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1.
PLoS Pathog ; 16(8): e1008780, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866188

RESUMO

Ubiquitin like protein 5 (UBL5) interacts with other proteins to regulate their function but differs from ubiquitin and other UBLs because it does not form covalent conjugates. Ubiquitin and most UBLs mediate the degradation of target proteins through the 26S proteasome but it is not known if UBL5 can also do that. Here we found that the UBL5s of rice and Nicotiana benthamiana interacted with rice stripe virus (RSV) p3 protein. Silencing of NbUBL5s in N. benthamiana facilitated RSV infection, while UBL5 overexpression conferred resistance to RSV in both N. benthamiana and rice. Further analysis showed that NbUBL5.1 impaired the function of p3 as a suppressor of silencing by degrading it through the 26S proteasome. NbUBL5.1 and OsUBL5 interacted with RPN10 and RPN13, the receptors of ubiquitin in the 26S proteasome. Furthermore, silencing of NbRPN10 or NbRPN13 compromised the degradation of p3 mediated by NbUBL5.1. Together, the results suggest that UBL5 mediates the degradation of RSV p3 protein through the 26S proteasome, a previously unreported plant defense strategy against RSV infection.


Assuntos
Proteínas de Plantas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proteínas Repressoras/metabolismo , Tenuivirus/metabolismo , Tabaco/metabolismo , Ubiquitinas/metabolismo , Proteínas Virais/metabolismo , Proteínas de Plantas/genética , Complexo de Endopeptidases do Proteassoma/genética , Proteínas Repressoras/genética , Tenuivirus/genética , Tabaco/genética , Ubiquitinas/genética , Proteínas Virais/genética
2.
PLoS Comput Biol ; 16(9): e1007740, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32881861

RESUMO

The circadian clock is a complex system that plays many important roles in most organisms. Previously, many mathematical models have been used to sharpen our understanding of the Arabidopsis clock, which brought to light the roles of each transcriptional and post-translational regulations. However, the presence of both regulations, instead of either transcription or post-translation, raised curiosity of whether the combination of these two regulations is important for the clock's system. In this study, we built a series of simplified oscillators with different regulations to study the importance of post-translational regulation (specifically, 26S proteasome degradation) in the clock system. We found that a simple transcriptional-based oscillator can already generate sustained oscillation, but the oscillation can be easily destroyed in the presence of transcriptional leakage. Coupling post-translational control with transcriptional-based oscillator in a feed-forward loop will greatly improve the robustness of the oscillator in the presence of basal leakage. Using these general models, we were able to replicate the increased variability observed in the E3 ligase mutant for both plant and mammalian clocks. With this insight, we also predict a plausible regulator of several E3 ligase genes in the plant's clock. Thus, our results provide insights into and the plausible importance in coupling transcription and post-translation controls in the clock system.


Assuntos
Relógios Circadianos/genética , Modelos Biológicos , Processamento de Proteína Pós-Traducional/genética , Transcrição Genética/genética , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Biologia Computacional , Retroalimentação Fisiológica , Regulação da Expressão Gênica de Plantas/genética , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
3.
Nat Commun ; 11(1): 4580, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32917864

RESUMO

Proteasomal machinery performs essential regulated protein degradation in eukaryotes. Classic proteasomes are symmetric, with a regulatory ATPase docked at each end of the cylindrical 20S. Asymmetric complexes are also present in cells, either with a single ATPase or with an ATPase and non-ATPase at two opposite ends. The mechanism that populates these different proteasomal complexes is unknown. Using archaea homologs, we construct asymmetric forms of proteasomes. We demonstrate that the gate conformation of the two opposite ends of 20S are coupled: binding one ATPase opens a gate locally, and also opens the opposite gate allosterically. Such allosteric coupling leads to cooperative binding of proteasomal ATPases to 20S and promotes formation of proteasomes symmetrically configured with two identical ATPases. It may also promote formation of asymmetric complexes with an ATPase and a non-ATPase at opposite ends. We propose that in eukaryotes a similar mechanism regulates the composition of the proteasomal population.


Assuntos
Archaea/metabolismo , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Adenosina Trifosfatases/metabolismo , Archaea/genética , Microscopia Crioeletrônica , Cinética , Modelos Moleculares , Complexo de Endopeptidases do Proteassoma/genética , Conformação Proteica , Thermoplasma/genética , Thermoplasma/metabolismo
4.
Nat Commun ; 11(1): 3904, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32764536

RESUMO

A major challenge in chemotherapy is chemotherapy resistance in cells lacking p53. Here we demonstrate that NIP30, an inhibitor of the oncogenic REGγ-proteasome, attenuates cancer cell growth and sensitizes p53-compromised cells to chemotherapeutic agents. NIP30 acts by binding to REGγ via an evolutionarily-conserved serine-rich domain with 4-serine phosphorylation. We find the cyclin-dependent phosphatase CDC25A is a key regulator for NIP30 phosphorylation and modulation of REGγ activity during the cell cycle or after DNA damage. We validate CDC25A-NIP30-REGγ mediated regulation of the REGγ target protein p21 in vivo using p53-/- and p53/REGγ double-deficient mice. Moreover, Phosphor-NIP30 mimetics significantly increase the growth inhibitory effect of chemotherapeutic agents in vitro and in vivo. Given that NIP30 is frequently mutated in the TCGA cancer database, our results provide insight into the regulatory pathway controlling the REGγ-proteasome in carcinogenesis and offer a novel approach to drug-resistant cancer therapy.


Assuntos
Autoantígenos/metabolismo , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/metabolismo , Proteína Supressora de Tumor p53/deficiência , Animais , Autoantígenos/genética , Ciclo Celular , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Resistencia a Medicamentos Antineoplásicos , Células HEK293 , Xenoenxertos , Humanos , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , Fosforilação , Complexo de Endopeptidases do Proteassoma/deficiência , Complexo de Endopeptidases do Proteassoma/genética , Proteína Supressora de Tumor p53/genética , Fosfatases cdc25/metabolismo
5.
Proc Natl Acad Sci U S A ; 117(32): 19190-19200, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32723828

RESUMO

The 26S proteasome, a self-compartmentalized protease complex, plays a crucial role in protein quality control. Multiple levels of regulatory systems modulate proteasomal activity for substrate hydrolysis. However, the destruction mechanism of mammalian proteasomes is poorly understood. We found that inhibited proteasomes are sequestered into the insoluble aggresome via HDAC6- and dynein-mediated transport. These proteasomes colocalized with the autophagic receptor SQSTM1 and cleared through selective macroautophagy, linking aggresomal segregation to autophagic degradation. This proteaphagic pathway was counterbalanced with the recovery of proteasomal activity and was critical for reducing cellular proteasomal stress. Changes in associated proteins and polyubiquitylation on inhibited 26S proteasomes participated in the targeting mechanism to the aggresome and autophagosome. The STUB1 E3 Ub ligase specifically ubiquitylated purified human proteasomes in vitro, mainly via Lys63-linked chains. Genetic and chemical inhibition of STUB1 activity significantly impaired proteasome processing and reduced resistance to proteasomal stress. These data demonstrate that aggresomal sequestration is the crucial upstream event for proteasome quality control and overall protein homeostasis in mammals.


Assuntos
Macroautofagia , Organelas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Células A549 , Humanos , Organelas/genética , Complexo de Endopeptidases do Proteassoma/genética , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
6.
Nucleic Acids Res ; 48(15): 8474-8489, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32652040

RESUMO

Highly toxic DNA double-strand breaks (DSBs) readily trigger the DNA damage response (DDR) in cells, which delays cell cycle progression to ensure proper DSB repair. In Saccharomyces cerevisiae, mitotic S phase (20-30 min) is lengthened upon DNA damage. During meiosis, Spo11-induced DSB onset and repair lasts up to 5 h. We report that the NH2-terminal domain (NTD; residues 1-66) of Rad51 has dual functions for repairing DSBs during vegetative growth and meiosis. Firstly, Rad51-NTD exhibits autonomous expression-enhancing activity for high-level production of native Rad51 and when fused to exogenous ß-galactosidase in vivo. Secondly, Rad51-NTD is an S/T-Q cluster domain (SCD) harboring three putative Mec1/Tel1 target sites. Mec1/Tel1-dependent phosphorylation antagonizes the proteasomal degradation pathway, increasing the half-life of Rad51 from ∼30 min to ≥180 min. Our results evidence a direct link between homologous recombination and DDR modulated by Rad51 homeostasis.


Assuntos
Quebras de DNA de Cadeia Dupla , Endodesoxirribonucleases/genética , Meiose/genética , Rad51 Recombinase/genética , Proteínas de Saccharomyces cerevisiae/genética , Dano ao DNA/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fosforilação/genética , Complexo de Endopeptidases do Proteassoma/genética , Domínios Proteicos/genética , Proteínas Serina-Treonina Quinases/genética , Proteólise , Saccharomyces cerevisiae/genética , beta-Galactosidase/genética
7.
Bratisl Lek Listy ; 121(5): 331-333, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32356429

RESUMO

IM: Alzheimer's disease (AD) is a progressive and fatal neurodegenerative disorder resulting in degeneration of certain neuronal structures in certain brain regions and severe neuronal loss, characterized by a pathological accumulation of senile amyloid plaques (SP) and neurofibrillary tangles (NFT) within the brain . Alzheimer's disease has been associated with Type 2 diabetes mellitus (T2DM) in recent years. We designed our study on the relationship between AD and T2DM. Genome screening studies in different populations had linked the chromosome 12q24 region to type 2 diabetes. Within this region, there is the PSMD9 gene encoding a transcriptional coactivator of insulin production. METHOD: The effect of PSMD9 gene E197G (rs14259) polymorphism on AD was investigated in29 Alzheimer's patients and 25 healthy controls, who were included in the study. RESULTS: In our study, it was determined that the variant of PSMD9 gene E197G (rs14259) did not cause genetic risk factor for Alzheimer's disease in Turkish population. CONCLUSIONS: Our study was the first to investigate the relationship between PSMD9 gene and Alzheimer's disease. A larger sample group is needed to investigate the contribution of the PSMD9 gene to Alzheimer's disease in further studies (Tab. 5, Ref. 8).


Assuntos
Doença de Alzheimer , Diabetes Mellitus Tipo 2 , Complexo de Endopeptidases do Proteassoma , Doença de Alzheimer/genética , Diabetes Mellitus Tipo 2/genética , Humanos , Placa Amiloide , Polimorfismo de Nucleotídeo Único , Complexo de Endopeptidases do Proteassoma/genética
8.
Nat Med ; 26(6): 909-918, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32472114

RESUMO

PD-1 blockade has transformed the management of advanced clear cell renal cell carcinoma (ccRCC), but the drivers and resistors of the PD-1 response remain incompletely elucidated. Here, we analyzed 592 tumors from patients with advanced ccRCC enrolled in prospective clinical trials of treatment with PD-1 blockade by whole-exome and RNA sequencing, integrated with immunofluorescence analysis, to uncover the immunogenomic determinants of the therapeutic response. Although conventional genomic markers (such as tumor mutation burden and neoantigen load) and the degree of CD8+ T cell infiltration were not associated with clinical response, we discovered numerous chromosomal alterations associated with response or resistance to PD-1 blockade. These advanced ccRCC tumors were highly CD8+ T cell infiltrated, with only 27% having a non-infiltrated phenotype. Our analysis revealed that infiltrated tumors are depleted of favorable PBRM1 mutations and enriched for unfavorable chromosomal losses of 9p21.3, as compared with non-infiltrated tumors, demonstrating how the potential interplay of immunophenotypes with somatic alterations impacts therapeutic efficacy.


Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Carcinoma de Células Renais/tratamento farmacológico , Neoplasias Renais/tratamento farmacológico , Nivolumabe/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Apresentação do Antígeno/genética , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/imunologia , Carcinoma de Células Renais/patologia , Deleção Cromossômica , Cromossomos Humanos Par 6 , Cromossomos Humanos Par 9/genética , Classe I de Fosfatidilinositol 3-Quinases/genética , Proteínas de Ligação a DNA/genética , Feminino , Imunofluorescência , Deleção de Genes , Genômica , Antígenos de Histocompatibilidade Classe II/genética , Histona Desmetilases/genética , Histona-Lisina N-Metiltransferase/genética , Humanos , Neoplasias Renais/genética , Neoplasias Renais/imunologia , Neoplasias Renais/patologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/patologia , Masculino , Pessoa de Meia-Idade , Mutação , PTEN Fosfo-Hidrolase/genética , Prognóstico , Complexo de Endopeptidases do Proteassoma/genética , Análise de Sequência de RNA , Serina-Treonina Quinases TOR/genética , Fatores de Transcrição/genética , Proteína 1 do Complexo Esclerose Tuberosa/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Sequenciamento Completo do Exoma
9.
Nat Commun ; 11(1): 1662, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-32245955

RESUMO

The proteasome is an essential protein-degradation machinery in eukaryotic cells that controls protein turnover and thereby the biogenesis and function of cell organelles. Chloroplasts import thousands of nuclear-encoded precursor proteins from the cytosol, suggesting that the bulk of plastid proteins is transiently exposed to the cytosolic proteasome complex. Therefore, there is a cytosolic equilibrium between chloroplast precursor protein import and proteasomal degradation. We show here that a shift in this equilibrium, induced by mild genetic proteasome impairment, results in elevated precursor protein abundance in the cytosol and significantly increased accumulation of functional photosynthetic complexes in protein import-deficient chloroplasts. Importantly, a proteasome lid mutant shows improved photosynthetic performance, even in the absence of an import defect, signifying that functional precursors are continuously degraded. Hence, turnover of plastid precursors in the cytosol represents a mechanism to constrain thylakoid membrane assembly and photosynthetic electron transport.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Cloroplastos/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Proteínas de Cloroplastos/metabolismo , Citosol/metabolismo , Mutação , Fotossíntese , Plantas Geneticamente Modificadas , Complexo de Endopeptidases do Proteassoma/metabolismo , Precursores de Proteínas/metabolismo , Proteólise , Estresse Fisiológico
10.
Biochim Biophys Acta Mol Cell Res ; 1867(7): 118714, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32246947

RESUMO

Ceramide kinase (CerK) phosphorylates ceramide to ceramide-1-phosphate (C1P). CerK is highly expressed in the brain, and its association with the neuronal function has been reported. Previous reports showed that the activity of CerK is regulated by post-translational modifications including phosphorylation, whereas the cellular fate of CerK protein and its role in neuronal functions have not been clearly elucidated. Therefore, we investigated these issues in PC12 cells. Treatment with nerve growth factor (NGF) for 6 h increased the formation of C1P but not CerK mRNA. Knockdown of CerK and overexpression of HA-tagged CerK down- and up-regulated the formation of C1P, respectively. In PC12-CerK-HA cells, serum withdrawal caused ubiquitination of CerK-HA protein and down-regulated both CerK-HA protein and C1P formation within 6 h, and these down-regulations were abolished by co-treatments with NGF or proteasome inhibitors such as MG132 and clasto-lactacystin. Microscopic analysis showed that treatment with the proteasome inhibitors increased CerK-HA in puncture structures, possibly endosomes and/or vesicles, in cells. Treatment with the lysosome inhibitors reduced serum withdrawal-induced down-regulation of CerK-HA protein but not C1P formation. When knockdown or overexpression of CerK was performed, Ca2+-induced release of [3H] noradrenaline was reduced or enhanced, respectively, but neurite extension was not modified. There was a positive correlation between noradrenaline release and formation of C1P and/or CerK-HA levels in NGF- and clasto-lactacystin-treated cells. These results suggest that levels of CerK were down-regulated by the ubiquitin/proteasome and lysosome pathways and the former pathway-sensitive pool of CerK was suggested to be linked with exocytosis in PC12 cells.


Assuntos
Exocitose/genética , Fator de Crescimento Neural/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Animais , Ciclo Celular , Proliferação de Células , Ceramidas , Lisossomos/genética , Lisossomos/metabolismo , Redes e Vias Metabólicas/genética , Fator de Crescimento Neural/metabolismo , Células PC12 , Fosforilação , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ratos
11.
Proc Natl Acad Sci U S A ; 117(14): 7776-7781, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32193351

RESUMO

The Saccharomyces cerevisiae protein Ddi1 and its homologs in higher eukaryotes have been proposed to serve as shuttling factors that deliver ubiquitinated substrates to the proteasome. Although Ddi1 contains both ubiquitin-interacting UBA and proteasome-interacting UBL domains, the UBL domain is atypical, as it binds ubiquitin. Furthermore, unlike other shuttling factors, Ddi1 and its homologs contain a conserved helical domain (helical domain of Ddi1, HDD) and a retroviral-like protease (RVP) domain. The RVP domain is probably responsible for cleavage of the precursor of the transcription factor Nrf1 in higher eukaryotes, which results in the up-regulation of proteasomal subunit genes. However, enzymatic activity of the RVP domain has not yet been demonstrated, and the function of Ddi1 remains poorly understood. Here, we show that Ddi1 is a ubiquitin-dependent protease, which cleaves substrate proteins only when they are tagged with long ubiquitin chains (longer than about eight ubiquitins). The RVP domain is inactive in isolation, in contrast to its retroviral counterpart. Proteolytic activity of Ddi1 requires the HDD domain and is stimulated by the UBL domain, which mediates high-affinity interaction with the polyubiquitin chain. Compromising the activity of Ddi1 in yeast cells results in the accumulation of polyubiquitinated proteins. Aside from the proteasome, Ddi1 is the only known endoprotease that acts on polyubiquitinated substrates. Ddi1 and its homologs likely cleave polyubiquitinated substrates under conditions where proteasome function is compromised.


Assuntos
Chaperonas Moleculares/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Ubiquitina/genética , Proteínas de Transporte Vesicular/genética , Poliubiquitina/genética , Complexo de Endopeptidases do Proteassoma/genética , Ligação Proteica/genética , Domínios Proteicos/genética , Homologia de Sequência
12.
Mol Cell ; 77(5): 1107-1123.e10, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-32142684

RESUMO

Mitochondria import nearly their entire proteome from the cytoplasm by translocating precursor proteins through the translocase of the outer membrane (TOM) complex. Here, we show dynamic regulation of mitochondrial import by the ubiquitin system. Acute pharmacological inhibition or genetic ablation of the mitochondrial deubiquitinase (DUB) USP30 triggers accumulation of Ub-substrates that are normally localized inside the mitochondria. Mitochondrial import of USP30 substrates is impaired in USP30 knockout (KO) cells, suggesting that deubiquitination promotes efficient import. Upstream of USP30, the E3 ligase March5 ubiquitinates mitochondrial proteins whose eventual import depends on USP30. In USP30 KOs, exogenous March5 expression induces accumulation of unimported translocation intermediates that are degraded by the proteasomes. In USP30 KO mice, TOM subunits have reduced abundance across multiple tissues. Together these data highlight how protein import into a subcellular compartment can be regulated by ubiquitination and deubiquitination by E3 ligase and DUB machinery positioned at the gate.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Tioléster Hidrolases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Animais , Transporte Biológico , Proteínas de Transporte/genética , Feminino , Células HEK293 , Células HeLa , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Tioléster Hidrolases/genética , Fatores de Tempo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
13.
PLoS One ; 15(3): e0229462, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32119722

RESUMO

Aging is a major risk factor for morbidity and mortality from cardiovascular causes in men. To better understand the cellular processes related to age-related cardiac complications, we undertook research aimed at comparative evaluation of genes expression and distribution of ß-catenin, CacyBP/SIP, galectin-3 and LMP7 in the heart of healthy men in different age groups. The study was conducted on the hearts of 12 men (organ donors) without a history of cardiovascular disease, who were divided into two age groups: men under and men over 45 years of age. On paraffin sections, immunohistochemical reactions were performed to detect ß-catenin, CacyBP/SIP, galectin-3 and immunoproteasome subunit LMP7. The expression of genes coding ß-catenin, CacyBP/SIP, galectin-3 and LMP7 was also evaluated by real-time PCR method. In the heart of men over 45 years old, both gene expression and immunoreactivity of ß-catenin, CacyBP/SIP, galectin-3 and LMP7 were stronger compared to younger individuals. The results of the presented studies suggest that ß-catenin, CacyBP/SIP, galectin-3 and immunoproteasomes might be involved in the internal regulation of heart homeostasis during ageing.


Assuntos
Envelhecimento/genética , Proteínas de Ligação ao Cálcio/genética , Galectina 3/genética , Miocárdio/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , beta Catenina/genética , Adulto , Distribuição por Idade , Idoso , Envelhecimento/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Galectina 3/metabolismo , Homeostase , Humanos , Masculino , Pessoa de Meia-Idade , Complexo de Endopeptidases do Proteassoma/metabolismo , Regulação para Cima , Adulto Jovem , beta Catenina/metabolismo
14.
Medicine (Baltimore) ; 99(13): e19482, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32221071

RESUMO

BACKGROUND: Insulin dependent diabetes mellitus (IDDM) is a kind of heterogeneous disease caused by the interaction of polygene inheritance and environmental factors. The LMP2 and LMP7 are 2 loci in LMP gene, and although genetic association between LMP2 and LMP7 polymorphisms were reported, the results are inconclusive. The aim of this study was to investigate the association between LMP2 and LMP7 polymorphisms and IDDM risk. METHODS: An exhaustive search was performed out through the electronic databases including PubMed, Embase, and Chinese National Knowledge Infrastructure (CNKI). The pooled odds ratio (OR) and 95% confidence interval (CI) were used to assess the strength association between LMP2 CfoI and LMP7 G37360T polymorphisms and IDDM risk. RESULTS: A total of 7 studies with 707 cases and 821 controls were included in the present study. The results indicated that the dominant model of LMP2 CfoI was significantly associated with IDDM in Asian population (OR = 1.96, 95% CI: 1.24-3.10, P = .004). In addition, the allelic and dominant models of LMP7 G37360T were associated with IDDM in Caucasian population (allelic model: OR = 0.69, 95% CI: 0.56-0.85, P = .0005; dominant model: OR = 0.67, 95% CI: 0.50-0.89, P = .007). CONCLUSIONS: The dominant model of LMP2 CfoI might be a risk factor for IDDM in Asian population. Whereas, the allelic and dominant models of LMP7 G37360T might be protective factors for IDDM in Caucasian population.


Assuntos
Cisteína Endopeptidases/genética , Diabetes Mellitus Tipo 1/genética , Complexo de Endopeptidases do Proteassoma/genética , Adolescente , Adulto , Fatores Etários , Alelos , Grupo com Ancestrais do Continente Asiático , Criança , Grupos Étnicos , Grupo com Ancestrais do Continente Europeu , Feminino , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Polimorfismo Genético , Fatores Sexuais , Adulto Jovem
15.
J Mol Biol ; 432(7): 2388-2404, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32147457

RESUMO

Well-studied structural motifs in Rad23 have been shown to bind polyubiquitin chains and the proteasome. These domains are predicted to enable Rad23 to transport polyubiquitylated (polyUb) substrates to the proteasome (Chen and Madura, 2002 [1]). The validation of this model, however, has been hindered by the lack of specific physiological substrates of Rad23. We report here that Rad23 can bind Ho-endonuclease (Ho-endo), a nuclear protein that initiates mating-type switching in Saccharomyces cerevisiae. We observed that the degradation of Ho-endo required export from the nucleus, in agreement with a previous report (Kaplun et al., 2003 [2]), and suggests that Rad23 can traffic proteins out of the nucleus. In agreement, the subcellular distribution of Rad23 is noticeably altered in genetic mutants that disrupt nucleocytoplasmic trafficking. Significantly, the location of Rad23 affected its binding to polyUb substrates. Mutations in nuclear export stabilized substrates, and caused accumulation in the nucleus. Importantly, Rad23 also accumulated in the nucleus in an export mutant, and bound to higher levels of polyUb proteins. In contrast, Rad23 is localized in the cytosol in rna1-1, a nucleocytoplasmic transport mutant, and it forms reduced binding to polyUb substrates. These and other studies indicate that substrates that are conjugated to polyubiquitin chains in the nucleus may rely on an export-dependent mechanism to be degraded by the proteasome. The evolutionary conservation of Rad23 and similar substrate-trafficking proteins predicts an important role for export in the turnover of nuclear proteins.


Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Ligação a DNA/metabolismo , Poliubiquitina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a DNA/genética , Mutação , Complexo de Endopeptidases do Proteassoma/genética , Ligação Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
16.
Metabolism ; 105: 154186, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32084429

RESUMO

BACKGROUND: Apolipoprotein A-I (ApoA-I) is involved in reverse cholesterol transport as a major component of HDL, but also conveys anti-thrombotic, anti-oxidative, anti-inflammatory and immune-regulatory properties that are pertinent to its protective roles in cardiovascular, inflammatory and malignant pathologies. Despite the pleiotropy in ApoA-I functions, the regulation of intracellular ApoA-I levels remains poorly explored. METHODS: HepG2 hepatoma cells and primary mouse hepatocytes were used as in vitro models to study the impact of genetic and chemical inhibitors of autophagy and the proteasome on ApoA-I by immunoblot, immunofluorescence and electron microscopy. Different growth conditions were implemented in conjunction with mTORC inhibitors to model the influence of nutrient scarcity versus sufficiency on ApoA-I regulation. Hepatic ApoA-I expression was also evaluated in high fat diet-fed mice displaying blockade in autophagy. RESULTS: Under nutrient-rich conditions, basal ApoA-I levels in liver cells are sustained by the balancing act of autophagy and of mTORC1-dependent de novo protein synthesis. ApoA-I proteolysis occurs through a canonical autophagic pathway involving Beclin1 and ULK1 and the receptor protein p62/SQSTM1 that targets ApoA-I to autophagosomes. However, upon aminoacid insufficiency, suppression of ApoA-I synthesis prevails, rendering mTORC1 inactivation dispensable for autophagy-mediated ApoA-I proteolysis. CONCLUSION: These data underscore the major contribution of post-transcriptional mechanisms to ApoA-I levels which differentially involve mTORC1-dependent signaling to protein synthesis and autophagy, depending on nutrient availability. Given the established role of ApoA-I in HDL-mediated reverse cholesterol transport, this mode of ApoA-I regulation may reflect a hepatocellular response to the organismal requirement for maintenance of cholesterol and lipid reserves under conditions of nutrient scarcity.


Assuntos
Apolipoproteína A-I/biossíntese , Autofagia/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Animais , Apolipoproteína A-I/genética , Colesterol/metabolismo , Dieta Hiperlipídica , Regulação da Expressão Gênica , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Metabolismo dos Lipídeos , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Camundongos , Hepatopatia Gordurosa não Alcoólica/genética , Estado Nutricional , Complexo de Endopeptidases do Proteassoma/genética
17.
Chemistry ; 26(36): 8022-8027, 2020 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-32105365

RESUMO

Messenger RNA display of peptides containing non-proteinogenic amino acids, referred to as RaPID system, has become one of the leading methods to express libraries consisting of more than trillion-members of macrocyclic peptides, which allows for discovering de novo bioactive ligands. Ideal macrocyclic peptides should have dissociation constants (KD ) as low as single-digit values in the nanomolar range towards a specific target of interest. Here, a twofold strategy to discover optimized macrocyclic peptides within this affinity regime is described. First, benzyl thioether cyclized peptide libraries were explored to identify tight binding hits. To obtain more insights into critical sequence information, sequence alignment was applied to guide rational mutagenesis for the improvement of their binding affinity. Using this twofold strategy, benzyl thioether macrocyclic peptide binders against Lys48-linked ubiquitin dimer (K48-Ub2) were successfully obtained that display KD values in the range 0.3-1.2 nm, which indicate binding two orders of magnitude stronger than those of macrocyclic peptides recently reported. Most importantly, this macrocyclic peptide also showed an improved cellular inhibition of the K48-Ub2 recognition by deubiquitinating enzymes and the 26S proteasome, resulting in the promotion of apoptosis in cancer cells.


Assuntos
Aminoácidos/química , Peptídeos/química , Complexo de Endopeptidases do Proteassoma/química , Humanos , Biblioteca de Peptídeos , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Alinhamento de Sequência , Ubiquitinas/genética
18.
Nat Commun ; 11(1): 896, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-32060274

RESUMO

Predicting the outcome of immunotherapy treatment in melanoma patients is challenging. Alterations in genes involved in antigen presentation and the interferon gamma (IFNγ) pathway play an important role in the immune response to tumors. We describe here that the overexpression of PSMB8 and PSMB9, two major components of the immunoproteasome, is predictive of better survival and improved response to immune-checkpoint inhibitors of melanoma patients. We study the mechanism underlying this connection by analyzing the antigenic peptide repertoire of cells that overexpress these subunits using HLA peptidomics. We find a higher response of patient-matched tumor infiltrating lymphocytes against antigens diferentially presented after immunoproteasome overexpression. Importantly, we find that PSMB8 and PSMB9 expression levels are much stronger predictors of melanoma patients' immune response to checkpoint inhibitors than the tumors' mutational burden. These results suggest that PSMB8 and PSMB9 expression levels can serve as important biomarkers for stratifying melanoma patients for immune-checkpoint treatment.


Assuntos
Melanoma/imunologia , Melanoma/terapia , Complexo de Endopeptidases do Proteassoma/genética , Apresentação do Antígeno , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/imunologia , Humanos , Imunoterapia , Interferon gama/genética , Interferon gama/imunologia , Melanoma/diagnóstico , Melanoma/genética , Prognóstico , Complexo de Endopeptidases do Proteassoma/imunologia
19.
Proc Natl Acad Sci U S A ; 117(10): 5298-5309, 2020 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-32094174

RESUMO

The 20S core particle (CP) proteasome is a molecular assembly catalyzing the degradation of misfolded proteins or proteins no longer required for function. It is composed of four stacked heptameric rings that form a barrel-like structure, sequestering proteolytic sites inside its lumen. Proteasome function is regulated by gates derived from the termini of α-rings and through binding of regulatory particles (RPs) to one or both ends of the barrel. The CP is dynamic, with an extensive allosteric pathway extending from one end of the molecule to catalytic sites in its center. Here, using methyl-transverse relaxation optimized spectroscopy (TROSY)-based NMR optimized for studies of high-molecular-weight complexes, we evaluate whether the pathway extends over the entire 150-Å length of the molecule. By exploiting a number of different labeling schemes, the two halves of the molecule can be distinguished, so that the effects of 11S RP binding, or the introduction of gate or allosteric pathway mutations at one end of the barrel can be evaluated at the distal end. Our results establish that while 11S binding and the introduction of key mutations affect each half of the CP allosterically, they do not further couple opposite ends of the molecule. This may have implications for the function of so-called "hybrid" proteasomes where each end of the CP is bound with a different regulator, allowing the CP to be responsive to both RPs simultaneously. The methodology presented introduces a general NMR strategy for dissecting pathways of communication in homo-oligomeric molecular machines.


Assuntos
Proteínas Arqueais/química , Complexo de Endopeptidases do Proteassoma/química , Thermoplasma/enzimologia , Regulação Alostérica , Proteínas Arqueais/genética , Domínio Catalítico/genética , Espectroscopia de Ressonância Magnética/métodos , Mutação , Complexo de Endopeptidases do Proteassoma/genética , Ligação Proteica , Thermoplasma/genética
20.
PLoS Pathog ; 16(2): e1008105, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32092124

RESUMO

Epstein-Barr virus (EBV) nuclear oncoprotein EBNA3C is essential for B-cell transformation and development of several B-cell lymphomas particularly those are generated in an immuno-compromised background. EBNA3C recruits ubiquitin-proteasome machinery for deregulating multiple cellular oncoproteins and tumor suppressor proteins. Although EBNA3C is found to be ubiquitinated at its N-terminal region and interacts with 20S proteasome, the viral protein is surprisingly stable in growing B-lymphocytes. EBNA3C can also circumvent autophagy-lysosomal mediated protein degradation and subsequent antigen presentation for T-cell recognition. Recently, we have shown that EBNA3C enhances autophagy, which serve as a prerequisite for B-cell survival particularly under growth deprivation conditions. We now demonstrate that proteasomal inhibition by MG132 induces EBNA3C degradation both in EBV transformed B-lymphocytes and ectopic-expression systems. Interestingly, MG132 treatment promotes degradation of two EBNA3 family oncoproteins-EBNA3A and EBNA3C, but not the viral tumor suppressor protein EBNA3B. EBNA3C degradation induced by proteasomal inhibition is partially blocked when autophagy-lysosomal pathway is inhibited. In response to proteasomal inhibition, EBNA3C is predominantly K63-linked polyubiquitinated, colocalized with the autophagy-lysosomal fraction in the cytoplasm and participated within p62-LC3B complex, which facilitates autophagy-mediated degradation. We further show that the degradation signal is present at the first 50 residues of the N-terminal region of EBNA3C. Proteasomal inhibition reduces the colony formation ability of this important viral oncoprotein, induces apoptotic cell death and increases transcriptional activation of both latent and lytic gene expression which further promotes viral reactivation from EBV transformed B-lymphocytes. Altogether, this study offers rationale to use proteasome inhibitors as potential therapeutic strategy against multiple EBV associated B-cell lymphomas, where EBNA3C is expressed.


Assuntos
Morte Celular Autofágica/efeitos dos fármacos , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Leupeptinas/farmacologia , Lisossomos/metabolismo , Proteínas Oncogênicas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Proteólise/efeitos dos fármacos , Animais , Antígenos Nucleares do Vírus Epstein-Barr/genética , Células HEK293 , Herpesvirus Humano 4/genética , Humanos , Lisossomos/genética , Camundongos , Proteínas Oncogênicas/genética , Complexo de Endopeptidases do Proteassoma/genética
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