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1.
Bioorg Med Chem ; 108: 117773, 2024 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-38850999

RESUMO

In this study, we have developedsmall molecule drug conjugates (SMDCs)consisting ofa prostate specific membrane antigen (PSMA) ligandand syringolin derivatives, which are potent proteasome inhibitors, to selectively deliver syringolin derivatives to prostate cancer cells. Two parent compounds were used for syringolin derivatives with different linkage sites. These SMDCs exhibited PSMA-expressing cell-selective cytotoxicity and they could potentially be used for safer treatment of cancer.


Assuntos
Antígenos de Superfície , Antineoplásicos , Glutamato Carboxipeptidase II , Inibidores de Proteassoma , Humanos , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/química , Inibidores de Proteassoma/síntese química , Glutamato Carboxipeptidase II/antagonistas & inibidores , Glutamato Carboxipeptidase II/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Antígenos de Superfície/metabolismo , Relação Estrutura-Atividade , Estrutura Molecular , Ensaios de Seleção de Medicamentos Antitumorais , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/síntese química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Masculino , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Neoplasias da Próstata/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo
2.
PLoS One ; 19(6): e0305350, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38861553

RESUMO

All-trans retinoic acid (ATRA), recognized as the principal and most biologically potent metabolite of vitamin A, has been identified for its inhibitory effects on hepatitis B virus (HBV) replication. Nevertheless, the underlying mechanism remains elusive. The present study reveals that ATRA induces E6-associated protein (E6AP)-mediated proteasomal degradation of HBx to suppress HBV replication in human hepatoma cells in a p53-dependent pathway. For this effect, ATRA induced promoter hypomethylation of E6AP in the presence of HBx, which resulted in the upregulation of E6AP levels in HepG2 but not in Hep3B cells, emphasizing the p53-dependent nature of this effect. As a consequence, ATRA augmented the interaction between E6AP and HBx, resulting in substantial ubiquitination of HBx and consequent reduction in HBx protein levels in both the HBx overexpression system and the in vitro HBV replication model. Additionally, the knockdown of E6AP under ATRA treatment reduced the interaction between HBx and E6AP and decreased the ubiquitin-dependent proteasomal degradation of HBx, which prompted a recovery of HBV replication in the presence of ATRA, as confirmed by increased levels of intracellular HBV proteins and secreted HBV levels. This study not only contributes to the understanding of the complex interactions between ATRA, p53, E6AP, and HBx but also provides an academic basis for the clinical employment of ATRA in the treatment of HBV infection.


Assuntos
Vírus da Hepatite B , Complexo de Endopeptidases do Proteassoma , Transativadores , Tretinoína , Proteína Supressora de Tumor p53 , Ubiquitina-Proteína Ligases , Proteínas Virais Reguladoras e Acessórias , Replicação Viral , Humanos , Proteínas Virais Reguladoras e Acessórias/metabolismo , Transativadores/metabolismo , Transativadores/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Replicação Viral/efeitos dos fármacos , Vírus da Hepatite B/fisiologia , Vírus da Hepatite B/efeitos dos fármacos , Tretinoína/farmacologia , Tretinoína/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Células Hep G2 , Regulação para Baixo/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Regiões Promotoras Genéticas , Metilação de DNA/efeitos dos fármacos , Linhagem Celular Tumoral
3.
Mol Cell ; 84(11): 2011-2013, 2024 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-38848689

RESUMO

In this issue of Molecular Cell, Yi et al.1 demonstrate that reduced mTORC1 activity induces the CTLH E3 ligase-dependent degradation of HMGCS1, an enzyme in the mevalonate pathway, thus revealing a unique connection between mTORC1 signaling and the degradation of a specific metabolic enzyme via the ubiquitin-proteasome system.


Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina , Complexo de Endopeptidases do Proteassoma , Transdução de Sinais , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteólise , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/genética , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/genética , Animais , Ácido Mevalônico/metabolismo , Ubiquitina/metabolismo
4.
Cancer Lett ; 596: 217020, 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-38849009

RESUMO

B7-H4 is an immune checkpoint crucial for inhibiting CD8+ T-cell activity. A clinical trial is underway to investigate B7-H4 as a potential immunotherapeutic agent. However, the regulatory mechanism of B7-H4 degradation via the ubiquitin-proteasome pathway (UPP) remains poorly understood. In this study, we discovered that proteasome inhibitors effectively increased B7-H4 expression, while EGFR-activating mutants promoted B7-H4 expression through the UPP. We screened B7-H4 binding proteins by co-immunoprecipitation and mass spectrometry and found that USP2a acted as a deubiquitinase of B7-H4 by removing K48- and K63-linked ubiquitin chains from B7-H4, leading to a reduction in B7-H4 degradation. EGFR mutants enhanced B7-H4 stability by upregulating USP2a expression. We further investigated the role of USP2a in tumor growth in vivo. Depletion of USP2a in L858R/LLC cells inhibited tumor cell proliferation, consequently suppressing tumor growth in immune-deficient nude mice by destabilizing downstream molecules such as Cyclin D1. In an immune-competent C57BL/6 mouse tumor model, USP2a abrogation facilitated infiltration of CD95+CD8+ effector T cells and hindered infiltration of Tim-3+CD8+ and LAG-3+CD8+ exhausted T cells by destabilizing B7-H4. Clinical lung adenocarcinoma samples showed a significant correlation between B7-H4 abundance and USP2a expression, indicating the contribution of the EGFR/USP2a/B7-H4 axis to tumor immunosuppression. In summary, this study elucidates the dual effects of USP2a in tumor growth by stabilizing Cyclin D1, promoting tumor cell proliferation, and stabilizing B7-H4, contributing to tumor immunosuppression. Therefore, USP2a represents a potential target for tumor therapy.


Assuntos
Adenocarcinoma de Pulmão , Receptores ErbB , Neoplasias Pulmonares , Camundongos Nus , Ubiquitina Tiolesterase , Inibidor 1 da Ativação de Células T com Domínio V-Set , Animais , Humanos , Receptores ErbB/metabolismo , Receptores ErbB/genética , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Camundongos , Inibidor 1 da Ativação de Células T com Domínio V-Set/genética , Inibidor 1 da Ativação de Células T com Domínio V-Set/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Adenocarcinoma de Pulmão/imunologia , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células , Mutação , Camundongos Endogâmicos C57BL , Linfócitos T CD8-Positivos/imunologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/genética
5.
Cell Death Dis ; 15(6): 409, 2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38862475

RESUMO

Low glucose is a common microenvironment for rapidly growing solid tumors, which has developed multiple approaches to survive under glucose deprivation. However, the specific regulatory mechanism remains largely elusive. In this study, we demonstrate that glucose deprivation, while not amino acid or serum starvation, transactivates the expression of DCAF1. This enhances the K48-linked polyubiquitination and proteasome-dependent degradation of Rheb, inhibits mTORC1 activity, induces autophagy, and facilitates cancer cell survival under glucose deprivation conditions. This study identified DCAF1 as a new cellular glucose sensor and uncovered new insights into mechanism of DCAF1-mediated inactivation of Rheb-mTORC1 pathway for promoting cancer cell survival in response to glucose deprivation.


Assuntos
Sobrevivência Celular , Glucose , Alvo Mecanístico do Complexo 1 de Rapamicina , Proteína Enriquecida em Homólogo de Ras do Encéfalo , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteína Enriquecida em Homólogo de Ras do Encéfalo/metabolismo , Proteína Enriquecida em Homólogo de Ras do Encéfalo/genética , Glucose/metabolismo , Linhagem Celular Tumoral , Autofagia , Ubiquitinação , Transdução de Sinais , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Células HEK293 , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética
6.
Diagn Pathol ; 19(1): 79, 2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38863002

RESUMO

BACKGROUND: Osteosarcoma is a bone tumor that is characterized by high malignancy and a high mortality rate, and that originates from primitive osteoblastic mesenchymal cells and is most common in rapidly growing long bones. PSMD14, also known as RPN11 or POH1, is a member of the JAMM isopeptidase family, which is able to remove the substrate protein ubiquitination label, thereby regulating the stability and function of the substrate protein. In this study, we explored the expression and potential biological significance of the PSMD14 deubiquitinating enzyme in osteosarcoma. METHODS: Immunohistochemical methods were used to detect the expression of PSMD14 in biopsies of 91 osteosarcoma patients, and the specimens were classified into high and low PSMD14 expression groups. The correlation between PSMD14 expression and clinical indicators and prognosis was compared.SiRNA was used to downregulate PSMD14 in two osteosarcoma cell lines (HOS and SJSA-1), and the effects of downregulation of PSMD14 on the viability, proliferation, and invasion ability of osteosarcoma cells were analyzed. RESULTS: We identified significant differences in recurrence, metastasis, and survival time of the osteosarcoma patients on the basis of PSMD14 expression. High expression of PSMD14 in osteosarcoma patients was associated with a low survival rate and high risk of metastasis and recurrence. Down-regulation of PSMD14 inhibited the viability, proliferation, and invasiveness of osteosarcoma cell lines. CONCLUSIONS: PSMD14 may be a new prognostic marker and therapeutic target for osteosarcoma.


Assuntos
Biomarcadores Tumorais , Neoplasias Ósseas , Osteossarcoma , Osteossarcoma/patologia , Osteossarcoma/mortalidade , Osteossarcoma/metabolismo , Osteossarcoma/genética , Humanos , Neoplasias Ósseas/patologia , Neoplasias Ósseas/mortalidade , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/genética , Masculino , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/análise , Feminino , Prognóstico , Linhagem Celular Tumoral , Adulto , Proliferação de Células , Complexo de Endopeptidases do Proteassoma/metabolismo , Adolescente , Adulto Jovem , Pessoa de Meia-Idade , Invasividade Neoplásica , Transativadores
7.
Sci Rep ; 14(1): 13037, 2024 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-38844605

RESUMO

The proteasome-associated deubiquitinase USP14 is a potential drug target. Using an inducible USP14 knockout system in colon cancer cells, we found that USP14 depletion impedes cellular proliferation, induces cell cycle arrest, and leads to a senescence-like phenotype. Transcriptomic analysis revealed altered gene expression related to cell division and cellular differentiation. USP14 knockout cells also exhibited changes in morphology, actin distribution, and expression of actin cytoskeletal components. Increased ubiquitin turnover was observed, offset by upregulation of polyubiquitin genes UBB and UBC. Pharmacological inhibition of USP14 with IU1 increased ubiquitin turnover but did not affect cellular growth or morphology. BioGRID data identified USP14 interactors linked to actin cytoskeleton remodeling, DNA damage repair, mRNA splicing, and translation. In conclusion, USP14 loss in colon cancer cells induces a transient quiescent cancer phenotype not replicated by pharmacologic inhibition of its deubiquitinating activity.


Assuntos
Proliferação de Células , Senescência Celular , Neoplasias Colorretais , Ubiquitina Tiolesterase , Humanos , Senescência Celular/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Ubiquitina Tiolesterase/metabolismo , Ubiquitina Tiolesterase/genética , Linhagem Celular Tumoral , Fenótipo , Complexo de Endopeptidases do Proteassoma/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Regulação Neoplásica da Expressão Gênica , Ubiquitina/metabolismo
8.
J Transl Med ; 22(1): 507, 2024 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-38802851

RESUMO

BACKGROUND: Gastric cancer (GC) ranks fifth in global cancer incidence and third in mortality rate among all cancer types. Circular RNAs (circRNAs) have been extensively demonstrated to regulate multiple malignant biological behaviors in GC. Emerging evidence suggests that several circRNAs derived from FNDC3B play pivotal roles in cancer. However, the role of circFNDC3B in GC remains elusive. METHODS: We initially screened circFNDC3B with translation potential via bioinformatics algorithm prediction. Subsequently, Sanger sequencing, qRT-PCR, RNase R, RNA-FISH and nuclear-cytoplasmic fractionation assays were explored to assess the identification and localization of circ0003692, a circRNA derived from FNDC3B. qRT-PCR and ISH were performed to quantify expression of circ0003692 in human GC tissues and adjacent normal tissues. The protein-encoding ability of circ0003692 was investigated through dual-luciferase reporter assay and LC/MS. The biological behavior of circ0003692 in GC was confirmed via in vivo and in vitro experiments. Additionally, Co-IP and rescue experiments were performed to elucidate the interaction between the encoded protein and c-Myc. RESULTS: We found that circ0003692 was significantly downregulated in GC tissues. Circ0003692 had the potential to encode a novel protein FNDC3B-267aa, which was downregulated in GC cells. We verified that FNDC3B-267aa, rather than circ0003692, inhibited GC migration in vitro and in vivo. Mechanistically, FNDC3B-267aa directly interacted with c-Myc and promoted proteasomal degradation of c-Myc, resulting in the downregulation of c-Myc-Snail/Slug axis. CONCLUSIONS: Our study revealed that the novel protein FNDC3B-267aa encoded by circ0003692 suppressed GC metastasis through binding to c-Myc and enhancing proteasome-mediated degradation of c-Myc. The study offers the potential applications of circ0003692 or FNDC3B-267aa as therapeutic targets for GC.


Assuntos
Fibronectinas , Metástase Neoplásica , Complexo de Endopeptidases do Proteassoma , Proteínas Proto-Oncogênicas c-myc , RNA Circular , Neoplasias Gástricas , Neoplasias Gástricas/patologia , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Humanos , RNA Circular/genética , RNA Circular/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Linhagem Celular Tumoral , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Animais , Fibronectinas/metabolismo , Regulação Neoplásica da Expressão Gênica , Masculino , Proteólise , Camundongos Nus , Sequência de Bases , Movimento Celular/genética , Feminino , Camundongos
9.
Mol Neurodegener ; 19(1): 44, 2024 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-38816762

RESUMO

Synucleinopathies are a group of neurodegenerative diseases characterized by the accumulation of α-synuclein (α-syn) in the brain, leading to motor and neuropsychiatric symptoms. Currently, there are no known cures for synucleinopathies, and treatments mainly focus on symptom management. In this study, we developed a single-domain antibody (sdAb)-based protein degrader with features designed to enhance proteasomal degradation of α-syn. This sdAb derivative targets both α-syn and Cereblon (CRBN), a substrate-receptor for the E3-ubiquitin ligase CRL4CRBN, and thereby induces α-syn ubiquitination and proteasomal degradation. Our results indicate that this therapeutic candidate enhances proteasomal degradation of α-syn, in addition to the endogenous lysosomal degradation machinery. By promoting proteasomal degradation of α-syn, we improved clearance of α-syn in primary culture and mouse models of synucleinopathy. These findings indicate that our sdAb-based protein degrader is a promising therapeutic candidate for synucleinopathies. Considering that only a small percentage of antibodies enter the brain, more potent sdAbs with greater brain entry than whole antibodies could enhance clinical benefits of antibody-based therapies.


Assuntos
Sinucleinopatias , alfa-Sinucleína , Animais , Sinucleinopatias/metabolismo , alfa-Sinucleína/metabolismo , alfa-Sinucleína/imunologia , Camundongos , Humanos , Anticorpos de Domínio Único , Modelos Animais de Doenças , Encéfalo/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo
10.
Mol Cell ; 84(11): 2166-2184.e9, 2024 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-38788716

RESUMO

Mammalian target of rapamycin (mTOR) senses changes in nutrient status and stimulates the autophagic process to recycle amino acids. However, the impact of nutrient stress on protein degradation beyond autophagic turnover is incompletely understood. We report that several metabolic enzymes are proteasomal targets regulated by mTOR activity based on comparative proteome degradation analysis. In particular, 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) synthase 1 (HMGCS1), the initial enzyme in the mevalonate pathway, exhibits the most significant half-life adaptation. Degradation of HMGCS1 is regulated by the C-terminal to LisH (CTLH) E3 ligase through the Pro/N-degron motif. HMGCS1 is ubiquitylated on two C-terminal lysines during mTORC1 inhibition, and efficient degradation of HMGCS1 in cells requires a muskelin adaptor. Importantly, modulating HMGCS1 abundance has a dose-dependent impact on cell proliferation, which is restored by adding a mevalonate intermediate. Overall, our unbiased degradomics study provides new insights into mTORC1 function in cellular metabolism: mTORC1 regulates the stability of limiting metabolic enzymes through the ubiquitin system.


Assuntos
Proliferação de Células , Hidroximetilglutaril-CoA Sintase , Alvo Mecanístico do Complexo 1 de Rapamicina , Proteólise , Ubiquitina-Proteína Ligases , Ubiquitinação , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Células HEK293 , Hidroximetilglutaril-CoA Sintase/metabolismo , Hidroximetilglutaril-CoA Sintase/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/genética , Ácido Mevalônico/metabolismo , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/genética , Transdução de Sinais , Degrons , Proteínas Adaptadoras de Transdução de Sinal
11.
Life Sci ; 349: 122732, 2024 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-38768775

RESUMO

Acetaminophen is a known antipyretic and non-opioid analgesic for mild pain and fever. Numerous studies uncover their hidden chemotherapeutics applications, including chronic cancer pain management. Acetaminophen also represents an anti-proliferative effect in some cancer cells. Few studies also suggest that the use of Acetaminophen can trigger apoptosis and impede cellular growth. However, Acetaminophen's molecular potential and precise mechanism against improper cellular proliferation and use as an effective anti-proliferative agent still need to be better understood. Here, our current findings show that Acetaminophen induces proteasomal dysfunctions, resulting in aberrant protein accumulation and mitochondrial abnormalities, and consequently induces cell apoptosis. We observed that the Acetaminophen treatment leads to improper aggregation of ubiquitylated expanded polyglutamine proteins, which may be due to the dysfunctions of proteasome activities. Our in-silico analysis suggests the interaction of Acetaminophen and proteasome. Furthermore, we demonstrated the accumulation of proteasome substrates and the depletion of proteasome activities after treating Acetaminophen in cells. Acetaminophen induces proteasome dysfunctions and mitochondrial abnormalities, leading to pro-apoptotic morphological changes and apoptosis successively. These results suggest that Acetaminophen can induce cell death and may retain a promising anti-proliferative effect. These observations can open new possible molecular strategies in the near future for developing and designing specific and effective proteasome inhibitors, which can be helpful in conjugation with other anti-tumor drugs for their better efficiency.


Assuntos
Acetaminofen , Apoptose , Mitocôndrias , Complexo de Endopeptidases do Proteassoma , Acetaminofen/farmacologia , Apoptose/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proliferação de Células/efeitos dos fármacos , Analgésicos não Narcóticos/farmacologia , Linhagem Celular Tumoral , Antineoplásicos/farmacologia
12.
Pharmacol Res ; 204: 107215, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38744399

RESUMO

The ubiquitinproteasome system (UPS) is the main mechanism responsible for the intracellular degradation of misfolded or damaged proteins. Under inflammatory conditions, the immunoproteasome, an isoform of the proteasome, can be induced, enhancing the antigen-presenting function of the UPS. Furthermore, the immunoproteasome also serves nonimmune functions, such as maintaining protein homeostasis and regulating signalling pathways, and is involved in the pathophysiological processes of various cardiovascular diseases (CVDs). This review aims to provide a comprehensive summary of the current research on the involvement of the immunoproteasome in cardiovascular diseases, with the ultimate goal of identifying novel strategies for the treatment of these conditions.


Assuntos
Doenças Cardiovasculares , Complexo de Endopeptidases do Proteassoma , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/imunologia , Doenças Cardiovasculares/imunologia , Doenças Cardiovasculares/metabolismo , Animais , Ubiquitina/metabolismo , Ubiquitina/imunologia , Transdução de Sinais
13.
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
14.
Biochemistry ; 63(11): 1423-1433, 2024 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-38743592

RESUMO

PGM1-linked congenital disorder of glycosylation (PGM1-CDG) is an autosomal recessive disease characterized by several phenotypes, some of which are life-threatening. Research focusing on the disease-related variants of the α-D-phosphoglucomutase 1 (PGM1) protein has shown that several are insoluble in vitro and expressed at low levels in patient fibroblasts. Due to these observations, we hypothesized that some disease-linked PGM1 protein variants are structurally destabilized and subject to protein quality control (PQC) and rapid intracellular degradation. Employing yeast-based assays, we show that a disease-associated human variant, PGM1 L516P, is insoluble, inactive, and highly susceptible to ubiquitylation and rapid degradation by the proteasome. In addition, we show that PGM1 L516P forms aggregates in S. cerevisiae and that both the aggregation pattern and the abundance of PGM1 L516P are chaperone-dependent. Finally, using computational methods, we perform saturation mutagenesis to assess the impact of all possible single residue substitutions in the PGM1 protein. These analyses identify numerous missense variants with predicted detrimental effects on protein function and stability. We suggest that many disease-linked PGM1 variants are subject to PQC-linked degradation and that our in silico site-saturated data set may assist in the mechanistic interpretation of PGM1 variants.


Assuntos
Fosfoglucomutase , Saccharomyces cerevisiae , Humanos , Fosfoglucomutase/metabolismo , Fosfoglucomutase/genética , Fosfoglucomutase/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteólise , Mutação de Sentido Incorreto , Ubiquitinação , Defeitos Congênitos da Glicosilação/genética , Defeitos Congênitos da Glicosilação/metabolismo , Estabilidade Proteica , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/genética
16.
Cell Death Dis ; 15(5): 373, 2024 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-38811535

RESUMO

The targeted elimination of radio- or chemotherapy-induced senescent cells by so-called senolytic substances represents a promising approach to reduce tumor relapse as well as therapeutic side effects such as fibrosis. We screened an in-house library of 178 substances derived from marine sponges, endophytic fungi, and higher plants, and determined their senolytic activities towards DNA damage-induced senescent HCT116 colon carcinoma cells. The Pan-PI3K-inhibitor wortmannin and its clinical derivative, PX-866, were identified to act as senolytics. PX-866 potently induced apoptotic cell death in senescent HCT116, MCF-7 mammary carcinoma, and A549 lung carcinoma cells, independently of whether senescence was induced by ionizing radiation or by chemotherapeutics, but not in proliferating cells. Other Pan-PI3K inhibitors, such as the FDA-approved drug BAY80-6946 (Copanlisib, Aliqopa®), also efficiently and specifically eliminated senescent cells. Interestingly, only the simultaneous inhibition of both PI3K class I alpha (with BYL-719 (Alpelisib, Piqray®)) and delta (with CAL-101 (Idelalisib, Zydelig®)) isoforms was sufficient to induce senolysis, whereas single application of these inhibitors had no effect. On the molecular level, inhibition of PI3Ks resulted in an increased proteasomal degradation of the CDK inhibitor p21WAF1/CIP1 in all tumor cell lines analyzed. This led to a timely induction of apoptosis in senescent tumor cells. Taken together, the senolytic properties of PI3K-inhibitors reveal a novel dimension of these promising compounds, which holds particular potential when employed alongside DNA damaging agents in combination tumor therapies.


Assuntos
Senescência Celular , Inibidor de Quinase Dependente de Ciclina p21 , Humanos , Senescência Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Células HCT116 , Complexo de Endopeptidases do Proteassoma/metabolismo , Apoptose/efeitos dos fármacos , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Células MCF-7 , Proteólise/efeitos dos fármacos , Células A549 , Wortmanina/farmacologia , Senoterapia/farmacologia , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Classe I de Fosfatidilinositol 3-Quinases/genética , Dano ao DNA/efeitos dos fármacos , Pirimidinas , Quinazolinas
17.
Nat Commun ; 15(1): 4584, 2024 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-38811577

RESUMO

Stimulator of interferon genes (STING) is a central component of the cytosolic nucleic acids sensing pathway and as such master regulator of the type I interferon response. Due to its critical role in physiology and its' involvement in a variety of diseases, STING has been a focus for drug discovery. Targeted protein degradation (TPD) has emerged as a promising pharmacology for targeting previously considered undruggable proteins by hijacking the cellular ubiquitin proteasome system (UPS) with small molecules. Here, we identify AK59 as a STING degrader leveraging HERC4, a HECT-domain E3 ligase. Additionally, our data reveals that AK59 is effective on the common pathological STING mutations, suggesting a potential clinical application of this mechanism. Thus, these findings introduce HERC4 to the fields of TPD and of compound-induced degradation of STING, suggesting potential therapeutic applications.


Assuntos
Proteínas de Membrana , Proteólise , Ubiquitina-Proteína Ligases , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Humanos , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteólise/efeitos dos fármacos , Células HEK293 , Animais , Mutação , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitinação
18.
J Cell Sci ; 137(11)2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38766715

RESUMO

Although protein aggregation can cause cytotoxicity, such aggregates can also form to mitigate cytotoxicity from misfolded proteins, although the nature of these contrasting aggregates remains unclear. We previously found that overproduction (op) of a three green fluorescent protein-linked protein (3×GFP) induces giant aggregates and is detrimental to growth. Here, we investigated the mechanism of growth inhibition by 3×GFP-op using non-aggregative 3×MOX-op as a control in Saccharomyces cerevisiae. The 3×GFP aggregates were induced by misfolding, and 3×GFP-op had higher cytotoxicity than 3×MOX-op because it perturbed the ubiquitin-proteasome system. Static aggregates formed by 3×GFP-op dynamically trapped Hsp70 family proteins (Ssa1 and Ssa2 in yeast), causing the heat-shock response. Systematic analysis of mutants deficient in the protein quality control suggested that 3×GFP-op did not cause a critical Hsp70 depletion and aggregation functioned in the direction of mitigating toxicity. Artificial trapping of essential cell cycle regulators into 3×GFP aggregates caused abnormalities in the cell cycle. In conclusion, the formation of the giant 3×GFP aggregates itself is not cytotoxic, as it does not entrap and deplete essential proteins. Rather, it is productive, inducing the heat-shock response while preventing an overload to the degradation system.


Assuntos
Proteínas de Fluorescência Verde , Proteínas de Choque Térmico HSP70 , Agregados Proteicos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteólise , Complexo de Endopeptidases do Proteassoma/metabolismo , Resposta ao Choque Térmico/genética , Dobramento de Proteína , Ciclo Celular/genética , Adenosina Trifosfatases
19.
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
20.
Biochim Biophys Acta Rev Cancer ; 1879(4): 189119, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38761982

RESUMO

Tumor recurrence is a mechanism triggered in sparse populations of cancer cells that usually remain in a quiescent state after strict stress and/or therapeutic factors, which is affected by a variety of autocrine and microenvironmental cues. Despite thorough investigations, the biology of dormant and/or cancer stem cells is still not fully elucidated, as for the mechanisms of their reawakening, while only the major molecular patterns driving the relapse process have been identified to date. These molecular patterns profoundly interfere with the elements of cellular proteostasis systems that support the efficiency of the recurrence process. As a major proteostasis machinery, we review the role of the ubiquitin-proteasome system (UPS) in tumor cell dormancy and reawakening, devoting particular attention to the functions of its components, E3 ligases, deubiquitinating enzymes and proteasomes in cancer recurrence. We demonstrate how UPS components functionally or mechanistically interact with the pivotal proteins implicated in the recurrence program and reveal that modulators of the UPS hold promise to become an efficient adjuvant therapy for eradicating refractory tumor cells to impede tumor relapse.


Assuntos
Recidiva Local de Neoplasia , Neoplasias , Complexo de Endopeptidases do Proteassoma , Ubiquitina , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Neoplasias/patologia , Neoplasias/metabolismo , Neoplasias/genética , Ubiquitina/metabolismo , Recidiva Local de Neoplasia/patologia , Animais , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/metabolismo , Microambiente Tumoral
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