Ubiquitin-related Modifier-1 Research Articles

Suspension Bead Loading (SBL): An Economical Protein Delivery Platform to Study URM1’s Behavior in Live Cells

AbstractUniquely modified synthetic proteins are difficult to produce in large quantities, which could limit their use in various in vitro settings and in cellular studies. In this study, we developed a method named “suspension bead loading” (SBL), to deliver protein molecules into suspended living cells using glass beads, which significantly reduces the amount of protein required for effective delivery. We investigated the delivery efficiency of functionally different proteins and evaluated the cytotoxic effect of our method and the chemical and functional integrity of the delivered protein. We utilized SBL to address questions related to ubiquitin‐related modifier 1 (URM1). Employing minimal protein quantities, SBL has enabled us to study its behavior within live cells under different redox conditions, including subcellular localization and conjugation patterns. We demonstrate that oxidative stress alters both the localization and conjugation pattern of URM1 in cells, highlighting its possible role in cellular response to such extreme conditions.

Suspension Bead Loading (SBL): An Economical Protein Delivery Platform to Study URM1's Behavior in Live Cells.

Uniquely modified synthetic proteins are difficult to produce in large quantities, which could limit their use in various in vitro settings and in cellular studies. In this study, we developed a method named "suspension bead loading" (SBL), to deliver protein molecules into suspended living cells using glass beads, which significantly reduces the amount of protein required for effective delivery. We investigated the delivery efficiency of functionally different proteins and evaluated the cytotoxic effect of our method and the chemical and functional integrity of the delivered protein. We utilized SBL to address questions related to ubiquitin-related modifier 1 (URM1). Employing minimal protein quantities, SBL has enabled us to study its behavior within live cells under different redox conditions, including subcellular localization and conjugation patterns. We demonstrate that oxidative stress alters both the localization and conjugation pattern of URM1 in cells, highlighting its possible role in cellular response to such extreme conditions.

Ubiquitin-Related Modifier 1 (URM-1) Modulates Cx43 in Breast Cancer Cell Lines.

Gap-junction-forming connexins are exquisitely regulated by post-translational modifications (PTMs). In particular, the PTM of connexin 43 (Cx43), a tumor suppressor protein, regulates its turnover and activity. Here, we investigated the interaction of Cx43 with the ubiquitin-related modifier 1 (URM-1) protein and its impact on tumor progression in two breast cancer cell lines, highly metastatic triple-negative MDA-MB-231 and luminal breast cancer MCF-7 cell lines. To evaluate the subsequent modulation of Cx43 levels, URM-1 was downregulated in these cells. The transcriptional levels of epithelial-to-mesenchymal transition (EMT) markers and the metastatic phenotype were assessed. We demonstrated that Cx43 co-localizes and interacts with URM-1, and URMylated Cx43 was accentuated upon cellular stress. The significant upregulation of small ubiquitin-like modifier-1 (SUMO-1) was also observed. In cells with downregulated URM-1, Cx43 expression significantly decreased, and SUMOylation by SUMO-1 was affected. The concomitant expression of EMT markers increased, leading to increased proliferation, migration, and invasion potential. Inversely, the upregulation of URM-1 increased Cx43 expression and reversed EMT-induced processes, underpinning a role for this PTM in the observed phenotypes. This study proposes that the URMylation of Cx43 in breast cancer cells regulates its tumor suppression properties and contributes to breast cancer cell malignancy.

URM1 Promoted Tumor Growth and Suppressed Apoptosis via the JNK Signaling Pathway in Hepatocellular Carcinoma.

ObjectiveUbiquitin-related modifier 1 (URM1) is a member of the ubiquitin-like regulator family, which acts as a post-translational protein modifier in the oxidative emergency response mechanism. Previous studies have shown that URM1 may be involved in the process of apoptosis and may play a role in JNK signaling pathway. In this study, we aimed to investigate the role and possible mechanism of URM1 in HCC progression.Patients and MethodsExpression of URM1 was determined in 90 pairs of matched liver cancer and adjacent non-cancerous tissues by immunohistochemistry. The impacts of URM1 on HCC cell proliferation, apoptosis, migration and invasion capacities were verified by CCK-8, colony formation, TUNEL staining, wound healing assay and transwell, respectively. Then, the effect of URM1 on subcutaneous tumor formation in vitro was explored by nude mouse xenograft model of liver cancer. Finally, the expression of apoptosis-related proteins was analyzed in URM1 knockdown samples by Western blotting.ResultsIn this study, compared with paired adjacent non-cancerous tissues, the expression of URM1 was higher in liver cancer tissues (P <0.01). Kaplan–Meier survival analysis showed that high URM1 expression was significantly associated with poor prognosis (P <0.05). Moreover, URM1 knockdown inhibited liver cancer cell proliferation and migration. Furthermore, URM1 knockdown promoted apoptosis of liver cancer cells. At the same time, URM1 knockdown inhibited tumor growth in nude mouse xenograft model of liver cancer. In addition, URM1 knockdown downregulated the expression of the apoptosis-related factors JNK1/2 and TP53 and upregulated the phosphorylation of JNK1/2 and P53.ConclusionIn summary, our results suggested that URM1 expression is increased in liver cancer tissues, and URM1 knockdown inhibits the proliferation and migration of liver cancer cells and accelerates apoptosis. High URM1 expression is associated with poor prognosis in patients with HCC.

Archaeal Tuc1/Ncs6 homolog required for wobble uridine tRNA thiolation is associated with ubiquitin-proteasome, translation, and RNA processing system homologs.

While cytoplasmic tRNA 2-thiolation protein 1 (Tuc1/Ncs6) and ubiquitin-related modifier-1 (Urm1) are important in the 2-thiolation of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) at wobble uridines of tRNAs in eukaryotes, the biocatalytic roles and properties of Ncs6/Tuc1 and its homologs are poorly understood. Here we present the first report of an Ncs6 homolog of archaea (NcsA of Haloferax volcanii) that is essential for maintaining cellular pools of thiolated tRNALys UUU and for growth at high temperature. When purified from Hfx. volcanii, NcsA was found to be modified at Lys204 by isopeptide linkage to polymeric chains of the ubiquitin-fold protein SAMP2. The ubiquitin-activating E1 enzyme homolog of archaea (UbaA) was required for this covalent modification. Non-covalent protein partners that specifically associated with NcsA were also identified including UbaA, SAMP2, proteasome activating nucleotidase (PAN)-A/1, translation elongation factor aEF-1α and a β-CASP ribonuclease homolog of the archaeal cleavage and polyadenylation specificity factor 1 family (aCPSF1). Together, our study reveals that NcsA is essential for growth at high temperature, required for formation of thiolated tRNALys UUU and intimately linked to homologs of ubiquitin-proteasome, translation and RNA processing systems.

Genetic polymorphisms and preliminary association analysis with production traits of the porcine SLC27A4 gene

Solute carrier family 27 (fatty acid transporter), member 4 (SLC27A4) is a fatty acyl-CoA synthetase producing very long chain fatty acid-CoA for lipid metabolic pathways, suggesting that the SLC27A4 gene is a potential candidate gene for traits related to fat deposition in animals. This study was conducted to sequence the genomic region from exon 6 to 12 of porcine SLC27A4 and detect polymorphisms by comparative sequencing. In silico mapping assigned SLC27A4 gene between gene COQ4 (coenzyme Q4 homolog) and URM1 (ubiquitin related modifier 1 homolog) on pig chromosome 1q24-q2.12 where significant QTL affecting backfat depth had previously been identified. Thirty six putative sites of variation were detected, of which 31 polymorphisms including 28 SNPs and 3 indels were located in the intronic region, and 5 in the exonic regions. The g.1777G>A (EU703769) in intron 8 was confirmed by PCR-RFLP using HpaII restriction enzyme and further genotyped in four Chinese native pig breeds (Meishan, Erhualian, Tongcheng and Qingping) and three western meat-type pig breeds (Duroc, Large White and Landrace). Allele G was exclusively present in Tongcheng and Qingping pigs and predominant in the other pig populations analyzed. Significant differences of backfat at rump, body weight at birth and average daily gain on weaning between the AG and GG genotype were observed in Landrace pig population (P < 0.05).

Solution structure of Urm1 and its implications for the origin of protein modifiers

Protein modifiers are involved in diverse biological processes and regulate the activity or function of target proteins by covalently conjugating to them. Although ubiquitin and a number of ubiquitin-like protein modifiers (Ubls) in eukaryotes have been identified, no protein modifier has been found in prokaryotes; thus, their evolutionary origin remains a puzzle. To infer the evolutionary relationships between the protein modifiers and sulfur carrier proteins, we solved the solution NMR structure of the Urm1 (ubiquitin-related modifier-1) protein from Saccharomyces cerevisiae. Both structural comparison and phylogenetic analysis of the ubiquitin superfamily, with emphasis on the Urm1 family, indicate that Urm1 is the unique "molecular fossil" that has the most conserved structural and sequence features of the common ancestor of the entire superfamily. The similarities of 3D structure and hydrophobic and electrostatic surface features between Urm1 and MoaD (molybdopterin synthase small subunit) suggest that they may interact with partners in a similar manner, and similarities between Urm1-Uba4 and MoaD-MoeB establish an evolutionary link between ATP-dependent protein conjugation in eukaryotes and ATP-dependent cofactor sulfuration.