Regulating the stability and localization of CDK inhibitor p27Kip1 via CSN6-COP1 axis

The COP9 signalosome subunit 6 (CSN6), which is involved in ubiquitin-mediated protein degradation, is overexpressed in many types of cancer. CSN6 is critical in causing p53 degradation and malignancy, but its target in cell cycle progression is not fully characterized. Constitutive photomorphogenic 1 (COP1) is an E3 ubiquitin ligase associating with COP9 signalosome to regulate important target proteins for cell growth. p27 is a critical G1 CDK inhibitor involved in cell cycle regulation, but its upstream regulators are not fully characterized. Here, we show that the CSN6-COP1 link is regulating p27^Kip1 stability, and that COP1 is a negative regulator of p27^Kip1. Ectopic expression of CSN6 can decrease the expression of p27^Kip1, while CSN6 knockdown leads to p27^Kip1 stabilization. Mechanistic studies show that CSN6 interacts with p27^Kip1 and facilitates ubiquitin-mediated degradation of p27^Kip1. CSN6-mediated p27 degradation depends on the nuclear export of p27^Kip1, which is regulated through COP1 nuclear exporting signal. COP1 overexpression leads to the cytoplasmic distribution of p27, thereby accelerating p27 degradation. Importantly, the negative impact of COP1 on p27 stability contributes to elevating expression of genes that are suppressed through p27 mediation. Kaplan-Meier analysis of tumor samples demonstrates that high COP1 expression was associated with poor overall survival. These data suggest that tumors with CSN6/COP1 deregulation may have growth advantage by regulating p27 degradation and subsequent impact on p27 targeted genes.     

新生隐球菌COP9复合体基因CSN6的敲除与鉴定

目的构建新生隐球菌COP9复合体蛋白元件Csn6的基因同源重组敲除框,并通过基因枪转化系统敲除CSN6基因。方法应用生物信息学方法获得COP9复合体蛋白元件的基因信息,采用套叠PCR的方法,构建包含报告基因NEO和CSN6基因ORF两侧上下游同源DNA片段的同源重组框。应用基因枪将其转化入新生隐球菌感受态细胞,通过PCR和DNA测序对遗传霉素(G418)耐受的阳性克隆子进行筛选与验证。结果成功构建了新生隐球菌基因突变株csn6裣。结论 COP9复合体亚基CSN6基因突变株的构建,为今后新生隐球菌COP9复合体的分子致病机制研究奠定基础。     

Crystal structure of the human CSN6 MPN domain

The mammalian COP9 signalosome is an eight-subunit (CSN1–CSN8) complex that plays essential roles in multiple cellular and physiological processes. CSN5 and CSN6 are the only two MPN (Mpr1-Pad1-N-terminal) domain-containing subunits in the complex. Unlike the CSN5 MPN domain, CSN6 lacks a metal-binding site and isopeptidase activity. Here, we report the crystal structure of the human CSN6 MPN domain. Each CSN6 monomer contains nine β sheets surrounded by three helices. Two forms of dimers are observed in the crystal structure. Interestingly, a domain swapping of β8 and β9 strands occurs between two neighboring monomers to complete a typical MPN fold. Analyses of the pseudo metal-binding motif in CSN6 suggest that the loss of two key histidine residues may contribute to the lack of catalytic activity in CSN6. Comparing the MPN domain of our CSN6 with that in the CSN complex shows that apart from the different β8–β9 conformation, they have minor conformational differences at two insertion regions (Ins-1 and Ins-2). Besides, the interacting mode of CSN6–CSN6 in our structure is distinct from that of CSN5–CSN6 in the CSN complex structure. Moreover, the functional implications for Ins-1 and Ins-2 are discussed.     

CSN7B defines a variant COP9 signalosome complex with distinct function in DNA damage response

1. Download : Download high-res image (144KB)
2. Download : Download full-size image

     

CSN5/Jab1 controls multiple events in the mammalian cell cycle

The COP9 signalosome (CSN) complex is critical for mammalian cell proliferation and survival, but it is not known how the CSN affects the cell cycle. In this study, MEFs lacking CSN5/Jab1 were generated using a CRE-flox system. MEFs ceased to proliferate upon elimination of CSN5/Jab1. Rescue experiments indicated that the JAMM domain of CSN5/Jab1 was essential. CSN5/Jab1-elimination enhanced the neddylation of cullins 1 and 4 and altered the expression of many factors including cyclin E and p53. CSN5/Jab1-elimination inhibited progression of the cell cycle at multiple points, seemed to initiate p53-independent senescence and increased the ploidy of cells. Thus, CSN5/Jab1 controls different events of the cell cycle, preventing senescence and endocycle as well as the proper progression of the somatic cell cycle.

Structured summary

MINT-8046253: Csn1 (uniprotkb:Q99LD4) physically interacts (MI:0914) with Csn5 (uniprotkb:O35864), Csn8 (uniprotkb:Q8VBV7), Csn3 (uniprotkb:O88543), Csn7b (uniprotkb:Q8BV13) and Csn6 (uniprotkb:O88545) by anti bait coimmunoprecipitation (MI:0006)     

Inhibition of neddylation induces mitotic defects and alters MKLP1 accumulation at the midbody during cytokinesis

The cullin-RING E3 ubiquitin ligases (CRLs) play crucial roles in modulating the stability of proteins in the cell and are, in turn, regulated by post-translational modification by the ubiquitin-like (Ubl) protein NEDD8. This process, termed neddylation, is reversible through the action of the COP9 signalosome (CSN); a multi-subunit metalloprotease conserved among eukaryotes that plays direct or indirect roles in DNA repair, cell signaling and cell cycle regulation in part through modulating the activity of the CRLs. Previously, inhibition of CRL neddylation by MLN4924, a small molecule inhibitor of the NEDD8-activating enzyme 1 (NAE1), was shown to induce interphase cell cycle arrest and cell death. Using fixed and living cell microscopy, we re-evaluated the cell cycle effects of inhibition of neddylation by MLN4924 in both asynchronous and mitotic cell populations. Consistent with previous studies, treatment of asynchronous cells with MLN4924 increased CDT1 expression levels, induced G2 arrest and increased nuclear size. However, in synchronized cells treated in mitosis, mitotic defects were observed including lagging chromosomes and binucleated daughter cells. Consistent with neddylation and deneddylation playing a role in cytokinesis, NEDD8, as well as subunits of the CSN, could be localized at the midbody and cleavage furrow. Finally, treatment of mitotic cells with MLN4924 induced the premature accumulation of MKLP1 at the cleavage furrow, a key regulator of cytokinesis, which was concomitant with increased abscission delay and failure. Thus, these studies uncover an uncharacterized mitotic effect of MLN4924 on MKLP1 accumulation at the midbody and support a role for neddylation during cytokinesis.

Abbreviations: CSN, COP9 Signalosome; MKLP1, mitotic kinesin-like protein 1; NEDD8, Neural precursor cell Expressed, Developmentally Down-regulated 8.     

The COP/DET/FUS proteins-regulators of eukaryotic growth and development

Eleven recessive mutant loci define the class of cop / det / fus mutants of Arabidopsis. The cop / det / fus mutants mimic the phenotype of light-grown seedlings when grown in the dark. At least four cop / det / fus mutants carry mutations in subunits of the COP9 signalosome, a multiprotein complex paralogous to the 'lid' subcomplex of the 26S proteasome. COP1, another COP/DET/FUS protein, is itself not a subunit of the COP9 signalosome. In the dark, COP1 accumulates in the nucleus where it is required for the degradation of the HY5 protein, a positive regulator of photomorphogenesis. In the light, COP1 is excluded from the nucleus and the constitutively nuclear HY5 protein can accumulate. Nuclear accumulation of COP1 and degradation of HY5 are impaired in the cop / det / fus mutants that carry mutations in subunits of the COP9 signalosome. Although the cellular function of the COP/DET/FUS proteins is not yet well understood, taken together the current findings suggest that the COP/DET/FUS proteins repress photomorphogenesis in the dark by mediating specific protein degradation.     

Building and remodelling Cullin–RING E3 ubiquitin ligases

Cullin–RING E3 ubiquitin ligases (CRLs) control a plethora of biological pathways through targeted ubiquitylation of signalling proteins. These modular assemblies use substrate receptor modules to recruit specific targets. Recent efforts have focused on understanding the mechanisms that control the activity state of CRLs through dynamic alterations in CRL architecture. Central to these processes are cycles of cullin neddylation and deneddylation, as well as exchange of substrate receptor modules to re‐sculpt the CRL landscape, thereby responding to the cellular requirements to turn over distinct proteins in different contexts. This review is focused on how CRLs are dynamically controlled with an emphasis on how cullin neddylation cycles are integrated with receptor exchange.     

COP9 signalosome controls the Carma1–Bcl10–Malt1 complex upon T‐cell stimulation

The Carma1–Bcl10–Malt1 (CBM) complex connects T‐cell receptor (TCR) signalling to the canonical IκB kinase (IKK)/NF (nuclear factor)‐κB pathway. Earlier studies have indicated that the COP9 signalosome (CSN), a pleiotropic regulator of the ubiquitin/26S proteasome system, controls antigen responses in T cells. The CSN is required for the degradation of the NF‐κB inhibitor IκBα, but other molecular targets involved in T‐cell signalling remained elusive. Here, we identify the CSN subunit 5 (CSN5) as a new interactor of Malt1 and Carma1. T‐cell activation triggers the recruitment of the CSN to the CBM complex, and CSN downregulation impairs TCR‐induced IKK activation. Furthermore, the CSN is required for maintaining the stability of Bcl10 in response to T‐cell activation. Taken together, our data provide evidence for a functional link between the evolutionarily conserved CSN and the adaptive immunoregulatory CBM complex in T cells.     

COP9信号传导体与调节盖子复合体的进化

COP9信号传导体和26S蛋白酶体的调节盖子复合体皆为含有8个亚基的蛋白复合体,在真核生物体中普遍存在,它们的相应亚基在大小和氨基酸序列上具有一一对应关系.从NCBI站点的所有数据库中获得了裂殖酵母、酿酒酵母、线虫、果蝇、哺乳动物和拟南芥等多种生物的复合体的亚基序列共8组.COP9信号传导体与调节盖子复合体相应亚基之间的氨基酸序列一致性大于12%,它们均具有一些保守的区域,而且保守位点分布均匀,表明它们来自于同一祖先.在基于氨基酸序列构建的系统发育树中,各组序列分别形成两个分支:一个分支由COP9信号传导体亚基和相似蛋白组成,另一分支由相应的调节盖子复合体亚基和相似蛋白构成.各个分支中单细胞生物的序列位于动、植物序列的根部,表明COP9信号传导体与调节盖子复合体的基因重复发生在真核单细胞生物和多细胞生物分化以前,并且二者的亚基基因沿各自的方向独立进化.几乎所有编码两个蛋白复合体的基因在基因组中均为单拷贝,第Ⅴ、Ⅵ组的亚基保守程度最高,暗示着它们在复合体中起着关键的作用.对COP9信号传导体和调节盖子复合体的相应亚基基因两两之间进行dN/dS的相关性分析,分别鉴定出21和15对亚基编码序列间具有显著的Pearson相关关系,推测其相应亚基间可能通过承担相互关联的重要的生物学功能而协同进化.     

COP9信号传导体与调节盖子复合体的进化

COP9信号传导体和26S蛋白酶体的调节盖子复合体皆为含有8个亚基的蛋白复合体,在真核生物体中普遍存在,它们的相应亚基在大小和氨基酸序列上具有一一对应关系。从NCBI站点的所有数据库中获得了裂殖酵母、酿酒酵母、线虫、果蝇、哺乳动物和拟南芥等多种生物的复合体的亚基序列共8组。COP9信号传导体与调节盖子复合体相应亚基之间的氨基酸序列一致性大于12％,它们均具有一些保守的区域,而且保守位点分布均匀,表明它们来自于同一祖先。在基于氨基酸序列构建的系统发育树中,各组序列分别形成两个分支：一个分支由COP9信号传导体亚基和相似蛋白组成,另一分支由相应的调节盖子复合体亚基和相似蛋白构成。各个分支中单细胞生物的序列位于动、植物序列的根部,表明COP9信号传导体与调节盖子复合体的基因重复发生在真核单细胞生物和多细胞生物分化以前,并且二者的亚基基因沿各自的方向独立进化。几乎所有编码两个蛋白复合体的基因在基因组中均为单拷贝,第Ⅴ、Ⅵ组的亚基保守程度最高,暗示着它们在复合体中起着关键的作用。对COP9信号传导体和调节盖子复合体的相应亚基基因两两之间进行dN／dS的相关性分析,分别鉴定出21和15对亚基编码序列间具有显著的Pearson相关关系,推测其相应亚基间可能通过承担相互关联的重要的生物学功能而协同进化。     

基因打靶技术:开启遗传学新纪元

基因打靶技术作为最有效的定向修饰小鼠基因的技术手段在揭示基因的生理功能、研究人类疾病的遗传机制以及寻找新的药物靶标的过程中发挥着重要的作用。近年来, 随着条件基因打靶技术的发展使基因失活可以限制在特定时段特定组织或细胞内。文章将主要介绍基因打靶技术的发展简史、近期进展以及在其他模式动物中的应用。