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1.
Biochem Biophys Res Commun ; 717: 150045, 2024 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-38718572

RESUMO

The ubiquitin-proteasome system (UPS) plays a key role in maintaining cellular protein homeostasis and participates in modulating various cellular functions. Target of rapamycin (TOR), a highly conserved Ser/Thr kinase found across species from yeasts to humans, forms two multi-protein complexes, TORC1 and TORC2, to orchestrate cellular processes crucial for optimal growth, survival, and stress responses. While UPS-mediated regulation of mammalian TOR complexes has been documented, the ubiquitination of yeast TOR complexes remains largely unexplored. Here we report a functional interplay between the UPS and TORC2 in Saccharomyces cerevisiae. Using avo3-2ts, a temperature-sensitive mutant of the essential TORC2 component Avo3 exhibiting TORC2 defects at restrictive temperatures, we obtained evidence for UPS-dependent protein degradation and downregulation of the TORC2 component Avo2. Our results established the involvement of the E3 ubiquitin ligase Ubr1 and its catalytic activity in mediating Avo2 degradation in cells with defective Avo3. Coimmunoprecipitation revealed the interaction between Avo2 and Ubr1, indicating Avo2 as a potential substrate of Ubr1. Furthermore, depleting Ubr1 rescued the growth of avo3-2ts cells at restrictive temperatures, suggesting an essential role of Avo2 in sustaining cell viability under heat stress and/or TORC2 dysfunction. This study uncovers a role of UPS in yeast TORC2 regulation, highlighting the impact of protein degradation control on cellular signaling.


Assuntos
Regulação para Baixo , Alvo Mecanístico do Complexo 2 de Rapamicina , Complexo de Endopeptidases do Proteassoma , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Ubiquitina-Proteína Ligases , Ubiquitina , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Proteólise , Ubiquitinação
2.
J Exp Clin Cancer Res ; 43(1): 142, 2024 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-38745188

RESUMO

BACKGROUND: Mounting evidences shows that the ubiquitin‒proteasome pathway plays a pivotal role in tumor progression. The expression of 26S proteasome non-ATPase regulatory subunit 9 (PSMD9) is correlated with recurrence and radiotherapy resistance in several tumor types. However, the role and mechanism of PSMD9 in hepatocellular carcinoma (HCC) progression remain largely unclear. METHODS: PSMD9 was identified as a prognosis-related biomarker for HCC based on analysis of clinical characteristics and RNA-seq data from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO) and the JP Project of the International Cancer Genome Consortium (ICGC-LIRI-JP). PSMD9 expression was analyzed in cancer tissues and adjacent noncancerous tissues via immunohistochemistry and Western blotting. Multiple in vivo and in vitro experimental techniques (such as CCK-8, colony formation, EdU, and Transwell assays; flow cytometry; Western blotting; quantitative RT-PCR; Coimmunoprecipitation assay and immunofluorescence confocal imaging) were used to assess the functions of PSMD9 in the pathogenesis of HCC. RESULTS: We found that the expression of PSMD9 was upregulated and associated with a poor prognosis in HCC patients. PSMD9 promoted HCC cell proliferation, migration, invasion and metastasis. Knockdown of PSMD9 significantly inhibited HCC cell proliferation by inducing G1/S cell cycle arrest and apoptosis. Mechanistically, we demonstrated that PSMD9 promoted HCC cell proliferation and metastasis via direct interaction with the E3 ubiquitin ligase c-Cbl, suppresses EGFR ubiquitination, influenced EGFR endosomal trafficking and degradation and subsequently activated ERK1/2 and Akt signaling. In addition, we showed that PSMD9 knockdown sensitized HCC cells to the tyrosine kinase inhibitor erlotinib in vitro and in vivo. CONCLUSIONS: Collectively, our results indicate that PSMD9 drives HCC progression and erlotinib resistance by suppressing c-Cbl mediated EGFR ubiquitination and therefore can be a potential therapeutic target for HCC.


Assuntos
Carcinoma Hepatocelular , Progressão da Doença , Receptores ErbB , Neoplasias Hepáticas , Proteínas Proto-Oncogênicas c-cbl , Transdução de Sinais , Humanos , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/genética , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Proteínas Proto-Oncogênicas c-cbl/genética , Receptores ErbB/metabolismo , Receptores ErbB/genética , Camundongos , Animais , Masculino , Feminino , Linhagem Celular Tumoral , Complexo de Endopeptidases do Proteassoma/metabolismo , Proliferação de Células , Prognóstico , Camundongos Nus , Apoptose , Pessoa de Meia-Idade , Movimento Celular
3.
Nat Commun ; 15(1): 3992, 2024 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-38734767

RESUMO

Visual proteomics attempts to build atlases of the molecular content of cells but the automated annotation of cryo electron tomograms remains challenging. Template matching (TM) and methods based on machine learning detect structural signatures of macromolecules. However, their applicability remains limited in terms of both the abundance and size of the molecular targets. Here we show that the performance of TM is greatly improved by using template-specific search parameter optimization and by including higher-resolution information. We establish a TM pipeline with systematically tuned parameters for the automated, objective and comprehensive identification of structures with confidence 10 to 100-fold above the noise level. We demonstrate high-fidelity and high-confidence localizations of nuclear pore complexes, vaults, ribosomes, proteasomes, fatty acid synthases, lipid membranes and microtubules, and individual subunits inside crowded eukaryotic cells. We provide software tools for the generic implementation of our method that is broadly applicable towards realizing visual proteomics.


Assuntos
Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Complexo de Endopeptidases do Proteassoma , Proteômica , Ribossomos , Software , Tomografia com Microscopia Eletrônica/métodos , Microscopia Crioeletrônica/métodos , Ribossomos/ultraestrutura , Ribossomos/metabolismo , Complexo de Endopeptidases do Proteassoma/ultraestrutura , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/química , Humanos , Proteômica/métodos , Poro Nuclear/ultraestrutura , Poro Nuclear/metabolismo , Microtúbulos/ultraestrutura , Microtúbulos/metabolismo , Ácido Graxo Sintases/metabolismo , Aprendizado de Máquina , Imageamento Tridimensional/métodos , Algoritmos , Processamento de Imagem Assistida por Computador/métodos
4.
J Cell Biol ; 223(8)2024 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-38767572

RESUMO

Proteasome activity is crucial for cellular integrity, but how tissues adjust proteasome content in response to catabolic stimuli is uncertain. Here, we demonstrate that transcriptional coordination by multiple transcription factors is required to increase proteasome content and activate proteolysis in catabolic states. Using denervated mouse muscle as a model system for accelerated proteolysis in vivo, we reveal that a two-phase transcriptional program activates genes encoding proteasome subunits and assembly chaperones to boost an increase in proteasome content. Initially, gene induction is necessary to maintain basal proteasome levels, and in a more delayed phase (7-10 days after denervation), it stimulates proteasome assembly to meet cellular demand for excessive proteolysis. Intriguingly, the transcription factors PAX4 and α-PALNRF-1 control the expression of proteasome among other genes in a combinatorial manner, driving cellular adaptation to muscle denervation. Consequently, PAX4 and α-PALNRF-1 represent new therapeutic targets to inhibit proteolysis in catabolic diseases (e.g., type-2 diabetes, cancer).


Assuntos
Fator 1 Nuclear Respiratório , Fatores de Transcrição Box Pareados , Complexo de Endopeptidases do Proteassoma , Proteólise , Animais , Masculino , Camundongos , Regulação da Expressão Gênica , Músculo Esquelético/metabolismo , Fatores de Transcrição Box Pareados/metabolismo , Fatores de Transcrição Box Pareados/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Camundongos Endogâmicos ICR , Fator 1 Nuclear Respiratório/genética , Fator 1 Nuclear Respiratório/metabolismo
5.
PLoS Biol ; 22(5): e3002550, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38768083

RESUMO

Alkenyl oxindoles have been characterized as autophagosome-tethering compounds (ATTECs), which can target mutant huntingtin protein (mHTT) for lysosomal degradation. In order to expand the application of alkenyl oxindoles for targeted protein degradation, we designed and synthesized a series of heterobifunctional compounds by conjugating different alkenyl oxindoles with bromodomain-containing protein 4 (BRD4) inhibitor JQ1. Through structure-activity relationship study, we successfully developed JQ1-alkenyl oxindole conjugates that potently degrade BRD4. Unexpectedly, we found that these molecules degrade BRD4 through the ubiquitin-proteasome system, rather than the autophagy-lysosomal pathway. Using pooled CRISPR interference (CRISPRi) screening, we revealed that JQ1-alkenyl oxindole conjugates recruit the E3 ubiquitin ligase complex CRL4DCAF11 for substrate degradation. Furthermore, we validated the most potent heterobifunctional molecule HL435 as a promising drug-like lead compound to exert antitumor activity both in vitro and in a mouse xenograft tumor model. Our research provides new employable proteolysis targeting chimera (PROTAC) moieties for targeted protein degradation, providing new possibilities for drug discovery.


Assuntos
Proteínas de Ciclo Celular , Oxindóis , Proteólise , Ubiquitina-Proteína Ligases , Humanos , Animais , Proteólise/efeitos dos fármacos , Camundongos , Ubiquitina-Proteína Ligases/metabolismo , Oxindóis/farmacologia , Oxindóis/metabolismo , Oxindóis/química , Proteínas de Ciclo Celular/metabolismo , Fatores de Transcrição/metabolismo , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Camundongos Nus , Células HEK293 , Relação Estrutura-Atividade , Complexo de Endopeptidases do Proteassoma/metabolismo , Azepinas/farmacologia , Azepinas/química , Azepinas/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/química , Feminino , Proteínas que Contêm Bromodomínio , Receptores de Interleucina-17
6.
Nat Commun ; 15(1): 4026, 2024 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-38740822

RESUMO

Unstable proteins are prone to form non-native interactions with other proteins and thereby may become toxic. To mitigate this, destabilized proteins are targeted by the protein quality control network. Here we present systematic studies of the cytosolic aspartoacylase, ASPA, where variants are linked to Canavan disease, a lethal neurological disorder. We determine the abundance of 6152 of the 6260 ( ~ 98%) possible single amino acid substitutions and nonsense ASPA variants in human cells. Most low abundance variants are degraded through the ubiquitin-proteasome pathway and become toxic upon prolonged expression. The data correlates with predicted changes in thermodynamic stability, evolutionary conservation, and separate disease-linked variants from benign variants. Mapping of degradation signals (degrons) shows that these are often buried and the C-terminal region functions as a degron. The data can be used to interpret Canavan disease variants and provide insight into the relationship between protein stability, degradation and cell fitness.


Assuntos
Amidoidrolases , Doença de Canavan , Proteólise , Humanos , Amidoidrolases/genética , Amidoidrolases/metabolismo , Doença de Canavan/genética , Doença de Canavan/metabolismo , Células HEK293 , Substituição de Aminoácidos , Mutação , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Estabilidade Proteica , Ubiquitina/metabolismo , Termodinâmica
7.
Nat Commun ; 15(1): 3736, 2024 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-38744818

RESUMO

The E3 SUMO ligase PIAS2 is expressed at high levels in differentiated papillary thyroid carcinomas but at low levels in anaplastic thyroid carcinomas (ATC), an undifferentiated cancer with high mortality. We show here that depletion of the PIAS2 beta isoform with a transcribed double-stranded RNA-directed RNA interference (PIAS2b-dsRNAi) specifically inhibits growth of ATC cell lines and patient primary cultures in vitro and of orthotopic patient-derived xenografts (oPDX) in vivo. Critically, PIAS2b-dsRNAi does not affect growth of normal or non-anaplastic thyroid tumor cultures (differentiated carcinoma, benign lesions) or cell lines. PIAS2b-dsRNAi also has an anti-cancer effect on other anaplastic human cancers (pancreas, lung, and gastric). Mechanistically, PIAS2b is required for proper mitotic spindle and centrosome assembly, and it is a dosage-sensitive protein in ATC. PIAS2b depletion promotes mitotic catastrophe at prophase. High-throughput proteomics reveals the proteasome (PSMC5) and spindle cytoskeleton (TUBB3) to be direct targets of PIAS2b SUMOylation at mitotic initiation. These results identify PIAS2b-dsRNAi as a promising therapy for ATC and other aggressive anaplastic carcinomas.


Assuntos
Mitose , Proteínas Inibidoras de STAT Ativados , Humanos , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteínas Inibidoras de STAT Ativados/genética , Animais , Linhagem Celular Tumoral , Camundongos , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/metabolismo , Interferência de RNA , Fuso Acromático/metabolismo , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Complexo de Endopeptidases do Proteassoma/metabolismo , Sumoilação , Carcinoma/genética , Carcinoma/metabolismo , Carcinoma/patologia , Feminino
8.
Int J Mol Sci ; 25(9)2024 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-38732146

RESUMO

The ubiquitin-proteasome system (UPS) is an essential mechanism responsible for the selective degradation of substrate proteins via their conjugation with ubiquitin. Since cardiomyocytes have very limited self-renewal capacity, as they are prone to protein damage due to constant mechanical and metabolic stress, the UPS has a key role in cardiac physiology and pathophysiology. While altered proteasomal activity contributes to a variety of cardiac pathologies, such as heart failure and ischemia/reperfusion injury (IRI), the environmental cues affecting its activity are still unknown, and they are the focus of this work. Following a recent study by Ciechanover's group showing that amino acid (AA) starvation in cultured cancer cell lines modulates proteasome intracellular localization and activity, we tested two hypotheses in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs, CMs): (i) AA starvation causes proteasome translocation in CMs, similarly to the observation in cultured cancer cell lines; (ii) manipulation of subcellular proteasomal compartmentalization is associated with electrophysiological abnormalities in the form of arrhythmias, mediated via altered intracellular Ca2+ handling. The major findings are: (i) starving CMs to AAs results in proteasome translocation from the nucleus to the cytoplasm, while supplementation with the aromatic amino acids tyrosine (Y), tryptophan (W) and phenylalanine (F) (YWF) inhibits the proteasome recruitment; (ii) AA-deficient treatments cause arrhythmias; (iii) the arrhythmias observed upon nuclear proteasome sequestration(-AA+YWF) are blocked by KB-R7943, an inhibitor of the reverse mode of the sodium-calcium exchanger NCX; (iv) the retrograde perfusion of isolated rat hearts with AA starvation media is associated with arrhythmias. Collectively, our novel findings describe a newly identified mechanism linking the UPS to arrhythmia generation in CMs and whole hearts.


Assuntos
Arritmias Cardíacas , Cálcio , Miócitos Cardíacos , Complexo de Endopeptidases do Proteassoma , Miócitos Cardíacos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Humanos , Cálcio/metabolismo , Animais , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/etiologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Estresse Fisiológico , Transporte Proteico , Ratos , Aminoácidos/metabolismo
9.
Int J Mol Sci ; 25(9)2024 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-38731881

RESUMO

Aging and age-related diseases are associated with a decline in the capacity of protein turnover. Intrinsically disordered proteins, as well as proteins misfolded and oxidatively damaged, prone to aggregation, are preferentially digested by the ubiquitin-independent proteasome system (UIPS), a major component of which is the 20S proteasome. Therefore, boosting 20S activity constitutes a promising strategy to counteract a decrease in total proteasome activity during aging. One way to enhance the proteolytic removal of unwanted proteins appears to be the use of peptide-based activators of the 20S. In this study, we synthesized a series of peptides and peptidomimetics based on the C-terminus of the Rpt5 subunit of the 19S regulatory particle. Some of them efficiently stimulated human 20S proteasome activity. The attachment of the cell-penetrating peptide TAT allowed them to penetrate the cell membrane and stimulate proteasome activity in HEK293T cells, which was demonstrated using a cell-permeable substrate of the proteasome, TAS3. Furthermore, the best activator enhanced the degradation of aggregation-prone α-synuclein and Tau-441. The obtained compounds may therefore have the potential to compensate for the unbalanced proteostasis found in aging and age-related diseases.


Assuntos
Envelhecimento , Complexo de Endopeptidases do Proteassoma , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Células HEK293 , Envelhecimento/metabolismo , Agregados Proteicos/efeitos dos fármacos , Proteólise/efeitos dos fármacos , alfa-Sinucleína/metabolismo , Peptídeos/farmacologia , Peptídeos/química , Peptídeos/metabolismo , Proteínas tau/metabolismo , Agregação Patológica de Proteínas/metabolismo , Peptidomiméticos/farmacologia , Peptidomiméticos/química
10.
Mol Cancer ; 23(1): 110, 2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38773495

RESUMO

Proteolysis-targeting chimeras (PROTACs) technology has garnered significant attention over the last 10 years, representing a burgeoning therapeutic approach with the potential to address pathogenic proteins that have historically posed challenges for traditional small-molecule inhibitors. PROTACs exploit the endogenous E3 ubiquitin ligases to facilitate degradation of the proteins of interest (POIs) through the ubiquitin-proteasome system (UPS) in a cyclic catalytic manner. Despite recent endeavors to advance the utilization of PROTACs in clinical settings, the majority of PROTACs fail to progress beyond the preclinical phase of drug development. There are multiple factors impeding the market entry of PROTACs, with the insufficiently precise degradation of favorable POIs standing out as one of the most formidable obstacles. Recently, there has been exploration of new-generation advanced PROTACs, including small-molecule PROTAC prodrugs, biomacromolecule-PROTAC conjugates, and nano-PROTACs, to improve the in vivo efficacy of PROTACs. These improved PROTACs possess the capability to mitigate undesirable physicochemical characteristics inherent in traditional PROTACs, thereby enhancing their targetability and reducing off-target side effects. The new-generation of advanced PROTACs will mark a pivotal turning point in the realm of targeted protein degradation. In this comprehensive review, we have meticulously summarized the state-of-the-art advancements achieved by these cutting-edge PROTACs, elucidated their underlying design principles, deliberated upon the prevailing challenges encountered, and provided an insightful outlook on future prospects within this burgeoning field.


Assuntos
Antineoplásicos , Neoplasias , Proteólise , Humanos , Proteólise/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Antineoplásicos/química , Animais , Complexo de Endopeptidases do Proteassoma/metabolismo , Terapia de Alvo Molecular , Ubiquitina-Proteína Ligases/metabolismo , Quimera de Direcionamento de Proteólise
11.
Bioorg Med Chem ; 106: 117733, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38704960

RESUMO

Development of selective or dual proteasome subunit inhibitors based on syringolin B as a scaffold is described. We focused our efforts on a structure-activity relationship study of inhibitors with various substituents at the 3-position of the macrolactam moiety of syringolin B analogue to evaluate whether this would be sufficient to confer subunit selectivity by using sets of analogues with hydrophobic, basic and acidic substituents, which were designed to target Met45, Glu53 and Arg45 embedded in the S1 subsite, respectively. The structure-activity relationship study using systematic analogues provided insight into the origin of the subunit-selective inhibitory activity. This strategy would be sufficient to confer subunit selectivity regarding ß5 and ß2 subunits.


Assuntos
Complexo de Endopeptidases do Proteassoma , Inibidores de Proteassoma , Relação Estrutura-Atividade , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/química , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/química , Inibidores de Proteassoma/síntese química , Humanos , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/metabolismo , Subunidades Proteicas/química , Estrutura Molecular
12.
Int J Mol Sci ; 25(8)2024 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-38673728

RESUMO

BTB and CNC homology 1 (BACH1) represses the expression of genes involved in the metabolism of iron, heme and reactive oxygen species. While BACH1 is rapidly degraded when it is bound to heme, it remains unclear how BACH1 degradation is regulated under other conditions. We found that FBXO22, a ubiquitin ligase previously reported to promote BACH1 degradation, polyubiquitinated BACH1 only in the presence of heme in a highly purified reconstitution assay. In parallel to this regulatory mechanism, TANK binding kinase 1 (TBK1), a protein kinase that activates innate immune response and regulates iron metabolism via ferritinophagy, was found to promote BACH1 degradation when overexpressed in 293T cells. While TBK1 phosphorylated BACH1 at multiple serine and threonine residues, BACH1 degradation was observed with not only the wild-type TBK1 but also catalytically impaired TBK1. The BACH1 degradation in response to catalytically impaired TBK1 was not dependent on FBXO22 but involved both autophagy-lysosome and ubiquitin-proteasome pathways judging from its suppression by using inhibitors of lysosome and proteasome. Chemical inhibition of TBK1 in hepatoma Hepa1 cells showed that TBK1 was not required for the heme-induced BACH1 degradation. Its inhibition in Namalwa B lymphoma cells increased endogenous BACH1 protein. These results suggest that TBK1 promotes BACH1 degradation in parallel to the FBXO22- and heme-dependent pathway, placing BACH1 as a downstream effector of TBK1 in iron metabolism or innate immune response.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica , Proteínas F-Box , Heme , Proteínas Serina-Treonina Quinases , Proteólise , Receptores Citoplasmáticos e Nucleares , Humanos , Heme/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas F-Box/metabolismo , Proteínas F-Box/genética , Células HEK293 , Ubiquitinação , Linhagem Celular Tumoral , Lisossomos/metabolismo , Autofagia , Complexo de Endopeptidases do Proteassoma/metabolismo
13.
BMC Cancer ; 24(1): 402, 2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38561760

RESUMO

BACKGROUND: Among the most common forms of cancer worldwide, breast cancer posed a serious threat to women. Recent research revealed a lack of oxygen, known as hypoxia, was crucial in forming breast cancer. This research aimed to create a robust signature with hypoxia-related genes to predict the prognosis of breast cancer patients. The function of hypoxia genes was further studied through cell line experiments. MATERIALS AND METHODS: In the bioinformatic part, transcriptome and clinical information of breast cancer were obtained from The Cancer Genome Atlas(TCGA). Hypoxia-related genes were downloaded from the Genecards Platform. Differentially expressed hypoxia-related genes (DEHRGs) were identified. The TCGA filtered data was evenly split, ensuring a 1:1 distribution between the training and testing sets. Prognostic-related DEHRGs were identified through Cox regression. The signature was established through the training set. Then, it was validated using the test set and external validation set GSE131769 from Gene Expression Omnibus (GEO). The nomogram was created by incorporating the signature and clinicopathological characteristics. The predictive value of the nomogram was evaluated by C-index and receiver operating characteristiccurve. Immune microenvironment and mutation burden were also examined. In the experiment part, the function of the two most significant hypoxia-related genes were further explored by cell-line experiments. RESULTS: In the bioinformatic part, 141 up-regulated and 157 down-regulated DEHRGs were screened out. A prognostic signature was constructed containing nine hypoxia genes (ALOX15B, CA9, CD24, CHEK1, FOXM1, HOTAIR, KCNJ11, NEDD9, PSME2) in the training set. Low-risk patients exhibited a much more favorable prognosis than higher-risk ones (P < 0.001). The signature was double-validated in the test set and GSE131769 (P = 0.006 and P = 0.001). The nomogram showed excellent predictive value with 1-year OS AUC: 0.788, 3-year OS AUC: 0.783, and 5-year OS AUC: 0.817. Patients in the high-risk group had a higher tumor mutation burden when compared to the low-risk group. In the experiment part, the down-regulation of PSME2 inhibited cell growth ability and clone formation capability of breast cancer cells, while the down-regulation of KCNJ11 did not have any functions. CONCLUSION: Based on 9 DEHRGs, a reliable signature was established through the bioinformatic method. It could accurately predict the prognosis of breast cancer patients. Cell line experiment indicated that PSME2 played a protective role. Summarily, we provided a new insight to predict the prognosis of breast cancer by hypoxia-related genes.


Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/genética , Prognóstico , Nomogramas , Hipóxia/genética , Oxigênio , Microambiente Tumoral/genética , Proteínas Adaptadoras de Transdução de Sinal , Complexo de Endopeptidases do Proteassoma
14.
Neuromolecular Med ; 26(1): 9, 2024 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-38568291

RESUMO

Aggregation of α-synuclein (α-syn) and α-syn cytotoxicity are hallmarks of sporadic and familial Parkinson's disease (PD). Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-dependent enhancement of the expression of the 20S proteasome core particles (20S CPs) and regulatory particles (RPs) increases proteasome activity, which can promote α-syn clearance in PD. Activation of peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α) may reduce oxidative stress by strongly inducing Nrf2 gene expression. In the present study, tetramethylpyrazine nitrone (TBN), a potent-free radical scavenger, promoted α-syn clearance by the ubiquitin-proteasome system (UPS) in cell models overexpressing the human A53T mutant α-syn. In the α-syn transgenic mice model, TBN improved motor impairment, decreased the products of oxidative damage, and down-regulated the α-syn level in the serum. TBN consistently up-regulated PGC-1α and Nrf2 expression in tested models of PD. Additionally, TBN similarly enhanced the proteasome 20S subunit beta 8 (Psmb8) expression, which is linked to chymotrypsin-like proteasome activity. Furthermore, TBN increased the mRNA levels of both the 11S RPs subunits Pa28αß and a proteasome chaperone, known as the proteasome maturation protein (Pomp). Interestingly, specific siRNA targeting of Nrf2 blocked TBN's effects on Psmb8, Pa28αß, Pomp expression, and α-syn clearance. In conclusion, TBN promotes the clearance of α-syn via Nrf2-mediated UPS activation, and it may serve as a potentially disease-modifying therapeutic agent for PD.


Assuntos
Fator 2 Relacionado a NF-E2 , Complexo de Endopeptidases do Proteassoma , Pirazinas , Humanos , Animais , Camundongos , Fator 2 Relacionado a NF-E2/genética , alfa-Sinucleína/genética , Camundongos Transgênicos , Ubiquitinas
15.
J Clin Immunol ; 44(4): 88, 2024 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-38578475

RESUMO

The ubiquitin-proteasome system (UPS) has a critical role in post-translational protein modification that is essential for the maintenance of all cellular functions, including immune responses. The proteasome complex is ubiquitously expressed and is responsible for degradation of short-lived structurally abnormal, misfolded and not-needed proteins that are targeted for degradation via ubiquitin conjugation. Over the last 14 years, an increasing number of human diseases have been linked to pathogenic variants in proteasome subunits and UPS regulators. Defects of the proteasome complex or its chaperons - which have a regulatory role in the assembly of the proteasome - disrupt protein clearance and cellular homeostasis, leading to immune dysregulation, severe inflammation, and neurodevelopmental disorders in humans. Proteasome-associated diseases have complex inheritance, including monogenic, digenic and oligogenic disorders and can be dominantly or recessively inherited. In this review, we summarize the current known genetic causes of proteasomal disease, and discuss the molecular pathogenesis of these conditions based on the function and cellular expression of mutated proteins in the proteasome complex.


Assuntos
Complexo de Endopeptidases do Proteassoma , Ubiquitina , Humanos , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Síndrome , Ubiquitina/metabolismo
16.
Protein Sci ; 33(5): e4975, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38588275

RESUMO

The deubiquitinase (DUB) ubiquitin-specific protease 14 (USP14) is a dual domain protein that plays a regulatory role in proteasomal degradation and has been identified as a promising therapeutic target. USP14 comprises a conserved USP domain and a ubiquitin-like (Ubl) domain separated by a 25-residue linker. The enzyme activity of USP14 is autoinhibited in solution, but is enhanced when bound to the proteasome, where the Ubl and USP domains of USP14 bind to the Rpn1 and Rpt1/Rpt2 units, respectively. No structure of full-length USP14 in the absence of proteasome has yet been presented, however, earlier work has described how transient interactions between Ubl and USP domains in USP4 and USP7 regulate DUB activity. To better understand the roles of the Ubl and USP domains in USP14, we studied the Ubl domain alone and in full-length USP14 by nuclear magnetic resonance spectroscopy and used small angle x-ray scattering and molecular modeling to visualize the entire USP14 protein ensemble. Jointly, our results show how transient interdomain interactions between the Ubl and USP domains of USP14 predispose its conformational ensemble for proteasome binding, which may have functional implications for proteasome regulation and may be exploited in the design of future USP14 inhibitors.


Assuntos
Complexo de Endopeptidases do Proteassoma , Ubiquitina , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Ubiquitina/química , Conformação Molecular , Modelos Moleculares
17.
J Exp Med ; 221(6)2024 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-38625151

RESUMO

In a genetic screen, we identified two viable missense alleles of the essential gene Midnolin (Midn) that were associated with reductions in peripheral B cells. Causation was confirmed in mice with targeted deletion of four of six MIDN protein isoforms. MIDN was expressed predominantly in lymphocytes where it augmented proteasome activity. We showed that purified MIDN directly stimulated 26S proteasome activity in vitro in a manner dependent on the ubiquitin-like domain and a C-terminal region. MIDN-deficient B cells displayed aberrant activation of the IRE-1/XBP-1 pathway of the unfolded protein response. Partial or complete MIDN deficiency strongly suppressed Eµ-Myc-driven B cell leukemia and the antiapoptotic effects of Eµ-BCL2 on B cells in vivo and induced death of Sp2/0 hybridoma cells in vitro, but only partially impaired normal lymphocyte development. Thus, MIDN is required for proteasome activity in support of normal lymphopoiesis and is essential for malignant B cell proliferation over a broad range of differentiation states.


Assuntos
Leucemia Linfocítica Crônica de Células B , Complexo de Endopeptidases do Proteassoma , Animais , Camundongos , Mutação , Proteínas Nucleares
18.
Cell Chem Biol ; 31(4): 625-626, 2024 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-38640899

RESUMO

In an interview with Samantha Nelson, a scientific editor of Cell Chemical Biology, the first and corresponding authors of the research article entitled Selective targeting of Plasmodium falciparum Hsp90 disrupts the 26S proteasome share more about the project and their perspectives on the field.


Assuntos
Plasmodium falciparum , Complexo de Endopeptidases do Proteassoma
19.
Int J Mol Sci ; 25(7)2024 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-38612788

RESUMO

Proteasome inhibitors are used in the therapy of several cancers, and clinical trials are underway for their use in the treatment of glioblastoma (GBM). However, GBM becomes resistant to chemotherapy relatively rapidly. Recently, the overexpression of ribonucleotide reductase (RNR) genes was found to mediate therapy resistance in GBM. The use of combinations of chemotherapeutic agents is considered a promising direction in cancer therapy. The present work aimed to evaluate the efficacy of the combination of proteasome and RNR inhibitors in yeast and GBM cell models. We have shown that impaired proteasome function results in increased levels of RNR subunits and increased enzyme activity in yeast. Co-administration of the proteasome inhibitor bortezomib and the RNR inhibitor hydroxyurea was found to significantly reduce the growth rate of S. cerevisiae yeast. Accordingly, the combination of bortezomib and another RNR inhibitor gemcitabine reduced the survival of DBTRG-05MG compared to the HEK293 cell line. Thus, yeast can be used as a simple model to evaluate the efficacy of combinations of proteasome and RNR inhibitors.


Assuntos
Glioblastoma , Saccharomyces cerevisiae , Humanos , Complexo de Endopeptidases do Proteassoma , Glioblastoma/tratamento farmacológico , Bortezomib/farmacologia , Células HEK293
20.
Cell Death Dis ; 15(4): 263, 2024 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-38615011

RESUMO

Abnormal cardiac fibrosis is the main pathological change of post-myocardial infarction (MI) heart failure. Although the E3 ubiquitin ligase FBXL8 is a key regulator in the cell cycle, cell proliferation, and inflammation, its role in post-MI ventricular fibrosis and heart failure remains unknown. FBXL8 was primarily expressed in cardiac fibroblasts (CFs) and remarkably decreased in CFs treated by TGFß and heart subjected to MI. The echocardiography and histology data suggested that adeno-associated viruses (AAV9)-mediated FBXL8 overexpression had improved cardiac function and ameliorated post-MI cardiac fibrosis. In vitro, FBXL8 overexpression prevented TGFß-induced proliferation, migration, contraction, and collagen secretion in CFs, while knockdown of FBXL8 demonstrated opposite effects. Mechanistically, FBXL8 interacted with Snail1 to promote Snail1 degradation through the ubiquitin-proteasome system and decreased the activation of RhoA. Moreover, the FBXL8ΔC3 binding domain was indispensable for Snail1 interaction and degradation. Ectopic Snail1 expression partly abolished the effects mediated by FBXL8 overexpression in CFs treated by TGFß. These results characterized the role of FBXL8 in regulating the ubiquitin-mediated degradation of Snail1 and revealed the underlying molecular mechanism of how MI up-regulated the myofibroblasts differentiation-inducer Snail1 and suggested that FBXL8 may be a potential curative target for improving post-MI cardiac function.


Assuntos
Insuficiência Cardíaca , Infarto do Miocárdio , Humanos , Complexo de Endopeptidases do Proteassoma , Infarto do Miocárdio/genética , Fator de Crescimento Transformador beta , Ubiquitinas
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