RESUMO
Deubiquitinases are proteases with a wide functional diversity that profoundly impact multiple biological processes. Among them, the ubiquitin-specific protease 36 (USP36) has been implicated in the regulation of nucleolar activity. However, its functional relevance in vivo has not yet been fully described. Here, we report the generation of an Usp36-deficient mouse model to examine the function of this enzyme. We show that Usp36 depletion is lethal in preimplantation mouse embryos, where it blocks the transition from morula to blastocyst during embryonic development. USP36 reduces the ubiquitination levels and increases the stability of the DEAH-box RNA helicase DHX33, which is critically involved in ribosomal RNA synthesis and mRNA translation. In agreement with this finding, O-propargyl-puromycin incorporation experiments, Northern blot, and electron microscopy analyses demonstrated the role of USP36 in ribosomal RNA and protein synthesis. Finally, we show that USP36 down-regulation alters cell proliferation in human cancer cells by inducing both apoptosis and cell cycle arrest, and that reducing DHX33 levels through short hairpin RNA interference has the same effect. Collectively, these results support that Usp36 is essential for cell and organism viability because of its role in ribosomal RNA processing and protein synthesis, which is mediated, at least in part, by regulating DHX33 stability.
Assuntos
Blastocisto , RNA Helicases DEAD-box/química , Enzimas Desubiquitinantes/fisiologia , RNA Helicases/química , Ubiquitina Tiolesterase/fisiologia , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Enzimas Desubiquitinantes/genética , Perda do Embrião , Humanos , Camundongos , Camundongos Knockout , Biossíntese de Proteínas , Estabilidade Proteica , RNA Ribossômico , Ubiquitina Tiolesterase/genéticaRESUMO
KRAS is the most frequently mutated oncogene in human cancer, but its therapeutic targeting remains challenging. Here, we report a synthetic lethal screen with a library of deubiquitinases and identify USP39, which encodes an essential splicing factor, as a critical gene for the viability of KRAS-dependent cells. We show that splicing fidelity inhibitors decrease preferentially the proliferation rate of KRAS-active cells. Moreover, depletion of DHX38, encoding an USP39-interacting splicing factor, also reduces the viability of these cells. In agreement with these results, USP39 depletion caused a significant reduction in pre-mRNA splicing efficiency, as demonstrated through RNA-seq experiments. Furthermore, we show that USP39 is up-regulated in lung and colon carcinomas and its expression correlates with KRAS levels and poor clinical outcome. Accordingly, our work provides critical information for the development of splicing-directed antitumor treatments and supports the potential of USP39-targeting strategies as the basis of new anticancer therapies.
Assuntos
Neoplasias do Colo/patologia , Neoplasias Pulmonares/patologia , Mutação/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteases Específicas de Ubiquitina/metabolismo , Animais , Apoptose , Western Blotting , Proliferação de Células , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Nus , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida , Células Tumorais Cultivadas , Proteases Específicas de Ubiquitina/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The gut microbiota is a complex community of different microbial species that influence many aspects of health. Consequently, shifts in the composition of gut microbiome have been proposed to exert negative effects on the host physiology, leading to the pathogenesis of various age-related disorders, including cardiovascular and neurological diseases, type 2 diabetes, obesity, non-alcoholic liver disease, and other pathological conditions. Thus, understanding how the gut microbiota influences the aging-related decline is particularly topical. Advances in next-generation sequencing techniques, together with mechanistic experiments in animal models, have provided substantial improvements in microbiome analysis. However, standardization and best practices are needed to limit experimental variation between different studies. Here, we detail a simple method for microbiota composition analysis in mouse fecal samples using 16S rRNA next-generation sequencing.
Assuntos
Diabetes Mellitus Tipo 2 , Microbioma Gastrointestinal , Microbiota , Camundongos , Animais , RNA Ribossômico 16S/genética , Microbiota/genética , Fezes , Microbioma Gastrointestinal/genéticaRESUMO
ADAM29 (a disintegrin and metalloprotease domain 29) is a member of the membrane-anchored ADAM family of proteins, which is highly expressed in testis and may mediate different physiological and pathological processes. Although the functions of many ADAM family members have been well characterized, the biological relevance of ADAM29 has remained largely unknown. Here, we report the generation of an Adam29-deficient mouse model to delve deeper into the in vivo functions of this ADAM family member. We show that ADAM29 depletion does not affect mice viability, development, or fertility, but somehow impinges on metabolism and energy expenditure. We also report herein that ADAM29 deficiency leads to an accelerated wound healing process, without affecting cell reprogramming in mouse-derived fibroblasts. Collectively, our findings provide new insights into ADAM29 biological functions, highlighting the importance of non-catalytic ADAM proteases.
RESUMO
The spindle assembly checkpoint (SAC) is an essential mechanism that ensures the accurate chromosome segregation during mitosis, thus preventing genomic instability. Deubiquitinases have emerged as key regulators of the SAC, mainly by determining the fate of proteins during cell cycle progression. Here, we identify USP49 deubiquitinase as a novel regulator of the spindle checkpoint. We show that loss of USP49 in different cancer cell lines impairs proliferation and increases aneuploidy. In addition, USP49-depleted cells overcome the arrest induced by the SAC in the presence of nocodazole. Finally, we report new binding partners of USP49, including ribophorin 1, USP44, and different centrins.
Assuntos
Pontos de Checagem da Fase M do Ciclo Celular , Fuso Acromático , Humanos , Fuso Acromático/metabolismo , Aneuploidia , Mitose , Enzimas Desubiquitinantes/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismoRESUMO
Giant tortoises are among the longest-lived vertebrate animals and, as such, provide an excellent model to study traits like longevity and age-related diseases. However, genomic and molecular evolutionary information on giant tortoises is scarce. Here, we describe a global analysis of the genomes of Lonesome George-the iconic last member of Chelonoidis abingdonii-and the Aldabra giant tortoise (Aldabrachelys gigantea). Comparison of these genomes with those of related species, using both unsupervised and supervised analyses, led us to detect lineage-specific variants affecting DNA repair genes, inflammatory mediators and genes related to cancer development. Our study also hints at specific evolutionary strategies linked to increased lifespan, and expands our understanding of the genomic determinants of ageing. These new genome sequences also provide important resources to help the efforts for restoration of giant tortoise populations.
Assuntos
Envelhecimento/genética , Genoma , Tartarugas/genética , Animais , Reparo do DNA/genética , Evolução Molecular , Células HEK293 , Humanos , Mediadores da Inflamação , Masculino , Neoplasias/genética , Filogenia , Densidade DemográficaRESUMO
Proteases play key roles in the execution and regulation of most if not all biological functions, and alterations in their activity, expression, or location are associated with multiple pathological conditions, including cancer and aging. In this regard, the use of RNA interference-based approaches to specifically target the expression of individual proteases constitutes an invaluable tool to identify enzymes involved in central aspects of these processes and to explore their potential as targets of therapeutic interventions. Here we describe simple protocols to optimize and monitor the specific silencing of cancer- and aging-related proteases.
Assuntos
Envelhecimento/metabolismo , Proteínas de Membrana/genética , Metaloendopeptidases/genética , Neoplasias/patologia , Interferência de RNA , Proteases Específicas de Ubiquitina/genética , Envelhecimento/genética , Apoptose/genética , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes/instrumentação , Técnicas de Silenciamento de Genes/métodos , Vetores Genéticos/genética , Humanos , Lentivirus/genética , Proteínas de Membrana/metabolismo , Metaloendopeptidases/metabolismo , Neoplasias/genética , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteases Específicas de Ubiquitina/metabolismoRESUMO
Ubiquitin-Specific Proteases (USPs) are deubiquitinating enzymes frequently deregulated in human malignancies. Here, we show that USP54 is overexpressed in intestinal stem cells and demonstrate that its downregulation in colorectal carcinoma cells impedes tumorigenesis. We have generated mutant mice deficient for this deubiquitinase, which are viable and fertile, and protected against chemically-induced colorectal carcinoma. Furthermore, we show that USP54 is upregulated in human colon cancer and associates with poor prognosis. In agreement with these results, Usp54 downregulation in mouse melanoma cells inhibits lung metastasis formation. Collectively, this work has uncovered the pro-tumorigenic properties of USP54, highlighting the importance of deubiquitinating enzymes as promising targets for the development of specific anti-cancer therapies.