Proteasome Activator REGγ Enhances Coxsackieviral Infection by Facilitating p53 Degradation

Coxsackievirus B3 (CVB3) is a small RNA virus associated with diseases such as myocarditis, meningitis, and pancreatitis. We have previously demonstrated that proteasome inhibition reduces CVB3 replication and attenuates virus-induced myocarditis. However, the underlying mechanisms by which the ubiquitin/proteasome system regulates CVB replication remain unclear. In this study, we investigated the role of REGγ, a member of the 11S proteasome activator, in CVB3 replication. We showed that overexpression of REGγ promoted CVB3 replication but that knockdown of REGγ led to reduced CVB3 replication. We further demonstrated that REGγ-mediated p53 proteolysis contributes, as least in part, to the proviral function of REGγ. Although total protein levels of REGγ remained unaltered after CVB3 infection, virus infection induced a redistribution of REGγ from the nucleus to the cytoplasm, rendering an opportunity for a direct interaction of REGγ with viral proteins and/or host proteins (e.g., p53), which controls viral growth and thereby enhances viral infectivity. Further analyses suggested a potential modification of REGγ by SUMO following CVB3 infection, which was verified by both in vitro and in vivo sumoylation assays. Sumoylation of REGγ may play a role in its nuclear export during CVB3 infection. Taken together, our results present the first evidence that the host REGγ pathway is utilized and modified during CVB3 infection to promote efficient viral replication.Viruses often adapt to the existing host cellular machinery to complete their own life cycle. The ubiquitin/proteasome system (UPS), a primary intracellular protein degradation system in eukaryotic cells, has emerged as a key modulator in viral infectivity and virus-mediated pathogenesis (6).Coxsackievirus B3 (CVB3) is a small RNA virus associated with diseases such as myocarditis, meningitis, and pancreatitis (36). We have previously studied the function and regulation of the UPS in CVB3 infection and CVB3-induced myocarditis (7, 16, 17, 33). We demonstrated that CVB3 utilizes and manipulates the host UPS to achieve successful replication (17, 33). We provided evidence that proteasome inhibition reduces CVB3 replication and attenuates virus-induced myocarditis (7). However, we recognize the potential toxicity of general inhibition of proteasome function as a therapeutic means. Further investigation to identify specific targets within the UPS utilized during CVB3 infection is urgently needed and will allow for more-precise targeting in drug therapy.The 20S proteasome is a multisubunit protease complex responsible for the degradation of misfolded proteins or short-lived regulatory proteins (16, 18). In the absence of proteasome activators, the 20S proteasome is latent and the protein substrates are barred from entering the 20S proteasome (16, 18). There are at least two families of proteasome activators, the 19S proteasome (also known as PA700) and the 11S proteasome (also known as REG or PA28) (16, 18). The 19S activator binds to proteasome to form the 26S proteasome, which primarily performs degradation of proteins in a ubiquitin-dependent manner.The REG activator binds to and activates the proteasome in an ATP-independent manner to promote mainly ubiquitin-independent protein degradation. Three classes of REG have been identified, REGα, REGβ, and REGγ. REGα/β forms a heteroheptamer which is mainly localized to the cytosol (16, 18). The level of REGα/β is inducible by gamma interferon, and the main function of REGα/β has been implicated in major histocompatibility complex (MHC) class I antigen presentation (16, 18). REGγ exists in a homoheptamer and is primarily found in the nucleus (16, 18). Although the functional significance of REGγ has not been fully defined, studies of REGγ-deficient mice reveal a role for REGγ in the regulation of cell cycle progression and cell survival/apoptosis (1, 27). These effects appear to be related to REGγ-mediated degradation of several important intracellular proteins, such as cyclin-dependent kinase inhibitors p21, p16, and p19 (2, 14) and tumor suppressor p53 (43). Moreover, an interaction between the REGγ system and the viral proteins has recently been reported. It was shown that REGγ binds to and regulates the stability and nuclear retention of hepatitis C core protein (26), contributing to hepatitis C core protein-induced insulin resistance and hepatocarcinoma (24, 25).We have previously reported that gene silencing of ubiquitin reduces viral protein synthesis and viral titers (33). However, such inhibitions are not as potent as by proteasome inhibition, suggesting that 11S proteasome-mediated proteasomal degradation may also play a role. In the present study, we seek to further understand the underlying mechanisms by which the UPS regulates CVB3 replication by investigating the interplay between REGγ and CVB3 infection and exploring the potential mechanisms of how REGγ controls CVB3 replication. Here, we provided the first evidence that the host REGγ pathway was utilized and modulated during CVB3 infection to promote efficient viral replication.     

Site-specific Acetylation of the Proteasome Activator REGγ Directs Its Heptameric Structure and Functions

The proteasome activator REGγ has been reported to promote degradation of steroid receptor coactivator-3 and cyclin-dependent kinase inhibitors p21, p16, and p19 in a ubiquitin- and ATP-independent manner. A recent comparative analysis of REGγ expression in mouse and human tissues reveals a unique pattern of REGγ in specific cell types, suggesting undisclosed functions and biological importance of this molecule. Despite the emerging progress made in REGγ-related studies, how REGγ function is regulated remains to be explored. In this study, we report for the first time that REGγ can be acetylated mostly on its lysine 195 (Lys-195) residue by CREB binding protein (CBP), which can be reversed by sirtuin 1 (SIRT1) in mammalian cells. Site-directed mutagenesis abrogated acetylation at Lys-195 and significantly attenuated the capability of REGγ to degrade its target substrates, p21 and hepatitis C virus core protein. Mechanistically, acetylation at Lys-195 is important for the interactions between REGγ monomers and ultimately influences REGγ heptamerization. Biological analysis of cells containing REGγ-WT or REGγ-K195R mutant indicates an impact of acetylation on REGγ-mediated regulation of cell proliferation and cell cycle progression. These findings reveal a previously unknown mechanism in the regulation of REGγ assembly and activity, suggesting a potential venue for the intervention of the ubiquitin-independent REGγ proteasome activity.     

蛋白酶体激活因子REGγ的研究进展

REGγ(PSME3,proteasome activator complex subunit 3)是20S蛋白酶体的一种重要激活因子,REGγ-蛋白酶体系统可以非泛素和非ATP依赖方式介导多种重要蛋白降解。研究发现,REGγ以降解不同底物蛋白的方式参与肿瘤的进程、免疫反应、神经性疾病等系列生理病理过程。REGγ的表达可被不同的信号刺激诱导。翻译后修饰也可调控REGγ蛋白的活性,如REGγ的SUMO修饰和乙酰化修饰,均对其降解底物的功能至关重要。因此,REGγ是一个受多水平调控、具多种功能的细胞核内蛋白。本文较系统地综述了REGγ研究的最新进展,阐述了REGγ在许多生理病理过程中的重要作用及其机理。     

α-synuclein fate: Proteasome or autophagy?

The accumulation of α-synuclein is critical for the development of Parkinson disease (PD), and unraveling the mechanisms that regulate α-synuclein levels is key to understanding the pathophysiology of the disease. We recently found that USP9X deubiquitinates α-synuclein, and that this process determines the partition of α-synuclein between the proteasomal and autophagy pathways. By manipulating USP9X levels, we observed that monoubiquitinated α-synuclein is degraded by the proteasome, whereas deubiquitination of α-synuclein favors its degradation by autophagy. As USP9X levels and activity are decreased in α-synucleinopathy brains, USP9X may now represent a novel target for PD.     

前列腺细胞REGⅣ基因功能的初步研究

目的:探讨人再生基因Ⅳ(REGIV)在前列腺细胞中的表达及意义.方法:构建REGIV基因的全序列过表达质粒.将全序列过表达质粒采用脂质体转染的方式转入前列腺细胞系PC-3中.应用Real-time-PCR方法检测REGIV基因mRNA表达,Westembloting检测REGIV基因蛋白质表达,MTT法分析细胞增殖活性.结果:通过荧光显微镜观察计数,细胞转染成功.REGIV基因的全序列过表达使REGIV基因mRNA的表达,蛋白表达提高,细胞增殖能力增强.结论:应用全序列过表达技术可以使前列腺癌REGIV表达水平特异性增高.前列腺癌增值能力的增强说明REGW可能与肿瘤快速增长有关.     

The Ubiquitin–Proteasome System in Retinal Health and Disease

The ubiquitin–proteasome system (UPS) is the main intracellular pathway for modulated protein turnover, playing an important role in the maintenance of cellular homeostasis. It also exerts a protein quality control through degradation of oxidized, mutant, denatured, or misfolded proteins and is involved in many biological processes where protein level regulation is necessary. This system allows the cell to modulate its protein expression pattern in response to changing physiological conditions and provides a critical protective role in health and disease. Impairments of UPS function in the central nervous system (CNS) underlie an increasing number of genetic and idiopathic diseases, many of which affect the retina. Current knowledge on the UPS composition and function in this tissue, however, is scarce and dispersed. This review focuses on UPS elements reported in the retina, including ubiquitinating and deubiquitinating enzymes (DUBs), and alternative proteasome assemblies. Known and inferred roles of protein ubiquitination, and of the related, SUMO conjugation (SUMOylation) process, in normal retinal development and adult homeostasis are addressed, including modulation of the visual cycle and response to retinal stress and injury. Additionally, the relationship between UPS dysfunction and human neurodegenerative disorders affecting the retina, including Alzheimer's, Parkinson's, and Huntington's diseases, are dealt with, together with numerous instances of retina-specific illnesses with UPS involvement, such as retinitis pigmentosa, macular degenerations, glaucoma, diabetic retinopathy (DR), and aging-related impairments. This information, though still basic and limited, constitutes a suitable framework to be expanded in incoming years and should prove orientative toward future therapy design targeting sight-affecting diseases with a UPS underlying basis.     

Phylogenomic Analysis of the α Proteasome Gene Family from Early-Diverging Eukaryotes

We employed a phylogenomic approach to study the evolution of α subunits of the proteasome gene family from early diverging eukaryotes. BLAST similarity searches of the Giardia lamblia genome identified all seven α proteasome genes characteristic of eukaryotes from the crown group. In addition, a PCR strategy for the amplification of multiple α subunit sequences generated single α proteasome products for representatives of the Kinetoplastida (Leishmania major), the Parabasalia (Trichomonas vaginalis), and the Microsporidia (Vairimorpha sp., Nosema sp., Endoreticulata sp., and Spraguea lophii). The kinetoplastid Trypanosoma cruzi and the eukaryote crown group Acanthamoeba castellanii yielded two distinct α proteasome genes each. The presence of seven distinct α proteasome genes in G. lamblia, one of the earliest-diverging eukaryotes, indicates that the α proteasome gene family evolved rapidly from a minimum of one gene in Archaea to seven or more in Eukarya. Results from the phylogenomic analysis are consistent with the idea that the Diplomonida (as represented by G. lamblia), the Kinetoplastida, the Parabasalia, and the Microsporidia diverged after the duplication events that originated the α proteasome gene family. A model for the early origin and evolution of the proteasome gene family is presented. Received: 14 February 2000 / Accepted: 14 August 2000     

Characterization of the Testis-specific Proteasome Subunit α4s in Mammals

The 26 S proteasome is responsible for regulated proteolysis in eukaryotic cells. It is composed of one 20 S core particle (CP) flanked by one or two 19 S regulatory particles. The CP is composed of seven different α-type subunits (α1-α7) and seven different β-type subunits, three of which are catalytic. Vertebrates encode four additional catalytic β subunits that are expressed predominantly in immune tissues and produce distinct subtypes of CPs particularly well suited for the acquired immune system. In contrast, the diversity of α subunits remains poorly understood. Recently, another α subunit, referred to as α4s, was reported. However, little is known about α4s. Here we provide a detailed characterization of α4s and the α4s-containing CP. α4s is exclusively expressed in germ cells that enter the meiotic prophase and is incorporated into the CP in place of α4. A comparison of structural models revealed that the differences in the primary sequences between α4 and α4s are located on the outer surface of the CP, suggesting that α4s interacts with specific molecules via these unique regions. α4s-containing CPs account for the majority of the CPs in mouse sperm. The catalytic β subunits in the α4s-containing CP are β1, β2, and β5, and immunosubunits are not included in the α4s-containing CP. α4s-containing CPs have a set of peptidase activities almost identical to those of α4-containing CPs. Our results provide a basis for understanding the role of α4s and male germ cell-specific proteasomes in mammals.     

REGγ基因下调对HaCat细胞的生物学影响

为了研究蛋白酶体激活因子REGγ对HaCat细胞增殖、凋亡、周期的影响,该研究通过瞬时转染建立了siN、siR细胞系,并利用流式细胞术、MTT、Western blot等实验技术检测细胞的处理效果。研究结果显示,通过小干扰RNA使REGγ低表达后,HaCat细胞内抑癌基因蛋白p53、细胞周期依赖性激酶抑制因子p21表达量增加,进而抑制了细胞周期和增殖、促进了细胞凋亡。     

Regulation of REGγ cellular distribution and function by SUMO modification

Discovery of emerging REGγ-regulated proteins has accentuated the REGγ-proteasome as an important pathway in multiple biological processes, including cell growth, cell cycle regulation, and apoptosis. However, little is known about the regulation of the REGγ-proteasome pathway. Here we demonstrate that REGγ can be SUMOylated in vitro and in vivo by SUMO-1, SUMO-2, and SUMO-3. The SUMO-E3 protein inhibitor of activated STAT (PIAS)1 physically associates with REGγ and promotes SUMOylation of REGγ. SUMOylation of REGγ was found to occur at multiple sites, including K6, K14, and K12. Mutation analysis indicated that these SUMO sites simultaneously contributed to the SUMOylation status of REGγ in cells. Posttranslational modification of REGγ by SUMO conjugation was revealed to mediate cytosolic translocation of REGγ and to cause increased stability of this proteasome activator. SUMOylation-deficient REGγ displayed attenuated ability to degrade p21(Waf//Cip1) due to reduced affinity of the REGγ SUMOylation-defective mutant for p21. Taken together, we report a previously unrecognized mechanism regulating the activity of the proteasome activator REGγ. This regulatory mechanism may enable REGγ to function as a more potent factor in protein degradation with a broader substrate spectrum.     

REGγ proteasome mediates degradation of the ubiquitin ligase Smurf1

The ubiquitin ligase Smad ubiquitination regulatory factor 1 (Smurf1) targets many proteins including Smad1/5 for ubiquitin-dependent proteasomal degradation. However, how Smurf1 is degraded remains unclear. Here we show that REGγ, an activator for the 20S proteasome-mediated protein degradation, interacts with Smurf1 and mediates its degradation. We provide evidence that depletion of REGγ stabilizes Smurf1 whereas overexpression of REGγ promotes the degradation of Smurf1. Interestingly both Smurf2 and Smurf1 are destabilized by the REGγ proteasome while the other members of Neural precursor cell-expressed developmentally downregulated gene 4 family were not affected. More importantly, we found that the REGγ proteasome-mediated degradation of Smurf1 results in degradation of Smad5. These findings reveal that the REGγ-proteasome targets a ubiquitin ligase for protein degradation.

Structured summary

MINT-7894509: CKIP (uniprotkb:Q53GL0) binds (MI:0407) to Smurf1 (uniprotkb:Q9HCE7) by pull down (MI:0096)MINT-7894494: REG gamma (uniprotkb:P61289) binds (MI:0407) to Smurf1 (uniprotkb:Q9HCE7) by pull down (MI:0096)MINT-7894523, MINT-7894543, MINT-7894481: REG gamma (uniprotkb:P61289) physically interacts (MI:0915) with Smurf1 (uniprotkb:Q9HCE7) by anti tag coimmunoprecipitation (MI:0007)MINT-7894558: Smurf1 (uniprotkb:Q9HCE7) physically interacts (MI:0915) with REG gamma (uniprotkb:P61289) by two hybrid (MI:0018)     

Structure–activity relationship of boronic acid derivatives of tyropeptin: Proteasome inhibitors

The structure–activity relationship of the boronic acid derivatives of tyropeptin, a proteasome inhibitor, was studied. Based on the structure of a previously reported boronate analog of tyropeptin (2), 41 derivatives, which have varying substructure at the N-terminal acyl moiety and P2 position, were synthesized. Among them, 3-phenoxyphenylacetamide 6 and 3-fluoro picolinamide 22 displayed the most potent inhibitory activity toward chymotryptic activity of proteasome and cytotoxicity, respectively. The replacement of the isopropyl group in the P2 side chain to H or Me had negligible effects on the biological activities examined in this study.     

Proteasome inhibition: an early or late event in nitric oxide-induced neuronal death?

Nitric oxide (NO), ubiquitously expressed in the central nervous system, has been perceived to be a potential neuromodulator. Employing cultured murine primary cortical neurons, NO resulted in an inhibition of the ubiquitin-proteasome system (UPS) with a dose- and time-dependent decrease in cell viability. This is consistent with a previous study that reported a dysfunction of UPS with consequential apoptotic death in macrophage cell with NO treatment. However, it cannot be unclear if the drop in UPS efficiency is directly imposed on by NO. Therefore by using microarray analysis, our study revealed an early down-regulation or non-significant differential expression of genes encoding UPS proteins in NOC-18 (NO donor)-treated neurons as compared to an observed elevation of corresponding gene expression genes in lactacystin (classical proteasome inhibitor)-treated neurons (conducted earlier). Furthermore, time-course analysis of proteasome activity in NOC-18-treated neurons demonstrated a late onset of reduction. This is intriguing as it is well established that in an exclusive proteasome dysfunction-induced cell death, a compensatory feedback mechanism will be activated with an initial and concerted up-regulation of genes encoding proteins involved in UPS as seen when neurons were treated with lactacystin. Thus, it is highly suggestive that NO-triggered neuronal death takes on a different signaling cascade from that of a classical proteasome inhibitor, and that the late reduction of proteasome activity is a downstream event following the activation of apoptotic cellular signaling cascade. In intracellular condition, the proteasome is not NO preferred primary target responsible for the trigger of the cell death machinery. In conclusion, we presented novel findings that shed light into NO-induced cell death signaling cascade, which would be important in understanding the pathogenesis of neurodegenerative disorders such as Parkinson's disease.     

PHOR1: A U-Box GA Signaling Component With a Role in Proteasome Degradation?

Recent identification of U-box proteins as E3 ubiquitin ligases suggests that the U-box arm-repeat protein PHOR1, for which we have demonstrated a role in GA signal transduction, may play a role in GA signaling by ubiquitinating one or more components of the GA response pathway to target them for proteasome degradation. Here we show that PHOR1 function in GA signaling is not exclusive of potato plants, but it is also conserved in Arabidopsis. Three PHOR1-homologs have been identified in this plant species, which would correspond to PHOR1-orthologs. Experimental evidence has recently been obtained for the involvement of proteasome-dependent protein degradation in GA-mediated destabilization of the SLN1 DELLA protein, thus pointing to this repressor as a likely substrate for ubiquitination by the PHOR1 ubiquitin ligase activity.     

人源蛋白酶体α亚基6(Proteasome subunit alpha 6)在酿酒酵母表面展示

为构建人源蛋白酶体α亚基6(α6)的酵母展示体系,研制其单克隆抗体用于抗体表位分析和研究泛素-蛋白酶体途径,建立绕过重组抗原表达及纯化制备、将展示重组抗原直接应用于抗体检测的方法.在酵母展示表达载体pICAS中引入His.tag标签,将编码α6的基因PSA6_HUMAN克隆到酵母表面展示载体pICAS-H上,用流式细胞仪检测其抗原表位活性,以表面展示α6的重组酵母细胞,结合酶联吸附免疫检测技术,建立酵母(yeast)-ELISA检测技术,应用于检测小鼠单克隆抗体及单抗效价.酵母细胞培养48h后获得抗原α6的高效表面展示,展示的α6具有良好的抗原活性和特异性,将α6的展示酵母用于yeast-ELISA的初步实验结果显示可有效检测和筛选到抗α6抗体.     

Dendritic Cell IL-1α and IL-1β Are Polyubiquitinated and Degraded by the Proteasome

IL-1α and β are key players in the innate immune system. The secretion of these cytokines by dendritic cells (DC) is integral to the development of proinflammatory responses. These cytokines are not secreted via the classical secretory pathway. Instead, 2 independent processes are required; an initial signal to induce up-regulation of the precursor pro-IL-1α and -β, and a second signal to drive cleavage and consequent secretion. Pro-IL-1α and -β are both cytosolic and thus, are potentially subject to post-translational modifications. These modifications may, in turn, have a functional outcome in the context of IL-1α and -β secretion and hence inflammation. We report here that IL-1α and -β were degraded intracellularly in murine bone marrow-derived DC and that this degradation was dependent on active cellular processes. In addition, we demonstrate that degradation was ablated when the proteasome was inhibited, whereas autophagy did not appear to play a major role. Furthermore, inhibition of the proteasome led to an accumulation of polyubiquitinated IL-1α and -β, indicating that IL-1α and -β were polyubiquitinated prior to proteasomal degradation. Finally, our investigations suggest that polyubiquitination and proteasomal degradation are not continuous processes but instead are up-regulated following DC activation. Overall, these data highlight that IL-1α and -β polyubiquitination and proteasomal degradation are central mechanisms in the regulation of intracellular IL-1 levels in DC.