Altered dynamics of ubiquitin hybrid proteins during tumor cell apoptosis

The ubiquitin hybrid genes Uba80 and Uba52 encode ubiquitin (Ub), which is fused to the ribosomal proteins S27a (RPS27a) and L40 (RPL40), respectively. Here, we show that these genes are preferentially over-expressed during hepatoma cell apoptosis. Experiments using the tet-inducible transgenic system revealed that over-expression of the ubiquitin hybrid genes sensitized the cells to apoptosis. Further analysis suggested that Ub, and not RPS27a or RPL40, was associated with apoptotic cell death. Cleavage-resistant mutation analysis revealed that the N-terminal portion and the last two amino acids (GG) of Ub are critical for cleavage at the junction between the two protein moieties. An apoptogenic stimulus enhances the nuclear targeting and aggregation of Ub in the nucleus, resulting in histone H2A deubiquitylation followed by abnormal ubiquitylation of the nuclear envelope and the lamina. These events accompany the apoptotic nuclear morphology in the late stage of apoptosis. Each fused RP is localized in the nucleoli. These results suggest a role for Ub hybrid proteins in the altered nuclear dynamics of Ub during tumor cell apoptosis induced by apoptogenic stimuli.     

昆虫杆状病毒泛素基因研究进展

昆虫杆状病毒是目前已知唯一编码泛素(ubiquitin)的病毒。迄今,已克隆了8种该类病毒的泛素基因。与真核生物Uba52(80)相似,这些基因在一个泛素分子的C末端都有不同长度的融合,其中斜纹夜蛾核多角体病毒(Spodopteralituramulticapsidnucleopolyhedrovirus,SpltMNPV)的ubiquitingp37基因是一个典型的融合基因。近年来,对苜蓿银纹夜蛾核多角体病毒(Autographacaliforniamulticapsidnucleopolyhedrovirus,AcMNPV)泛素的定位与功能研究取得了重要进展 。     

Characterization and expression of two chicken cDNAs encoding ubiquitin fused to ribosomal proteins of 52 and 80 amino acids

We have determined the complete nucleotide sequence of two chicken cDNAs, Ub-t52 and Ub-t80, encoding ubiquitin fused to ribosomal proteins of 52 and 80 amino acids. The deduced amino acid sequences of the ribosomal proteins are identical or very similar to the homologous human and rat proteins and to the corresponding proteins of other species. Unexpectedly, the ubiquitin moiety of the Ub-t52 protein showed two amino acid substitutions: serine-20 has been replaced by asparagine and serine-57 by alanine. Ubiquitin is a protein strongly conserved during evolution, with no changes in sequence previously reported in vertebrates. Ub-t52 and Ub-t80 are highly expressed in early embryogenesis and during postmitotic stages of spermatogenesis, in parallel with the expression of the polyubiquitin gene UbII. Whereas the 5′ untranslated regions (5′UTRs) of the chicken polyubiquitin mRNAs showed marked differences in mature testes in relation to somatic tissues, no differences were observed in the 5′UTRs of the ubiquitin-ribosomal protein mRNAs. These mRNAs possess a 5′-terminal oligopyrimidine tract that could be used as a mechanism to postpone translation during postmitotic stages of spermatogenesis, as has been proposed in quiescent cells.     

Ubiquitin--conserved protein or selfish gene?

The posttranslational modifier ubiquitin is encoded by a multigene family containing three primary members, which yield the precursor protein polyubiquitin and two ubiquitin moieties, Ub(L40) and Ub(S27), that are fused to the ribosomal proteins L40 and S27, respectively. The gene encoding polyubiquitin is highly conserved and, until now, those encoding Ub(L40) and Ub(S27) have been generally considered to be equally invariant. The evolution of the ribosomal ubiquitin moieties is, however, proving to be more dynamic. It seems that the genes encoding Ub(L40) and Ub(S27) are actively maintained by homologous recombination with the invariant polyubiquitin locus. Failure to recombine leads to deterioration of the sequence of the ribosomal ubiquitin moieties in several phyla, although this deterioration is evidently constrained by the structural requirements of the ubiquitin fold. Only a few amino acids in ubiquitin are vital for its function, and we propose that conservation of all three ubiquitin genes is driven not only by functional properties of the ubiquitin protein, but also by the propensity of the polyubiquitin locus to act as a 'selfish gene'.     

一种新的遍在蛋白融合基因Uba256的克隆及表达

斜纹夜蛾核多角体病毒 (Spodopteralituramulticapsidnucleopolyhedrovirus ,SpltMNPV)的Uba2 5 6基因是已知昆虫病毒基因组中惟一编码遍在蛋白与GP37融合蛋白的基因。通过PCR方法扩增得到该基因及其N端的遍在蛋白编码区与C端的GP37编码区。将这 3个基因片段分别克隆至质粒pBV2 2 0与pQE30 ,构建得到重组质粒pB VUBCP、pQEUB与pQECP。pBVUBCP转化大肠杆菌DH5α ,经热激诱导表达了一条 38kD的蛋白带 ,证明Uba2 5 6表达的蛋白在大肠杆菌中没有发生剪切。pQEUB、pQECP转化大肠杆菌M15 ,经IPTG诱导 ,Western印迹分析结果表明遍在蛋白与GP37蛋白获得高效表达。以Ni2 NTA偶联抗体检测证明所表达的蛋白质均为融合蛋白质。纯化的融合蛋白质免疫新西兰大白兔可诱导产生特异抗体。ELISA和Western印迹检测结果显示 ,SpltMNPV遍在蛋白抗体及GP37抗体均与表达的融合蛋白质呈阳性反应 ,表明所表达的融合蛋白质仍保持原有蛋白质的免疫原性。以获得的抗体对感染SpltMNPV 72h的Sl zsu 1细胞进行检测 ,发现仅有一条 34kD的蛋白质带与GP37抗体发生特异反应 ;而有 4条分子量分别为 14、2 4、33、5 1kD的蛋白质带与遍在蛋白抗体发生特异反应 ,表明Uba2 5 6基因表达的蛋白质在宿主细胞内发生了剪切。     

The yeast ubiquitin genes: a family of natural gene fusions.

Ubiquitin is a 76-residue protein highly conserved among eukaryotes. Conjugation of ubiquitin to intracellular proteins mediates their selective degradation in vivo. We describe a family of four ubiquitin-coding loci in the yeast Saccharomyces cerevisiae. UB11, UB12 and UB13 encode hybrid proteins in which ubiquitin is fused to unrelated ('tail') amino acid sequences. The ubiquitin coding elements of UB11 and UB12 are interrupted at identical positions by non-homologous introns. UB11 and UB12 encode identical 52-residue tails, whereas UB13 encodes a different 76-residue tail. The tail amino acid sequences are highly conserved between yeast and mammals. Each tail contains a putative metal-binding, nucleic acid-binding domain of the form Cys-X2-4-Cys-X2-15-Cys-X2-4-Cys, suggesting that these proteins may function by binding to DNA. The fourth gene, UB14, encodes a polyubiquitin precursor protein containing five ubiquitin repeats in a head-to-tail, spacerless arrangement. All four ubiquitin genes are expressed in exponentially growing cells, while in stationary-phase cells the expression of UB11 and UB12 is repressed. The UB14 gene, which is strongly inducible by starvation, high temperatures and other stresses, contains in its upstream region strong homologies to the consensus 'heat shock box' nucleotide sequence. Elsewhere we show that the essential function of the UB14 gene is to provide ubiquitin to cells under stress.     

The E1 ubiquitin-activating enzyme Uba1 in Drosophila controls apoptosis autonomously and tissue growth non-autonomously

Ubiquitination is an essential process regulating turnover of proteins for basic cellular processes such as the cell cycle and cell death (apoptosis). Ubiquitination is initiated by ubiquitin-activating enzymes (E1), which activate and transfer ubiquitin to ubiquitin-conjugating enzymes (E2). Conjugation of target proteins with ubiquitin is then mediated by ubiquitin ligases (E3). Ubiquitination has been well characterized using mammalian cell lines and yeast genetics. However, the consequences of partial or complete loss of ubiquitin conjugation in a multi-cellular organism are not well understood. Here, we report the characterization of Uba1, the only E1 in Drosophila. We found that weak and strong Uba1 alleles behave genetically differently with sometimes opposing phenotypes. Whereas weak Uba1 alleles protect cells from cell death, clones of strong Uba1 alleles are highly apoptotic. Strong Uba1 alleles cause cell cycle arrest which correlates with failure to reduce cyclin levels. Surprisingly, clones of strong Uba1 mutants stimulate neighboring wild-type tissue to undergo cell division in a non-autonomous manner giving rise to overgrowth phenotypes of the mosaic fly. We demonstrate that the non-autonomous overgrowth is caused by failure to downregulate Notch signaling in Uba1 mutant clones. In summary, the phenotypic analysis of Uba1 demonstrates that impaired ubiquitin conjugation has significant consequences for the organism, and may implicate Uba1 as a tumor suppressor gene.     

Cytoplasmic ribosomal protein genes of the fission yeast Schizosaccharomyces pombe display a unique promoter type: a suggestion for nomenclature of cytoplasmic ribosomal proteins in databases.

We identified 34 new ribosomal protein genes in the Schizosaccharomyces pombe database at the Sanger Centre coding for 30 different ribosomal proteins. All contain the Homol D-box in their promoter. We have shown that Homol D is, in this promoter type, the TATA-analogue. Many promoters contain the Homol E-box, which serves as a proximal activation sequence. Furthermore, comparative sequence analysis revealed a ribosomal protein gene encoding a protein which is the equivalent of the mammalian ribosomal protein L28. The budding yeast Saccharomyces cerevisiae has no L28 equivalent. Over the past 10 years we have isolated and characterized nine ribosomal protein (rp) genes from the fission yeast S.pombe . This endeavor yielded promoters which we have used to investigate the regulation of rp genes. Since eukaryotic ribosomal proteins are remarkably conserved and several rp genes of the budding yeast S.cerevisiae were sequenced in 1985, we probed DNA fragments encoding S.cerevisiae ribosomal proteins with genomic libraries of S.pombe . The deduced amino acid sequence of the different isolated rp genes of fission yeast share between 65 and 85% identical amino acids with their counterparts of budding yeast.     

Mechanistic studies on activation of ubiquitin and di-ubiquitin-like protein, FAT10, by ubiquitin-like modifier activating enzyme 6, Uba6

Uba6 is a homolog of the ubiquitin-activating enzyme, Uba1, and activates two ubiquitin-like proteins (UBLs), ubiquitin and FAT10. In this study, biochemical and biophysical experiments were performed to understand the mechanisms of how Uba6 recognizes two distinct UBLs and catalyzes their activation and transfer. Uba6 is shown to undergo a three-step activation process and form a ternary complex with both UBLs, similar to what has been observed for Uba1. The catalytic mechanism of Uba6 is further supported by inhibition studies using a mechanism-based E1 inhibitor, Compound 1, which forms covalent adducts with both ubiquitin and FAT10. In addition, pre-steady state kinetic analysis revealed that the rates of UBL-adenylate (step 1) and thioester (step 2) formation are similar between ubiquitin and FAT10. However, distinct kinetic behaviors were also observed for ubiquitin and FAT10. FAT10 binds Uba6 with much higher affinity than ubiquitin while demonstrating lower catalytic activity in both ATP-PP(i) exchange and E1-E2 transthiolation assays. Also, Compound 1 is less potent with FAT10 as the UBL compared with ubiquitin in ATP-PP(i) exchange assays, and both a slow rate of covalent adduct formation and weak adduct binding to Uba6 contribute to the diminished potency observed for FAT10. Together with expression level analysis in IM-9 cells, this study sheds light on the potential role of cytokine-induced FAT10 expression in regulating Uba6 pathways.     

Ubiquitins (polyubiquitin and ubiquitin extension protein) in marine sponges: cDNA sequence and phylogenetic analysis

The complete nucleotide sequences of two Suberites domuncula cDNAs and one Sycon raphanus cDNA, all encoding ubiquitin, have been determined. One cDNA from S. domuncula codes for polyubiquitin with four tandemly repeated monomeric units and the second cDNA encodes ubiquitin fused to a ribosomal protein of 78 amino acids (aa). S. domuncula possesses at least one additional polyubiquitin gene, from which the last two monomers were also sequenced. All analysed genes from S. domuncula encode identical ubiquitin proteins, with only one aa difference (Ala 19) to the human/higher animals ubiquitin (Pro 19). Ubiquitin in S. domuncula is identical with the ubiquitin found in another Demospongia, Geodia cydonium. The cDNA from S. raphanus encodes polyubiquitin with seven tandemly repeated units. All these gene monomers code for the same ubiquitin, which differs from the human/higher animals ubiquitin only at position 24 (Asp in Sycon, Glu in others). However, ubiquitin from S. raphanus (Calcarea) shows two aa differences (positions 19 and 24), when compared with the ubiquitin sequences from the two Demospongiae. In a phylogenetic tree constructed by multiple sequence alignment of all sponge ubiquitin gene monomers so far identified, all monomers from the same species cluster together, with the clear exception of the monomer from S. domuncula ribosomal protein fusion gene. This monomer branches off first from the tree and forms a separate line; this gives evidence for a very ancient split of ubiquitin-ribosomal-protein fusion genes from polyubiquitin encoding genes and their long separate coexistence in eukaryotes. The ubiquitin extension protein from S. domuncula is 78 aa long, displays all characteristics of 76–81 aa long ribosomal fusion proteins and shows 78% identity in the first 73 aa with the human S27a protein. However, its C-terminal sequence: 69-GLTYVYKKSD-78 is more similar to the plant consensus (69-GLTYVYQ/NK-76), than to the higher animal consensus (69-CLTYCFNK-76). This protein isolated from a sponge, belonging to the phylogenetically oldest multicellular animals, the Porifera, branches off first from the phylogenetic tree of metazoan ubiquitin extension proteins of the small ribosomal subunits.     

Pollen-specific gene expression in transgenic plants: coordinate regulation of two different tomato gene promoters during microsporogenesis

To investigate the regulation of gene expression during male gametophyte development, we analyzed the promoter activity of two different genes (LAT52 and LAT59) from tomato, isolated on the basis of their anther-specific expression. In transgenic tomato, tobacco and Arabidopsis plants containing the LAT52 promoter region fused to the beta-glucuronidase (GUS) gene, GUS activity was restricted to pollen. Transgenic tomato, tobacco and Arabidopsis plants containing the LAT59 promoter region fused to GUS also showed very high levels of GUS activity in pollen. However, low levels of expression of the LAT59 promoter construct were also detected in seeds and roots. With both constructs, the appearance of GUS activity in developing anthers was correlated with the onset of microspore mitosis and increased progressively until anthesis (pollen shed). Our results demonstrate co-ordinate regulation of the LAT52 and LAT59 promoters in developing microspores and suggest that the mechanisms that regulate pollen-specific gene expression are evolutionarily conserved.     

眼镜王蛇泛素融合蛋白基因和核糖体蛋白L30基因的克隆及分析

从广西产眼镜王蛇（Ophiophagus hannah）毒腺中抽提总RNA,经mRNA纯化后构建眼镜王蛇毒腺cDNA文库。从所构建的cDNA文库中,随机筛选200个克隆测序,得到两个在进化上高度保守的基因：泛素融合蛋白基因（GenBank登录号为AF297036）和核糖体蛋白L30基因（GenBank登录号是AF297033）。前者cDNA的开放阅读框为387bp,后者为348bp。前者编码128个氨基酸残基组成的泛素融合蛋白前体;后者编码115个氨基酸残基组成的核糖体蛋白L30前体。由cDNA序列推导出的氨基酸序列分析表明,泛素融合蛋白前体包括N-末端的泛素结构域（76个氨基酸残基）和C-末端的核糖体蛋白L40结构域（52个氨基酸残基）。该蛋白为一高碱性蛋白,C末端含有一个“锌指”模式结构。与16个物种比较的结果表明,眼镜王蛇与脊椎动物的泛素融合蛋白氨基酸序列相似度较高,具有高度的保守性。     

Solution structure and dynamics of Ufm1, a ubiquitin-fold modifier 1

The ubiquitin-fold modifier 1 (Ufm1) is one of various ubiquitin-like modifiers and conjugates to target proteins in cells through Uba5 (E1) and Ufc1 (E2). The Ufm1-system is conserved in metazoa and plants, suggesting its potential roles in various multicellular organisms. Herein, we analyzed the solution structure and dynamics of human Ufm1 (hsUfm1) by nuclear magnetic resonance spectroscopy. Although the global fold of hsUfm1 is similar to those of ubiquitin (Ub) and NEDD8, the cluster of acidic residues conserved in Ub and NEDD8 does not exist on the Ufm1 surface. 15N spin relaxation data revealed that the amino acid residues of hsUfm1 exhibiting conformational fluctuations form a cluster at the C-terminal segment and its spatial proximity, which correspond to the versatile ligand-binding sites of Ub and other ubiquitin-like proteins (Ubls). We suggest that Ub and other Ubl-modifiers share a common feature of potential conformational multiplicity, which might be associated with the broad ligand specificities of these proteins.     

Pineapple translation factor SUI1 and ribosomal protein L36 promoters drive constitutive transgene expression patterns in Arabidopsis thaliana

The availability of a variety of promoter sequences is necessary for the genetic engineering of plants, in basic research studies and for the development of transgenic crops. In this study, the promoter and 5′ untranslated regions of the evolutionally conserved protein translation factor SUI1 gene and ribosomal protein L36 gene were isolated from pineapple and sequenced. Each promoter was translationally fused to the GUS reporter gene and transformed into the heterologous plant system Arabidopsis thaliana. Both the pineapple SUI1 and L36 promoters drove GUS expression in all tissues of Arabidopsis at levels comparable to the CaMV35S promoter. Transient assays determined that the pineapple SUI1 promoter also drove GUS expression in a variety of climacteric and non-climacteric fruit species. Thus the pineapple SUI1 and L36 promoters demonstrate the potential for using translation factor and ribosomal protein genes as a source of promoter sequences that can drive constitutive transgene expression patterns.