Biomedical subjects

Kwang-Hyun Baek

Publications and source records attributed to Kwang-Hyun Baek.

At least 19 recordsLinked to original sources

PubMed2006

Hyaluronan- and RNA-binding deubiquitinating enzymes of USP17 family members associated with cell viability.

BACKGROUND: Protein degradation by the ubiquitin system plays a crucial role in numerous cellular signaling pathways. Deubiquitination, a reversal of ubiquitination, has been recognized as an important regulatory step in the ubiquitin-dependent degradation pathway. RESULTS: While identifying putative ubiquitin specific protease (USP) enzymes that contain a conserved Asp (I) domain in humans, 4 USP17 subfamily members, highly homologous to DUB-3, have been found (USP17K, USP17L, USP17M, and USP17N), from human chorionic villi. Expression analysis showed that USP17 transcripts are highly expressed in the heart, liver, and pancreas and are expressed moderately in various human cancerous cell lines. Amino acid sequence analysis revealed that they contain the highly conserved Cys, His, and Asp domains which are responsible for the deubiquitinating activity. Biochemical enzyme assays indicated that they have deubiquitinating activity. Interestingly, the sequence analysis showed that these proteins, with exception of USP17N, contain the putative hyaluronan/RNA binding motifs, and cetylpyridinium chloride (CPC)-precipitation analysis confirmed the association between these proteins and intracellular hyaluronan and RNA. CONCLUSION: Here, we report that the overexpression of these proteins, with exception of USP17N, leads to apoptosis, suggesting that the hyaluronan and RNA binding motifs in these enzymes play an important role in regulating signal transduction involved in cell death.

Amino Acid Sequence↗

PubMed2006

The expression of Usp42 during embryogenesis and spermatogenesis in mouse.

Mouse Usp42, a novel ubiquitin specific protease gene, was isolated from mouse embryonic stem cells. It consists of 1,324 amino acids with a predicted molecular weight of 146kDa and contains the conserved Cys, Asp (I), His and Asn/Asp (II) domains defined as one of characteristics for deubiquitinating enzymes. RT-PCR analysis showed that the Usp42 transcript is expressed in NIH3T3 cells, B- and T-lymphocytes, and L1210 cells. Northern blot analysis revealed that Usp42 is expressed mainly in brain, lung, thymus and testis, and at mouse E10.5 the most during embryonic development. Usp42 expression rises from 2 weeks after birth to round-spermatid stage and decreases from condensing-spermatid stage during spermatogenesis. Deubiquitinating enzyme assays demonstrated that Usp42 can cleave ubiquitin from ubiquitinated substrates in vitro and in vivo. Taken all together, it is suggested that Usp42 is one of typical deubiquitinating enzymes, which may play an important role in mouse embryogenesis and spermatogenesis.

Amino Acid Sequence↗

PubMed2006

Single nucleotide polymorphism in exon 17 of the insulin receptor gene is not associated with polycystic ovary syndrome in a Korean population.

OBJECTIVE: To assess the association between the single nucleotide polymorphism of the insulin receptor (INSR) gene and polycystic ovary syndrome (PCOS) in a Korean population. DESIGN: Case-control study. SETTING: University-based hospital. PATIENT(S): One hundred seventy-four patients with PCOS and 93 healthy women as controls. MAIN OUTCOME MEASURE(S): Frequency of three genotypes for single nucleotide polymorphism found in exon 17 of INSR gene. RESULT(S): The high frequency of the T allele was shown both in patient and control groups. The frequency of C allele, which known as a normal allele, was slightly higher in the patient group than in the control group. CONCLUSION(S): The C/T polymorphism in exon 17 of the INSR gene is not associated with susceptibility of PCOS in a Korean population.

Adult↗

PubMed2006

Proteomic analysis of recurrent spontaneous abortion: Identification of an inadequately expressed set of proteins in human follicular fluid.

Recurrent spontaneous abortion (RSA), defined as the loss of three or more consecutive pregnancies prior to the 20th week of gestation, affects up to 5% of the child-bearing population. To investigate the proteins associated with RSA, the protein expression in human follicular fluid was analyzed using 2-DE. Follicular fluid contains a variety of biologically important proteins for oocyte fertilization and follicle maturation in the mammalian reproductive process. Therefore, it can be used as a provisional source for identifying proteins involved in RSA. In this study, we identified five aberrantly expressed proteins (complement component C3c chain E, fibrinogen gamma, antithrombin, angiotensinogen, and hemopexin precursor) in follicular fluid from RSA patients with MALDI-TOF-MS and nano-LC MS/MS. Western blot analysis confirmed that the protein expression level of fibrinogen gamma and antithrombin was less in follicular fluid from RSA patients than those from normal controls. Semiquantitative RT-PCR and real-time PCR analyses revealed that mRNA level of these coagulation factors was also decreased significantly in chorionic villi of RSA patients compared with normal samples. Taken all together, it is likely that coagulation factors (fibrinogen gamma and antithrombin) play an important role in maintaining the normal pregnancy.

Abortion, Habitual↗

PubMed2006

Molecular structure and organization of the wheat genomic manganese superoxide dismutase gene.

The genomic structure of a manganese superoxide dismutase (MnSOD) gene in wheat was elucidated by sequencing a clone from a BAC library of a stripe rust resistant wheat line. The clone was identified by hybridization with a wheat MnSOD cDNA. The gene consisted of 6 exons interrupted by 5 introns with a total length of 4770 nucleotides from the start codon to the termination codon. The wheat MnSOD gene was the longest among those sequenced from plant species. The transcription initiation site was preceded by a G+C-rich promoter without a TATA or CAAT box. The promoter contained many putative cis-acting regulatory elements, including an abscisic acid (ABA)-responsive element, a stress-responsive element, and a GC-repeat, as well as several other structural features in common with the promoter of the rice MnSOD gene. A Stowaway-like transposable element was found in intron 5 of the wheat MnSOD gene, but further investigation revealed the transposable element was not present in all copies of the MnSOD genes.

Base Sequence↗

PubMed2006

Cytokine-regulated protein degradation by the ubiquitination system.

The ubiquitin-mediated protein degradation pathway exerts a wide spectrum of effects and modulates a variety of biological processes including cell cycle progression, transcriptional regulation, signal transduction, antigen presentation, apoptosis (or programmed cell death), oncogenesis, preimplantation, and DNA repair. Recently, the importance of deubiquitination mechanism has been emerged as an essential regulatory step to control these cellular mechanisms for homeostasis. Even though a number of deubiquitinating enzymes have recently been isolated, relatively little is known about their substrates and biological functions. Identified from yeast to human, deubiquitinating (DUB) enzymes are classified into the ubiquitin C-terminal hydrolase (UCH), the ubiquitin-specific processing proteases (UBP or USP), Jab1/Pad1/MPN domain containing metallo-enzymes (JAMM), Otu domain ubiquitin-aldehyde binding proteins (OTU), and Ataxin-3/Josephin domain containing proteins (Ataxin-3/Josephin). Several members of a novel DUB subfamily induced by cytokines in murine lymphocytes have recently been identified. In addition, human DUB enzyme DUB-3, highly homologous to USP17 and induced by cytokines interleukin (IL)-4 and IL-6, has been recently isolated and showed that it has significant homology to the known murine DUB subfamily members. Interestingly, both murine DUB and human USP17 subfamily members are localized and clustered on murine chromosome 7 and on human chromosomes 4 and 8, respectively. This review introduces the reader to provide a great understanding of cytokine-inducible DUB enzymes in both mouse and human, and new insights into DUB subfamily members.

Amino Acid Sequence↗

PubMed2006

Molecular cloning of rHAUSP encoding a deubiquitinating enzyme in rat testis.

The tumor suppressor protein p53 is stabilized by the herpes-virus-associated ubiquitin-specific protease (HAUSP), a deubiquitinating enzyme. We previously isolated and characterized a mouse orthologue of HAUSP, mHAUSP. In this study, we have identified a rat orthologue of HAUSP, rHAUSP, from the rat testis by RT-PCR using primers used for cloning mHAUSP. rHAUSP cDNA encodes 3,312 bp and 1,103 amino acids with a molecular weight of approximately 135 kDa containing highly conserved Cys, Asp (I), His, and Asn/Asp (II) domains characteristic of the ubiquitin-specific processing proteases. pI value of rHAUSP is 5.31. In vivo and in vitro deubiquitinating enzyme assays demonstrated that rHAUSP has deubiquitinating enzymatic activity. The over-expression of rHAUSP induced cell death of cervical adenocarcinoma cells.

Amino Acid Sequence↗

PubMed2006

HAUSP as a therapeutic target for hematopoietic tumors (review).

p53, one of the most important tumor suppressor proteins, plays an essential role in regulating the cell cycle and apoptosis by sensing the integrity of genome. Therefore, the level of p53 protein is critical for normal cellular homeostasis, and is known to be subtly regulated by ubiquitination and deubiquitination systems. Numerous genetic alterations of p53 have been reported in all types of tumors. In hematopoietic tumors, the mutations of p53 gene are rare compared with solid tumors, which showed more than 50% frequency for p53 mutations. According to this characteristic feature of hematological tumors, the therapeutic strategy for targeting the level of p53 may be valuable in anti-cancer treatment of hematological tumors. Herein, we deal with the post-translational regulation of p53 via its specific ubiquitinating enzymes (Mdm2, Mdmx, COP1, Pirh2, ARF-BP1/Mule, and CHIP) and a deubiquitinating enzyme, herpesvirus-associated ubiquitin-specific protease (HAUSP). In this article, we review the regulatory mechanism of p53 via ubiquitination and deubiquitination system and suggest the several possible therapeutic strategies of targeting HAUSP, a deubiquitinating enzyme for p53, for treating hematopoietic tumors.

Endopeptidases↗

PubMed2006

Disruption of protein-protein interaction in the Mgl-1 oncoprotein.

Mammalian homologues of the Lethal giant larvae (Lgl) tumor suppressor gene have been identified and these homologues can complement the yeast double mutant of Sop1 and Sop2, the yeast homologue of Lgl, as reported previously. In the absence of these genes in yeast, cellular viability is affected at restrictive temperature and salt environments. Members of this family contain five or more of the WD-40 repeat motifs, which is known to be involved in protein-protein interaction. In order to investigate the biochemical roles for conserved amino acids within the most conserved WD-40 repeat motif amongst these family members, we generated deletion mutants for five conserved amino acids (G450, H451, D453, W459 and D460) in mouse Lgl-1 (Mgl-1), located between 450-460 amino acids. We found that the deletion mutants of Mgl-1, DeltaG450 and DeltaD453, were not capable of complementing yeast mutants of Sop1 and Sop2 at restrictive temperature and high salt environments. These results indicate that the WD-40 repeat motif is important for cellular viability by regulating temperature-sensitivity and salt tolerance in yeast.

Amino Acid Motifs↗

PubMed2005

The expression patterns of deubiquitinating enzymes, USP22 and Usp22.

Deubiquitinating enzymes regulate a number of cellular mechanisms including pre-implantation, growth and differentiation, oncogenesis, cell cycle progression, transcriptional activation, and signal transduction. In this study, we have identified a novel human deubiquitinating enzyme gene, USP22, and its mouse homologue, Usp22. They encode 525 amino acids (approximate MW: 60kDa) and contains Cys, Asp (I), His and Asp/Asn (II), the highly conserved domains of the UBP family of deubiquitinating enzymes. The biochemical assay revealed that they have deubiquitinating enzyme activity. Northern blot analysis for USP22 showed moderate expression in various organs including human heart and skeletal muscle, and weak expression in lung and liver. However, Usp22 is expressed strongly in brain and weakly in other organs. We investigated the expression level of Usp22 mRNA and the localization during implantation and early pregnancy by in situ hybridization. Interestingly, Northern blot analysis showed the strong expression of Usp22 between embryonic days E10.5 and E12.5. Whole mount in situ hybridization staining revealed that Usp22 was expressed in the midbrain, forebrain, hindbrain and dorsal root ganglia of embryos at E12.5. Embryos at E12.5 showed the pronounced expression of Usp22 during the early embryonic development, although its expression was not detectable in the gut, liver and heart.

Amino Acid Sequence↗

PubMed2005

Protein kinase A phosphorylates and regulates dimerization of 14-3-3 epsilon.

Recognition of phosphorylated serine/threonine-containing motifs by 14-3-3 depends on the dimerization of 14-3-3. However, the molecular cues that control 14-3-3 dimerization are not well understood. In order to identify proteins that control 14-3-3 dimerization, we analyzed proteins that have effects on 14-3-3 dimerization and report that protein kinase A (PKA) phosphorylates 14-3-3zeta at a specific residue (Ser58). Phosphorylation by PKA leads to modulation of 14-3-3zeta dimerization and affect its interaction with partner proteins. Substitution of Ser58 to Ala completely abolished phosphorylation of 14-3-3zeta by PKA. A phospho-mimic mutant of 14-3-3zeta, Ser58 to Glu substitution, failed to form homodimers, showed reduced interaction with 14-3-3epsilon and p53, and could not enhance transcriptional activity of p53. Moreover, activation of PKA decreases and inhibition of PKA increases the dimerization of 14-3-3zeta and the functional interaction of 14-3-3zeta with p53. Therefore, our results suggest that PKA is a new member of protein kinases that can phosphorylate and impair the function of 14-3-3.

14-3-3 Proteins↗

PubMed2005

Differential expression of manganese superoxide dismutase sequence variants in near isogenic lines of wheat during cold acclimation.

Numerous sequence variants of wheat (Triticum aestivum L.) manganese superoxide dismutase (MnSOD) genes have been found. Quantitative real-time PCR was used to measure the expression levels of three MnSOD genes distinguished by a variable amino acid, and three genes distinguished by sequence variation in the 3' untranslated region (3' UTR), in wheat plants grown at 20 degrees C and cold-acclimated for 1-4 weeks at 2 degrees C. The amino acid variants did not differ significantly in expression levels, however, differential expression of genes differing in the 3' UTR was observed. Diploid wheat-related species also carried sequence variants of MnSOD, with differing levels of expression, suggesting diversification of the MnSOD gene family occurred prior to the polyploidization events of hexaploid wheat.

3' Untranslated Regions↗

PubMed2005

Differential mRNA stability to endogenous ribonucleases of the coding region and 3' untranslated regions of wheat (Triticum aestivum L.) manganese superoxide dismutase genes.

The sequences of the 3' untranslated region (UTR) of the manganese superoxide dismutase (MnSOD) genes in wheat (Triticum aestivum) were found to be quite variable with different predicted thermostabilities. The degradation rates of the 3' UTR variants and the coding region were measured following exposure to endogenous nucleases. The degradation rates of the 3' UTR variants for 15 min were not significantly different, meaning the degradation rates of the 3' UTR variants were not directly related to the thermostabilities. However, the degradation rate of the coding region was significantly faster than those of the 3' UTR variants. Further investigation revealed the coding region seemed to have specific sites for degradation, indicating a possibility of increasing MnSOD expression by the degradation site alteration.

3' Untranslated Regions↗

PubMed2005

HAUSP, a deubiquitinating enzyme for p53, is polyubiquitinated, polyneddylated, and dimerized.

The tumor suppressor protein p53 is ubiquitinated and neddylated by MDM2 and then degraded by 26S proteasome. However, p53 is stabilized by the HAUSP (Herpes-virus-associated ubiquitin-specific protease) deubiquitinating enzyme. In this study, we discovered that rat HAUSP (rHAUSP) is polyubiquitinated, polyneddylated, and dimerized using co-immunoprecipitation assays. This suggests that rHAUSP may function as a dimer or multimer and is also degraded through the proteasome-mediated degradation. Transfection of rHAUSP into RGC-Lac-Z cell line with the integrated p53 response element revealed that rHAUSP contributed to p53 stabilization, and a rHAUSP (C224S) mutant contributed to p53 destabilization in a dose-dependent manner.

Animals↗

PubMed2005

The WD-40 repeat motif of Lgl tumor suppressor proteins associated with salt tolerance and temperature sensitivity.

We have recently identified mammalian homologues of lethal giant larvae (Lgl) tumor suppressor gene, rat Rgl-1 and bovine Bgl-1, and demonstrated that they can complement yeast double mutants lacking Sop1 and Sop2, yeast homologues of Lgl. These gene products are capable of regulating cellular viability in restrictive salt and temperature environments. Since Lgl family members contain the WD-40 repeat motif, we investigated its cellular functions using mouse homologue Mgl-1 in the absence of Sop1 and Sop2 in yeasts by complementation. Interestingly, mutant forms of Mgl-1 at the conserved glycine at position 450 and aspartic acid at position 453 in the most conserved WD-40 repeat motif were not able to complement, indicating that these amino acids are critical for regulating salt tolerance and temperature sensitivity in yeast. These results shed light on the important regulation of cytoskeletal complex for cellular polarity within eukaryotic cells.

Adaptation, Physiological↗

PubMed2005

Deubiquitinating enzyme USP36 contains the PEST motif and is polyubiquitinated.

The ubiquitin-mediated protein degradation pathway has been emphasized for the regulation of numerous cellular mechanisms and the significance of deubiquitination, mediated by deubiquitinating (DUB) enzymes, has been emerging as an essential regulatory step to control these cellular mechanisms. Previously, we demonstrated a human DUB enzyme, HeLa DUB-1, expressed in human ovarian cancer cells. Here, we report human USP36, which has the extension of the C-terminal region of HeLa DUB-1 and has conserved amino acid domains as previously shown in other DUBs. Human USP36, encoding a DUB enzyme, was isolated from ovarian cancer cells using RT-PCR and characterized. We identified DUB enzyme activity of USP36 by analyzing its capability to cleave the ubiquitin. Interestingly, structural and immunoprecipitation analyses revealed for the first time that USP36 contains the PEST motif and is polyubiquitinated.

Amino Acid Motifs↗

PubMed2005

Long oligonucleotide microarrays in wheat: evaluation of hybridization signal amplification and an oligonucleotide-design computer script.

A computer script was written in the Perl language to design equal-length long oligonucleotides from DNA sequences. The script allows the user to specify G + C content, melting temperature, self-complementarity, the maximum number of contiguous duplicate bases, whether to start with the first start codon and whether to report reverse complements. Microarrays were fabricated with 95 oligonucleotides (60 mers) representing 41 genes. The microarray was interrogated with cDNA from roots and shoots of two near-isogenic lines and a commercial cultivar of Triticum aestivum L. (hexaploid wheat) challenged with cold temperature, hot temperature, or the biological control bacterium Pseudomonas fluorescens. Self-complementarity of the oligonucleotides was negatively correlated with signal intensity in 23 of 54 arrays (39%; P <0.01). Tyramide signal amplification was essential for signal generation and detection. Genes involved in signal transduction pathways responded similarly following exposure to cold, heat and P. fluorescens, suggesting intersection of the pathways involved in response to these disparate stress factors. Microarray results were corroborated by quantitative real-time PCR in 75% of samples assayed. We conclude that long oligonucleotide microarrays for interrogation with cDNA from hexaploid wheat should be constructed from oligonucleotides having minimal self complementarity that also meet user-specified requirements of length, G + C content and melting temperature; multiple oligonucleotides should be used to represent each gene; and Tyramide signal amplification is useful in wheat oligonucleotide microarray studies.

Base Sequence↗

PubMed2005

Trophoblast apoptosis is increased in women with evidence of TH1 immunity.

Interferon-gamma secretion from peripheral blood mononuclear cells (PBMCs) of women with unexplained recurrent spontaneous abortions was increased by trophoblast stimulation. Apoptosis was significantly increased in trophoblast cells cultured with the supernatant of PBMCs from women with evidence of T(H)1 immunity to trophoblast stimulation.

Abortion, Habitual↗