The implication of SUMO in intrinsic and innate immunity

Since its discovery, SUMOylation has emerged as a key post-translational modification involved in the regulation of host-virus interactions. SUMOylation has been associated with the replication of a large number of viruses, either through the direct modification of viral proteins or through the modulation of cellular proteins implicated in antiviral defense. SUMO can affect protein function via covalent or non-covalent binding. There is growing evidence that SUMO regulates several host proteins involved in intrinsic and innate immunity, thereby contributing to the process governing interferon production during viral infection; as well as the interferon-activated Jak/STAT pathway. Unlike the interferon-mediated innate immune response, intrinsic antiviral resistance is mediated by constitutively expressed antiviral proteins (defined as restriction factors), which confer direct viral resistance through a variety of mechanisms. The aim of this review is to evaluate the role of SUMO in intrinsic and innate immunity; highlighting the involvement of the TRIM family proteins, with a specific focus on the mechanism through which SUMO affects i- interferon production upon viral infection, ii-interferon Jak/STAT signaling and biological responses, iii-the relationship between restriction factors and RNA viruses.     

Broad spectrum identification of cellular small ubiquitin-related modifier (SUMO) substrate proteins

Reversible covalent modification of proteins with a small ubiquitin-related modifier (SUMO) is emerging as an important system contributing to dynamic regulation of protein function. To enhance our understanding of the cell regulatory systems impacted by sumoylation, we used affinity chromatography-coupled high pressure liquid chromatography/tandem mass spectrometry for unbiased identification of candidate cellular SUMO substrate proteins. Here we describe the identification of 21 candidate sumoylated proteins from whole-cell lysates of HEK-293 cells. The nature of the proteins identified is consistent with a role for sumoylation in diverse cell regulatory systems but highlights regulation of chromatin organization and gene expression as major systems targeted by the sumoylation machinery.     

SUMO在转录中的抑制作用

许多调控基因转录的重要蛋白质能被SUMO (small ubiquitin-related modifier)化修饰,这些蛋白质包括转录因子,转录辅助因子和染色质修饰酶.SUMO化修饰对底物蛋白的活性产生影响,在大多数情况下,与转录活性的抑制有关.最近,对SUMO化调控转录的机制有了新的认识,认为SUMO化的一个重要作用是促进转录因子与转录抑制因子之间的相互作用.另一方面,已经发现转录共抑制因子HDAC (组蛋白去乙酰化酶)可以作为SUMO化的底物、效应因子和调控因子,说明乙酰化和SUMO化之间复杂的相互作用对基因转录调控起着非常重要的作用.     

Simultaneous knockdown of BRAF and expression of INK4A in melanoma cells leads to potent growth inhibition and apoptosis

Abnormal BRAF and p16INK4A co-exist in 60% of melanomas. BRAF mutation also occurs in 80% of benign nevi where it turns-on p16INK4A resulting in proliferative senescence; loss of p16INK4A removes the inhibitory block leading to melanoma development. Since only melanomas with wild-type BRAF have amplified CDK4 and cyclin D1 genes, p16INK4A-CDK4/6-cyclin D pathway is viewed as linearly downstream of BRAF. Thus, co-occurrence of aberrant BRAF and INK4A may be remnant of changes during melanoma formation without functional significance. To explore this notion, we simultaneously knocked down BRAF (via siRNA) and expressed INK4A cDNA in melanoma cells and observed enhanced growth inhibition. Notably, although each alone had no statistically significant effect on apoptosis, co-expression of BRAF siRNA and INK4A cDNA caused potent apoptosis, which was associated with up-regulation of BIM and down-regulation of BCL2. Our results suggest that aberrant BRAF and INK4A cooperate to promote proliferation and survival of melanoma cells.     

小泛素相关修饰物SUMO研究进展

蛋白质翻译后修饰对改变蛋白功能、活性或定位都起着非常重要的作用,泛素及其相似蛋白的修饰是其中一种重要形式。与其他诸如磷酸化、乙酰化、糖基化等不同的是,泛素及其相似蛋白的修饰基团本身即是一个小的多肽,通过异肽键与靶蛋白Lys侧链ε-NH2相连,其中小泛素相关修饰物(small ubiquitin—related modifier,SUMO)与蛋白的共价连接是一种新的广泛存在的翻译后修饰形式。SUMO是广泛存在于真核生物中高度保守的蛋白家族,在脊椎动物中有三个SUMO基因,称为SUMO-1,-2,-3,与泛素在二级结构上极其相似,且催化修饰过程的酶体系也具有很高的同源性。然而,与泛素化介导的蛋白酶降解途径不同,SUMO化修饰发挥着更为广泛的功能,如核质转运、细胞周期调控、信号转导、转录活性调控等。     

SUMO modification of PCNA is controlled by DNA

Post-translational modification by the ubiquitin-like protein SUMO is often regulated by cellular signals that restrict the modification to appropriate situations. Nevertheless, many SUMO-specific ligases do not exhibit much target specificity, and--compared with the diversity of sumoylation substrates--their number is limited. This raises the question of how SUMO conjugation is controlled in vivo. We report here an unexpected mechanism by which sumoylation of the replication clamp protein, PCNA, from budding yeast is effectively coupled to S phase. We find that loading of PCNA onto DNA is a prerequisite for sumoylation in vivo and greatly stimulates modification in vitro. To our surprise, however, DNA binding by the ligase Siz1, responsible for PCNA sumoylation, is not strictly required. Instead, the stimulatory effect of DNA on conjugation is mainly attributable to DNA binding of PCNA itself. These findings imply a change in the properties of PCNA upon loading that enhances its capacity to be sumoylated.     

Modification of Akt by SUMO conjugation regulates alternative splicing and cell cycle

Akt/PKB is a key signaling molecule in higher eukaryotes and a crucial protein kinase in human health and disease. Phosphorylation, acetylation, and ubiquitylation have been reported as important regulatory post-translational modifications of this kinase. We describe here that Akt is modified by SUMO conjugation, and show that lysine residues 276 and 301 are the major SUMO attachment sites within this protein. We found that phosphorylation and SUMOylation of Akt appear as independent events. However, decreasing Akt SUMOylation levels severely affects the role of this kinase as a regulator of fibronectin and Bcl-x alternative splicing. Moreover, we observed that the Akt mutant (Akt E17K) found in several human tumors displays increased levels of SUMOylation and also an enhanced capacity to regulate fibronectin splicing patterns. This splicing regulatory activity is completely abolished by decreasing Akt E17K SUMO conjugation levels. Additionally, we found that SUMOylation controls Akt regulatory function at G?/S transition during cell cycle progression. These findings reveal SUMO conjugation as a novel level of regulation for Akt activity, opening new areas of exploration related to the molecular mechanisms involved in the diverse cellular functions of this kinase.     

Enhanced protein expression in mammalian cells using engineered SUMO fusions: secreted phospholipase A2

SUMOylation, the covalent attachment of SUMO (small ubiquitin-like modifier), is a eukaryotic post-translational event that has been demonstrated to play a critical role in several biological processes. When used as an N-terminal tag or fusion partner, SUMO has been shown to enhance functional protein production significantly by improving folding, solubility, and stability. We have engineered several SUMOs and, through their fusion, developed a system for enhancing the expression and secretion of complex proteins. To demonstrate the fidelity of this fusion technology, secreted phospholipase A(2) proteins (sPLA(2)) were produced using HEK-293T and CHO-K1 cells. Five mouse sPLA(2) homologs were expressed and secreted in mammalian cell cultures using SUMO or SUMO-derived, N-terminal fusion partners. Mean and median increases of 43- and 18-fold, respectively, were obtained using novel SUMO mutants that are resistant to digestion by endogenous deSUMOylases.     

Regulation of the tumor suppressor PTEN by SUMO

The crucial function of the PTEN tumor suppressor in multiple cellular processes suggests that its activity must be tightly controlled. Both, membrane association and a variety of post-translational modifications, such as acetylation, phosphorylation, and mono- and polyubiquitination, have been reported to regulate PTEN activity. Here, we demonstrated that PTEN is also post-translationally modified by the small ubiquitin-like proteins, small ubiquitin-related modifier 1 (SUMO1) and SUMO2. We identified lysine residue 266 and the major monoubiquitination site 289, both located within the C2 domain required for PTEN membrane association, as SUMO acceptors in PTEN. We demonstrated the existence of a crosstalk between PTEN SUMOylation and ubiquitination, with PTEN-SUMO1 showing a reduced capacity to form covalent interactions with monoubiquitin and accumulation of PTEN-SUMO2 conjugates after inhibition of the proteasome. Moreover, we found that virus infection induces PTEN SUMOylation and favors PTEN localization at the cell membrane. Finally, we demonstrated that SUMOylation contributes to the control of virus infection by PTEN.     

A genome-wide analysis of sumoylation-related biological processes and functions in human nucleus

Protein sumoylation is an important reversible post-translational modification of proteins in the nucleus, and it orchestrates a variety of the cellular processes. Genome-wide analysis of functional abundance and distribution of Small Ubiquitin-related MOdifier (SUMO) substrates may shed a light on how sumoylation is involved in nuclear biological processes and functions. Two interesting questions about sumoylation have emerged: (1) how many SUMO substrates exist in mammalian proteomes, such as human and mouse, (2) and what are their functions and how are they involved in a variety of biological processes? To address these two questions,we present an in silico genome-scale analysis for SUMO substrates in human. Based on the pattern recognition and phylogenetic conservation, we retrieved a list of 2683 potential SUMO substrates conserved in both human and mouse. Then, by functional enrichment analysis, we surveyed the over-represented GO terms and functional domains of them against the whole human proteome. Besides the consistence between our analyses and in vivo or in vitro work, the in silico predicted candidates also point to several potential roles of sumoylation, e.g., perception of sound. These potential SUMO substrates in human are of great value for further in vivo or in vitro experimental analysis.     

小分子泛素相关修饰物SUMO及其融合表达

小分子泛素相关修饰物(small ubiquitin-related modifier,SUMO)是一类具有高度保守序列的低分子量蛋白.在蛋白质的融合表达中,SUMO作为融合标签得到了广泛应用.与其他传统融合标签如谷胱甘肽-S-转移酶、二硫键形成蛋白A、硫氧还蛋白等相比,SUMO具有防止蛋白降解、促进蛋白折叠、能被SUMO蛋白酶专一性识别切割并在目的蛋白上不会留下氨基酸残基等优点.本文综述SUMO融合蛋白及其配套的SUMO蛋白酶在融合表达中的优势和应用.     

Dynamic regulation of innate immunity by ubiquitin and ubiquitin-like proteins

Protein post-translational modifications (PTMs) are central to the host innate immune regulations. Dynamically, PTMs fine-tune the spatial and temporary responses of immune- and non-immune-cells, in accordance with extracellular and intracellular stresses. Ubiquitin and ubiquitin-like proteins (Ubls) are emerging as the important multi-functional signals, controlling the activation, stability, affinity and location of many signaling proteins. Recent investigations, at the molecular-cellular-animal models, have shed new light on the versatility of the ubiquitin, SUMO and ISG15, for shaping the strength and duration of the innate immune responses. This review summarizes our current knowledge on the functions and regulatory mechanisms of the ubiquitin and Ubls in the innate immunity, the first line of host defense against microbial infection.     

SUMO and SUMOylation in plants

The traditional focus on the central dogma of molecular biology, from gene through RNA to protein, has now been replaced by the recognition of an additional mechanism. The new regulatory mechanism, post-translational modifications to proteins, can actively alter protein function or activity introducing additional levels of functional complexity by altering cellular and sub-cellular location, protein interactions and the outcome of biochemical reaction chains. Modifications by ubiquitin (Ub) and ubiquitin-like modifiers systems are conserved in all eukaryotic organisms. One of them, small ubiquitin-like modifier (SUMO) is present in plants. The SUMO mechanism includes several isoforms of proteins that are involved in reactions of sumoylation and de-sumoylation. Sumoylation affects several important processes in plants. Outstanding among those are responses to environmental stresses. These may be abiotic stresses, such as phosphate deficiency, heat, low temperature, and drought, or biotic stressses, as well including defense reactions to pathogen infection. Also, the regulations of flowering time, cell growth and development, and nitrogen assimilation have recently been added to this list. Identification of SUMO targets is material to characterize the function of sumoylation or desumoylation. Affinity purification and mass spectrometric identification have been done lately in plants. Further SUMO noncovalent binding appears to have function in other model organisms and SUMO interacting proteins in plants will be of interest to plant biologists who dissect the dynamic function of SUMO. This review will discuss results of recent insights into the role of sumoylation in plants.     

类泛素化修饰蛋白SUMO1的表达纯化及抗体制备

SUMO是近年发现的类泛素化修饰蛋白,可通过异肽键共价连接到靶蛋白上,影响靶蛋白的细胞内定位、稳定性及与其它生物大分子的相互作用. 为研究蛋白质的SUMO化修饰,本文表达并利用亲和层析的方法纯化了重组的人SUMO1,制备了兔抗hSUMO1的多克隆抗体. 经ELISA和免疫印迹检测,获得了灵敏度高、特异性好的抗体,可用于SUMO化修饰靶蛋白的鉴定及SUMO化修饰的生物学功能研究.     

BH3-mimetic gossypol-induced autophagic cell death in mutant BRAF melanoma cells with high expression of p21

Aims

The aim of the present study was to identify the potential therapeutic effects of BH3-mimetic gossypol on melanoma cells with acquired resistance to BRAF inhibitors.

Main methods

The IC₅₀ values of gossypol were determined using MTT assays in three melanoma cell lines with different resistances to BRAF inhibitor. The effects of gossypol on three melanoma cell lines were further examined by immunoblotting analysis, cell cycle analysis, flow cytometric apoptotic assay and autophagy assay. The functional role of autophagy in gossypol-induced growth inhibition was investigated using siRNA-mediated knockdown of Beclin-1.

Key findings

Gossypol retained its efficacy in BRAF-V600E melanoma clones with acquired resistance to BRAF inhibitors through a mechanism independent of MEK–ERK inhibition. Gossypol caused G₂/M arrest in both BRAF mutant A375P and A375P/Mdr cells with high expression of p21^Cip1, regardless of their drug resistance. Interestingly, we determined that the lack of gossypol-induced mitotic arrest in BRAF-WT-harboring SK-MEL-2 cells was associated with a low level of p21^Cip1 expression. In addition, gossypol preferentially induced autophagy and apoptosis in the gossypol-sensitive cells and not in the gossypol-resistant SK-MEL-2 cells. In particular, alleviation of autophagy by knockdown of Beclin-1 partially caused a resistance to gossypol-induced cell cycle arrest at G₂/M in BRAF-V600E cells with a concomitant decreased induction of apoptosis.

Significance

Taken together, these results suggest that gossypol may exhibit potential for the treatment of BRAF inhibitor-resistant tumors, but a functional p21^Cip1 is a prerequisite for a positive response to its clinical application.     

吉林双阳型梅花鹿sentrin/SUMO的发现

为了确定梅花鹿未知的编码区cDNA,我们利用TaKaRa公司的cDNA 合成试剂盒及PCR cDNA文库试剂盒,构建了吉林双阳型梅花鹿子宫PCR cDNA文库。将文库的PCR产物克隆入pGEM-Teasy载体并进行测序后,应用BLAST网络服务对测得的序列在GenBank数据库中进行同源性比较。结果显示构建的PCR cDNA文库中包含有不同长度的cDNA片段,而且自该文库中我们发现了一与人sentrin-1/SUMO-1（small ubiquitin-related modifier 1）高度同源的全编码区cDNA序列。此序列已在Genbank登录,登录号为AF 242526。这说明我们自梅花鹿子宫PCR cDNA文库中发现了梅花鹿的sentrin/SUMO基因。 Abstract: In order to identify unknown encoding cDNAs of Cervus nioppon Temminck (sika deer),we constructed a cDNA library of uterus from Jilin-Shuangyang Cervus nippon Temminck using PCR cDNA library kit.PCR products of the library were cloned into pGEM-Teasy vectors and the cDNAs were sequenced and analyzed by nucleotide homology comparison against GenBank Database using the BLAST network service.The results showed that the cDNA library contained cDNA fragments of different lengths and a full length encoding cDNA highly homologous to human sentrin-1/SUMO-1 (small ubiquitin-related modifier 1) was identified.The cDNA was deposited in GenBank under the accession number AF 242526.These show that Cervus nippon Temminck-derived sentrin/SUMO gene has been discovered from PCR cDNA library of uterus from Cervus nippon Temminck.     

Generation of cytotoxic effector cells against human melanoma

Metastatic or tumor-draining lymph nodes from six of nine melanoma patients undergoing lymph node dissection for metastatic melanoma generated cytotoxic T cells against autologous melanoma when these lymph node cells were treated by in vitro sensitization and recombinant interleukin-2 (IL-2). During the initial lymphocyte culture (2–6 weeks), cross-reactivity with autologous tumor cells, K562 and Daudi cells was usually noted. Cold-target inhibition assay with K562 and Daudi showed K562/Daudi-associated antigens on melanoma cells. During the later phase of lymphocyte culture with repeated in vitro sensitization (over 6–10 weeks), cytotoxicity was noted against autologous and allogeneic melanoma cells but not against K562, Daudi cells or autologous fibroblasts. Repeated in vitro sensitization resulted in the selection of specific cytotoxic lymphocytes against melanoma. Cold-target inhibition assay with autologous and allogeneic melanoma cells revealed shared and individual antigens. Using blocking monoclonal antibodies, MHC-restricted killing was noted in the autologous system. Further, both the autologous and allogeneic systems could be mediated through adhesion molecules such as ICAM-1 and LFA-3 on melanoma cells and LFA-1 on T cells. This study suggests that a constellation of cytotoxic effector cells and melanoma-associated antigens may be pivotal in tumor killing. Thus, future adoptive immunotherapy should modulate and enhance this complex interaction.This work was supported by an Elsa, U. Pardee Foundation grant, the Arizona Chronic Disease Research Commission grant and partly by grant CA23074 from the National Institutes of Health, Bethesda, MD, 20892