Evidence for functional conservation of a mammalian homologue of the light-responsive plant protein COP1.

Identified in Arabidopsis as a repressor of light-regulated development, the COP1 (constitutively photomorphogenic 1) protein is characterized by a RING-finger motif and a WD40 repeat domain [1]. The subcellular localization of COP1 is light-dependent. COP1 acts within the nucleus to repress photomorphogenic development, but light inactivates COP1 and diminishes its nuclear abundance [2]. Here, we report the identification of a mammalian COP1 homologue that contains all the structural features present in Arabidopsis COP1 (AtCOP1). When expressed in plant cells, a fusion protein comprising mammalian COP1 and beta-glucuronidase (GUS) responded to light by changing its subcellular localization pattern in a manner similar to AtCOP1. Whereas the mammalian COP1 was unable to rescue the defects of Arabidopsis cop1 mutants, expression of the amino-terminal half of mammalian COP1 in Arabidopsis interfered with endogenous COP1 function, resulting in a hyperphotomorphogenic phenotype. Therefore, the regulatory modules in COP1 proteins that are responsible for the signal-dependent subcellular localization are functionally conserved between higher plants and mammals, suggesting that mammalian COP1 may share a common mode of action with its plant counterpart in regulating development and cellular signaling.     

动植物中COP1泛素连接酶介导的信号转导与蛋白质稳态调控

COP1 E3泛素连接酶最初是在植物中作为光形态建成的关键抑制因子被发现和广泛研究的，是植物生长发育和环境适应过程中的核心“开关”。光受体接收外界环境信号后传递给COP1,COP1再靶向调控下游核心转录因子，从而完成光形态建成等生命过程。在哺乳动物中，尽管大部分光受体都消失，但COP1仍在代谢调控和肿瘤发生过程中靶向重要的转录因子。通过比较动植物中COP1调控过程的异同发现，哺乳动物中COP1所感知的上游信号几乎是未知的，其中COP1结合CRL4形成的复合体E3泛素连接酶的组装机制调控仍不清楚。植物中光是其主要能量来源和COP1的主要上游信号，而作为动物的主要能量来源，葡萄糖和相关激素很可能也是动物COP1的上游信号。同时，通过总结医学研究中针对蛋白质泛素化相关过程的丰富靶点和相关药物，可以为植物COP1等E3泛素连接酶的研究提供有效工具。COP1在细胞生命过程调控中至关重要，其功能和作用机制随着进化而产生多样性，尚有待继续深入探究。     

植物COP1蛋白的结构与功能

组成型光形态建成 1 (constitutivelyphotomorphogenic 1 ,COP1 )蛋白是一个分子量为 76kD的核蛋白 ,它由 3个特殊的结构域组成即环形锌指结合域、卷曲螺旋形结构域和WD_40重复序列 ,并含有一个核定位信号和一个新型细胞质定位信号 ,它是一个光形态建成的抑制子 ,是一个光调控植物发育的分子开关。当植物在暗环境下生长时 ,COP1蛋白聚集在细胞核内 ,抑制光形态的建成 ,而在光环境下 ,COP1蛋白则分散到细胞质中 ,解除其抑制作用 ,恢复光形态建成。COP1蛋白在细胞内的核质分布受多个因素的影响 ,核内COP1通过与特异转录因子相互作用来调节光形态建成。继从植物中分离鉴定出COP1蛋白之后 ,动物体内也发现有COP1蛋白的存在 ,提示COP1蛋白可能在调节动物和植物的发育及信号转导等方面具有共同作用模式。     

植物COP1蛋白的结构与功能

组成型光形态建成1(constitutively photomorphogenic 1,COP1)蛋白是一个分子量为76 kD的核蛋白,它由3个特殊的结构域组成即环形锌指结合域、卷曲螺旋形结构域和WD_40 重复序列,并含有一个核定位信号和一个新型细胞质定位信号,它是一个光形态建成的抑制子,是一个光调控植物发育的分子开关。当植物在暗环境下生长时,COP1蛋白聚集在细胞核内,抑制光形态的建成,而在光环境下,COP1蛋白则分散到细胞质中,解除其抑制作用,恢复光形态建成。COP1蛋白在细胞内的核质分布受多个因素的影响,核内COP1通过与特异转录因子相互作用来调节光形态建成。继从植物中分离鉴定出COP1蛋白之后,动物体内也发现有COP1蛋白的存在,提示COP1蛋白可能在调节动物和植物的发育及信号转导等方面具有共同作用模式。     

Evidence for FUS6 as a component of the nuclear-localized COP9 complex in Arabidopsis.

The pleiotropic CONSTITUTIVE PHOTOMORPHOGENIC (COP), DEETIOLATED (DET), and FUSCA (FUS) loci are essential regulatory genes involved in the light control of seedling developmental patterns in Arabidopsis. Although COP1, DET1, COP9, and FUS6 (also called COP11) have been cloned, their biochemical activities and interactions remain elusive. We have recently suggested that multiple pleiotropic COP, DET, and FUS genes may encode subunits of a large regulatory complex. In this study, we generated specific antibodies against Arabidopsis FUS6 and show that accumulation of both COP9 and FUS6 is coordinated in the pleiotropic cop, det, and fus mutant backgrounds and in wild-type plants throughout development. Both COP9 and FUS6 cofractionated into identical high molecular mass fractions in an analytical gel filtration assay, and neither was found in its monomeric form. Moreover, antibodies raised against either COP9 or FUS6 selectively coimmunoprecipitated both proteins. We have also developed an Arabidopsis protoplast immunolocalization assay and demonstrated that the COP9 complex is localized in the nucleus and that its nuclear localization is not affected by light conditions or tissue types. The integrated genetic and biochemical results strongly support the conclusion that both COP9 and FUS6 are components of the nuclear-localized COP9 complex. Therefore, we have provided the strongest evidence for the conclusion that at least some of the pleiotropic COP, DET, and FUS loci act in the same signaling pathway.     

Fungal development and the COP9 signalosome

The conserved COP9 signalosome (CSN) multiprotein complex is located at the interface between cellular signaling, protein modification, life span and the development of multicellular organisms. CSN is required for light-controlled responses in filamentous fungi. This includes the circadian rhythm of Neurospora crassa or the repression of sexual development by light in Aspergillus nidulans. In contrast to plants and animals, CSN is not essential for fungal viability. Therefore fungi are suitable models to study CSN composition, activity and cellular functions and its role in light controlled development.     

Genome-Wide Identification,In Silico Characterization of AtCOP1-Targeting Regulatory Proteins Network and their Expression Profiling in The COP1 Downregulated Arabidopsis thaliana

The functional diversification of the RING-finger constitutive photomorphogenesis 1 (COP1) enzyme highly depends on its (in)direct interaction with regulatory proteins involved in the Arabidopsis photomorphogenesis signaling pathways. In last decade, only a few AtCOP1-ligand complexes have been investigated using functional analysis and proved by biochemical interaction analysis, despite much more have been remaining unclear. Identifying the functions of COP1 will undoubtedly provide an opportunity for the discovery of novel COP1 targets either triggered on COP1 or targeted by COP1, which their characteristics have not been reported so far. Here, we have focused on the tertiary structure analysis of COP1 protein and characterization of its potent ligands based on the protein–protein interaction characteristics of the known AtCOP1-based protein complexes, which their interaction with COP1 were biochemically approved. Based on motif analysis and molecular docking results, a total of 88 regulatory proteins with different confidence were identified to be interactive and co-regulated with AtCOP1 E3 ligase. The correlation regulatory network analysis reveals that COP1 functions as a master switch in controlling the Arabidopsis growth and development. The up/down regulations of the potent COP1-ligands gene expression levels in cop1 knockdown T0 plants indicate the COP1 expanded roles in a wide diversity of biological processes. These characteristics consist of the photomorphogenesis signaling pathway, cell cycle regulation, cell division, histone H3-K27 methylation, stomata formation, and iron starvation. Meanwhile, the VPS11 transporter probably transports the COP1 into the endosome, lysosome, and vacuole between the nucleus and cytoplasm during the photomorphogenesis signaling pathway in darkness condition.

     

Organization of protein complexes under photomorphogenic UV-B in Arabidopsis

Low-fluence and long-wavelength UV-B light promotes photomorphogenic development in Arabidopsis. CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) is a positive regulator in this pathway while it is a negative regulator of the traditional photomorphogenesis triggered by far-red and visible light. We have recently reported the mechanism by which the switch of COP1 function is accomplished in distinct light contexts. In response to photomorphogenic UV-B, the photoactivated UV RESISTANCE LOCUS 8 (UVR8) associates with the COP1- SUPRESSOR OF PHYA (SPA) core complexes, resulting in the physical and functional disassociation of COP1-SPA from the CULLIN4-DAMAGED DNA BINDING PROTEIN 1 (CUL4-DDB1) E3 scaffold. These UV-B dependent UVR8-COP1-SPA complexes promote the stability and activity of ELONGATED HYPOCOTYL 5 (HY5), and eventually cause COP1 to switch from repressing to promoting photomorphogenesis. In addition, it is possible that CUL4-DDB1 might simultaneously recruit alternative DDB1 BINDING WD40 (DWD) proteins to repress this UV-B-specific signaling. Further investigation is required, however, to verify this hypothesis. Overall, the coordinated organization of various protein complexes facilitates an efficient and balanced UV-B signaling.     

An Arabidopsis SUMO E3 Ligase,SIZ1, Negatively Regulates Photomorphogenesis by Promoting COP1 Activity

COP1 (CONSTITUTIVE PHOTOMORPHOGENIC 1), a ubiquitin E3 ligase, is a central negative regulator of photomorphogenesis. However, how COP1 activity is regulated by post-translational modifications remains largely unknown. Here we show that SUMO (small ubiquitin-like modifier) modification enhances COP1 activity. Loss-of-function siz1 mutant seedlings exhibit a weak constitutive photomorphogenic phenotype. SIZ1 physically interacts with COP1 and mediates the sumoylation of COP1. A K193R substitution in COP1 blocks its SUMO modification and reduces COP1 activity in vitro and in planta. Consistently, COP1 activity is reduced in siz1 and the level of HY5, a COP1 target protein, is increased in siz1. Sumoylated COP1 may exhibits higher transubiquitination activity than does non-sumoylated COP1, but SIZ1-mediated SUMO modification does not affect COP1 dimerization, COP1-HY5 interaction, and nuclear accumulation of COP1. Interestingly, prolonged light exposure reduces the sumoylation level of COP1, and COP1 mediates the ubiquitination and degradation of SIZ1. These regulatory mechanisms may maintain the homeostasis of COP1 activity, ensuing proper photomorphogenic development in changing light environment. Our genetic and biochemical studies identify a function for SIZ1 in photomorphogenesis and reveal a novel SUMO-regulated ubiquitin ligase, COP1, in plants.