A novel polyubiquitin structure in Cercozoa and Foraminifera: evidence for a new eukaryotic supergroup

Ubiquitin is a 76 amino acid protein with a remarkable degree of evolutionary conservation. Ubiquitin plays an essential role in a large number of eukaryotic cellular processes by targeting proteins for proteasome-mediated degradation. Most ubiquitin genes are found as head-to-tail polymers whose products are posttranslationally processed to ubiquitin monomers. We have characterized polyubuiquitin genes from the photosynthetic amoeboflagellate Chlorarachnion sp. CCMP 621 (also known as Bigelowiella natans) and found that they deviate from the canonical polyubiquitin structure in having an amino acid insertion at the junction between each monomer, suggesting that polyubiquitin processing in this organism is unique among eukaryotes. The gene structure indicates that processing likely cleaves monomers at the amino terminus of the insertion. We examined the phylogenetic distribution of the insertion by sequencing polyubiquitin genes from several other eukaryotic groups and found it to be confined to Cercozoa (including Chlorarachnion, Lotharella, Cercomonas, and Euglypha) and Foraminifera (including Reticulomyxa and Haynesina). This character strongly suggests that Cercozoa and Foraminifera are close relatives and form a new "supergroup" of eukaryotes.     

The novel functions of ubiquitination in signaling

Ubiquitin is best known for its function in targeting proteins for degradation by the proteasome. Recent studies have revealed several new functions of ubiquitin that are independent of proteasomal degradation. These functions include the novel signaling roles of ubiquitin in DNA repair and the activation of protein kinases such as IkappaB kinase. In both cases, a novel form of polyubiquitin chain linked through lysine-63 of ubiquitin plays an important regulatory role. Monoubiquitination also has signaling roles that are distinct from those of polyubiquitination, as illustrated from the studies of DNA repair. Thus, polyubiquitination and monoubiquitination have emerged as important signaling mechanisms that control diverse physiological and pathological processes.     

Essential factors determining codon usage in ubiquitin genes

Summary Ubiquitin is ubiquitous in all eukaryotes and its amino acid sequence shows extreme conservation. Ubiquitin genes comprise direct repeats of the ubiquitin coding unit with no spacers. The nucleotide sequences coding for 13 ubiquitin genes from 11 species reported so far have been compiled and analyzed. The G+C content of codon third base reveals a positive linear correlation with the genome G+C content of the corresponding species. The slope strongly suggests that the overall G+C content of codons of polyubiquitin genes clearly reflects the genome G+C content by AT/GC substitutions at the codon third position. The G+C content of ubiquitin codon third base also shows a positive linear correlation with the overall G+C content of coding regions of compiled genes, indicating the codon choices among synonymous codons reflect the average codon usage pattern of corresponding species. On the other hand, the monoubiquitin gene, which is different from the polyubiquitin gene in gene organization, gene expression, and function of the encoding protein, shows a different codon usage pattern compared with that of the polyubiquitin gene. From comparisons of the levels of synonymous substitutions among ubiquitin repeats and the homology of the amino acid sequence of the tail of monomeric ubiquitin genes, we propose that the molecular evolution of ubiquitin genes occurred as follows: Plural primitive ubiquitin sequences were dispersed on genome in ancestral eukaryotes. Some of them situated in a particular environment fused with the tail sequence to produce monomeric ubiquitin genes that were maintained across species. After divergence of species, polyubiquitin genes were formed by duplication of the other primitive ubiquitin sequences on different chromosomes. Differences in the environments in which ubiquitin genes are embedded reflect the differences in codon choice and in gene expression pattern between poly- and monomeric ubiquitin genes.     

The mouse polyubiquitin gene Ubb is essential for meiotic progression

Ubiquitin is encoded in mice by two polyubiquitin genes, Ubb and Ubc, that are considered to be stress inducible and two constitutively expressed monoubiquitin (Uba) genes. Here we report that targeted disruption of Ubb results in male and female infertility due to failure of germ cells to progress through meiosis I and hypogonadism. In the absence of Ubb, spermatocytes and oocytes arrest during meiotic prophase, before metaphase of the first meiotic division. Although cellular ubiquitin levels are believed to be maintained by a combination of functional redundancy among the four ubiquitin genes, stress inducibility of the two polyubiquitin genes, and ubiquitin recycling by proteasome-associated isopeptidases, our results indicate that ubiquitin is required for and consumed during meiotic progression. The striking similarity of the meiotic phenotype in Ubb^−/− germ cells to the sporulation defect in fission yeast (Schizosaccharomyces pombe) lacking a polyubiquitin gene suggests that a meiotic role of the polyubiquitin gene has been conserved throughout eukaryotic evolution.     

Ubiquitin: not just for proteasomes anymore

Ubiquitin is a small protein that can be covalently linked to itself or other proteins, either as single ubiquitin molecules or as chains of polyubiquitin. Addition of ubiquitin to a target protein requires a series of enzymatic activities (by ubiquitin-activating, -conjugating and -ligating enzymes). The first function attributed to ubiquitin was the covalent modification of misfolded cytoplasmic proteins, thereby directing proteasome-dependent proteolysis. More recently, additional functions have been ascribed to ubiquitin and ubiquitin-related proteins. Ubiquitin directs specific proteins through the endocytic pathway by modifying cargo proteins, and possibly also components of the cytoplasmic protein trafficking machinery.     

Ubiquitin family proteins and their relationship to the proteasome: a structural perspective

Many biological processes rely on targeted protein degradation, the dysregulation of which contributes to the pathogenesis of various diseases. Ubiquitin plays a well-established role in this process, in which the covalent attachment of polyubiquitin chains to protein substrates culminates in their degradation via the proteasome. The three-dimensional structural topology of ubiquitin is highly conserved as a domain found in a variety of proteins of diverse biological function. Some of these so-called "ubiquitin family proteins" have recently been shown to bind components of the 26S proteasome via their ubiquitin-like domains, thus implicating proteasome activity in pathways other than protein degradation. In this chapter, we provide a structural perspective of how the ubiquitin family of proteins interacts with the proteasome.     

UbiA, the major polyubiquitin locus in Caenorhabditis elegans, has unusual structural features and is constitutively expressed.

Ubiquitin is a multifunctional 76-amino-acid protein which plays critical roles in many aspects of cellular metabolism. In Caenorhabditis elegans, the major source of ubiquitin RNA is the polyubiquitin locus, UbiA. UbiA is transcribed as a polycistronic mRNA which contains 11 tandem repeats of ubiquitin sequence and possesses a 2-amino-acid carboxy-terminal extension on the final repeat. The UbiA locus possesses several unusual features not seen in the ubiquitin genes of other organisms studied to date. Mature UbiA mRNA acquires a 22-nucleotide leader sequence via a trans-splicing reaction involving a 100-nucleotide splice leader RNA derived from a different chromosome. UbiA is also unique among known polyubiquitin genes in containing four cis-spliced introns within its coding sequence. Thus, UbiA is one of a small class of genes found in higher eucaryotes whose heterogeneous nuclear RNA undergoes both cis and trans splicing. The putative promoter region of UbiA contains a number of potential regulatory elements: (i) a cytosine-rich block, (ii) two sequences resembling the heat shock regulatory element, and (iii) a palindromic sequence with homology to the DNA-binding site of the mammalian steroid hormone receptor. The expression of the UbiA gene has been studied under various heat shock conditions and has been monitored during larval moulting and throughout the major stages of development. These studies indicate that the expression of the UbiA gene is not inducible by acute or chronic heat shock and does not appear to be under nutritional or developmental regulation in C. elegans.     

泛素蛋白磷酸化修饰的功能与解析

泛素化是存在于真核生物中一种重要的翻译后修饰过程,参与调控包括蛋白质降解在内的多种生命活动。实现这一调控过程需要将一个由76个氨基酸组成的泛素蛋白共价连接到底物蛋白上。同时,泛素本身也存在多种翻译后修饰,包括泛素化、磷酸化、乙酰化等,进一步丰富了泛素的修饰类型,决定了底物蛋白不同的命运。近年来,伴随着第65位丝氨酸磷酸化泛素蛋白参与调控线粒体自噬这一突破性进展,泛素蛋白其余磷酸化位点的功能研究也获得越来越多的关注。本文根据目前已有的国内外研究和报道,总结了泛素蛋白已知的磷酸化修饰位点,梳理了泛素蛋白第12位和66位苏氨酸、第57位和65位丝氨酸等位点的磷酸化修饰对其生物物理特性带来的改变,并对相应修饰位点所涉及的生物学功能调控进行了综述。     

Concerted evolution in protists: Recent homogenization of a polyubiquitin gene in Trichomonas vaginalis

Ubiquitin is a 76-amino-acid protein with a remarkably high degree of conservation between all known sequences. Ubiquitin genes are almost always multicopy in eukaryotes, and often are found as polyubiquitin genes—fused tandem repeats which are coexpressed. Seventeen ubiquitin sequences from the amitochondrial protist Trichomonas vaginalis have been examined here, including an 11-repeat fragment of a polyubiquitin gene. These sequences reveal a number of interesting features that are not seen in other eukaryotes. The predicted amino acid sequences lack several universally conserved residues, and individual units do not always encode identical peptides as is usually the case. On the nucleotide level, these repeats are in general highly variable, but one region in the polyubiquitin is extremely homogeneous, with seven repeats absolutely identical. Such extended stretches of homogeneity have never been observed in ubiquitin genes and since substitutions are common in other coding units, it is likely that these repeats are the product of a very recent homogenization or amplification. Correspondence to: P.J. Keeling     

Analysis of ubiquitin E3 ligase activity using selective polyubiquitin binding proteins

The ubiquitin proteasome pathway controls the cellular degradation of ~80-90% of the proteome in a highly regulated manner. In this pathway, E3 ligases are responsible for the conjugation of ubiquitin to protein substrates which can lead to their destruction by the 26S proteasome. Aberrant E3 ligases have been implicated in several diseases and are widely recognized as attractive targets for drug discovery. As researchers continue to characterize E3 ligases, additional associations with various disease states are being exposed. The availability of assays that allow rapid analysis of E3 ligase activity is paramount to both biochemical studies and drug discovery efforts aimed at E3 ligases. To address this need, we have developed a homogenous assay for monitoring ubiquitin chain formation using Tandem Ubiquitin Binding Entities (TUBEs). TUBEs bind selectively to polyubiquitin chains versus mono-ubiquitin thus enabling the detection of polyubiquitin chains in the presence of mono-ubiquitin. This assay reports on the proximity between the protein substrate and TUBEs as a result of polyubiquitin chain formation by an E3 ligase. This homogenous assay is a step forward in streamlining an approach for characterizing and quantitating E3 ligase activity in a rapid and cost effective manner. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.     

Modulation of HIV-1 Rev protein abundance and activity by polyubiquitination with unconventional Lys-33 branching

The HIV-1 Rev protein plays a key role in virus replication by allowing export to the cytoplasm of unspliced or singly-spliced RNAs. In this report, we investigated whether Rev is modified by ubiquitination or sumoylation. Whereas no evidence of sumoylation was obtained, transient expression experiments showed that ubiquitin conjugates to Rev as high molecular weight polyubiquitin chains. Mutation of the three lysine residues of Rev showed that the site of ubiquitin conjugation is Lys-115. Experiments with ubiquitin mutants including a single lysine at every seven possible position indicated that branching of the polyubiquitin chains mainly involves Lys-33. Mutation of Rev Lys-115 to arginine reduces markedly the steady state amount of the protein, but does not impair its ability to export RNA via the Rev response element. These observations support the notion that polyubiquitination of Rev stabilizes the viral protein but hinders its activity.     

Budding yeast Dsk2 protein forms a homodimer via its C-terminal UBA domain

Budding yeast Dsk2 is a family of UbL-UBA proteins that can interact with both polyubiquitin and the proteasome, and is thereby thought to function as a shuttle protein in the ubiquitin-proteasome pathway. Here we show that Dsk2 can homodimerize via its C-terminal UBA domain in the absence of ubiquitin. Dsk2 mutants defective in the UBA domain do not dimerize and do not bind polyubiquitin. The expression of Dsk2 UBA mutants fails to restore the growth defect caused by DSK2 disruption although that of wild-type Dsk2 can restore the defect. These results suggest that Dsk2 homodimerization via the UBA domain plays a role in regulating polyubiquitin binding in the ubiquitin-proteasome pathway.     

Neddylation dysfunction in Alzheimer's disease

Ubiquitin‐dependent proteolysis is a major mechanism that downregulates misfolded proteins or those that have finished a programmed task. In the last two decades, neddylation has emerged as a major regulatory pathway for ubiquitination. Central to the neddylation pathway is the amyloid precursor protein (APP)‐binding protein APP‐BP1, which together with Uba3, plays an analogous role to the ubiquitin‐activating enzyme E1 in nedd8 activation. Activated nedd8 covalently modifies and activates a major class of ubiquitin ligases called Cullin‐RING ligases (CRLs). New evidence suggests that neddylation also modifies Type‐1 transmembrane receptors such as APP. Here we review the functions of neddylation and summarize evidence suggesting that dysfunction of neddylation is involved in Alzheimer's disease.     

Ubiquitin in stressed chicken embryo fibroblasts

Ubiquitin, a small 76-amino acid protein which is highly conserved in eukaryotic cells, occurs in several forms other than the free polypeptide. Among these are protein conjugates in which ubiquitin is covalently linked in lysylpeptide bond to lysl residues of other proteins and fusion proteins in which the amino-terminal domain is the precise ubiquitin sequence. Ubiquitin plays a role in cellular proteolytic degradation and in chromatin structure and has been postulated to be involved in the induction of a set of proteins which function during the cellular response to various kinds of environmental stress. We have measured the various forms of ubiquitin in cultures of chicken embryo fibroblasts under normal growth conditions and after treatment with a thermal or chemical stress. Levels of free ubiquitin fell slightly, ubiquitin conjugate levels rose shortly after stress began, and both then increased substantially as one of the cell's ubiquitin-encoding genes was activated by stress. The level of a protein synthesized as the carboxyl-terminal domain of one ubiquitin fusion protein was unchanged by a heat stress. The most dramatic effect was seen in the rapid disappearance of the ubiquitinated form of histone H2A, one of the major ubiquitin conjugates in cells in the interphase portion of their growth cycle. A significant rise in protein turnover was detected as a result of the stress, but occurred only when cells were removed from the stress condition. These results suggest that ubiquitin plays an important role both during and after stress, but fails to support hypotheses for ubiquitin and proteolysis in the activation of stress genes.     

Polyubiquitin gene expression and structural properties of the ubi4-2 gene in Petroselinum crispum

Ubiquitin is an omnipresent protein found in all eukaryotes so far analysed. It is involved in several important processes, including protein turnover, chromosome structure and stress response. Parsley (Petroselinum crispum) contains at least two active polyubiquitin (ubi4) genes encoding hexameric precursor proteins. The deduced amino acid sequences of the ubiquitin monomers are identical to one another and to ubiquitin sequences from several other plant species. Analysis of the promoter region of one ubi4 gene revealed putative regulatory elements. In parsley plants, the ubi4 mRNAs were the predominant ubiquitin mRNAs and were present at comparable levels in all plant organs tested. In cultured parsley cells, high levels of ubiquitin gene expression remained unaffected by heat shock, elicitor or light treatment.     

Ubiquitin in homeostasis,development and disease

Ubiquitin is the most phylogenetically conserved protein known. This 8,500 Da polypeptide can be covalently attached to cellular proteins as a posttranslational modification. In most cases, the addition of multiple ubiquitin adducts to a protein targets it for rapid degradation by a multisubunit protease known as the 26S proteasome. While the ubiquitin/26S proteasome pathway is responsible for the degradation of the bulk of cellular proteins during homeostasis, it may also be responsible for the rapid loss of protein during the programmed death of certain cells, such as skeletal muscle during insect metamorphosis. In addition, alterations in the expression and regulation of ubiquitin may play significant roles in pathological disorders. For example, dramatic increases in ubiquitin and ubiquitin-protein conjugates are observed in a wide variety of neurodegenerative disorders, including Alzheimer's disease. Patients suffering from the autoimmune disease systemic lupus erythematosus generate antibodies reacting with ubiquitin and ubiquitinated histones. At present, it is not known whether these changes in ubiquitin expression and regulation initiate pathological changes in these diseases or if they are altered as a consequence of these disorders.