RESUMO
Deubiquitinating enzymes (DUBs) are important regulators of ubiquitin signaling. Here, we report the discovery of deubiquitinating activity in ZUFSP/C6orf113. High-resolution crystal structures of ZUFSP in complex with ubiquitin reveal several distinctive features of ubiquitin recognition and catalysis. Our analyses reveal that ZUFSP is a novel DUB with no homology to any known DUBs, leading us to classify ZUFSP as the seventh DUB family. Intriguingly, the minimal catalytic domain does not cleave polyubiquitin. We identify two ubiquitin binding domains in ZUFSP: a ZHA (ZUFSP helical arm) that binds to the distal ubiquitin and an atypical UBZ domain in ZUFSP that binds to polyubiquitin. Importantly, both domains are essential for ZUFSP to selectively cleave K63-linked polyubiquitin. We show that ZUFSP localizes to DNA lesions, where it plays an important role in genome stability pathways, functioning to prevent spontaneous DNA damage and also promote cellular survival in response to exogenous DNA damage.
Assuntos
Núcleo Celular/enzimologia , Dano ao DNA , Enzimas Desubiquitinantes/metabolismo , Instabilidade Genômica , Poliubiquitina/metabolismo , Sítios de Ligação , Sobrevivência Celular , Enzimas Desubiquitinantes/química , Enzimas Desubiquitinantes/genética , Células HEK293 , Células HeLa , Humanos , Células Jurkat , Lisina , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-Atividade , Especificidade por Substrato , UbiquitinaçãoRESUMO
Protein ubiquitylation is a dynamic post-translational modification that can be reversed by deubiquitylating enzymes (DUBs). It is unclear how the small number (â¼100) of DUBs present in mammalian cells regulate the thousands of different ubiquitylation events. Here, we analysed annotated transcripts of human DUBs and found â¼300 ribosome-associated transcripts annotated as protein coding, which thus increases the total number of DUBs. By using USP35, a poorly studied DUB, as a case study, we provide evidence that alternative isoforms contribute to the functional expansion of DUBs. We show that there are two different USP35 isoforms that localise to different intracellular compartments and have distinct functions. Our results reveal that isoform 1 is an anti-apoptotic factor that inhibits staurosporine- and TNF-related apoptosis-inducing ligand (TRAIL; also known as TNFSF10)-induced apoptosis. In contrast, USP35 isoform 2 is an integral membrane protein of the endoplasmic reticulum (ER) that is also present at lipid droplets. Manipulations of isoform 2 levels cause rapid ER stress, likely through deregulation of lipid homeostasis, and lead to cell death. Our work highlights how alternative isoforms provide functional expansion of DUBs and sets directions for future research.This article has an associated First Person interview with the first author of the paper.
Assuntos
Endopeptidases/metabolismo , Isoformas de Proteínas/metabolismo , Ubiquitina Tiolesterase/metabolismo , Apoptose , Endopeptidases/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Células HeLa , Humanos , Isoformas de Proteínas/genética , Transporte Proteico , Ubiquitina/metabolismo , Ubiquitina Tiolesterase/genética , UbiquitinaçãoRESUMO
Glargine is an analog of Insulin currently being produced by recombinant DNA technology using two different hosts namely Escherichia coli and Pichia pastoris. Production from E. coli involves the steps of extraction of inclusion bodies by cell lysis, refolding, proteolytic cleavage and purification. In P. pastoris, a single-chain precursor with appropriate disulfide bonding is secreted to the medium. Downstream processing currently involves use of trypsin which converts the precursor into two-chain final product. The use of trypsin in the process generates additional impurities due to presence of Lys and Arg residues in the Glargine molecule. In this study, we describe an alternate approach involving over-expression of endogenous Kex2 proprotein convertase, taking advantage of dibasic amino acid sequence (Arg-Arg) at the end of B-chain of Glargine. KEX2 gene over-expression in Pichia was accomplished by using promoters of varying strengths to ensure production of greater levels of fully functional two-chain Glargine product, confirmed by HPLC and mass analysis. In conclusion, this new production process involving Kex2 protease over-expression improves the downstream process efficiency, reduces the levels of impurities generated and decreases the use of raw materials.
Assuntos
Biotecnologia/métodos , Expressão Gênica , Insulina de Ação Prolongada/metabolismo , Peptídeo Hidrolases/metabolismo , Pichia/metabolismo , Tecnologia Farmacêutica/métodos , Cromatografia Líquida de Alta Pressão , Insulina Glargina , Insulina de Ação Prolongada/genética , Insulina de Ação Prolongada/isolamento & purificação , Espectrometria de Massas , Peptídeo Hidrolases/genética , Pichia/genética , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
BACKGROUND: Placental dysfunction is one of the main causes of preeclampsia and hypertensive disorders of pregnancy. MATERIAL AND METHODS: This is a prospective comparative study done on 30 pregnant women with pre-eclampsia and another 30 pregnant women as controls. In all these subjects the elasticity of the placenta was measured. RESULTS: The results obtained showed that there was a significant difference in SWV values between the two groups (p value = 0.001). The mean SWV value of normal pregnant women was 0.99 m/ second as opposed to 1.99 m/second in pre-eclamptic pregnant women. CONCLUSION: Sonoelastography is a promising noninvasive tool in the detection of preeclampsia with high diagnostic accuracy. The SWV values measured at the central zones of the placenta with a cut-off value of 1.325 m/s identify the presence of pre-eclampsia with high sensitivity and specificity.