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At least 19 recordsLinked to original sources
PubMed2026

Molecular mechanism of HUWE1-HAPSTR1-USP7-mediated ubiquitin chain amplification on nuclear proteins.

Rapid protein turnover is essential for cellular stress adaptation. HUWE1 (HECT, UBA, and WWE domain containing 1), a large HECT-type E3 ligase, regulates many short-lived stress-responsive proteins, yet the mechanisms underlying its substrate selectivity remain unclear. Here, we reveal that HUWE1 functions as a ubiquitin chain amplifier that captures pre-ubiquitinated substrates and amplifies the degradation signal by assembling long ubiquitin chains containing K11-K48 branch points, a process regulated by its partners HUWE1-associated protein stress response 1 (HAPSTR1) and USP7 (ubiquitin-specific-processing protease 7). Structural and biochemical analyses show that HAPSTR1 engages HUWE1's ubiquitin-binding motifs to drive nuclear import and modulate substrate recruitment. A cryo-EM structure of the HUWE1-USP7 complex reveals a bidirectional regulatory mechanism: HUWE1 activates USP7's catalytic activity, while USP7 modulates HUWE1 conformational states. Global proteomic analyses demonstrate that this axis drives extensive remodeling of the short-lived nuclear proteome. These findings establish the HUWE1-HAPSTR1-USP7 complex as a key ubiquitin code modifier, providing a molecular rationale for HUWE1 dysregulation in neurodevelopmental disorders and cancer.

Ubiquitin-Specific Peptidase 7↗
PubMed2006

The two human homologues of yeast UFD2 ubiquitination factor, UBE4A and UBE4B, are located in common neuroblastoma deletion regions and are subject to mutations in tumours.

Chromosomes 11q and 1p are commonly deleted in advanced-stage neuroblastomas and are therefore assumed to contain tumour suppressor genes involved in the development of this cancer. The two UFD2 yeast gene human homologues, UBE4A and UBE4B, involved in the ubiquitin/proteasome pathway, are located in 11q and 1p, respectively. UBE4B has previously been analysed for mutations and one mutation in the splice donor site of exon 9, c.1439 + 1G > C, was found in a neuroblastoma tumour with fatal outcome. We speculated that the homologue UBE4A might be involved in an alternative tumourigenesis pathway. The coding exons of UBE4A were therefore sequenced. One putative missense mutation (1028T > C, leading to I343T, residing in exon 8) was found in neuroblastoma tumour 20R8; this finding was confirmed by sequencing in both directions. The change, isoleucine (non-polar) to threonine (polar), was situated in a highly conserved amino acid region. In addition, two novel variants were also found in intronic sequences of UBE4A. It might be speculated that the proteins generated from UBE4B and UBE4A are involved in protecting the cell from environmental stress and that inactivation of either of them could contribute to malignancy.

Amino Acid Sequence↗
PubMedDate not supplied

Identification of an E3 ubiquitin-protein ligase in the softshell clam (Mya arenaria).

Softshell clams (Mya arenaria) were exposed to dioxin in controlled laboratory experiments in order to study their molecular response to dioxin exposure. A complementary DNA (cDNA) fragment with sequence similarity to E3 ubiquitin-protein ligase appeared to be upregulated in dioxin-exposed clams compared to controls. E3 covalently ligates ubiquitin onto a protein, targeting it for degradation. Our findings suggest that the ubiquitin-mediated proteolytic pathway in the softshell clam may be activated by dioxin exposure. Because the clam E3-predicted amino acid sequence is most similar to a specific vertebrate E3 protein (E6-AP), we hypothesize that dioxin may stimulate ubiquitin-mediated degradation of cell-cycle regulatory proteins, such as the tumor suppressor p53, which promotes cell proliferation. This pathway has been observed in human cervical cancer. Partial cDNA sequence of the clam E3 has been identified using the differential display polymerase chain reaction (ddPCR) and RACE (Rapid Amplification of cDNA Ends) PCR; the full-length sequence is currently being determined. Discovering the molecular mechanism(s) stimulated by dioxin exposure in this invertebrate model may contribute to a better understanding of the effects of dioxin on marine organisms.

Amino Acid Sequence↗
PubMed2000

Identification of a novel isopeptidase with dual specificity for ubiquitin- and NEDD8-conjugated proteins.

Covalent conjugation of proteins by ubiquitin or ubiquitin-like molecules is an important form of post-translational modification and plays a critical role in many cellular processes. Similar to the concept of phosphorylation and dephosphorylation, these conjugates are regulated by a large number of deconjugating enzymes. Here, we report the cloning of a 2,141-base pair DNA fragment from human placenta cDNA library by a strategy that involves expressed sequence tag data base searching, polymerase chain reaction, and rapid amplification of cDNA ends. Nucleotide sequence analysis revealed that the cloned cDNA contains an open reading frame of 1,143 base pairs encoding a novel protease, USP21, which is composed of 381 residues with a calculated molecular mass of 43 kDa. The human USP21 gene is located on chromosome 1q21 and encodes a member of the ubiquitin-specific protease family with highly conserved Cys and His domains. The activity and specificity of USP21 were determined by using a COS cell expression system in vivo. We showed that USP21 is capable of removing ubiquitin from ubiquitinated proteins as expected. Furthermore, USP21 is capable of removing NEDD8 from NEDD8 conjugates but has no effect on Sentrin-1 conjugates. As expected from its biochemical activity, overexpression of USP21 has a profound growth inhibitory effect on U2OS cells. Thus, USP21 is the first ubiquitin-specific protease shown to have dual specificity for both ubiquitin and NEDD8 and may play an important role in the regulation of cell growth.

Amino Acid Sequence↗
PubMed2006

Detection of aberrations of ubiquitin-conjugating enzyme E2C gene (UBE2C) in advanced colon cancer with liver metastases by DNA microarray and two-color FISH.

Using DNA microarrays, the expression profiles of 1,700 genes in the primary tumor, liver metastases and paired normal tissue obtained from nine patients with advanced colorectal cancer was studied. Twenty genes were upregulated and only one gene was downregulated in the primary tumors. In the liver metastases, 39 genes were upregulated and only three genes were downregulated. There was no significant difference in gene expression between the primary tumors and the liver metastases. The most highly overexpressed gene in both the primary tumors and the liver metastases was the ubiquitin-conjugating enzyme E2C gene (UBE2C), located at 20q13.1. Additionally, two-color FISH analysis using probes for the region 20q13.1 and the chromosome 20 centromere revealed that amplification at 20q13.1 had occurred in 5 of 10 (50%) colon cancers. Comparison between the levels of gene expression and FISH results revealed that UBE2C expression is significantly changed by amplification at 20q13.1, suggesting genomic amplification as one mechanism of increased UBE2C expression. Our results showing aberrations in levels of gene expression and locus copy number of UBE2C suggest that this gene may play an important role in tumor progression leading to advanced colon cancer with liver metastasis.

Centromere↗
PubMed2004

Discrimination of melanocytic tumors by cDNA array hybridization of tissues prepared by laser pressure catapulting.

Gene expression profiling by cDNA array analysis in melanoma is hampered by the need for large amounts of RNA to prepare reliable probes for array hybridization. On the other hand, for ex vivo analysis of malignant cells from melanocytic tumors laser pressure catapulting is an essential prerequisite to obtain noncontaminated melanocytic preparations; however, laser pressure catapulting prepared material provides only nanogram amounts of RNA. In this study we present an approach to overcome these limitations by combining laser pressure catapulting and real-time polymerase chain reaction based SMART cDNA amplification technology. Reproducible and reliable hybridization patterns from about 500 laser pressure catapulting prepared cell equivalents from 22 cases of melanocytic tumors were generated using array analysis. Univariate analysis revealed significant differences of the expression pattern of melanocytic nevi, melanomas, and melanoma metastases. Multivariate analysis with four genes being the best univariate discriminative features (tyrosinase related protein 2, translation initiation factor 2 gamma, ubiquitine conjugating enzyme E2I and one expressed sequence tag) allowed clustering of nevi, melanomas, and melanoma metastases with an accuracy of 82%. Data validation was performed by additional quantitative reverse transcription-polymerase chain reaction (TaqMan-reverse transcription-polymerase chain reaction). Taken together, this study shows, that (1) array analysis is feasible on tumors with rather low cell numbers, and (2) differences in expression profiles allow discrimination between benign and malignant lesions. Expression patterns of marker genes defined in unequivocal histopathologic entities may improve the diagnostic and prognostic assessment of difficult melanocytic lesions, which is still the hardest problem in dermatopathology.

Biopsy↗
PubMed1995

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.

Amino Acid Sequence↗
PubMed2001

DUB-2A, a new member of the DUB subfamily of hematopoietic deubiquitinating enzymes.

Protein ubiquitination is an important regulator of cytokine-activated signal transduction pathways and hematopoietic cell growth. Protein ubiquitination is controlled by the coordinate action of ubiquitin-conjugating enzymes and deubiquitinating enzymes. Recently a novel family of genes encoding growth-regulatory deubiquitinating enzymes (DUB-1 and DUB-2) has been identified. DUBs are immediate-early genes and are induced rapidly and transiently in response to cytokine stimuli. By means of polymerase chain reaction amplification with degenerate primers for the DUB-2 complementary DNA, 3 murine bacterial artificial chromosome (BAC) clones that contain DUB gene sequences were isolated. One BAC contained a novel DUB gene (DUB-2A) with extensive homology to DUB-2. Like DUB-1 and DUB-2, the DUB-2A gene consists of 2 exons. The predicted DUB-2A protein is highly related to other DUBs throughout the primary amino acid sequence, with a hypervariable region at its C-terminus. In vitro, DUB-2A had functional deubiquitinating activity; mutation of its conserved amino acid residues abolished this activity. The 5' flanking sequence of the DUB-2A gene has a hematopoietic-specific functional enhancer sequence. It is proposed that there are at least 3 members of the DUB subfamily (DUB-1, DUB-2, and DUB-2A) and that different hematopoietic cytokines induce specific DUB genes, thereby initiating a cytokine-specific growth response. (Blood. 2001;98:636-642)

Animals↗
PubMed1997

An Acanthamoeba polyubiquitin gene and application of its promoter to the establishment of a transient transfection system.

We have isolated and sequenced a 2388 bp polyubiquitin encoding genomic DNA from Acanthamoeba encompassing two complete and one incomplete ubiquitin units. Codon usage frequency shows extreme bias. The deduced amino acid sequences of each unit are identical to each other and the same as that deduced from a previously sequenced Acanthamoeba castellanii cDNA. The upstream region of this gene, which contained some putative regulatory modules, was recovered by PCR (polymerase chain reaction) amplification and subcloning. This upstream fragment was ligated to the CAT (chloramphenicol acetyltransferase) gene in a eukaryotic expression plasmid and successfully applied to the establishment of an Acanthamoeba transient transfection system. Transfection was performed by electroporation and the optimal voltage was 4500 volts/cm at capacitance 25 microF. DEAE-dextran (25 microg/ml) added into the electroporation buffer increased the transfection efficiency by about 45%. The CAT activity was proportional to the amount of DNA transfected and reached the peak level 48 h after transfection. CAT assays showed that the polyubiquitin gene upstream fragment contains a functional promoter which is about 2.5 times as strong as a viral RSV-LTR promoter when driving CAT expression in Acanthamoeba.

Acanthamoeba↗
PubMed2006

Upregulation of dopamine D2 receptors in dopaminergic drug-naive patients with Parkin gene mutations.

Medicated patients with Parkinsonism and parkin gene mutations have been reported to show a significant decrease in striatal dopamine D2 receptors (D2R) in comparison to medicated idiopathic Parkinson's disease (IPD) patients with similar age and disease severity. The aim of this study was to verify whether the genetic defect per se is responsible for this decrease. We have studied with [11C]raclopride (RAC) positron emission tomography (PET) in a group of 14 sporadic patients with parkin-linked Parkinsonism, 6 of whom had never received levodopa or dopamine agonists. The remaining 8 patients had been treated with levodopa for at least 5 years. Presynaptic striatal [18F]dopa storage was not significantly different between these two groups of patients. In untreated parkin-positive patients, significant putaminal increases in RAC-binding potential (BP) were found in comparison to an age-matched healthy control group by using a classical region of interest approach and statistical parametric mapping. In contrast, levodopa-treated parkin-positive patients showed significant decreases in RAC-BP in the caudate and putamen when compared to an age-matched healthy control group. The RAC PET findings revealed that striatal D2R upregulation occurs in dopaminergic drug-naive parkin-positive patients, in a similar fashion to the upregulation reported in drug-naive IPD. D2R downregulation observed in medicated parkin-positive patients, therefore, is not caused primarily by the genetic defect itself. Parkin-positive patients appear to have a greater susceptibility to the exposure to dopaminergic medication than IPD patients, which in turn might be an indirect effect of their genetic mutation.

Adult↗
PubMed2004

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.

Amino Acid Sequence↗
PubMed2005

No association of the codon 55 methionine to valine polymorphism in the SUMO4 gene with Graves' disease.

OBJECTIVE: A functional polymorphism at codon 55 of the small ubiquitin-like modifier-4 (SUMO4) gene (methionine to valine; M55V) has recently been associated with type 1 diabetes mellitus (T1D). We aimed to establish whether this locus also contributes towards the genetic susceptibility to Graves' disease (GD) and autoimmune Addison's disease. DESIGN: A case-control analysis was performed using genomic DNA samples from 595 unrelated white GD subjects, 104 white autoimmune Addison's disease subjects and 467 healthy white control subjects. The SUMO4 M55V single nucleotide polymorphism (SNP) was genotyped using polymerase chain reaction (PCR) amplification followed by digestion with the restriction enzyme MseI. RESULTS: There was no association of the SUMO4 M55V alleles with either GD, thyroid-associated orbitopathy or autoimmune Addison's disease when compared to controls; P = 0.28, 0.46 and 0.91, respectively, by chi2 testing. CONCLUSION: We cannot confirm a generalized role for SUMO4 in autoimmune endocrinopathy. The SUMO4 codon 55 methionine to valine polymorphism may be exclusively associated with susceptibility to T1D, or the effect of the locus in GD and Addison's disease may be much less than that found in T1D patients.

Addison Disease↗
PubMed2003

Refined mapping of 1q32 amplicons in malignant gliomas confirms MDM4 as the main amplification target.

We previously reported on the amplification and overexpression of the mouse double minute 4 homolog gene (MDM4) from 1q32 in a subset of malignant gliomas (Riemenschneider et al., Cancer Res 1999;59:6091-6). More recently, amplification and overexpression of the neighboring contactin 2 gene (CNTN2) was reported in individual malignant gliomas without MDM4 amplification (Rickman et al., Cancer Res 2001;61:2162-8). To address the question of whether 1q32 carries 2 independent amplification targets or a common target other than MDM4 and CNTN2, we analyzed primary malignant gliomas for amplification and overexpression of 17 different genes from this region. Our results indicate a single region of amplification that comprises the genes MDM4, GAC1, PIK3C2B and PEPP3, with only MDM4 amplification being invariably associated with overexpression. CNTN2 was found to be coamplified with MDM4 in 3 malignant gliomas but overexpressed in only 1 of these tumors. No CNTN2 amplification was detected in any of 102 malignant gliomas without MDM4 amplification. Our data therefore corroborate the notion that MDM4 is the main amplification target on 1q32 in malignant gliomas. However, coamplification and overexpression of adjacent genes may provide an additional growth advantage in some malignant gliomas with MDM4 amplification.

Adult↗
PubMed1994

Isolation and characterization of the gene encoding histone H2A from Trypanosoma cruzi.

In the present paper we report the isolation and characterization of the sequence of two genomic DNA fragments coding for the histone H2A of Trypanosoma cruzi. An analysis of the predicted amino acid sequence shows the presence of the amino-terminal motif characteristic of the H2A histones proteins and the Lys-Lys motif reported to be the site for the ubiquitin attachment. Southern blots of total parasite DNA probed with the H2A sequence suggested that the T. cruzi histone H2A gene is encoded in two independent gene clusters. The molecular karyotyping of the parasite indicated that these two clusters locate in a single chromosome of about 700 kb in length. The T. cruzi H2A mRNA is polyadenylated as are the basal histone mRNAs of higher eukaryotes and the histone mRNAs of yeast. By polymerase chain reaction amplification and sequencing and by S1 mapping we determined respectively the 5' and 3' end of the gene showing that the miniexon is added to the mRNA 71 nucleotides upstream of the ATG initiation codon and that the polyadenylation site locates in nucleotide position 773-775 close to invert repeats.

Amino Acid Sequence↗
PubMed2004

Real-time PCR quantification of bovine lactase mRNA: localization in the gastrointestinal tract of milk-fed calves.

Lactase is a disaccharidase that is present in the brush-border membrane of the small intestine, hydrolyzes lactose to glucose and galactose, and is therefore important in milk-fed animals. Assays based on quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) in the bovine species have not yet been described. Therefore, we have developed an RT-PCR assay for the quantification of lactase mRNA levels and have tested its suitability in the bovine gastrointestinal tract of seven 5-d-old milk-fed calves. Primers for RT-PCR amplification of bovine lactase mRNA were designed in the 100% identical regions of species (rats, rabbits, humans) from which lactase sequences were available. Lactase mRNA was expressed relative to mean levels of 4 housekeeping genes (glyceraldehyde-3-phosphate dehydrogenase, beta-actin, ubiquitin, and 18S). The presence of lactase mRNA along the entire gastrointestinal tract was evaluated in samples that consisted of whole gut walls (mucosa plus submucosa). Furthermore, mRNA levels of lactase were measured in fractionized layers of jejunal and ileal mucosa (mainly containing villus tips or crypts) and ileal lamina propria (mainly containing Peyer's patches). Agarose gel electrophoresis of the lactase PCR product revealed a single band that corresponded to the single-amplified product as predicted by the melting curve analysis of the PCR. The amplified partial-bovine lactase sequence showed 87% similarity with human and rabbit sequences and 82% similarity with the rat sequence. Lactase mRNA was present in whole walls (consisting of mucosa and submucosa) of the entire small intestine, but was absent in esophagus, rumen, fundus, pylorus, and colon. Furthermore, lactase mRNA was detected in fractionized villus and crypt layers of jejunum and ileum, but levels were higher in the jejunum in villus than in crypt fractions. No lactase mRNA was detectable in the lamina propria fraction of the ileum containing mainly Peyer's patches. In conclusion, the developed RT-PCR method allows study of lactase mRNA levels.

Animals↗
PubMed2001

Isolation and characterization of a novel cDNA, UBAP1, derived from the tumor suppressor locus in human chromosome 9p21-22.

PURPOSE: To clone the putative tumor suppressor gene(s) in a refined region at 9p21-22 undergoing loss of heterozygosity in nasopharyngeal carcinoma (NPC). METHODS: We systematically screened the expression patterns of 25 novel ESTs (expressed sequence tags) in a minimal common deleted region of 9p21-22 in NPC. One of these ESTs was found down-regulated in NPC. Subsequently, the corresponding gene sequence of this EST was established by cDNA cloning and RACE (rapid amplification of cDNA end) procedures. Furthermore, a mouse homologue of this gene was identified. The expression of this gene was examined using Northern blot or reverse transcription-polymerase chain reaction (RT-PCR) in various human and mouse tissues. A limited screen for mutation of coding sequence of this novel human gene was undertaken using RT-PCR and direct sequencing analysis. RESULTS: A novel gene was cloned. This gene is a new member of the UBA domain family, so we named it UBAPI for ubiquitin-associated protein 1 (HUGO Gene Nomenclature Committee-approved symbol). Northern blot and RT-PCR analysis demonstrate a ubiquitous pattern of gene expression in human and mouse tissues. The direct sequencing analysis of the coding region of hUBAP1 following RT-PCR failed to reveal any mutations in a preliminary screen of NPC cell line HNE1 and primary nasopharyngeal carcinoma samples. CONCLUSIONS: We cloned a novel gene UBAPI, which is highly conserved between human and mouse. Clearly, as a novel member of UBA domain protein family and taking its map location into account, a more extensive analysis is essential to establish whether subtle mutations are present in nasopharyngeal carcinomas.

Amino Acid Sequence↗
PubMed1996

Genomic analysis of human multigene families using chromosome-specific vectorette PCR.

We report a technique for the rapid determination of genomic structure of individual members of human interspersed multigene families which circumvents the requirement for genomic clone isolation. In this approach, vectorette libraries were constructed from human/rodent somatic cell hybrid DNA harbouring single members of the gene family. Using these libraries as PCR templates with nested gene-specific primers in combination with a common vectorette primer resulted in the amplification of gene-specific products suitable for the subsequent determination of intron/exon structure. We have applied this technique to characterise members of two gene families.

Chromosomes↗
PubMed2002

Quantification of housekeeping transcript levels during the development of bovine preimplantation embryos.

In mammals, the study of gene expression in the preimplantation embryo has been difficult because the standard procedures used to quantify mRNA generally require large amounts of starting material. The development of protocols using different quantitative strategies generally involving the polymerase chain reaction (PCR) has provided new tools for exploration of gene expression in preimplantation embryos. However, the use of an internal standard, often referred as a housekeeping gene, is essential to normalize the mRNA levels. RNA levels of eight housekeeping genes were quantified using real time PCR throughout the preimplantation period of the bovine embryo to find the most suitable gene to be used as standard. Histone H2a was the best internal standard because the transcript levels were constant across the preimplantation period. Linear amplification of antisense RNA using the T7 promotor for in vitro transcription of the entire RNA pool was evaluated as a suitable way to preamplify the starting material prior to quantification and was effective in providing accurate RNA abundance profiles throughout the preimplantation period. However, the amplification appears to be template dependent because the amplification factors were higher for some genes.

Actins↗