Interspecific Comparison in the Frequency of Concerted Evolution at the Polyubiquitin Gene Locus

The polyubiquitin gene, encoding tandemly repeated multiple ubiquitins, constitutes a uniquitin gene subfamily. It has been demonstrated that polyubiquitin genes are subject to concerted evolution; namely, the individual ubiquitin coding units contained within a polyubiquitin gene are more similar to one another than they are to the ubiquitin coding units in the orthologous gene from other species. However there has been no comprehensive study on the concerted evolution of polyubiquitin genes in a wide range of species, because the relationships (orthologous or paralogous) among multiple polyubiquitin genes from different species have not been extensively analyzed yet. In this report, we present the results of analyzing the nucleotide sequence of polyubiquitin genes of mammals, available in the DDBJ/EMBL/GenBank nucleotide sequence databases, in which we found that there are two groups of polyubiquitin genes in an orthologous relationship. Based on this result, we analyzed the concerted evolution of the polyubiquitin gene in various species and compared the frequency of concerted evolutionary events interspecifically by taking into consideration that the rate of synonymous substitution at the polyubiquitin gene locus may vary depending on species. We found that the concerted evolutionary events in polyubiquitin genes have been more frequent in rats and Chinese hamsters than those in humans, cows, and sheep. The guinea pig polyubiquitin gene was an intermediate example. The frequency of concerted evolution in the mouse gene was unexpectedly low compared to that of other rodent genes. Received: 18 January 2000 / Accepted: 26 April 2000     

The mitochondrial pathway and reactive oxygen species are critical contributors to interferon-α/β-mediated apoptosis in Ubp43-deficient hematopoietic cells

Stress associated proteins (SAPs) in plants contain A20-type zinc finger (A20_ZF) domains and are involved with abiotic stress response. A20-type zinc finger domains in animals reportedly recognize ubiquitin as a regulatory signal in cell. However, it remains unclear whether A20_ZF domains in plants perform similar roles. AtSAP5, a SAP from Arabidopsis thaliana, exhibits a unique sequence feature among 10 AtSAPs harboring A20_ZF domains. The highly conserved diaromatic patch is replaced by the dialipathic patch. Here we investigated whether AtSAP5 recognizes ubiquitin and the roles of the dialipathic patch in ubiquitin binding in vitro. GST pulldown assay reveals that AtSAP5 binds polyubiquitin rather than monoubiquitin. AtSAP5 shows preferences for linear and K63-linked polyubiquitin chains to K48-linked one. The A20_ZF domain of AtSAP5 is sufficient for linkage-specific polyubiquitin recognition. The dialipathic patch in AtSAP5 plays an important role in K48-linked polyubiquitin recognition. Taken together, our results suggest that AtSAP5 participates in polyubiquitin recognition in plants and that the dialipathic patch in AtSAP5 is critical in binding K48-linked polyubiquitn chains.     

Characterization of the nucleotide sequence of a polyubiquitin gene (PUBC1) from Arabian camel, Camelus dromedarius

Molecular amplification and sequencing of genomic DNA that encodes camel polyubiquitin (PUBC1) was performed by a polymerase chain reaction (PCR) using various sets of primers. The amplification generated a number of DNA fragments, which were sequenced and compared with the polyubiquitin coding sequences of various species. One DNA fragment that conformed to 325 bp was found to be 95 and 88% homologous to the sequences of human polyubiquitin B and C, respectively. The DNA translated into 108 amino acids that corresponded to two fused units of ubiquitin with no intervening sequence, which indicates that it is a polyubiquitin and contains at least two units of ubiquitin. Although, variations were found in the nucleotide sequence when compared to those of other species, the amino acid sequence was 100% homologous to the polyubiquitin sequences of humans, mice, and rats. This is the first report of the polyubiquitin DNA coding sequence and its corresponding amino acid sequence from camels, amplified using direct genomic DNA preparations.     

Insights into the molecular composition of endogenous unanchored polyubiquitin chains

The diverse influences of ubiquitin, mediated by its post-translational covalent modification of other proteins, have been extensively investigated. However, more recently roles for unanchored (nonsubstrate linked) polyubiquitin chains have also been proposed. Here we describe the use of ubiquitin-binding domains to affinity purify endogenous unanchored polyubiquitin chains and their subsequent characterization by mass spectrometry (MS). Using the A20 Znf domain of the ubiquitin receptor ZNF216 we isolated a protein from skeletal muscle shown by a combination of nanoLC-MS and LC-MS/MS to represent an unmodified and unanchored K48-linked ubiquitin dimer. Selective purification of unanchored polyubiquitin chains using the Znf UBP (BUZ) domain of USP5/isopeptidase-T allowed the isolation of K48 and K11-linked ubiquitin dimers, as well as revealing longer chains containing as many as 15 ubiquitin moieties, which include the K48 linkage. Top-down nanoLC-MS/MS of the A20 Znf-purified ubiquitin dimer generated diagnostic ions consistent with the presence of the K48 linkage, illustrating for the first time the potential of this approach to probe connectivity within endogenous polyubiquitin modifications. As well as providing initial proteomic insights into the molecular composition of endogenous unanchored polyubiquitin chains, this work also represents the first definition of polyubiquitin chain length in vivo.     

Activation of polyubiquitin gene expression during developmentally programmed cell death

Ubiquitin, a highly conserved 76 amino acid protein, plays a role in targeting intracellular proteins for degradation. Ubiquitin expression was examined during the developmentally programmed atrophy and degeneration of the intersegmental muscles (ISMs) in the hawk-moth, Manduca sexta. A clone containing nine repeats of the ubiquitin coding sequence was isolated from an ISM cDNA library and was used as a probe to examine polyubiquitin expression during development. When the ISMs became committed to degenerate, polyubiquitin gene expression increased dramatically. Injection of 20-hydroxyecdysone, which delays degeneration in this system, prevented the increase in polyubiquitin mRNA. The expression of polyubiquitin occurred without apparent activation of the cell's heat shock response. These data suggest that ubiquitin plays a role in programmed cell death.     

Cloning of a polyubiquitin gene fromNicotiana tabacum and comparison to other polyubiquitin genes

Using a tobacco cDNA clone as a probe, a genomic clone named TUQG-4, coding for a tobacco polyubiquitin protein with the five head-to-tail repeats of ubiquitin monomer was isolated. The five ubiquitin units were completely conserved except for the extra phenylalanine at the carboxy terminus of the last ubiquitin monomer. The putative open reading frame identified from the nucleotide sequence showed two possible intron sequences in the coding region for the first ubiquitin monomer. When the amino acid sequence deduced from the nucleotide sequence of TUQG-4 was compared to the amino acid sequences coded by other polyubiquitin genes of tobacco, there were three or four amino acid differences in the sequence. When the nucleotide sequences coding for the ubiquitin monomers were compared for various species origins, the degree of identity was at the highest between the ubiquitin monomers in one polyubiquitin and did not reflect the distance of the phylogenetic relationship.     

Two different classes of E2 ubiquitin-conjugating enzymes are required for the mono-ubiquitination of proteins and elongation by polyubiquitin chains with a specific topology

RING (really interesting new gene) and U-box E3 ligases bridge E2 ubiquitin-conjugating enzymes and substrates to enable the transfer of ubiquitin to a lysine residue on the substrate or to one of the seven lysine residues of ubiquitin for polyubiquitin chain elongation. Different polyubiquitin chains have different functions. Lys(48)-linked chains target proteins for proteasomal degradation, and Lys(63)-linked chains function in signal transduction, endocytosis and DNA repair. For this reason, chain topology must be tightly controlled. Using the U-box E3 ligase CHIP [C-terminus of the Hsc (heat-shock cognate) 70-interacting protein] and the RING E3 ligase TRAF6 (tumour-necrosis-factor-receptor-associated factor 6) with the E2s Ubc13 (ubiquitin-conjugating enzyme 13)-Uev1a (ubiquitin E2 variant 1a) and UbcH5a, in the present study we demonstrate that Ubc13-Uev1a supports the formation of free Lys(63)-linked polyubiquitin chains not attached to CHIP or TRAF6, whereas UbcH5a catalyses the formation of polyubiquitin chains linked to CHIP and TRAF6 that lack specificity for any lysine residue of ubiquitin. Therefore the abilities of these E2s to ubiquitinate a substrate and to elongate polyubiquitin chains of a specific topology appear to be mutually exclusive. Thus two different classes of E2 may be required to attach a polyubiquitin chain of a particular topology to a substrate: the properties of one E2 are designed to mono-ubiquitinate a substrate with no or little inherent specificity for an acceptor lysine residue, whereas the properties of the second E2 are tailored to the elongation of a polyubiquitin chain using a defined lysine residue of ubiquitin.     

Glycosylation is required for maintenance of functional voltage-activated channels in growing neocortical neurons of the rat.

Voltage-activated currents were studied in whole-cell patch-clamped rat neocortical neurons growing in culture and treated with tunicamycin (TU), an inhibitor of protein N-glycosylation. The size of the Na+ current decreased progressively in the presence of TU (1-2 microM). This decrease was faster in growing 5-14 day-old neurons (to ca. 40% of control after 24 hours of treatment) than in fully grown 20-40-day-old neurons (to ca. 40% of control after 68 hours of treatment). The fast transient K+ current (A-current) was abolished, and the delayed rectifier K+ current was markedly reduced by a 24 hour treatment with TU (1-2 microM) in growing neurons. In contrast, in fully grown neurons these currents were unaffected by the same TU treatment. The size of the Ca2+ current was significantly reduced following a 24 hour treatment with TU (1-2 microM) in neurons at early stages of differentiation, but remained stable in 20-40-day-old neurons. It is concluded that protein glycosylation, presumably of the channel proteins themselves, is important for the functional expression of voltage-activated channels in embryonic cortical neurons during the early stages of cell growth in culture; the channels become less dependent on glycosylation in mature neurons.     

预测转录单位基础上的原核生物启动子预测

启动子及转录单位预测对于了解基因间的功能及相互间的调节关系具有重要的作用 ,这方面的研究一直是生物信息学的一个重要方向 ,但预测的准确率一直都很有限。本文建立了在转录单位预测基础上进行原核生物启动子预测的新方法 ,首先根据基因间距离、功能关系及多基因比对结果来进行转录单位预测 ,得到了比较理想的结果 ,而且对于研究得比较透和研究得较少的基因组都适用。其后在转录单位预测结果基础上进行启动子预测则采用了隐Markov链模型 ,并在Markov链中考虑状态驻留时间。结果显示 ,该方法能有效地预测出启动子序列及其位置 ,准确率达到 70 %以上。     

Rad23 ubiquitin-associated domains (UBA) inhibit 26 S proteasome-catalyzed proteolysis by sequestering lysine 48-linked polyubiquitin chains

Most substrates of the 26 S proteasome are recognized only following conjugation to a Lys48-linked polyubiquitin chain. Rad23 is one member of a family of proteins that possesses an N-terminal ubiquitin-like domain (UbL) and a C-terminal ubiquitin-associated domain(s) (UBA). Recent studies have shown that UbLs interact with 26 S proteasomes, whereas UBAs bind polyubiquitin chains. These biochemical properties suggest that UbL-UBA proteins may shuttle polyubiquitinated substrates to proteasomes. Here we show that contrary to prediction from this model, the effect of human Rad23A on the degradation of polyubiquitinated substrates catalyzed by purified proteasomes is exclusively inhibitory. Strong inhibition is dependent on the presence of both UBAs, independent of the UbL, and can be explained by competition between the UBA domains and the proteasome for binding to substrate-linked polyubiquitin chains. The UBA domains bind Lys48-linked polyubiquitin chains in strong preference to Lys63 or Lys29-linked chains, leading to selective inhibition of the assembly and disassembly of Lys48-linked chains. These results place constraints on the mechanism(s) by which UbL-UBA proteins promote proteasome-catalyzed proteolysis and reveal new properties of UBA domains.     

Recognition of polyubiquitin isoforms by the multiple ubiquitin binding modules of isopeptidase T

The conjugation of polyubiquitin to target proteins acts as a signal that regulates target stability, localization, and function. Several ubiquitin binding domains have been described, and while much is known about ubiquitin binding to the isolated domains, little is known with regard to how the domains interact with polyubiquitin in the context of full-length proteins. Isopeptidase T (IsoT/USP5) is a deubiquitinating enzyme that is largely responsible for the disassembly of unanchored polyubiquitin in the cell. IsoT has four ubiquitin binding domains: a zinc finger domain (ZnF UBP), which binds the proximal ubiquitin, a UBP domain that forms the active site, and two ubiquitin-associated (UBA) domains whose roles are unknown. Here, we show that the UBA domains are involved in binding two different polyubiquitin isoforms, linear and K48-linked. Using isothermal titration calorimetry, we show that IsoT has at least four ubiquitin binding sites for both polyubiquitin isoforms. The thermodynamics of the interactions reveal that the binding is enthalpy-driven. Mutation of the UBA domains suggests that UBA1 and UBA2 domains of IsoT interact with the third and fourth ubiquitins in both polyubiquitin isoforms, respectively. These data suggest that recognition of the polyubiquitin isoforms by IsoT involves considerable conformational mobility in the polyubiquitin ligand, in the enzyme, or in both.     

Diverse polyubiquitin interaction properties of ubiquitin-associated domains

The ubiquitin-associated (UBA) domain occurs frequently in proteins involved in ubiquitin-dependent signaling pathways. Although polyubiquitin chain binding is considered to be a defining feature of the UBA domain family, the generality of this property has not been established. Here we have surveyed the polyubiquitin interaction properties of 30 UBA domains, including 16 of 17 occurrences in budding yeast. The UBA domains sort into four classes that include linkage-selective polyubiquitin binders and domains that bind different chains (and monoubiquitin) in a nondiscriminatory manner; one notable class ( approximately 30%) did not bind any ubiquitin ligand surveyed. The properties of a given UBA domain are conserved from yeast to mammals. Their functional relevance is further suggested by the ability of an ectopic UBA domain to alter the specificity of a deubiquitylating enzyme in a predictable manner. Conversely, non-UBA sequences can modulate the interaction properties of a UBA domain.     

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.     

Identification of a novel 29-linked polyubiquitin binding protein, Ufd3, using polyubiquitin chain analogues

Lysine 48-linked polyubiquitin chains are the best understood form of polyubiquitin and are necessary for the function of the ubiquitin-proteasome system. However, other forms of polyubiquitin (e.g., K29- and K63-linked chains) are also present in vivo. Less is known about the functional roles of these linkages or the proteins specifically interacting with these forms of polyubiquitin. Use of native polyubiquitin chains to identify binding proteins is complicated by the difficulties of synthesis and stability. Here, we report the synthesis of a nonhydrolyzable analogue of 29-linked polyubiquitin chains on an affinity support and its use in identifying proteins that bind 29-linked polyubiquitin chains. The 29-linked Ub4 resin was stable and tightly bound recombinant human Isopeptidase T (USP5), a deubiquitinating enzyme known to bind the 29-linked polyubiquitin chains. Two high affinity interactors of the 29-linked polyubiquitin analogues were identified from Saccharomyces cerevisiae lysates. They were identified as Ubp14, the yeast ortholog of Isopeptidase T, and Ufd3, a member of the ubiquitin-fusion degradation pathway with unknown function. Purified recombinant Ufd3 bound to the resin as well, confirming that Ufd3 is a novel binding partner of polyubiquitin. These results demonstrate the efficacy of using polyubiquitin analogue affinity supports to identify novel binding partners of specifically linked polyubiquitin chains. Identification of these proteins will lead to a greater understanding of the physiological relevance of different polyubiquitin linkages.     

Covalent Modification of the NF-κB Essential Modulator (NEMO) by a Chemical Compound Can Regulate Its Ubiquitin Binding Properties in Vitro

Post-translational modification by ubiquitin plays important roles in multiple physiological and pathological processes. Ubiquitin-binding proteins play a critical role in recognizing and relaying polyubiquitin-based signaling. NEMO (NF-κB Essential Modulator) is a central player in canonical NF-κB signaling whose major function is to bind to Lys-63- and/or M1- (or linear) linked polyubiquitin chains generated in response to cell stimulation. Here we show that Withaferin A (WA), a steroidal lactone, causes a change in NEMO''s interaction with specific types of polyubiquitin chains in vitro. WA induces full-length recombinant NEMO to bind to long Lys-48-linked polyubiquitin chains but not tetra-ubiquitin species. Significantly, the UBAN (ubiquitin binding in ABIN and NEMO) domain, essential for the ability of NEMO to bind M1/Lys-63-linked polyubiquitin, is dispensable for the WA-induced gain-of-function activity. Mass spectrometric analysis demonstrated that WA covalently modifies NEMO on a cysteine residue within the C-terminal zinc finger (ZF) domain. Point mutations to the ZF can reverse the WA-induced Lys-48-polyubiquitin binding phenotype. Our study demonstrates the feasibility of directly altering the ubiquitin interaction properties of an ubiquitin-binding protein by a chemical compound, thereby shedding light on a novel drug class to potentially alter polyubiquitin-based cellular processes.     

Novel polyubiquitin imaging system,PolyUb-FC,reveals that K33-linked polyubiquitin is recruited by SQSTM1/p62

Ubiquitin chains are formed with 8 structurally and functionally distinct polymers. However, the functions of each polyubiquitin remain poorly understood. We developed a polyubiquitin-mediated fluorescence complementation (PolyUb-FC) assay using Kusabira Green (KG) as a split fluorescent protein. The PolyUb-FC assay has the advantage that monoubiquitination is nonfluorescent and chain-specific polyubiquitination can be directly visualized in living cells without using antibodies. We applied the PolyUb-FC assay to examine K33-linked polyubiquitin. We demonstrated that SQSTM1/p62 puncta colocalized with K33-linked polyubiquitin and this interaction was modulated by the ZRANB1/TRABID-K29 and -K33 linkage-specific deubiquitinase (DUB). We further showed that the colocalization of K33-linked polyubiquitin and MAP1LC3/LC3 (microtubule associated protein 1 light chain 3) puncta was impaired by SQSTM1/p62 deficiency. Taken together, these findings provide novel insights into how atypical polyubiquitin is recruited by SQSTM1/p62. Finally, we developed an inducible-PolyUb-FC system for visualizing chain-specific polyubiquitin. The PolyUb-FC will be a useful tool for analyzing the dynamics of atypical polyubiquitin chain generation.