Usp Protein Research Articles

MiR-206 alleviates LPS-induced inflammatory injury in cardiomyocytes via directly targeting USP33 to inhibit the JAK2/STAT3 signaling pathway.

Previous reports have confirmed that miR-206 participates in inflammatory cardiomyopathy, but its definite mechanism remains elusive. This study aims to elucidate the potential mechanism of miR-206 in septic cardiomyopathy (SCM). The primary mouse cardiomyocytes were isolated and exposed to lipopolysaccharides (LPS) to construct a septic injury model in vitro. Then, the gene transcripts and protein levels were detected by RT-qPCR and/or Western blot assay. Cell proliferation, apoptosis, and inflammatory responses were evaluated by CCK-8/EdU, flow cytometry, and ELISA assays, respectively. Dual luciferase assay, Co-IP, and ubiquitination experiments were carried out to validate the molecular interactions among miR-206, USP33, and JAK2/STAT3 signaling. miR-206 was significantly downregulated, but USP33 was upregulated in LPS-induced cardiomyocytes. Gain-of-function of miR-206 elevated the proliferation but suppressed the inflammatory responses and apoptosis in LPS-induced cardiomyocytes. USP33, as a member of the USP protein family, was confirmed to be a direct target of miR-206 and could catalyze deubiquitination of JAK2 to activate JAK2/STAT3 signaling. Rescue experiments presented that neither upregulation of USP33 nor JAK2/STAT3 signaling activation considerably reversed the protective effects of miR-206 upregulation in LPS-induced cardiomyocytes. The above data showed that miR-206 protected cardiomyocytes from LPS-induced inflammatory injuries by targeting the USP33/JAK2/STAT3 signaling pathway, which might be a novel target for SCM treatment.

Hotspots of Sequence Variability in Gut Microbial Genes Encoding Pro-Inflammatory Factors Revealed by Oligotyping.

The gut microbiota has been implicated in a number of normal and disease biological processes. Recent studies have identified a subset of gut bacterial genes as potentially involved in inflammatory processes. In this work, we explore the sequence variability for some of these bacterial genes using a combination of deep sequencing and oligotyping, a data analysis application that identifies mutational hotspots in short stretches of DNA. The genes for pks island, tcpC and usp, all harbored by certain strains of E. coli and all implicated in inflammation, were amplified by PCR directly from stool samples and subjected to deep amplicon sequencing. For comparison, the same genes were amplified from individual bacterial clones. The amplicons for pks island and tcpC from stool samples showed minimal levels of heterogeneity comparable with the individual clones. The amplicons for usp from stool samples, by contrast, revealed the presence of five distinct oligotypes in two different regions. Of these, the oligotype GT was found to be present in the control uropathogenic clinical isolate and also detected in stool samples from individuals with colorectal cancer (CRC). Mutational hotspots were mapped onto the USP protein, revealing possible substitutions around Leu110, Glu114, and Arg115 in the middle of the pyocin domain (Gln110, Gln114, and Thr115 in most healthy samples), and also Arg218 in the middle of the nuclease domain (His218 in the uropathogenic strain). All of these results suggest that a level of variability within bacterial pro-inflammatory genes could explain differences in bacterial virulence and phenotype.

The Gene Encoding the Universal Stress Protein AtUSP is Regulated by Phytohormones and Involved in Seed Germination of Arabidopsis thaliana.

For the first time, the organ-specific expression pattern of the AtUSP (At3g58450) gene, which also undergoes hormonal regulation, was shown. The USP protein encoded by this gene is involved in seed germination of Arabidopsis thaliana and, unlike abscisic acid, stimulates this process.

Characterization of the universal stress protein F from atypical enteropathogenic Escherichia coli and its prevalence in Enterobacteriaceae.

Atypical enteropathogenic Escherichia coli (aEPEC) are heterogeneous strains in terms of serotypes, adherence patterns and the presence of novel virulence factors. This heterogeneity is intriguing, promoting studies trying to characterize these novel proteins and to better comprehend this pathotype group. In a previous study analyzing low‐molecular mass proteomes of four representative aEPEC strains of three different adhesion phenotypes, we classified proteins according to their annotated function, with most of them being involved in metabolism and transport; while some of them were classified as hypothetical proteins. The majority of the hypothetical proteins were homologue products of genes identified in the genome of enterohemorrhagic E. coli. One of the hypothetical proteins was annotated as Z2335, with orthologue in EPEC, and by bioinformatics analysis, this protein was revealed to be the universal stress protein F (UspF). Thus, herein we successfully obtained a recombinant UspF protein from aEPEC, which is a α/β, ATP‐binding protein involved in stress response, with comparable protein production among the four studied strains, but showing noteworthy differences when cultivated in different stress conditions, also present in other enterobacterial species, such as Shigella sonnei and Citrobacter freundii. Furthermore, our results confirm that the Usp protein superfamily encompasses a conserved group of proteins involved in stress resistance in aEPEC and other Enterobacteriaceae.

Crystal structure and functional implications of the tandem-type universal stress protein UspE from Escherichia coli.

BackgroundThe universal stress proteins (USP) family member UspE is a tandem-type USP that consists of two Usp domains. The UspE expression levels of the Escherichia coli (E. coli) become elevated in response to oxidative stress and DNA damaging agents, including exposure to mitomycin C, cadmium, and hydrogen peroxide. It has been shown that UspA family members are survival factors during cellular growth arrest. The structures and functions of the UspA family members control the growth of E. coli in animal hosts. While several UspA family members have known structures, the structure of E. coli UspE remains to be elucidated.ResultsTo understand the biochemical function of UspE, we have determined the crystal structure of E. coli UspE at 3.2 Å resolution. The asymmetric unit contains two protomers related by a non-crystallographic symmetry, and each protomer contains two tandem Usp domains. The crystal structure shows that UspE is folded into a fan-shaped structure similar to that of the tandem-type Usp protein PMI1202 from Proteus mirabilis, and it has a hydrophobic cavity that binds its ligand. Structural analysis revealed that E. coli UspE has two metal ion binding sites, and isothermal titration calorimetry suggested the presence of two Cd2+ binding sites with a Kd value of 38.3–242.7 μM. Structural analysis suggested that E. coli UspE has two Cd2+ binding sites (Site I: His117, His 119; Site II: His193, His244).ConclusionThe results show that the UspE structure has a hydrophobic pocket. This pocket is strongly bound to an unidentified ligand. Combined with a previous study, the ligand is probably related to an intermediate in lipid A biosynthesis. Subsequently, sequence analysis found that UspE has an ATP binding motif (Gly269- X2-Gly272-X9-Gly282-Asn) in its C-terminal domain, which was confirmed by in vitro ATPase activity monitored using Kinase-Glo® Luminescent Kinase Assay. However, the residues constituting this motif were disordered in the crystal structure, reflecting their intrinsic flexibility. ITC experiments revealed that the UspE probably has two Cd2+ binding sites. The His117, His 119, His193, and His244 residues within the β-barrel domain are necessary for Cd2+ binding to UspE protein. As mentioned above, USPs are associated with several functions, such as cadmium binding, ATPase function, and involvement in lipid A biosynthesis by some unknown way.

Sumoylation modulates 20-hydroxyecdysone signaling by maintaining USP protein levels in Drosophila

The nuclear receptor complex for the insect steroid hormone, 20-hydroxyecdysone (20E), is a heterodimer of EcR and USP. It has been shown that Drosophila EcR and USP can be sumoylated in mammalian cells, but it is unknown whether EcR-USP sumoylation naturally occurs in Drosophila. In Drosophila cells, USP, but not EcR, was sumoylated by Smt3, the only Drosophila SUMO protein. The presence of EcR enhanced USP sumoylation, which is further enhanced by 20E treatment. In addition to the Lys20 sumoylation site, five potential acceptor lysine residues in USP were predicted and verified. Mutation of the USP sumoylation sites or reduction of smt3 expression by RNAi attenuated 20E-induced reporter activity. Moreover, in the salivary glands, reducing smt3 expression by RNAi decreased 20E-induced reporter activity, gene expression, and autolysosome formation. Importantly, at least partially, the smt3 RNAi-mediated reduction in 20E signaling resulted from decreased protein levels of USP. In conclusion, sumoylation modulates 20E signaling by maintaining USP protein levels in Drosophila.

Homodimerization propensity of the intrinsically disordered N-terminal domain of Ultraspiracle from Aedes aegypti

The mosquito Aedes aegypti is the principal vector of dengue, one of the most devastating arthropod-borne viral infections in humans. The isoform specific A/B region, called the N-terminal domain (NTD), is hypervariable in sequence and length and is poorly conserved within the Ultraspiracle (Usp) family. The Usp protein together with ecdysteroid receptor (EcR) forms a heterodimeric complex. Up until now, there has been little data on the molecular properties of the isolated Usp-NTD. Here, we describe the biochemical and biophysical properties of the recombinant NTD of the Usp isoform B (aaUsp-NTD) from A. aegypti. These results, in combination with in silico bioinformatics approaches, indicate that aaUsp-NTD exhibits properties of an intrinsically disordered protein (IDP). We also present the first experimental evidence describing the dimerization propensity of the isolated NTD of Usp. These characteristics also appear for other members of the Usp family in different species, for example, in the Usp-NTD from Drosophila melanogaster and Bombyx mori. However, aaUsp-NTD exhibits the strongest homodimerization potential. We postulate that the unique dimerization of the NTD might be important for Usp function by providing an additional platform for interactions, in addition to the nuclear receptor superfamily dimerization via DNA binding domains and ligand binding domains that has already been extensively documented. Furthermore, the unique NTD–NTD interaction that was observed might contribute new insight into the dimerization propensities of nuclear receptors.

Uropathogenic specific protein gene, highly distributed in extraintestinal uropathogenic Escherichia coli, encodes a new member of H-N-H nuclease superfamily

BackgroundThe uropathogenic specific protein (Usp) and three OrfU proteins (OrfU1, OrfU2 and OrfU3) are encoded in the putative small pathogenicity island which is closely associated with Uropathogenic Escherichia coli. Although homology search revealed that Usp and OrfUs have a homology with nuclease-type bacteriocins, which possess H-N-H nuclease motif, and immunity proteins respectively, the molecular activity of these proteins was never investigated. In this study, we try to over-express Usp in E. coli, purify Usp and characterize its molecular activity.MethodRecombinant Usp protein was expressed in E. coli BL21(DE3) cells together with 6× Histidine tagged OrfU1 (OrfU1-His) protein, and purified with affinity chromatography using Ni2+ chelating agarose. The nuclease activity of the purified Usp was examined in vitro by using plasmid DNA as a substrate. The importance of H-N-H motif in nuclease activity of Usp was examined by site-directed mutagenesis study.ResultsWe revealed that pET expression vector encoding Usp alone could not be maintained in E. coli BL21(DE3), and insertion of the orfUs as well as usp in the constructed plasmid diminished the toxic effect, suggesting that co-expressed OrfUs masked the activity of Usp. To purify Usp protein, we employed the expression vector encoding untagged Usp together with OrfU1-His. A tight complex formation could be observed between Usp and OrfU1-His, which allowed the purification of Usp in a single chromatographic step: binding of Usp/OrfU1-His complex to Ni2+ chelating agarose followed by elution of Usp from the complex with denaturing reagent. The purified free Usp was found to have the nuclease activity, and the activity was constitutively higher than Usp/OrfU1-His complex. H-N-H motif, which is found in various types of nucleases including a subfamily of nuclease-type bacteriocin, had been identified in the C-terminal region of Usp. Site-directed mutagenesis study showed that the H-N-H motif in Usp is indispensable for its nuclease activity.ConclusionThis is the first evidence of the molecular activity of the new member of H-N-H superfamily and lays the foundation for the biological characterization of Usp and its inhibitor protein, OrfUs.

Crystal structure of the tandem-type universal stress protein TTHA0350 from Thermus thermophilus HB8

The genome sequence of an extremely thermophilic bacterium, Thermus thermophilus HB8, revealed that TTHA0350 is a tandem-type universal stress protein (Usp) consisting of two Usp domains. Usp proteins, which are characterized by a conserved domain consisting of 130-160 amino acids, are inducibly expressed under a large number of stress conditions. The N-terminal domain of TTHA0350 contains a motif similar to the consensus ATP-binding one (G-2 x-G-9x-G-(S/T)), but the C-terminal one seems to lack the consensus motif. In order to determine its structural properties, we determined the crystal structures of TTHA0350 in the unliganded form and TTHA0350•2ATP at 2.50 and 1.70 Å resolution, respectively. This is the first structure determination of a Usp family protein in both unliganded and ATP-liganded forms. TTHA0350 is folded into a fan-shaped structure which is similar to that of tandem-type Usp protein Rv2623 from Mycobacterium tuberculosis. However, the dimer assembly with C2-symmetry in TTHA0350 is quite different from that with D2-symmetry in Rv2623. The X-ray structure showed that not only the N-terminal but also the C-terminal domain binds one ATP, although the ATP-binding motif could not be detected in the C-terminal domain. The loop interacting with ATP in the C-terminal domain is in a conformation quite different from that in the N-terminal domain.

Regulatory elements in the juvenile hormone binding protein gene from Galleria mellonella — Topography of binding sites for Usp and EcRDBD

The juvenile hormone binding protein (JHBP) plays a key role in the protection and transport of the hormone to target tissues. In this report the sequence of the jhbp promoter comprising about 2000 bp is characterized. Using a minimized false positive algorithm, six putative regulatory elements, Hunchback, Heat shock factor binding element, Ultrabithorax, Broad-Complex Z3, Elf-1 and Chorion factor 1/ultraspiracle (CF1/Usp) were found in the distal promoter of the jhbp gene. Proteins from nuclear extract of Galleria mellonella fat body form four specific complexes with probe containing TATA box, five complexes with Inr probe and one protein complex with DPE probe. EMSA and footprinting analyses showed that one of the three CF1/Usp elements (starting at -1053) has an exceptionally high affinity to Usp protein. An unknown, high-affinity Usp/EcRDBD-binding element (TCAACA-AAC-TGTTCA), distinct from 20-hydroxyecdysone response elements, was identified in the jhbp gene promoter, based on a footprinting assay. Deletions of jhbp promoter in the regions containing the CF1/Usp elements enhance the transcriptional activity of luciferase reporter gene in the Trichoplusia ni High Five cell line. Obtained data suggest that jhbp promoter is TATA- and Inr-driven, CF1/Usp elements exhibit inhibitory effect on jhbp expression, and an interaction between Usp and DNA relies on recognition of the consensus sequence (GGGTCA) and on ionic interactions of several phosphate groups outside from this element.

66: Subtyping of Uropathogenic Escherichia Coli According to Genetic Mosaicism in the Pathogenicity Island Encoding USP Protein

66: Subtyping of Uropathogenic Escherichia Coli According to Genetic Mosaicism in the Pathogenicity Island Encoding USP Protein

The novel ethylene-regulated gene OsUsp1 from rice encodes a member of a plant protein family related to prokaryotic universal stress proteins.

Using subtractive hybridization a submergence-induced gene was identified from deepwater rice, OsUsp1, that encodes a homologue of the bacterial universal stress protein family. Sequence analysis revealed that OsUSP1 is most closely related to the bacterial MJ0577-type of ATP-binding USP proteins which have been suggested to act as a molecular switch. USP protein homologues appear to be ubiquitous in plants and are encoded by gene families, but are absent in animal species. In the youngest internode of deepwater rice plants, OsUsp1 expression was very strongly induced within 1 h of submergence. Elevated transcript levels were observed in dividing cells, in expanding cells and in differentiated tissue indicating that USP1 mediates a general process. Gene induction was shown to be regulated by ethylene with a highly similar expression pattern to that observed with submergence treatment. Based on sequence information and on expression data it is hypothesized that OsUSP1 plays a role in ethylene-mediated stress adaptation in rice.

Structural and sequence diversity of the pathogenicity island of uropathogenic Escherichia coli which encodes the USP protein

A total of 321 uropathogenic Escherichia coli (UPEC) strains and 12 strains of E. coli isolated from stool samples of healthy individuals, which were previously shown to be positive in colony hybridization test using the usp (encoding for the uropathogenic-specific protein) DNA probe, were examined by PCR amplification to determine the size of the usp gene and the pathogenicity island (PI). Three types of size variation were observed for the usp gene and four types for the PI. Sequencing analysis of the PIs from seven representative strains (six UPEC and one from a normal healthy individual) revealed that the usp genes can be classified into two groups, each having different sequences in the 3′-terminal region. The peptides encoded by the three open reading frames (ORFs) downstream of usp had identical 23 amino acid residues in the C-terminal region. The subregion encoding these small ORFs has a mosaic structure constituted of six segments. The positions of these segments vary from strain to strain, and in some strains, two to four segments are deleted. This indicates that rearrangements occur frequently in this region and the mosaic arrangement apparently contributes to the size variation observed in the PCR examination of the usp genes and PIs.

Structural and sequence diversity of the pathogenicity island of uropathogenic Escherichia coli which encodes the USP protein.

A total of 321 uropathogenic Escherichia coli (UPEC) strains and 12 strains of E. coli isolated from stool samples of healthy individuals, which were previously shown to be positive in colony hybridization test using the usp (encoding for the uropathogenic-specific protein) DNA probe, were examined by PCR amplification to determine the size of the usp gene and the pathogenicity island (PI). Three types of size variation were observed for the usp gene and four types for the PI. Sequencing analysis of the PIs from seven representative strains (six UPEC and one from a normal healthy individual) revealed that the usp genes can be classified into two groups, each having different sequences in the 3'-terminal region. The peptides encoded by the three open reading frames (ORFs) downstream of usp had identical 23 amino acid residues in the C-terminal region. The subregion encoding these small ORFs has a mosaic structure constituted of six segments. The positions of these segments vary from strain to strain, and in some strains, two to four segments are deleted. This indicates that rearrangements occur frequently in this region and the mosaic arrangement apparently contributes to the size variation observed in the PCR examination of the usp genes and PIs.

Expression and Function of the ultraspiracle (usp) Gene during Development of Drosophila melanogaster

The usp locus encodes a member of the nuclear hormone receptor superfamily in Drosophila melanogaster that interacts with EcR (ecdysone receptor) to mediate ecdysteroid-induced gene expression. A 2.7-kb usp mRNA was detected at all developmental times tested, although its abundance varied. Among premetamorphic stages, both the 2.7-kb transcript and Usp protein attained their highest levels in the late third larval instar. The 2.7-kb usp transcript was also found in adult stages and a 1.2-kb transcript was detected in the polyadenylated RNA fraction of both mature adult females and early embryos. Aneuploids carrying two usp mutant alleles and a putative variegating usp+ allele often developed deformities of the adult wing disc that apparently resulted from mutational disruption of usp activity before metamorphosis and whose frequency was affected by maternal genotype. Both of the recessive lethal usp mutations associated with this "cleft thorax" phenotype involved substitutions of conserved arginine residues in the DNA-binding domain, although the frequency of the phenotype was not the same for the two alleles. Both mutant proteins retained the ability to form heterodimers with EcR in vitro but showed reduced affinity for an ecdysone response element.