumumab, two PIs, two immunomodulatory drugs and autologous stem cell transplant. Additionally, one and two patients received prior treatment with B-cell maturation antigen targeted bi-specific antibody and chimeric antigen receptor modified T-cell, respectively. In the last line of treatment, these six patients showed progression during CFZ based regimens with low-dose CFZ, namely 20 or 27 mg/m 2. We therefore increased the CFZ dose to 36 or 56 mg/m 2 and the doses of agents other than CFZ in the combination regimens remained the same. High-dose CFZ dose recaptured response in all six patients with four and two patients that achieved partial remission and very good partial remission, respectively, and the progression free survival ranged from 1-13 months. Conclusion In summary, high-dose CFZ, namely ≥ 36mg/m 2, showed more effective proteasome inhibition via blocking β5 and β2 subunits, while low-dose CFZ could not achieve a sufficient inhibition of β2 subunit. We provided the first in human evidence that high-dose CFZ could recapture response in RRMM patients resistant to low-dose CFZ by co-inhibiting the β2 subunit activity of proteasome complex. Figure 1 Figure 1. Disclosures Einsele: Janssen, Celgene/BMS, Amgen, GSK, Sanofi: Consultancy, Honoraria, Research Funding.

Cellular processes are tightly regulated by functional protein networks, which are composed of numerous protein-protein interactions (PPIs). Proteins interact with their binding partners through distinct mechanisms, while defects of these machineries have been consequently implicated in the development of various diseases, such as cancer, neurodegeneration and immunological disorders. Interactions between proteins contribute to a complicated network involving many signaling pathways, and these interactions are either permanent or transient depending on the circumstances. Stable interactions assemble relatively stable complexes, which are mainly responsible for cellular functions under unstressed conditions, such as the proteasome complex, nuclear pore complex and histone octamer. On the other hand, transient proteins interactions allow cells to respond to both intracellular signals and extracellular stimuli timely and efficiently. To understand how PPIs are precisely regulated, it is critical to know how proteins and their binding partners interact in precise orientations. It is now well accepted that post-translational modifications (PTMs) of certain amino acid residues can be recognized and bound by specific motifs. PTMs such as methylation, acetylation, phosphorylation or ubiquitination on certain protein amino acid residues located on or around the interaction surfaces may interfere the electrical property, hydrophobicity, structure of proteins and provide anchors or obstacles for intermolecular binding. This is of great importance in regulating both permanent and transient interactions, which are indispensable for the coordination of temporal and spatial adaption. Abnormal PTMs can result in aberrant protein interaction network, cause systematic dysfunctions and ultimately lead to diseases such as cancer. Therefore, inhibitors design based on PTMs regulated PPIs may have good therapeutic prospects for cancer treatment. Recent studies have begun to show that, with the right tools, certain classes of PTMs regulated PPI can yield to the efforts to develop inhibitors to disrupt the interactions between proteins and their partners, and the first PTMs regulated PPI inhibitors have reached clinical development. In this review, we described the research leading to these breakthroughs, briefly summarize the vital roles played by several common PTMs in PPIs regulation and highlight the existence of mechanisms and structural basis, and explore their roles in deciding whether the PPIs are specific or not. Besides, we also explore and discuss emerging effective research strategies for PTMs in PPIs regulation.

Background: Multiple myeloma (MM) patients with suboptimal response to induction therapy or early relapse, classified as the functional high-risk (FHR) patients, have been shown to have poor outcomes. However, the current risk stratification at diagnosis has not been able to accurately identify these patients. In clinical practice, we saw patients who were not identified as being high-risk at diagnosis having refractory disease or early relapse. The aim of our study is to evaluate the genomic profile of FHR MM patients at diagnosis. Method: We evaluated newly-diagnosed MM patients in the CoMMpass dataset and divided them into 3 groups: genomic high-risk (GHR) group for patients with t(4;14) or t(14;16) or del17p13 and TP53 mutation or 1q gain and International Staging System (ISS) stage 3; FHR group for patients who were refractory to induction therapy or had early relapse within 12 months and without the markers of GHR group; and standard-risk (SR) group for patients who did not fulfil both criteria. We evaluated the genomic profile based on the differentially expressed genes (DEG), copy number aberrations (CNA), mutational signatures (MS), and gene set enrichment analysis (GSEA). Results: Of 512 evaluable patients, there were 345 patients in the SR group, 106 patients in the GHR group, and 61 patients in the FHR group. On the survival analysis, both FHR and GHR groups had significantly poorer outcomes as compared with the SR group, with FHR group being the worst (FHR: HR=5.19, p=3.42x10-11;GHR: HR=3.55, p=3.5x10-8) (Fig A) The DEGs in FHR and GHR groups were distinct. FHR patients were enriched for genes linked to centromeres, mitosis, DNA repair, C2H2-type zinc finger proteins, and proteasome complex. On the other hand, GHR patients were enriched for genes linked to ribosomal proteins, immunoglobulin proteins, and cell-cell junctions. As FHR patients could not be identified at diagnosis by clinical and genetic parameters, we applied established gene expression signatures of high-risk disease, including proliferation (PI), chromosomal instability (CIN70, CINSARC, CINGEC), centrosome (CI), cell death (HZDCD), and others (EMC92, HMCL7, IFM15, UAMS70, and UAMS80), to see if they could be used. Interestingly, none of these could identify all FHR patients. The best amongst these signatures was able to identify one-third of these patients. Indeed, about one-third of patients were not classified as high-risk by any of these signatures (Fig B). We next explored the use of machine learning methods on the 453 DEGs to identify a predictive model for the FHR group. Our predictive model based on random forest technique resulted in 19 significant genes with accuracy of 0.88, specificity of 0.94, sensitivity of 0.47, AUC - ROC of 0.70, F1 score of 0.48, and Matthews correlation coefficient of 0.42 (Fig C). In terms of CNA, FHR group was predominantly hyperdiploid. On the other hand, GHR group was mostly non-hyperdiploid with fewer gains of odd-numbered chromosomes, more pronounced 13q deletion, and increased 1q gain, as compared with FHR (p=1.45×10-10) and SR groups (p Mutation analysis revealed that IL6-JAK-STAT3 pathway had more mutated genes per patient in FHR group, while estrogen response, KRAS, and WNT β catenin signaling pathways had more mutated genes per patient in GHR group. GHR also had higher mutational load (p=0.00331), and FGFR3 (p=1.63×10-11), PRKD2 (p=2.82×10-7), and TP53 (p=8.7×10-6) were predominantly mutated in GHR group as compared with others. MS analysis using SigProfiler with Catalogue Of Somatic Mutations in Cancer (COSMIC) reference catalogue (version 3.1) showed that as compared with SR group, FHR group had increased activity in the defective homologous recombination-based DNA damage repair signature (SBS3), while GHR group had increased activity in the AID/APOBEC family of cytidine deaminase signatures (SBS2, SBS13). Interestingly, GSEA showed enrichment in cell cycle related gene sets (G2M, E2F) in FHR group and estrogen response gene sets in GHR group (Fig D). Conclusion: Identifying FHR MM patients at diagnosis represents an unmet clinical need and in this study, we report preliminary results in developing a high-specificity classifier. Our study also shows that FHR patients have specific MS and deregulate genes in unique pathways and biological processes as compared with GHR patients. This may provide insights into the biology and potential therapeutics of this group. Figure 1 Disclosures Chng: Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria; Abbvie: Honoraria.

The proteasome complex is the main responsible of proteolytic degradation of cytosolic proteins, generating the C-terminus of MHC I-restricted peptide ligands and CD8 T cell epitopes. Prediction of proteasomal cleavage sites is thus relevant for predicting CD8 T cell epitopes. We previously reported an $n$ -gram based method to predict proteasomal cleavage sites named PCPS, which is implemented for free public use online at http://imed.med.ucm.es/pcps/. Here, we used a set of 59 Hepatitis C Virus (HCV) CD8 T-cell epitopes to analyze cleavage predictions by PCPS and compared them with those provided by a related method implemented in NetChop web server. PCPS clearly outperformed NetChop in sensitivity (0.88 and 0.78, respectively); it identified much better than NetChop that the C-terminus of tested CD8 $T$ cell epitopes likely resulted from proteasomal cleavage. However, PCPS identified additional preferential cleavage sites within the tested epitopes more often than NetChop, which resulted in lower specificity (0.57 vs 0.66, respectively). Proteasomal cleavage site predictions are used to enhance CD8 T cell epitopes by discarding peptides resulting from peptide-MHC I binding models that do not have a C-terminus compatible with proteasome production. Thereby, we have now implemented proteasomal cleavage site predictions provided by PCPS $n$ -grams in RANKPEP, a tool to identify peptides presented by MHC molecules, allowing to filter out peptides predicted to bind to MHC I molecules that are unlikely to result from proteasomal cleavage. RANKPEP is available for free public use online at http://imed.med.ucm.es/Tools/rankpep_new.html.

The ubiquitin-proteasome system (UPS) and autophagy are two major degradative pathways of proteins in eukaryotic cells. As about 30% of newly synthesized proteins are known to be misfolded under normal cell conditions, the precise and timely operation of the UPS and autophagy to remove them as well as their tightly controlled regulation, is so important for proper cell function and survival. In the UPS, target proteins are labeled by small proteins called ubiquitin, which are then transported to the proteasome complex for degradation. Alternatively, many greatly damaged proteins are believed to be delivered to the lysosome for autophagic degradation. Although these autophagy and UPS pathways have not been considered to be directly related, many recent studies proposed their close link and dynamic interconversion. In this review, we’ll focus on the several regulatory molecules that function in both UPS and autophagy and their crosstalk. Among the proposed multiple modulators, we will take a closer look at the so-called main connector of UPS-autophagy regulation, p62. Last, the functional role of p62 in the mitophagy and its implication for the pathogenesis of Parkinson''s disease, one of the major neurodegenerative diseases, will be briefly reviewed. [BMB Reports 2020; 53(1): 56-63]

Proteasomes are highly abundant and conserved protease complexes that eliminate unwanted proteins in the cells. As a single-chain ATP-independent nuclear proteasome activator, proteasome activator 200 (PA200) associates with 20S core particle to form proteasome complex that catalyzes polyubiquitin-independent degradation of acetylated histones, thus playing a pivotal role in DNA repair and spermatogenesis. Here, we present cryo–electron microscopy (cryo-EM) structures of the human PA200-20S complex and PA200 at 2.72 Å and 3.75 Å, respectively. PA200 exhibits a dome-like architecture that caps 20S and uses its C-terminal YYA (Tyr-Tyr-Ala) to induce the α-ring rearrangements and partial opening of the 20S gate. Our structural data also indicate that PA200 has two openings formed by numerous positively charged residues that respectively bind (5,6)-bisdiphosphoinositol tetrakisphosphate (5,6[PP]2-InsP4) and inositol hexakisphosphate (InsP6) and are likely to be the gates that lead unfolded proteins through PA200 and into the 20S. Besides, our structural analysis of PA200 found that the bromodomain (BRD)-like (BRDL) domain of PA200 shows considerable sequence variation in comparison to other human BRDs, as it contains only 82 residues because of a short ZA loop, and cannot be classified into any of the eight typical human BRD families. Taken together, the results obtained from this study provide important insights into human PA200-induced 20S gate opening for substrate degradation and the opportunities to explore the mechanism for its recognition of H4 histone in acetylation-mediated proteasomal degradation., Proteasomes are highly abundant and conserved protease complexes that eliminate unwanted proteins in the cells. The cryo-EM structures of PA200 and the PA200-20S proteasome complex reveal two openings on PA200 which bind inositol phosphates as cofactors, and novel insights into PA200-induced gate-opening of 20S.

Fluctuations in ambient temperature along with the presence of pathogenic microorganisms can induce important cellular changes that alter the homeostasis of ectothermic fish. The aim of this study was to evaluate how sudden or gradual changes in environmental temperature together with the administration of Piscirickettsia salmonis modulate the transcription of genes involved in cellular stress response in the liver of Eleginops maclovinus. Fish were subjected to the following experimental conditions in duplicate: C− 12 °C: Injection only with culture medium, C+ 12 °C: Injection with P. salmonis, AM 18 °C: Injection only with culture medium under acclimation at 18 °C, AB 18 °C: Injection with P. salmonis under acclimation at 18 °C, SM 18 °C: Injection only with culture medium and thermal shock at 18 °C and SB 18 °C: Injection with P. salmonis and thermal shock at 18 °C and sampling at 4-, 8-, 12-, 16- and 20-day post injection (dpi). The genes implied in the heat shock response (HSP70, HSC70, HSP90, and GRP78), apoptosis pathway (BAX and SMAC/Diablo), ubiquitination (E2, E3, ubiquitin, and CHIP), and 26 proteasome complex (PSMB7, PSMC1, and PSMA2) showed expression profiles dependent on time and type of injection applied. All the genes greatly increased their expression levels at day 16 and showed moderate increases at day 20, except for PSMA2 which showed a higher increase between 4- and 12-day post challenges. Our results suggest that the changes observed at the final days of the experiment are due to temperature more than P. salmonis.

Atmospheric pollution has major health effects on directly exposed subjects but intergenerational consequences are poorly characterized. We previously reported that diesel engine exhaust (DE) could lead to structural changes in the placenta of in utero exposed rabbits (first generation, F1). The effects of maternal exposure to DE were further studied on second-generation (F2) rabbits. Pregnant F0 females were exposed to filtered, diluted DE (1 mg/m3, median particle diameter: 69 nm) or clean filtered air (controls) for 2 h/day, 5 days/week by nose-only exposure during days 3–27 post-conception (dpc). Adult female offspring (F1) were mated to control males: F1 tissues and F2 foeto-placental units were collected at 28 dpc and placental structure and gene expression (microarray) analysed. Fatty acid profiles were determined in foetal and maternal plasma, maternal liver and placenta. In F1, compared to controls, hepatic neutral lipid contents were increased in exposed animals without change in the blood biochemistry. In F2, the placental lipid contents were higher, with higher monounsaturated fatty acids and reduced pro-inflammatory arachidonic acid (AA), without placental structural changes. Conversely, the proportion of anti-inflammatory n-3 polyunsaturated fatty acids in F2 plasma was increased while that of AA was decreased. Gene set enrichment analyses (GSEA) of F2 placenta transcriptomic data identified that the proteasome complex and ubiquitin pathways genes were over-represented and ion channel function and inflammation pathways genes were under-represented in exposed animals. These preliminary results demonstrate that diesel engine exhaust exposure and in utero indirect exposure should be considered as a programming factor within the context of the DOHaD (Developmental Origins of Health and Disease) with a probable intergenerational transmission.

One of the enigmas in the ubiquitin (Ub) field is the requirement for a poly-Ub chain as a proteasomal targeting signal. The canonical chain appears to be longer than the distance between the two Ub-binding proteasomal receptors. Furthermore, genetic manipulation has shown that one receptor subunit is sufficient, which suggests that a single Ub can serve as a degradation signal. To shed light on this mystery, we chemically synthesized tetra-Ub, di-Ub (K(48)-based), and mono-Ub adducts of HA-α-globin, where the distal or proximal Ub moieties were tagged differentially with either Myc or Flag. When incubated in a crude cell extract, the distal Ub moiety in the tetra-Ub adduct was mostly removed by deubiquitinating enzymes (DUBs) and reconjugated to other substrates in the extract. In contrast, the proximal moiety was most likely degraded with the substrate. The efficacy of degradation was proportionate to the chain length; while tetra-Ub globin was an efficient substrate, with mono-Ub globin, we observed rapid removal of the Ub moiety with almost no degradation of the free globin. Taken together, these findings suggest that the proximal moieties are necessary for securing the association of the substrate with the proteasome along the proteolytic process, whereas the distal moieties are important in protecting the proximal moieties from premature deubiquitination. Interestingly, when the same experiment was carried out using purified 26S proteasome, mono- and tetra-Ub globin were similarly degraded, highlighting the roles of the entire repertoire of cellular DUBs in regulating the degradation of proteasomal substrates.