The emerging roles of ubiquitin-like protein Urm1 in eukaryotes

The ubiquitin related modifier Urm1 protein was firstly identified in the yeast Saccharomyces cerevisiae, and was later found to play important roles in different eukaryotes. By the assistance of an E1-like activation enzyme Uba4, Urm1 can function as a modifier to target proteins, called urmylation. The thioredoxin peroxidase Ahp1 was the only identified Urm1 target in the early time. Recently, many other Urm1 targets were identified, which is important for us to fully understand functions of urmylation. Urm1 can also function as a sulfur carrier to play a key role in tRNAs thiolation. Mechanisms of the Urm1 in protein and RNA modifications were finely revealed in the past few years. Biological and physiological functions of Urm1 were also found in different organisms. In this review, we will summarize these emerging progresses.     

The dual role of ubiquitin-like protein Urm1 as a protein modifier and sulfur carrier

The ubiquitin-related modifier Urm1 can be covalently conjugated to lysine residues of other proteins, such as yeast Ahp1 and human MOCS3, through a mechanism involving the E1-like protein Uba4 (MOCS3 in humans). Similar to ubiquitination, urmylation requires a thioester intermediate and forms isopeptide bonds between Urm1 and its substrates. In addition, the urmylation process can be significantly enhanced by oxidative stress. Recent findings have demonstrated that Urm1 also acts as a sulfur carrier in the thiolation of eukaryotic tRNA via a mechanism that requires the formation of a thiocarboxylated Urm1. This role is very similar to that of prokaryotic sulfur carriers such as MoaD and ThiS. Evidence strongly supports the hypothesis that Urm1 is the molecular fossil in the evolutionary link between prokaryotic sulfur carriers and eukaryotic ubiquitin-like proteins. In the present review, we discuss the dual role of Urm1 in protein and tRNA modification.     

A protein conjugation system in yeast with homology to biosynthetic enzyme reaction of prokaryotes

Protein conjugation, such as ubiquitination, is the process by which the C-terminal glycine of a small modifier protein is covalently attached to target protein(s) through sequential reactions with an activating enzyme and conjugating enzymes. Here we report on a novel protein conjugation system in yeast. A newly identified ubiquitin related modifier, Urm1 is a 99-amino acid protein terminated with glycine-glycine. Urm1 is conjugated to target proteins, which requires the C-terminal glycine of Urm1. At the first step of this reaction, Urm1 forms a thioester with a novel E1-like protein, Uba4. Deltaurm1 and Deltauba4 cells showed a temperature-sensitive growth phenotype. Urm1 and Uba4 show similarity to prokaryotic proteins essential for molybdopterin and thiamin biosynthesis, although the Urm1 system is not involved in these pathways. This is the fifth conjugation system in yeast, following ubiquitin, Smt3, Rub1, and Apg12, but it is unique in respect to relation to prokaryotic enzyme systems. This fact may provide an important clue regarding evolution of protein conjugation systems in eukaryotic cells.     

The sulfurtransferase activity of Uba4 presents a link between ubiquitin-like protein conjugation and activation of sulfur carrier proteins

Because of mechanistic parallels in the activation of ubiquitin and the biosynthesis of several sulfur-containing cofactors, we have characterized the human Urm1 and Saccharomyces cerevisiae Uba4 proteins, which are very similar in sequence to MOCS2A and MOCS3, respectively, two proteins essential for the biosynthesis of the molybdenum cofactor (Moco) in humans. Phylogenetic analyses of MOCS3 homologues showed that Uba4 is the MOCS3 homologue in yeast and thus the only remaining protein of the Moco biosynthetic pathway in this organism. Because of the high levels of sequence identity of human MOCS3 and yeast Uba4, we purified Uba4 and characterized the catalytic activity of the protein in detail. We demonstrate that the C-terminal domain of Uba4, like MOCS3, has rhodanese activity and is able to transfer the sulfur from thiosulfate to cyanide in vitro. In addition, we were able to copurify stable heterotetrameric complexes of Uba4 with both human Urm1 and MOCS2A. The N-terminal domain of Uba4 catalyzes the activation of either MOCS2A or Urm1 by formation of an acyl-adenylate bond. After adenylation, persulfurated Uba4 was able to form a thiocarboxylate group at the C-terminal glycine of either Urm1 or MOCS2A. The formation of a thioester intermediate between Uba4 and Urm1 or MOCS2A was not observed. The functional similarities between Uba4 and MOCS3 further demonstrate the evolutionary link between ATP-dependent protein conjugation and ATP-dependent cofactor sulfuration.     

NEDD化修饰系统及其在肿瘤发展中的作用

目前已经鉴定出17种类泛素蛋白（ubiquitin like proteins,UBLs）,这些蛋白与底物的结合方式与泛素相似．根据进化特征,可将UBLs分为9类,分别为：NEDD8、SUMO、ISG15、FUB1、FAT10、Atg8、Atg12、Urm1和UFM1．NEDD8是目前研究最多的UBLs之一,与泛素的氨基酸序列具有高度相似性．NEDD化修饰是一种动态的可逆蛋白质翻译后修饰方式,可以将NEDD8共价结合到靶蛋白之上,也可以将NEDD8从靶蛋白上去除．NEDD化修饰对蛋白功能具有重要的调节作用,如改变蛋白质的空间构象、阻碍底物与其它蛋白质的相互作用和招募与NEDD8相互作用的蛋白等．最新研究表明,NEDD化与肿瘤的发生发展密切相关,但具体的机制还不清楚．本文将就NEDD化修饰在肿瘤发展过程中的作用机制做一综述．     

Attachment of the ubiquitin-related protein Urm1p to the antioxidant protein Ahp1p

Urm1p is a ubiquitin-related protein that serves as a posttranslational modification of other proteins. Urm1p conjugation has been implicated in the budding process and in nutrient sensing. Here, we have identified the first in vivo target for the urmylation pathway as the antioxidant protein Ahp1p. The attachment of Urm1p to Ahp1p requires the E1 for the urmylation pathway, Uba4p. Loss of the urmylation pathway components results in sensitivity to a thiol-specific oxidant, as does loss of Ahp1p, implying that urmylation has a role in an oxidative-stress response. Moreover, treatment of cells with thiol-specific oxidants affects the abundance of Ahp1p-Urm1p conjugates. These results suggest that the conjugation of Urm1p to Ahp1p could regulate the function of Ahp1p in antioxidant stress response in Saccharomyces cerevisiae.     

Three-dimensional structure of the AAH26994.1 protein from Mus musculus, a putative eukaryotic Urm1

We have used NMR spectroscopy to determine the solution structure of protein AAH26994.1 from Mus musculus and propose that it represents the first three-dimensional structure of a ubiquitin-related modifier 1 (Urm1) protein. Amino acid sequence comparisons indicate that AAH26994.1 belongs to the Urm1 family of ubiquitin-like modifier proteins. The best characterized member of this family has been shown to be involved in nutrient sensing, invasive growth, and budding in yeast. Proteins in this family have only a weak sequence similarity to ubiquitin, and the structure of AAH26994.1 showed a much closer resemblance to MoaD subunits of molybdopterin synthases (known structures are of three bacterial MoaD proteins with 14%-26% sequence identity to AAH26994.1). The structures of AAH26994.1 and the MoaD proteins each contain the signature ubiquitin secondary structure fold, but all differ from ubiquitin largely in regions outside of this fold. This structural similarity bolsters the hypothesis that ubiquitin and ubiquitin-related proteins evolved from a protein-based sulfide donor system of the molybdopterin synthase type.     

1H, 13C and 15N resonance assignment of Urm1 from Trypanosoma brucei

Urm1 (ubiquitin-related modifier), involved in diverse biological processes in yeast, is proved to be a “molecular fossil” in ubiquitin superfamily. Here we report the resonance assignment of Urm1 from Trypanosoma brucei.     

The CsdA cysteine desulphurase promotes Fe/S biogenesis by recruiting Suf components and participates to a new sulphur transfer pathway by recruiting CsdL (ex‐YgdL), a ubiquitin‐modifying‐like protein

Cysteine desulphurases are primary sources of sulphur that can eventually be used for Fe/S biogenesis or thiolation of various cofactors and tRNA. Escherichia coli contains three such enzymes, IscS, SufS and CsdA. The importance of IscS and SufS in Fe/S biogenesis is well established. The physiological role of CsdA in contrast remains uncertain. We provide here additional evidences for a functional redundancy between the three cysteine desulphurases in vivo. In particular, we show that a deficiency in isoprenoid biosynthesis is the unique cause of the lethality of the iscS sufS mutant. Moreover, we show that CsdA is engaged in two separate sulphur transfer pathways. In one pathway, CsdA interacts functionally with SufE–SufBCD proteins to assist Fe/S biogenesis. In another pathway, CsdA interacts with CsdE and a newly discovered protein, which we called CsdL, resembling E1‐like proteins found in ubiquitin‐like modification systems. We propose this new pathway to allow synthesis of an as yet to be discovered thiolated compound.     

Structure and interaction of ubiquitin‐associated domain of human Fas‐associated factor 1

Fas‐associated factor (FAF)‐1 is a multidomain protein that was first identified as a member of the Fas death‐inducing signaling complex, but later found to be involved in various biological processes. Although the exact mechanisms are not clear, FAF1 seems to play an important role in cancer, asbestos‐induced mesotheliomas, and Parkinson's disease. It interacts with polyubiquitinated proteins, Hsp70, and p97/VCP (valosin‐containing protein), in addition to the proteins of the Fas‐signaling pathway. We have determined the crystal structure of the ubiquitin‐associated domain of human FAF1 (hFAF1‐UBA) and examined its interaction with ubiquitin and ubiquitin‐like proteins using nuclear magnetic resonance. hFAF1‐UBA revealed a canonical three‐helical bundle that selectively binds to mono‐ and di‐ubiquitin (Lys48‐linked), but not to SUMO‐1 (small ubiquitin‐related modifier 1) or NEDD8 (neural precursor cell expressed, developmentally down‐regulated 8). The interaction between hFAF1‐UBA and di‐ubiquitin involves hydrophobic interaction accompanied by a transition in the di‐ubiquitin conformation. These results provide structural insight into the mechanism of polyubiquitin recognition by hFAF1‐UBA.     

GdX/UBL4A null mice exhibit mild kyphosis and scoliosis accompanied by dysregulation of osteoblastogenesis and chondrogenesis

GdX, also named ubiquitin‐like protein 4A, is a ubiquitin‐domain protein characterized by a ubiquitin‐like domain that regulates the movement of misfolded proteins from the endoplasmic reticulum membrane to proteasome. However, its function in skeletal biology remains unclear. Here, we report that GdX plays a crucial role in skeletal development as mice lacking GdX exhibit skeletal dysplasias, mild kyphosis, and scoliosis. During embryonic stage, GdX knockout mice display decreased bone mineral density and trabecular bone accompanied by delayed osteogenic formation. GdX knockout mice also have blended spine and small body size. At the molecular level, GdX knockout mice showed perturbed expression of osteogenesis‐related genes and cartilage developmental genes, indicative of altered differentiation of mesenchymal cell lineage. Collectively, our results uncovered GdX as a novel regulator in bone development and a potential candidate gene for skeletal dysplasias.     

MAPL is a new mitochondrial SUMO E3 ligase that regulates mitochondrial fission

The modification of proteins by the small ubiquitin‐like modifier (SUMO) is known to regulate an increasing array of cellular processes. SUMOylation of the mitochondrial fission GTPase dynamin‐related protein 1 (DRP1) stimulates mitochondrial fission, suggesting that SUMOylation has an important function in mitochondrial dynamics. The conjugation of SUMO to its substrates requires a regulatory SUMO E3 ligase; however, so far, none has been functionally associated with the mitochondria. By using biochemical assays, overexpression and RNA interference experiments, we characterized the mitochondrial‐anchored protein ligase (MAPL) as the first mitochondrial‐anchored SUMO E3 ligase. Furthermore, we show that DRP1 is a substrate for MAPL, providing a direct link between MAPL and the fission machinery. Importantly, the large number of unidentified mitochondrial SUMO targets suggests a global role for SUMOylation in mitochondrial function, placing MAPL as a crucial component in the regulation of multiple conjugation events.     

RBR E3‐ligases at work

The RING‐in‐between‐RING (RBR) E3s are a curious family of ubiquitin E3‐ligases, whose mechanism of action is unusual in several ways. Their activities are auto‐inhibited, causing a requirement for activation by protein‐protein interactions or posttranslational modifications. They catalyse ubiquitin conjugation by a concerted RING/HECT‐like mechanism in which the RING1 domain facilitates E2‐discharge to directly form a thioester intermediate with a cysteine in RING2. This short‐lived, HECT‐like intermediate then modifies the target. Uniquely, the RBR ligase HOIP makes use of this mechanism to target the ubiquitin amino‐terminus, by presenting the target ubiquitin for modification using its distinctive LDD region.     

Overexpression of ubiquitin‐like LpHUB1 gene confers drought tolerance in perennial ryegrass

HUB1, also known as Ubl5, is a member of the subfamily of ubiquitin‐like post‐translational modifiers. HUB1 exerts its role by conjugating with protein targets. The function of this protein has not been studied in plants. A HUB1 gene, LpHUB1, was identified from serial analysis of gene expression data and cloned from perennial ryegrass. The expression of this gene was reported previously to be elevated in pastures during the summer and by drought stress in climate‐controlled growth chambers. Here, pasture‐type and turf‐type transgenic perennial ryegrass plants overexpressing LpHUB1 showed improved drought tolerance, as evidenced by improved turf quality, maintenance of turgor and increased growth. Additional analyses revealed that the transgenic plants generally displayed higher relative water content, leaf water potential, and chlorophyll content and increased photosynthetic rate when subjected to drought stress. These results suggest HUB1 may play an important role in the tolerance of perennial ryegrass to abiotic stresses.     

Solution structure of the N‐terminal domain of DC‐UbP/UBTD2 and its interaction with ubiquitin

DC‐UbP/UBTD2 is a ubiquitin (Ub) domain‐containing protein first identified from dendritic cells, and is implicated in ubiquitination pathway. The solution structure and backbone dynamics of the C‐terminal Ub‐like (UbL) domain were elucidated in our previous work. To further understand the biological function of DC‐UbP, we then solved the solution structure of the N‐terminal domain of DC‐UbP (DC‐UbP_N) and studied its Ub binding properties by NMR techniques. The results show that DC‐UbP_N holds a novel structural fold and acts as a Ub‐binding domain (UBD) but with low affinity. This implies that the DC‐UbP protein, composing of a combination of both UbL and UBD domains, might play an important role in regulating protein ubiquitination and delivery of ubiquitinated substrates in eukaryotic cells.     

The retroviral proteinase active site and the N-terminus of Ddi1 are required for repression of protein secretion

The Ddi1 protein of the yeast Saccharomyces cerevisiae is involved in numerous interactions with the ubiquitin system, which may be mediated by its N-terminal ubiquitin like domain and its C-terminal ubiquitin associated domain. Ddi1 also contains a central region with all the features of a retroviral aspartic proteinase, which was shown to be important in cell-cycle control. Here we demonstrate an additional role for this domain, along with the N-terminal region, in protein secretion. These results further substantiate the hypothesis that Ddi1 functions in vivo as a catalytically-active aspartic proteinase.