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1.
Cell ; 186(4): 732-747.e16, 2023 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-36803603

RESUMO

Hematopoietic stem cells (HSCs) have a number of unique physiologic adaptations that enable lifelong maintenance of blood cell production, including a highly regulated rate of protein synthesis. Yet, the precise vulnerabilities that arise from such adaptations have not been fully characterized. Here, inspired by a bone marrow failure disorder due to the loss of the histone deubiquitinase MYSM1, characterized by selectively disadvantaged HSCs, we show how reduced protein synthesis in HSCs results in increased ferroptosis. HSC maintenance can be fully rescued by blocking ferroptosis, despite no alteration in protein synthesis rates. Importantly, this selective vulnerability to ferroptosis not only underlies HSC loss in MYSM1 deficiency but also characterizes a broader liability of human HSCs. Increasing protein synthesis rates via MYSM1 overexpression makes HSCs less susceptible to ferroptosis, more broadly illustrating the selective vulnerabilities that arise in somatic stem cell populations as a result of physiologic adaptations.


Assuntos
Ferroptose , Células-Tronco Hematopoéticas , Humanos , Endopeptidases/metabolismo , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Transativadores/metabolismo , Proteases Específicas de Ubiquitina/metabolismo
2.
Cell ; 185(21): 3913-3930.e19, 2022 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-36198316

RESUMO

Although women experience significantly higher tau burden and increased risk for Alzheimer's disease (AD) than men, the underlying mechanism for this vulnerability has not been explained. Here, we demonstrate through in vitro and in vivo models, as well as human AD brain tissue, that X-linked ubiquitin specific peptidase 11 (USP11) augments pathological tau aggregation via tau deubiquitination initiated at lysine-281. Removal of ubiquitin provides access for enzymatic tau acetylation at lysines 281 and 274. USP11 escapes complete X-inactivation, and female mice and people both exhibit higher USP11 levels than males. Genetic elimination of usp11 in a tauopathy mouse model preferentially protects females from acetylated tau accumulation, tau pathology, and cognitive impairment. USP11 levels also strongly associate positively with tau pathology in females but not males. Thus, inhibiting USP11-mediated tau deubiquitination may provide an effective therapeutic opportunity to protect women from increased vulnerability to AD and other tauopathies.


Assuntos
Doença de Alzheimer , Tauopatias , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Caracteres Sexuais , Tauopatias/genética , Tauopatias/patologia , Tioléster Hidrolases/genética , Proteases Específicas de Ubiquitina , Proteínas tau/genética
3.
Immunity ; 57(3): 528-540.e6, 2024 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-38417442

RESUMO

RNA splicing is involved in cancer initiation and progression, but how it influences host antitumor immunity in the metabolically abnormal tumor microenvironment (TME) remains unclear. Here, we demonstrate that lactate modulates Foxp3-dependent RNA splicing to maintain the phenotypic and functional status of tumor-infiltrating regulatory T (Treg) cells via CTLA-4. RNA splicing in Treg cells was correlated with the Treg cell signatures in the TME. Ubiquitin-specific peptidase 39 (USP39), a component of the RNA splicing machinery, maintained RNA-splicing-mediated CTLA-4 expression to control Treg cell function. Mechanistically, lactate promoted USP39-mediated RNA splicing to facilitate CTLA-4 expression in a Foxp3-dependent manner. Moreover, the efficiency of CTLA-4 RNA splicing was increased in tumor-infiltrating Treg cells from patients with colorectal cancer. These findings highlight the immunological relevance of RNA splicing in Treg cells and provide important insights into the environmental mechanism governing CTLA-4 expression in Treg cells.


Assuntos
Neoplasias , Linfócitos T Reguladores , Humanos , Antígeno CTLA-4 , Fatores de Transcrição Forkhead/genética , Ácido Láctico/metabolismo , Linfócitos do Interstício Tumoral , Neoplasias/genética , Neoplasias/metabolismo , Microambiente Tumoral , Proteases Específicas de Ubiquitina/metabolismo
4.
Cell ; 166(1): 152-66, 2016 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-27368102

RESUMO

Through a network of progressively maturing vesicles, the endosomal system connects the cell's interior with extracellular space. Intriguingly, this network exhibits a bilateral architecture, comprised of a relatively immobile perinuclear vesicle "cloud" and a highly dynamic peripheral contingent. How this spatiotemporal organization is achieved and what function(s) it curates is unclear. Here, we reveal the endoplasmic reticulum (ER)-located ubiquitin ligase Ring finger protein 26 (RNF26) as the global architect of the entire endosomal system, including the trans-Golgi network (TGN). To specify perinuclear vesicle coordinates, catalytically competent RNF26 recruits and ubiquitinates the scaffold p62/sequestosome 1 (p62/SQSTM1), in turn attracting ubiquitin-binding domains (UBDs) of various vesicle adaptors. Consequently, RNF26 restrains fast transport of diverse vesicles through a common molecular mechanism operating at the ER membrane, until the deubiquitinating enzyme USP15 opposes RNF26 activity to allow vesicle release into the cell's periphery. By drawing the endosomal system's architecture, RNF26 orchestrates endosomal maturation and trafficking of cargoes, including signaling receptors, in space and time.


Assuntos
Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Neoplasias/metabolismo , Linhagem Celular Tumoral , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Proteína Sequestossoma-1/metabolismo , Vesículas Transportadoras/metabolismo , Proteases Específicas de Ubiquitina/metabolismo
5.
Nat Immunol ; 18(1): 54-63, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27721430

RESUMO

Genes and pathways in which inactivation dampens tissue inflammation present new opportunities for understanding the pathogenesis of common human inflammatory diseases, including inflammatory bowel disease, rheumatoid arthritis and multiple sclerosis. We identified a mutation in the gene encoding the deubiquitination enzyme USP15 (Usp15L749R) that protected mice against both experimental cerebral malaria (ECM) induced by Plasmodium berghei and experimental autoimmune encephalomyelitis (EAE). Combining immunophenotyping and RNA sequencing in brain (ECM) and spinal cord (EAE) revealed that Usp15L749R-associated resistance to neuroinflammation was linked to dampened type I interferon responses in situ. In hematopoietic cells and in resident brain cells, USP15 was coexpressed with, and functionally acted together with the E3 ubiquitin ligase TRIM25 to positively regulate type I interferon responses and to promote pathogenesis during neuroinflammation. The USP15-TRIM25 dyad might be a potential target for intervention in acute or chronic states of neuroinflammation.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Malária Cerebral/imunologia , Inflamação Neurogênica/imunologia , Fatores de Transcrição/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Encefalomielite Autoimune Experimental/tratamento farmacológico , Células HEK293 , Humanos , Imunidade Inata , Interferon Tipo I/metabolismo , Malária Cerebral/tratamento farmacológico , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Terapia de Alvo Molecular , Glicoproteína Mielina-Oligodendrócito/imunologia , Inflamação Neurogênica/tratamento farmacológico , Fragmentos de Peptídeos/imunologia , Plasmodium berghei/imunologia , Fatores de Transcrição/genética , Proteases Específicas de Ubiquitina/genética
6.
Nat Immunol ; 17(3): 259-68, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26808229

RESUMO

The proinflammatory cytokines interleukin 12 (IL-12) and IL-23 connect innate responses and adaptive immune responses and are also involved in autoimmune and inflammatory diseases. Here we describe an epigenetic mechanism for regulation of the genes encoding IL-12 (Il12a and Il12b; collectively called 'Il12' here) and IL-23 (Il23a and Il12b; collectively called 'Il23' here) involving the deubiquitinase Trabid. Deletion of Zranb1 (which encodes Trabid) in dendritic cells inhibited induction of the expression of Il12 and Il23 by Toll-like receptors (TLRs), which impaired the differentiation of inflammatory T cells and protected mice from autoimmune inflammation. Trabid facilitated TLR-induced histone modifications at the promoters of Il12 and Il23, which involved deubiqutination and stabilization of the histone demethylase Jmjd2d. Our findings highlight an epigenetic mechanism for the regulation of Il12 and Il23 and establish Trabid as an innate immunological regulator of inflammatory T cell responses.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Encefalomielite Autoimune Experimental/genética , Epigênese Genética , Interleucina-12/genética , Interleucina-23/genética , Proteases Específicas de Ubiquitina/genética , Animais , Diferenciação Celular , Imunoprecipitação da Cromatina , Encefalomielite Autoimune Experimental/imunologia , Citometria de Fluxo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Immunoblotting , Imunoprecipitação , Interleucina-12/imunologia , Interleucina-23/imunologia , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Camundongos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Receptores Toll-Like/metabolismo , Proteases Específicas de Ubiquitina/imunologia , Dedos de Zinco/genética , Dedos de Zinco/imunologia
7.
Mol Cell ; 79(2): 304-319.e7, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32679077

RESUMO

Accurate regulation of innate immunity is necessary for the host to efficiently respond to invading pathogens and avoid excessive harmful immune pathology. Here we identified OTUD3 as an acetylation-dependent deubiquitinase that restricts innate antiviral immune signaling. OTUD3 deficiency in mice results in enhanced innate immunity, a diminished viral load, and morbidity. OTUD3 directly hydrolyzes lysine 63 (Lys63)-linked polyubiquitination of MAVS and thus shuts off innate antiviral immune response. Notably, the catalytic activity of OTUD3 relies on acetylation of its Lys129 residue. In response to virus infection, the acetylated Lys129 is removed by SIRT1, which promptly inactivates OTUD3 and thus allows timely induction of innate antiviral immunity. Importantly, acetyl-OTUD3 levels are inversely correlated with IFN-ß expression in influenza patients. These findings establish OTUD3 as a repressor of MAVS and uncover a previously unknown regulatory mechanism by which the catalytic activity of OTUD3 is tightly controlled to ensure timely activation of antiviral defense.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Imunidade Inata , Influenza Humana/imunologia , Proteases Específicas de Ubiquitina/fisiologia , Células A549 , Acetilação , Adulto , Animais , Enzimas Desubiquitinantes/metabolismo , Feminino , Células HEK293 , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Ubiquitinação
8.
Mol Cell ; 75(3): 483-497.e9, 2019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31253574

RESUMO

In mammals, ∼100 deubiquitinases act on ∼20,000 intracellular ubiquitination sites. Deubiquitinases are commonly regarded as constitutively active, with limited regulatory and targeting capacity. The BRCA1-A and BRISC complexes serve in DNA double-strand break repair and immune signaling and contain the lysine-63 linkage-specific BRCC36 subunit that is functionalized by scaffold subunits ABRAXAS and ABRO1, respectively. The molecular basis underlying BRCA1-A and BRISC function is currently unknown. Here we show that in the BRCA1-A complex structure, ABRAXAS integrates the DNA repair protein RAP80 and provides a high-affinity binding site that sequesters the tumor suppressor BRCA1 away from the break site. In the BRISC structure, ABRO1 binds SHMT2α, a metabolic enzyme enabling cancer growth in hypoxic environments, which we find prevents BRCC36 from binding and cleaving ubiquitin chains. Our work explains modularity in the BRCC36 DUB family, with different adaptor subunits conferring diversified targeting and regulatory functions.


Assuntos
Proteína BRCA1/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Enzimas Desubiquitinantes/genética , Chaperonas de Histonas/genética , Neoplasias/genética , Sítios de Ligação/genética , Proteínas de Transporte/genética , Núcleo Celular/genética , Núcleo Celular/imunologia , Citoplasma/genética , Citoplasma/imunologia , Quebras de DNA de Cadeia Dupla , Reparo do DNA/imunologia , Enzimas Desubiquitinantes/imunologia , Células HeLa , Humanos , Imunidade Celular/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Neoplasias/imunologia , Proteínas Associadas à Matriz Nuclear/genética , Ligação Proteica/genética , Ubiquitina/genética , Proteases Específicas de Ubiquitina/genética , Ubiquitinação/genética
9.
Proc Natl Acad Sci U S A ; 121(33): e2404883121, 2024 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-39102535

RESUMO

Transcription factor ELONGATED HYPOCOTYL5 (HY5) is the central hub for seedling photomorphogenesis. E3 ubiquitin (Ub) ligase CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) inhibits HY5 protein accumulation through ubiquitination. However, the process of HY5 deubiquitination, which antagonizes E3 ligase-mediated ubiquitination to maintain HY5 homeostasis has never been studied. Here, we identified that Arabidopsis thaliana deubiquitinating enzyme, Ub-SPECIFIC PROTEASE 14 (UBP14) physically interacts with HY5 and enhances its protein stability by deubiquitination. The da3-1 mutant lacking UBP14 function exhibited a long hypocotyl phenotype, and UBP14 deficiency led to the failure of rapid accumulation of HY5 during dark to light. In addition, UBP14 preferred to stabilize nonphosphorylated form of HY5 which is more readily bound to downstream target genes. HY5 promoted the expression and protein accumulation of UBP14 for positive feedback to facilitate photomorphogenesis. Our findings thus established a mechanism by which UBP14 stabilizes HY5 protein by deubiquitination to promote photomorphogenesis in A. thaliana.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Fatores de Transcrição de Zíper de Leucina Básica , Regulação da Expressão Gênica de Plantas , Ubiquitinação , Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Proteases Específicas de Ubiquitina/metabolismo , Proteases Específicas de Ubiquitina/genética , Estabilidade Proteica/efeitos da radiação , Luz , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Hipocótilo/crescimento & desenvolvimento , Hipocótilo/metabolismo , Hipocótilo/genética
10.
Proc Natl Acad Sci U S A ; 121(34): e2315759121, 2024 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-39145935

RESUMO

Ubiquitination status of proliferating cell nuclear antigen (PCNA) is crucial for regulating DNA lesion bypass. After the resolution of fork stalling, PCNA is subsequently deubiquitinated, but the underlying mechanism remains undefined. We found that the N-terminal domain of ATAD5 (ATAD5-N), the largest subunit of the PCNA-unloading complex, functions as a scaffold for Ub-PCNA deubiquitination. ATAD5 recognizes DNA-loaded Ub-PCNA through distinct DNA-binding and PCNA-binding motifs. Furthermore, ATAD5 forms a heterotrimeric complex with UAF1-USP1 deubiquitinase, facilitating the deubiquitination of DNA-loaded Ub-PCNA. ATAD5 also enhances the Ub-PCNA deubiquitination by USP7 and USP11 through specific interactions. ATAD5 promotes the distinct deubiquitination process of UAF1-USP1, USP7, and USP11 for poly-Ub-PCNA. Additionally, ATAD5 mutants deficient in UAF1-binding had increased sensitivity to DNA-damaging agents. Our results ultimately reveal that ATAD5 and USPs cooperate to efficiently deubiquitinate Ub-PCNA prior to its release from the DNA in order to safely deactivate the DNA repair process.


Assuntos
ATPases Associadas a Diversas Atividades Celulares , Proteínas de Ligação a DNA , Antígeno Nuclear de Célula em Proliferação , Ubiquitina Tiolesterase , Peptidase 7 Específica de Ubiquitina , Ubiquitinação , ATPases Associadas a Diversas Atividades Celulares/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Humanos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Ubiquitina Tiolesterase/metabolismo , Ubiquitina Tiolesterase/genética , Peptidase 7 Específica de Ubiquitina/metabolismo , Peptidase 7 Específica de Ubiquitina/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Tioléster Hidrolases/metabolismo , Tioléster Hidrolases/genética , Ubiquitina/metabolismo , Dano ao DNA , Ligação Proteica , Proteases Específicas de Ubiquitina
11.
PLoS Pathog ; 20(1): e1011943, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38215174

RESUMO

Deubiquitinases (DUBs) remove ubiquitin from substrates and play crucial roles in diverse biological processes. However, our understanding of deubiquitination in viral replication remains limited. Employing an oncogenic human herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) to probe the role of protein deubiquitination, we found that Ovarian tumor family deubiquitinase 4 (OTUD4) promotes KSHV reactivation. OTUD4 interacts with the replication and transcription activator (K-RTA), a key transcription factor that controls KSHV reactivation, and enhances K-RTA stability by promoting its deubiquitination. Notably, the DUB activity of OTUD4 is not required for K-RTA stabilization; instead, OTUD4 functions as an adaptor protein to recruit another DUB, USP7, to deubiquitinate K-RTA and facilitate KSHV lytic reactivation. Our study has revealed a novel mechanism whereby KSHV hijacks OTUD4-USP7 deubiquitinases to promote lytic reactivation, which could be potentially harnessed for the development of new antiviral therapies.


Assuntos
Herpesvirus Humano 8 , Proteínas Imediatamente Precoces , Sarcoma de Kaposi , Humanos , Proteínas Imediatamente Precoces/metabolismo , Peptidase 7 Específica de Ubiquitina/genética , Peptidase 7 Específica de Ubiquitina/metabolismo , Transativadores/genética , Herpesvirus Humano 8/genética , Replicação Viral , Regulação Viral da Expressão Gênica , Ativação Viral , Proteases Específicas de Ubiquitina/metabolismo
12.
Nat Immunol ; 15(6): 562-70, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24777531

RESUMO

Deubiquitinases (DUBs) are a new class of drug targets, although the physiological function of only few DUBs has been characterized. Here we identified the DUB USP15 as a crucial negative regulator of T cell activation. USP15 stabilized the E3 ubiquitin ligase MDM2, which in turn negatively regulated T cell activation by targeting the degradation of the transcription factor NFATc2. USP15 deficiency promoted T cell activation in vitro and enhanced T cell responses to bacterial infection and tumor challenge in vivo. USP15 also stabilized MDM2 in cancer cells and regulated p53 function and cancer-cell survival. Our results suggest that inhibition of USP15 may both induce tumor cell apoptosis and boost antitumor T cell responses.


Assuntos
Fatores de Transcrição NFATC/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/imunologia , Células Th1/imunologia , Proteases Específicas de Ubiquitina/imunologia , Transferência Adotiva , Animais , Apoptose/imunologia , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Sobrevivência Celular , Células HCT116 , Humanos , Leupeptinas/farmacologia , Listeria monocytogenes/imunologia , Listeriose/imunologia , Ativação Linfocitária/imunologia , Melanoma Experimental/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas c-mdm2/genética , Evasão Tumoral , Proteína Supressora de Tumor p53/imunologia , Proteases Específicas de Ubiquitina/genética , Ubiquitinação/genética , Ubiquitinação/imunologia
13.
PLoS Biol ; 21(12): e3002446, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38134227

RESUMO

Tumor metastasis is the major cause of breast cancer morbidity and mortality. It has been reported that the F-box protein FBXO3 functions as an E3 ubiquitin ligase in regulating various biological processes, including host autoimmune, antiviral innate immunity, and inflammatory response. However, the role of FBXO3 in tumor metastasis remains elusive. We have previously shown that ΔNp63α is a common inhibitory target in oncogene-induced cell motility and tumor metastasis. In this study, we show that FBXO3 plays a vital role in PI3K-mediated breast cancer metastasis independent of its E3 ligase activity and ΔNp63α in breast cancer cells and in mouse. FBXO3 can bind to and stabilize USP4, leading to Twist1 protein stabilization and increased breast cancer cell migration and tumor metastasis. Mechanistically, FBXO3 disrupts the interaction between USP4 and aspartyl aminopeptidase (DNPEP), thereby protecting USP4 from DNPEP-mediated degradation. Furthermore, p110αH1047R facilitates the phosphorylation and stabilization of FBXO3 in an ERK1-dependent manner. Knockdown of either FBXO3 or USP4 leads to significant inhibition of PI3K-induced breast cancer metastasis. Clinically, elevated expression of p110α/FBXO3/USP4/Twist1 is associated with poor overall survival (OS) and recurrence-free survival (RFS) of breast cancer patients. Taken together, this study reveals that the FBXO3-USP4-Twist1 axis is pivotal in PI3K-mediated breast tumor metastasis and that FBXO3/USP4 may be potential therapeutic targets for breast cancer treatment.


Assuntos
Neoplasias da Mama , Melanoma , Neoplasias Cutâneas , Animais , Feminino , Humanos , Camundongos , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Fosfatidilinositol 3-Quinases/metabolismo , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Ubiquitinação
14.
J Immunol ; 212(2): 295-301, 2024 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-38054892

RESUMO

Cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) detects cytoplasmic microbial DNA and self-DNA from genomic instability, initiates innate immunity, and plays fundamental roles in defense against viruses and the development of various diseases. The cellular cGAS level determines the magnitude of the response to DNA. However, the underlying mechanisms of the control of cGAS stability, especially its feedback regulation during viral infection, remain largely unknown. In this study, we show that viral infection induces the expression of the UAF1-USP1 deubiquitinase complex in primary peritoneal macrophages (PMs) of C57BL/6J mice. UAF1-USP interacts with cGAS, selectively cleaves its K48-linked polyubiquitination, and thus stabilizes its protein expression in PMs and HEK293T cells. Concordantly, the UAF1-USP1 deubiquitinase complex enhances cGAS-dependent type I IFN responses in PMs. Uaf1 deficiency and ML323 (a specific inhibitor of UAF1-USP1 deubiquitinase complex) attenuates cGAS-triggered antiviral responses and facilitates viral replication both in vitro and in vivo. Thus, our study uncovers a positive feedback mechanism of cGAS-dependent antiviral responses and suggests the UAF1-USP1 complex as a potential target for the treatment of diseases caused by aberrant cGAS activation.


Assuntos
Proteases Específicas de Ubiquitina , Viroses , Animais , Humanos , Camundongos , Antivirais , DNA , Células HEK293 , Camundongos Endogâmicos C57BL , Proteínas Nucleares/genética , Nucleotidiltransferases/genética , Proteases Específicas de Ubiquitina/metabolismo
15.
Cell ; 147(1): 223-34, 2011 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-21962518

RESUMO

Autophagy is an important intracellular catabolic mechanism that mediates the degradation of cytoplasmic proteins and organelles. We report a potent small molecule inhibitor of autophagy named "spautin-1" for specific and potent autophagy inhibitor-1. Spautin-1 promotes the degradation of Vps34 PI3 kinase complexes by inhibiting two ubiquitin-specific peptidases, USP10 and USP13, that target the Beclin1 subunit of Vps34 complexes. Beclin1 is a tumor suppressor and frequently monoallelically lost in human cancers. Interestingly, Beclin1 also controls the protein stabilities of USP10 and USP13 by regulating their deubiquitinating activities. Since USP10 mediates the deubiquitination of p53, regulating deubiquitination activity of USP10 and USP13 by Beclin1 provides a mechanism for Beclin1 to control the levels of p53. Our study provides a molecular mechanism involving protein deubiquitination that connects two important tumor suppressors, p53 and Beclin1, and a potent small molecule inhibitor of autophagy as a possible lead compound for developing anticancer drugs.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Benzilaminas/farmacologia , Endopeptidases/metabolismo , Quinazolinas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina Tiolesterase/metabolismo , Animais , Autofagia , Proteína Beclina-1 , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Humanos , Camundongos , Proteases Específicas de Ubiquitina , Ubiquitinação
16.
Cell ; 146(6): 918-30, 2011 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-21925315

RESUMO

Inhibitors of DNA binding (IDs) antagonize basic-helix-loop-helix (bHLH) transcription factors to inhibit differentiation and maintain stem cell fate. ID ubiquitination and proteasomal degradation occur in differentiated tissues, but IDs in many neoplasms appear to escape degradation. We show that the deubiquitinating enzyme USP1 promotes ID protein stability and stem cell-like characteristics in osteosarcoma. USP1 bound, deubiquitinated, and thereby stabilized ID1, ID2, and ID3. A subset of primary human osteosarcomas coordinately overexpressed USP1 and ID proteins. USP1 knockdown in osteosarcoma cells precipitated ID protein destabilization, cell-cycle arrest, and osteogenic differentiation. Conversely, ectopic USP1 expression in mesenchymal stem cells stabilized ID proteins, inhibited osteoblastic differentiation, and enhanced proliferation. Consistent with USP1 functioning in normal mesenchymal stem cells, USP1-deficient mice were osteopenic. Our observations implicate USP1 in preservation of the stem cell state that characterizes osteosarcoma and identify USP1 as a target for differentiation therapy.


Assuntos
Endopeptidases/metabolismo , Proteínas Inibidoras de Diferenciação/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Neoplásicas/citologia , Osteossarcoma/patologia , Animais , Proteínas de Arabidopsis , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Proteases Específicas de Ubiquitina , Ubiquitinação
17.
Mol Cell ; 70(4): 602-613.e3, 2018 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-29775578

RESUMO

The proteolysis-assisted protein quality control system guards the proteome from potentially detrimental aberrant proteins. How miscellaneous defective proteins are specifically eliminated and which molecular characteristics direct them for removal are fundamental questions. We reveal a mechanism, DesCEND (destruction via C-end degrons), by which CRL2 ubiquitin ligase uses interchangeable substrate receptors to recognize the unusual C termini of abnormal proteins (i.e., C-end degrons). C-end degrons are mostly less than ten residues in length and comprise a few indispensable residues along with some rather degenerate ones. The C-terminal end position is essential for C-end degron function. Truncated selenoproteins generated by translation errors and the USP1 N-terminal fragment from post-translational cleavage are eliminated by DesCEND. DesCEND also targets full-length proteins with naturally occurring C-end degrons. The C-end degron in DesCEND echoes the N-end degron in the N-end rule pathway, highlighting the dominance of protein "ends" as indicators for protein elimination.


Assuntos
Processamento de Proteína Pós-Traducional , Receptores de Citocinas/metabolismo , Selenoproteínas/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Ubiquitina/metabolismo , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Domínios Proteicos , Proteólise , Receptores de Citocinas/genética , Proteases Específicas de Ubiquitina/genética
18.
Mol Cell ; 72(6): 925-941.e4, 2018 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-30576655

RESUMO

BRCA1-deficient tumor cells have defects in homologous-recombination repair and replication fork stability, resulting in PARP inhibitor sensitivity. Here, we demonstrate that a deubiquitinase, USP1, is upregulated in tumors with mutations in BRCA1. Knockdown or inhibition of USP1 resulted in replication fork destabilization and decreased viability of BRCA1-deficient cells, revealing a synthetic lethal relationship. USP1 binds to and is stimulated by fork DNA. A truncated form of USP1, lacking its DNA-binding region, was not stimulated by DNA and failed to localize and protect replication forks. Persistence of monoubiquitinated PCNA at the replication fork was the mechanism of cell death in the absence of USP1. Taken together, USP1 exhibits DNA-mediated activation at the replication fork, protects the fork, and promotes survival in BRCA1-deficient cells. Inhibition of USP1 may be a useful treatment for a subset of PARP-inhibitor-resistant BRCA1-deficient tumors with acquired replication fork stabilization.


Assuntos
Proteína BRCA1/deficiência , Neoplasias da Mama/enzimologia , Replicação do DNA , DNA de Neoplasias/biossíntese , Proteases Específicas de Ubiquitina/metabolismo , Neoplasias do Colo do Útero/enzimologia , Animais , Proteína BRCA1/genética , Sítios de Ligação , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proliferação de Células , Sobrevivência Celular , DNA de Neoplasias/genética , Resistência a Medicamentos , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Camundongos Nus , Mutação , Desnaturação de Ácido Nucleico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ligação Proteica , Proteases Específicas de Ubiquitina/antagonistas & inibidores , Proteases Específicas de Ubiquitina/genética , Ubiquitinação , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Mol Cell ; 72(5): 813-822.e4, 2018 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-30526872

RESUMO

Aberrant proteins can be deleterious to cells and are cleared by the ubiquitin-proteasome system. A group of C-end degrons that are recognized by specific cullin-RING ubiquitin E3 ligases (CRLs) has recently been identified in some of these abnormal polypeptides. Here, we report three crystal structures of a CRL2 substrate receptor, KLHDC2, in complex with the diglycine-ending C-end degrons of two early-terminated selenoproteins and the N-terminal proteolytic fragment of USP1. The E3 recognizes the degron peptides in a similarly coiled conformation and cradles their C-terminal diglycine with a deep surface pocket. By hydrogen bonding with multiple backbone carbonyls of the peptides, KLHDC2 further locks in the otherwise degenerate degrons with a compact interface and unexpected high affinities. Our results reveal the structural mechanism by which KLHDC2 recognizes the simplest C-end degron and suggest a functional necessity of the E3 to tightly maintain the low abundance of its select substrates.


Assuntos
Antígenos de Neoplasias/química , Glicilglicina/química , Selenoproteínas/química , Proteases Específicas de Ubiquitina/química , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Baculoviridae/genética , Baculoviridae/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glicilglicina/metabolismo , Células HEK293 , Humanos , Cinética , Simulação de Acoplamento Molecular , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Selenoproteínas/genética , Selenoproteínas/metabolismo , Spodoptera , Especificidade por Substrato , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismo
20.
Mol Cell ; 69(3): 505-516.e5, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29395066

RESUMO

Ubiquitination is a major mechanism that regulates numerous cellular processes, including autophagy, DNA damage signaling, and inflammation. While hundreds of ubiquitin ligases exist to conjugate ubiquitin onto substrates, approximately 100 deubiquitinases are encoded by the human genome. Thus, deubiquitinases are likely regulated by unidentified mechanisms to target distinct substrates and cellular functions. Here, we demonstrate that the deubiquitinase OTUD4, which nominally encodes a K48-specific deubiquitinase, is phosphorylated near its catalytic domain, activating a latent K63-specific deubiquitinase. Besides phosphorylation, this latter activity requires an adjacent ubiquitin-interacting motif, which increases the affinity of OTUD4 for K63-linked chains. We reveal the Toll-like receptor (TLR)-associated factor MyD88 as a target of this K63 deubiquitinase activity. Consequently, TLR-mediated activation of NF-κB is negatively regulated by OTUD4, and macrophages from Otud4-/- mice exhibit increased inflammatory signaling upon TLR stimulation. Our results reveal insights into how a deubiquitinase may modulate diverse processes through post-translational modification.


Assuntos
Fator 88 de Diferenciação Mieloide/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Animais , Enzimas Desubiquitinantes/genética , Enzimas Desubiquitinantes/metabolismo , Células HEK293 , Humanos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Proteólise , Transdução de Sinais , Receptores Toll-Like , Ubiquitina/metabolismo , Ubiquitinação
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