The c-Jun N-terminal Kinases (JNKs) play important roles in cell responses to stress or growth factor stimulation. The JNK1α1 isoform shares >90% identity with a predominantly neuronal JNK3α1 isoform, Show more
The c-Jun N-terminal Kinases (JNKs) play important roles in cell responses to stress or growth factor stimulation. The JNK1α1 isoform shares >90% identity with a predominantly neuronal JNK3α1 isoform, but JNK3α1 also includes a distinctive 38 amino acid N-terminal sequence. To address the outstanding question of the potential for these JNK isoforms to have different binding partners that mediate different biological actions, the work presented here refined the yeast two-hybrid approach to identify and categorize binding partners for JNK1α1 and JNK3α1. Specifically, site-directed mutagenesis of the JNK1α1 common docking (CD) domain that mediates typical JNK-binding domain (JBD)-dependent interactions, truncation of the distinctive JNK3 N-terminal domain (i.e. ΔN JNK3α1), and interaction evaluation in the yeast two-hybrid system defined the interacting partners as either JNK1-specific interactors (ATF7, FUS, KCNE4, PIAS1, SHANK1, TKT), typical JBD-dependent interactors shared by JNK1α1 and JNK3α1 (AKAP6, BMPR2, EEF1A1, GFAP, GRIP2, GTF2F1, HDAC2, MAP1B, MYO9B, PTPN2, RABGAP1, RUSC2, SUMO1, SYPL1, TOPBP1, ZNF668), or JNK3-specific partners (ATXN1, NNAT, PTGDS) dependent on interaction with the JNK3 N-terminal extension. The interacting partners ATF7, AKAP6, and ATXN1 were explored further as representatives of these different classes. Two potential JBDs were identified in ATF7 as important for its interaction with JNK1α1, but additionally an interaction between ATF7 and ΔN JNK3α1 was shown to be JBD-dependent, suggesting that the JNK3α1 N-terminus prevents interaction with some proteins. For the shared partner AKAP6, one of the multiple potential JBDs predicted by sequence analysis was important for the AKAP6-JNK interaction in the yeast screening system as well as in mammalian cells. Finally, the ATXN1-JNK3α1 interaction was dependent on the JNK3α1 N-terminus in a mammalian cell context. These studies therefore highlight a diversity of potential JNK-interacting partners with both JBD-dependent as well as JBD-independent modes of interaction. Show less
Qing Hu, Wei-xian Chen, Shan-liang Zhong+6 more · 2014 · Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine · Springer · added 2026-04-24
MicroRNA-452 (miRNA-452) was overexpressed in docetaxel-resistant human breast cancer MCF-7 cells (MCF-7/DOC). However, its role in modulating the sensitivity of breast cancer cells to docetaxel (DOC) Show more
MicroRNA-452 (miRNA-452) was overexpressed in docetaxel-resistant human breast cancer MCF-7 cells (MCF-7/DOC). However, its role in modulating the sensitivity of breast cancer cells to docetaxel (DOC) remains unclear. The aim of this study is to investigate the role of miRNA-452 in the sensitivity of breast cancer cells to DOC.Real-time quantitative PCR (RT-qPCR) were used to identify the differential expression of miRNA-452 between MCF-7/DOC and MCF-7 cells. MiRNA-452 mimic was transfected into MCF-7 cells and miRNA-452 inhibitor was transfected into MCF-7/DOC cells. The role of miRNA-452 in these transfected cells was evaluated using RT-qPCR, MTT assay, and flow cytometry assay. The relationship of miRNA-452 and its predictive target gene "anaphase-promoting complex 4" (APC4) was analyzed by RT-qPCR and Western blot.MiRNA-452 showed significantly higher expression (78.9-folds) in MCF-7/DOC cells compared to parental MCF-7 cells. The expression of miRNA-452 in the mimic transfected MCF-7 cells was upregulated 212.2-folds (P < 0.05) compared to its negative control (NC), and the half maximal inhibitory concentration (IC50) value of DOC (1.98 ± 0.15 μM) was significantly higher than that in its NC (0.85 ± 0.08 μM, P < 0.05) or blank control (1.01 ± 0.19 μM, P < 0.05). Furthermore, its apoptotic rate (6.3 ± 1.3 %) was distinctly decreased compared with that in its NC (23.8 ± 6.6 %, P < 0.05) or blank control (18.6 ± 4.7 %, P < 0.05). In contrast, the expression of miRNA-452 in the inhibitor-transfected MCF-7/DOC cells was downregulated 0.58-fold (P < 0.05) compared to its NC, the IC50 value of DOC (44.5 ± 3.2 μM) was significantly lower than that in its NC (107.3 ± 6.63 μM, P < 0.05) or blank control (102.22 ± 11.34 μM, P < 0.05), and the apoptotic rate (45.5 ± 10.8 %) was distinctly increased compared with its NC (9.9 ± 2.2 %, P < 0.05) and blank control (9.4 ± 2.5 %, P < 0.05). Further, there was an inverse association between miRNA-452 and APC4 expression in breast cancer cells in vitro.Dysregulation of miRNA-452 involved in the DOC resistance formation of breast cancer cells may be, in part, via targeting APC4. Show less
LC/MS quantification of multiple plasma proteins that differ by several orders of magnitude in concentration from a single sample is challenging. We present a strategy that allows the simultaneous det Show more
LC/MS quantification of multiple plasma proteins that differ by several orders of magnitude in concentration from a single sample is challenging. We present a strategy that allows the simultaneous determination of the concentration and turnover kinetics of higher and lower abundant proteins from a single digestion mixture. Our attention was directed at a cluster of proteins that interact to affect the absorption and interorgan lipid trafficking. We demonstrate that apos involved in TG metabolism such as apoC2, C3, E, and A4 (micromolar concentration), and apoB48 and apoA5 (single-digit nanomolar concentration) can be quantified from a single digestion mixture. A high degree of correlation between LC/MS and immunobased measurements for apoC2, C3, E, and B48 was observed. Moreover, apoA5 fractional synthesis rate was measured in humans for the first time. Finally, the method can be directly applied to studies involving nonhuman primates because peptide sequences used in the method are conserved between humans and nonhuman primates. Show less
Alterations or mutations in the lipoprotein lipase (LPL) gene contribute to severe hypertriglyceridemia (HTG). This study reported on two patients in a Chinese family with LPL gene mutations and sever Show more
Alterations or mutations in the lipoprotein lipase (LPL) gene contribute to severe hypertriglyceridemia (HTG). This study reported on two patients in a Chinese family with LPL gene mutations and severe HTG and acute pancreatitis. Two patients with other five family members were included in this study for DNA-sequences of hyperlipidemia-related genes (such as LPL, APOC2, APOA5, LMF1, and GPIHBP1) and 43 healthy individuals and 70 HTG subjects were included for the screening of LPL gene mutations. Both patients were found to have a compound heterozygote for a novel LPL gene mutation (L279V) and a known mutation (A98T). Furthermore, one HTG subject out of 70 was found to carry this novel LPL L279V mutation. The data from this study showed that compound heterozygote mutations of A98T and L279V inactivate lipoprotein lipase enzymatic activity and contribute to severe HTG and acute pancreatitis in two Chinese patients. Further study will investigate how these LPL gene mutations genetically inactivate the LPL enzyme. Show less
Our objective was to evaluate the effect of gene polymorphisms of apolipoprotein C3 (APOC3) on the development of non-alcoholic fatty liver disease (NAFLD) in different populations. We performed a met Show more
Our objective was to evaluate the effect of gene polymorphisms of apolipoprotein C3 (APOC3) on the development of non-alcoholic fatty liver disease (NAFLD) in different populations. We performed a meta-analysis of all relevant studies published in the literature. A total of 115 clinical trials or reports were identified, but only seven trials met our inclusion criteria. A meta-analysis was performed according to the Cochrane Reviewers' Handbook recommendations. Five hospital-based and two population-based case-control studies were included in the final analysis. The overall frequency of APOC3 gene polymorphisms was 67.5% (1177/1745) in NAFLD and 68.8% (988/1437) in controls. The summary odds ratio for the association of gene polymorphisms of APOC3 and the risk of NAFLD was 1.03 (95% CI: 0.89-1.22),which was not statistically significant (P > 0.05). Our meta-analysis, while not ruling out possible publication bias, showed no association between gene polymorphisms of APOC3 and the risk of NAFLD development in different populations in the world. Show less
To investigate the relationship between Apolipoprotein C3 (APOC3) (-455T>C) polymorphism and nonalcoholic fatty liver disease (NAFLD) in the Southern Chinese Han population. In this prospective case-c Show more
To investigate the relationship between Apolipoprotein C3 (APOC3) (-455T>C) polymorphism and nonalcoholic fatty liver disease (NAFLD) in the Southern Chinese Han population. In this prospective case-control study, we recruited 300 NAFLD patients and 300 healthy controls to a cohort representing Southern Chinese Han population at The First Affiliated Hospital, Sun Yat-sen University, from January to December 2012. Polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing were used to genotype the APOC3 (-455T>C) variants. After adjusting for age, gender, and body-mass index, TC and CC genotypes were found to increase the susceptibility to NAFLD compared to the TT genotype, with adjusted odds ratios (ORs) of 1.77 (95%CI: 1.16-2.72) and 2.80 (95%CI: 1.64-4.79), respectively. Further stratification analysis indicated that carriers of the CC genotype was more susceptible to insulin resistance (IR) than those of the TT genotype, with an OR of 3.24 (95%CI: 1.52-6.92). The CC genotype also was associated with a significantly higher risk of hypertension, hypertriglyceridemia, and low levels of high-density lipoprotein cholesterol (HDL) (P < 0.05). No association was found between the APOC3 (-455T>C) polymorphism and obesity, impaired glucose tolerance, hyperuricemia, hypercholesterolemia, or high levels of low-density lipoprotein cholesterol (LDL) (P > 0.05). APOC3 (-455T>C) genetic variation is involved in the susceptibility to developing NAFLD, IR, hypertension, hypertriglyceridemia, and low HDL in the Southern Chinese Han population. Show less
The analysis of rich catalogues of genetic variation from population-based sequencing provides an opportunity to screen for functional effects. Here we report a rare variant in APOC3 (rs138326449-A, m Show more
The analysis of rich catalogues of genetic variation from population-based sequencing provides an opportunity to screen for functional effects. Here we report a rare variant in APOC3 (rs138326449-A, minor allele frequency ~0.25% (UK)) associated with plasma triglyceride (TG) levels (-1.43 s.d. (s.e.=0.27 per minor allele (P-value=8.0 × 10(-8))) discovered in 3,202 individuals with low read-depth, whole-genome sequence. We replicate this in 12,831 participants from five additional samples of Northern and Southern European origin (-1.0 s.d. (s.e.=0.173), P-value=7.32 × 10(-9)). This is consistent with an effect between 0.5 and 1.5 mmol l(-1) dependent on population. We show that a single predicted splice donor variant is responsible for association signals and is independent of known common variants. Analyses suggest an independent relationship between rs138326449 and high-density lipoprotein (HDL) levels. This represents one of the first examples of a rare, large effect variant identified from whole-genome sequencing at a population scale. Show less
Vitamin A status regulates obesity development, hyperlipidemia, and hepatic lipogenic gene expression in Zucker fatty (ZF) rats. The development of hyperlipidemia in acne patients treated with retinoi Show more
Vitamin A status regulates obesity development, hyperlipidemia, and hepatic lipogenic gene expression in Zucker fatty (ZF) rats. The development of hyperlipidemia in acne patients treated with retinoic acid (RA) has been attributed to the induction of apolipoprotein C-III expression. To understand the role of retinoids in the development of hyperlipidemia in ZF rats, the expression levels of several selected RA-responsive genes in the liver and isolated hepatocytes from Zucker lean (ZL) and ZF rats were compared using real-time PCR. The Rarb and Srebp-1c mRNA levels are higher in the liver and isolated hepatocytes from ZF than ZL rats. The Apoc3 mRNA level is only higher in the isolated hepatocytes from ZF than ZL rats. To determine whether dynamic RA production acutely regulates Apoc3 expression, its mRNA levels in response to retinoid treatments or adenovirus-mediated overexpression of hepatocyte nuclear factor 4 alpha (HNF4α) and chicken ovalbumin upstream-transcription factor II (COUP-TFII) were analyzed. Retinoid treatments for 2-6 h did not induce the expression of Apoc3 mRNA. The overexpression of HNF4α or COUP-TFII induced or inhibited Apoc3 expression, respectively. We conclude that short-term retinoid treatments could not induce Apoc3 mRNA expression, which is regulated by HNF4α and COUP-TFII in hepatocytes. Show less
Yan Pu, Peng Chen, Bin Zhou+5 more · 2014 · Biomarkers : biochemical indicators of exposure, response, and susceptibility to chemicals · added 2026-04-24
AXIN1 is a central component of Wnt signalling pathway which is essential for embryonic development. To investigate whether polymorphisms of AXIN1 contribute to ASD susceptibility. Three tag SNPs (rs1 Show more
AXIN1 is a central component of Wnt signalling pathway which is essential for embryonic development. To investigate whether polymorphisms of AXIN1 contribute to ASD susceptibility. Three tag SNPs (rs12921862, rs370681 and rs1805105) in AXIN1 were genotyped in 208 ASD patients and 302 healthy controls using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in a Chinese population. Significantly increased ASD risk was observed to be associated with the A allele of rs12921862 (p < 0.0001, OR = 3.096, 95% CI = 2.037-4.717). Increased ASD risk was observed to be associated with rs370681 in a codominant (p = 0.043, OR = 1.52, 95% CI = 1.04-2.22) and overdominant model (p = 0.016, OR = 1.57, 95% CI = 1.08-2.27). rs12921862 and rs370681 may contribute to ASD susceptibility. Show less
Genome-wide association study has reported a number of genes as being associated with ankylosing spondylitis (AS) in Caucasian European populations and Chinese Han population. The aim of the study was Show more
Genome-wide association study has reported a number of genes as being associated with ankylosing spondylitis (AS) in Caucasian European populations and Chinese Han population. The aim of the study was to investigate whether single nucleotide polymorphisms (SNPs) covering the 21q22 region are associated with AS in the Chinese Guangxi Zhuang population. A case-control study was performed in unrelated patients with AS (n = 315) and age-, sex-, and ethnicity-matched controls (n = 630) from Guangxi Zhuang ethnic group. All patients met the modified New York criteria for AS. TaqMan genotyping assay was used to genotype cases and controls for 17 tag SNPs covering 21q22. After multiple-testing correction, significant association with AS was not observed in all SNP, but one block haplotype was significantly associated with AS. The pairwise analysis of the rs8126528/rs2150414/rs6517532 alleles found that the G-A-A haplotype (OR 2.92, 95 % CI 1.48-3.55; p = 0.0002, permuted p = 0.0332) significantly increased the risk of AS in comparison with the G-A-G, A-A-A and G-G-A carriers. In conclusion, the study results define a novel risk haplotypes in 21q22 that was associated with AS in the Chinese Guangxi Zhuang population. The findings was consistent with previous genetic and functional studies that point at variants of the BRWD1 and/or PSMG1 loci as interesting genetic factors contributing to AS. Show less
Apoptosis constitutes a system for the removal of aged, or damaged cells, which is regulated by the interplay of pro-apoptotic and antiapoptotic proteins. Previous study has shown that Juvenile Batten Show more
Apoptosis constitutes a system for the removal of aged, or damaged cells, which is regulated by the interplay of pro-apoptotic and antiapoptotic proteins. Previous study has shown that Juvenile Batten disease protein, CLN3, is antiapoptotic gene in NT2 neuronal precursor cells and a few types of cancers. However, in colorectal cancer, whether CLN3 also play its antiapoptotic role and the effect of targeted controlling CLN3 on the biological behavior of human colorectal cancer cell is unknown. We employed the sequence-specific siRNA silencing the CLN3 gene and investigated its effects on growth and apoptosis of colorectal cancer HCT116 cells, which has highest elevation of CLN3 expression among four colorectal cancer cell lines. After CLN3 specific siRNA transfection, mRNA and protein expression levels of CLN3 in HCT116 cells were noticeably decreased. Moreover, CLN3-siRNA inhibited the proliferation of colorectal cancer cells, promoted their apoptosis and induced G0/G1 cell cycle arrest. Our current study demonstrated that CLN3 was expressed in colorectal cancer cells at a high frequency. Moreover, CLN3 down-regulation with RNA interference can inhibit proliferation, apoptosis, and cell cycle progression of colorectal cancer cells. Our study represented a potential new approach to understanding the role of CLN3 in cancer and provides a potential novel strategy colorectal cancer therapy. Show less
Juvenile neuronal ceroid lipofuscinosis (JNCL or CLN3 disease) is an autosomal recessive lysosomal storage disease resulting from mutations in the CLN3 gene that encodes a lysosomal membrane protein. Show more
Juvenile neuronal ceroid lipofuscinosis (JNCL or CLN3 disease) is an autosomal recessive lysosomal storage disease resulting from mutations in the CLN3 gene that encodes a lysosomal membrane protein. The disease primarily affects the brain with widespread intralysosomal accumulation of autofluorescent material and fibrillary gliosis, as well as the loss of specific neuronal populations. As an experimental treatment for the CNS manifestations of JNCL, we have developed a serotype rh.10 adeno-associated virus vector expressing the human CLN3 cDNA (AAVrh.10hCLN3). We hypothesized that administration of AAVrh.10hCLN3 to the Cln3(Δex7/8) knock-in mouse model of JNCL would reverse the lysosomal storage defect, as well as have a therapeutic effect on gliosis and neuron loss. Newborn Cln3(Δex7/8) mice were administered 3 × 10(10) genome copies of AAVrh.10hCLN3 to the brain, with control groups including untreated Cln3(Δex7/8) mice and wild-type littermate mice. After 18 months, CLN3 transgene expression was detected in various locations throughout the brain, particularly in the hippocampus and deep anterior cortical regions. Changes in the CNS neuronal lysosomal accumulation of storage material were assessed by immunodetection of subunit C of ATP synthase, luxol fast blue staining, and periodic acid-Schiff staining. For all parameters, Cln3(Δex7/8) mice exhibited abnormal lysosomal accumulation, but AAVrh.10hCLN3 administration resulted in significant reductions in storage material burden. There was also a significant decrease in gliosis in AAVrh.10hCLN3-treated Cln3(Δex7/8) mice, and a trend toward improved neuron counts, compared with their untreated counterparts. These data demonstrate that AAVrh.10 delivery of a wild-type cDNA to the CNS is not harmful and instead provides a partial correction of the neurological lysosomal storage defect of a disease caused by a lysosomal membrane protein, indicating that this may be an effective therapeutic strategy for JNCL and other diseases in this category. Show less
Retinitis pigmentosa (RP) is a devastating form of retinal degeneration, with significant social and professional consequences. Molecular genetic information is invaluable for an accurate clinical dia Show more
Retinitis pigmentosa (RP) is a devastating form of retinal degeneration, with significant social and professional consequences. Molecular genetic information is invaluable for an accurate clinical diagnosis of RP due to its high genetic and clinical heterogeneity. Using a gene capture panel that covers 163 of the currently known retinal disease genes, including 48 RP genes, we performed a comprehensive molecular screening in a collection of 123 RP unsettled probands from a wide variety of ethnic backgrounds, including 113 unrelated simplex and 10 autosomal recessive RP (arRP) cases. As a result, 61 mutations were identified in 45 probands, including 38 novel pathogenic alleles. Interestingly, we observed that phenotype and genotype were not in full agreement in 21 probands. Among them, eight probands were clinically reassessed, resulting in refinement of clinical diagnoses for six of these patients. Finally, recessive mutations in CLN3 were identified in five retinal degeneration patients, including four RP probands and one cone-rod dystrophy patient, suggesting that CLN3 is a novel non-syndromic retinal disease gene. Collectively, our results underscore that, due to the high molecular and clinical heterogeneity of RP, comprehensive screening of all retinal disease genes is effective in identifying novel pathogenic mutations and provides an opportunity to discover new genotype-phenotype correlations. Information gained from this genetic screening will directly aid in patient diagnosis, prognosis, and treatment, as well as allowing appropriate family planning and counseling. Show less
Yi-Ying Lee, Chien-Feng Li, Ching-Yih Lin+6 more · 2014 · Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine · Springer · added 2026-04-24
Locally advanced rectal cancers are currently treated with neoadjuvant concurrent chemoradiotherapy (CCRT) followed by surgery, but stratification of risk and final outcomes remain suboptimal. In view Show more
Locally advanced rectal cancers are currently treated with neoadjuvant concurrent chemoradiotherapy (CCRT) followed by surgery, but stratification of risk and final outcomes remain suboptimal. In view of the fact that glutamine metabolism is usually altered in cancer, we profiled and validated the significance of genes involved in this pathway in rectal cancers treated with CCRT. From a published transcriptome of rectal cancers (GSE35452), we focused on glutamine metabolic process-related genes (GO:0006541) and found upregulation of carbamoyl phosphate synthetase 1 (CPS1) gene most significantly predicted poor response to CCRT. We evaluated the expression levels of CPS1 using immunohistochemistry to analyze tumor specimens obtained during colonoscopy from 172 rectal cancer patients. Expression levels of CPS1 were further correlated with major clinicopathological features and survivals in this validation cohort. To further confirm CPS1 expression levels, Western blotting was performed for human colon epithelial primary cell (HCoEpiC) and four human colon cancer cells, including HT29, SW480, LoVo, and SW620. CPS1 overexpression was significantly related to advanced posttreatment tumor (T3, T4; P = 0.006) and nodal status (N1, N2; P < 0.001), and inferior tumor regression grade (P = 0.004). In survival analyses, CPS1 overexpression was significantly associated with shorter disease-specific survival (DSS) and metastasis-free survival (MeFS). Furthermore, using multivariate analysis, it was also independently predictive of worse DSS (P = 0.021, hazard ratio = 2.762) and MeFS (P = 0.004, hazard ratio = 3.897). CPS1 protein expression, as detected by Western blotting, is more abundant in colon cancer cells than nonneoplastic HCoEpiC. Overexpression of CPS1 is associated with poor therapeutic response and adverse outcomes among rectal cancer patients receiving CCRT, justifying the potential theranostic value of CPS1 for such patients. Show less
Asialoglycoprotein receptor (ASGPR)-ligand-based separation combined with identification with Hep Par 1 or pan-cytokeratin (P-CK) antibody have been demonstrated to detect circulating tumor cells (CTC Show more
Asialoglycoprotein receptor (ASGPR)-ligand-based separation combined with identification with Hep Par 1 or pan-cytokeratin (P-CK) antibody have been demonstrated to detect circulating tumor cells (CTCs) in hepatocellular carcinoma (HCC). The aim of this study was to develop an improved enrichment and identification system that allows the detection of all types of HCC CTCs. The specificity of the prepared anti-ASGPR monoclonal antibody was characterized. HCC cells were bound by ASGPR antibody and subsequently magnetically isolated by second antibody-coated magnetic beads. Isolated HCC cells were identified by immunofluorescence staining using a combination of anti-P-CK and anti-carbamoyl phosphate synthetase 1 (CPS1) antibodies. Blood samples spiked with HepG2 cells were used to determine recovery and sensitivity. CTCs were detected in blood samples from HCC patients and other patients. ASGPR was exclusively expressed in human hepatoma cell line, normal hepatocytes and HCC cells in tissue specimens detected by the ASGPR antibody staining. More HCC cells could be identified by the antibody cocktail for CPS1 and P-CK compared with a single antibody. The current approach obtained a higher recovery rate of HepG2 cells and more CTC detection from HCC patients than the previous method. Using the current method CTCs were detected in 89% of HCC patients and no CTCs were found in the other test subjects. Our anti-ASGPR antibody could be used for specific and efficient HCC CTC enrichment, and anti-P-CK combined with anti-CPS1 antibodies is superior to identification with one antibody alone in the sensitivity for HCC CTC detection. Show less
We developed a multiplex PCR assay capable of identifying two capsular polysaccharide synthesis sequence types (sequence type 258 [ST258] cps-1 and cps-2) in epidemic Klebsiella pneumoniae ST258 strai Show more
We developed a multiplex PCR assay capable of identifying two capsular polysaccharide synthesis sequence types (sequence type 258 [ST258] cps-1 and cps-2) in epidemic Klebsiella pneumoniae ST258 strains. The assay performed with excellent sensitivity (100%) and specificity (100%) for identifying cps types in 60 ST258 K. pneumoniae sequenced isolates. The screening of 419 ST258 clonal isolates revealed a significant association between cps type and K. pneumoniae carbapenemase (KPC) variant: cps-1 is largely associated with KPC-2, while cps-2 is primarily associated with KPC-3. Show less
We report the identification and characterization of a five-carbon protein posttranslational modification (PTM) called lysine glutarylation (Kglu). This protein modification was detected by immunoblot Show more
We report the identification and characterization of a five-carbon protein posttranslational modification (PTM) called lysine glutarylation (Kglu). This protein modification was detected by immunoblot and mass spectrometry (MS), and then comprehensively validated by chemical and biochemical methods. We demonstrated that the previously annotated deacetylase, sirtuin 5 (SIRT5), is a lysine deglutarylase. Proteome-wide analysis identified 683 Kglu sites in 191 proteins and showed that Kglu is highly enriched on metabolic enzymes and mitochondrial proteins. We validated carbamoyl phosphate synthase 1 (CPS1), the rate-limiting enzyme in urea cycle, as a glutarylated protein and demonstrated that CPS1 is targeted by SIRT5 for deglutarylation. We further showed that glutarylation suppresses CPS1 enzymatic activity in cell lines, mice, and a model of glutaric acidemia type I disease, the last of which has elevated glutaric acid and glutaryl-CoA. This study expands the landscape of lysine acyl modifications and increases our understanding of the deacylase SIRT5. Show less
Circulating homocysteine levels (tHcy), a product of the folate one carbon metabolism pathway (FOCM) through the demethylation of methionine, are heritable and are associated with an increased risk of Show more
Circulating homocysteine levels (tHcy), a product of the folate one carbon metabolism pathway (FOCM) through the demethylation of methionine, are heritable and are associated with an increased risk of common diseases such as stroke, cardiovascular disease (CVD), cancer and dementia. The FOCM is the sole source of de novo methyl group synthesis, impacting many biological and epigenetic pathways. However, the genetic determinants of elevated tHcy (hyperhomocysteinemia), dysregulation of methionine metabolism and the underlying biological processes remain unclear. We conducted independent genome-wide association studies and a meta-analysis of methionine metabolism, characterized by post-methionine load test tHcy, in 2,710 participants from the Framingham Heart Study (FHS) and 2,100 participants from the Vitamin Intervention for Stroke Prevention (VISP) clinical trial, and then examined the association of the identified loci with incident stroke in FHS. Five genes in the FOCM pathway (GNMT [p = 1.60 × 10(-63)], CBS [p = 3.15 × 10(-26)], CPS1 [p = 9.10 × 10(-13)], ALDH1L1 [p = 7.3 × 10(-13)] and PSPH [p = 1.17 × 10(-16)]) were strongly associated with the difference between pre- and post-methionine load test tHcy levels (ΔPOST). Of these, one variant in the ALDH1L1 locus, rs2364368, was associated with incident ischemic stroke. Promoter analyses reveal genetic and epigenetic differences that may explain a direct effect on GNMT transcription and a downstream affect on methionine metabolism. Additionally, a genetic-score consisting of the five significant loci explains 13% of the variance of ΔPOST in FHS and 6% of the variance in VISP. Association between variants in FOCM genes with ΔPOST suggest novel mechanisms that lead to differences in methionine metabolism, and possibly the epigenome, impacting disease risk. These data emphasize the importance of a concerted effort to understand regulators of one carbon metabolism as potential therapeutic targets. Show less
We discuss here a new approach to detecting hepatotoxicity by employing concentration changes of liver-specific blood proteins during disease progression. These proteins are capable of assessing the b Show more
We discuss here a new approach to detecting hepatotoxicity by employing concentration changes of liver-specific blood proteins during disease progression. These proteins are capable of assessing the behaviors of their cognate liver biological networks for toxicity or disease perturbations. Blood biomarkers are highly desirable diagnostics as blood is easily accessible and baths virtually all organs. Fifteen liver-specific blood proteins were identified as markers of acetaminophen (APAP)-induced hepatotoxicity using three proteomic technologies: label-free antibody microarrays, quantitative immunoblotting, and targeted iTRAQ mass spectrometry. Liver-specific blood proteins produced a toxicity signature of eleven elevated and four attenuated blood protein levels. These blood protein perturbations begin to provide a systems view of key mechanistic features of APAP-induced liver injury relating to glutathione and S-adenosyl-L-methionine (SAMe) depletion, mitochondrial dysfunction, and liver responses to the stress. Two markers, elevated membrane-bound catechol-O-methyltransferase (MB-COMT) and attenuated retinol binding protein 4 (RBP4), report hepatic injury significantly earlier than the current gold standard liver biomarker, alanine transaminase (ALT). These biomarkers were perturbed prior to onset of irreversible liver injury. Ideal markers should be applicable for both rodent model studies and human clinical trials. Five of these mouse liver-specific blood markers had human orthologs that were also found to be responsive to human hepatotoxicity. This panel of liver-specific proteins has the potential to effectively identify the early toxicity onset, the nature and extent of liver injury and report on some of the APAP-perturbed liver networks. Show less
Pediatric osteosarcoma is characterized by multiple somatic chromosomal lesions, including structural variations (SVs) and copy number alterations (CNAs). To define the landscape of somatic mutations Show more
Pediatric osteosarcoma is characterized by multiple somatic chromosomal lesions, including structural variations (SVs) and copy number alterations (CNAs). To define the landscape of somatic mutations in pediatric osteosarcoma, we performed whole-genome sequencing of DNA from 20 osteosarcoma tumor samples and matched normal tissue in a discovery cohort, as well as 14 samples in a validation cohort. Single-nucleotide variations (SNVs) exhibited a pattern of localized hypermutation called kataegis in 50% of the tumors. We identified p53 pathway lesions in all tumors in the discovery cohort, nine of which were translocations in the first intron of the TP53 gene. Beyond TP53, the RB1, ATRX, and DLG2 genes showed recurrent somatic alterations in 29%-53% of the tumors. These data highlight the power of whole-genome sequencing for identifying recurrent somatic alterations in cancer genomes that may be missed using other methods. Show less
Optimal molecular markers for detecting colorectal cancer (CRC) in a blood-based assay were evaluated. A matched (by variables of age and sex) case-control design (111 CRC and 227 non-cancer samples) Show more
Optimal molecular markers for detecting colorectal cancer (CRC) in a blood-based assay were evaluated. A matched (by variables of age and sex) case-control design (111 CRC and 227 non-cancer samples) was applied. Total RNAs isolated from the 338 blood samples were reverse-transcribed, and the relative transcript levels of candidate genes were analyzed. The training set was made of 162 random samples of the total 338 samples. A logistic regression analysis was performed, and odds ratios for each gene were determined between CRC and non-cancer. The samples (n = 176) in the testing set were used to validate the logistic model, and an inferred performance (generality) was verified. By pooling 12 public microarray datasets(GSE 4107, 4183, 8671, 9348, 10961, 13067, 13294, 13471, 14333, 15960, 17538, and 18105), which included 519 cases of adenocarcinoma and 88 controls of normal mucosa, we were able to verify the selected genes from logistic models and estimate their external generality. The logistic regression analysis resulted in the selection of five significant genes (P < 0.05; MDM2, DUSP6, CPEB4, MMD, and EIF2S3), with odds ratios of 2.978, 6.029, 3.776, 0.538 and 0.138, respectively. The five-gene model performed stably for the discrimination of CRC cases from controls in the training set, with accuracies ranging from 73.9% to 87.0%, a sensitivity of 95% and a specificity of 95%. In addition, a good performance in the test set was obtained using the discrimination model, providing 83.5% accuracy, 66.0% sensitivity, 92.0% specificity, a positive predictive value of 89.2% and a negative predictive value of 73.0%. Multivariate logistic regressions analyzed 12 pooled public microarray data sets as an external validation. Models that provided similar expected and observed event rates in subgroups were termed well calibrated. A model in which MDM2, DUSP6, CPEB4, MMD, and EIF2S3 were selected showed the result in logistic regression analysis (H-L P = 0.460, R2= 0.853, AUC = 0.978, accuracy = 0.949, specificity = 0.818 and sensitivity = 0.971). A novel gene expression profile was associated with CRC and can potentially be applied to blood-based detection assays. Show less
Hereditary multiple exostoses (HME) is an autosomal dominant disease. The classical paradigm of mutation screening seeks to relate alterations in the exostosin glycosyltransferase genes, EXT1 and EXT2 Show more
Hereditary multiple exostoses (HME) is an autosomal dominant disease. The classical paradigm of mutation screening seeks to relate alterations in the exostosin glycosyltransferase genes, EXT1 and EXT2, which are responsible for over 70% of HME cases. However, the pathological significance of the majority of these mutations is often unclear. In a Chinese family with HME, EXT1 and EXT2 genes were screened by direct sequencing. The consequence of a detected mutant was predicted by in silico analysis and confirmed by mRNA analysis. The EXT1 and EXT2 mRNA and protein levels and the HS patterns in the HME patients were compared with those in healthy controls. A heterozygous transition (c.743+1G>A) in the EXT2 gene, which co-segregated with the HME phenotype in this family, was identified. The G residue at position +1 in intron 4 of EXT2 was predicted to be a 5' donor splice site. The mRNA analysis revealed an alternative transcript with a cryptic splice site 5 bp downstream of the wild-type site, which harbored a premature stop codon. However, the predicted truncated protein was not detected by western blot analysis. Decay of the mutant mRNA was shown by clone sequencing and quantification analysis. The corresponding downregulation of the EXT2 mRNA will contribute to the abnormal EXT1/EXT2 ratio and HS pattern that were detected in the patients with HME. The heterozygous mutation c.743+1G>A in the EXT2 gene causes HME as a result of abnormal splicing, mRNA decay, and the resulting haploinsufficiency of EXT2. Show less
Hereditary multiple exostoses (HME) is an autosomal dominant bone disorder characterized by the presence of multiple benign cartilage-capped tumors. EXT1 located on chromosome 8q23-q24 and EXT2 locate Show more
Hereditary multiple exostoses (HME) is an autosomal dominant bone disorder characterized by the presence of multiple benign cartilage-capped tumors. EXT1 located on chromosome 8q23-q24 and EXT2 located on 11p11-p12 are the main disease-causing genes which are responsible for ~90% of HME cases. Mutations of EXT1 or EXT2 result in insufficient heparan sulfate biosynthesis, which facilitates chondrocyte proliferation, boosts abnormal bone growth of neighboring regions, causes multiple exostoses, and ultimately leads to possible malignant transformation. A family who displayed typical features of HME was enrolled in the present study. Mutation screening by Sanger sequencing identified a novel heterozygous nonsense mutation c.1902C>A (p.Tyr634X) in the EXT1 gene exclusively in all 3 patients, which is located in the glycosyltransferase domain and results in the truncation of 112 amino acids at the C-terminus of the EXT1 protein. Thus, the present study identified a novel disease-causing EXT1 mutation in a pedigree with HME, which provides additional evidence for developing quick and accurate genetic tools for HME diagnosis. Show less
The aim of this study was to examine the association of the genetic variants in the fatty acid desaturase (FADS) gene cluster with erythrocyte phospholipid fatty acids (PLFA), and their relation to ri Show more
The aim of this study was to examine the association of the genetic variants in the fatty acid desaturase (FADS) gene cluster with erythrocyte phospholipid fatty acids (PLFA), and their relation to risk for type 2 diabetes mellitus (T2DM) in Han Chinese. Seven hundred and fifty-eight patients with T2DM and 400 healthy individuals were recruited. The erythrocyte PLFA and single-nucleotide polymorphism were determined by standard method. Minor allele homozygotes and heterozygotes of rs174575 and rs174537 had lower PL 20:4 ω-6 levels in healthy individuals. Minor allele homozygotes and heterozygotes of rs174455 in FADS3 gene had lower levels of 22:5 ω-3, 20:4 ω-6, and Δ5desaturase activity in patients with T2DM. Erythrocyte membrane PL 18:3 ω-3 (P for trend = 0.002), 22:5 ω-3 (P for trend < 0.001), ω-3 polyunsaturated fatty acid (P for trend < 0.001), and ω-3:ω-6 (P for trend < 0.001) were significantly inversely associated with risk for T2DM. Genetic variants in the FADS gene cluster are associated with altered erythrocyte PLFAs. High levels of PL 18:3 ω-3, 22:5 ω-3, and total ω-3 polyunsaturated fatty acid were associated with low risk for T2DM. Show less
Carcinoids are slow-growing neuroendocrine tumors (NETs) that are characterized by hormone overproduction; surgery is currently the only option for treatment. Activation of the Notch pathway has previ Show more
Carcinoids are slow-growing neuroendocrine tumors (NETs) that are characterized by hormone overproduction; surgery is currently the only option for treatment. Activation of the Notch pathway has previously been shown to have a role in tumor suppression in NETs. The marine-derived thiodepsipeptide thiocoraline was investigated in vitro in two carcinoid cell lines (BON and H727). Carcinoid cells treated with nanomolar concentrations of thiocoraline resulted in a decrease in cell proliferation and an alteration of malignant phenotype evidenced by decrease of NET markers, achaete-scute complex like-1, chromogranin A and neurospecific enolase. Western blotting analysis demonstrated the activation of Notch1 on the protein level in BON cells. Additionally, thiocoraline activated downstream Notch targets HES1, HES5 and HEY2. Thiocoraline effectively suppressed carcinoid cell growth by promoting cell cycle arrest in BON and H727 cells. An in vivo study demonstrated that thiocoraline, formulated with polymeric micelles, slowed carcinoid tumor progression. Thus the therapeutic potential of thiocoraline, which induced activation of the Notch pathway, in carcinoid tumors was demonstrated. Show less
Ning Shi, Xia Guo, Shi-You Chen · 2014 · Molecular biology of the cell · American Society for Cell Biology · added 2026-04-24
Transforming growth factor-β (TGF-β) plays an important role in smooth muscle (SM) differentiation, but the downstream target genes regulating the differentiation process remain largely unknown. In th Show more
Transforming growth factor-β (TGF-β) plays an important role in smooth muscle (SM) differentiation, but the downstream target genes regulating the differentiation process remain largely unknown. In this study, we identified olfactomedin 2 (Olfm2) as a novel regulator mediating SM differentiation. Olfm2 was induced during TGF-β-induced SM differentiation of human embryonic stem cell-derived mesenchymal cells. Olfm2 knockdown suppressed TGF-β-induced expression of SM markers, including SM α-actin, SM22α, and SM myosin heavy chain, whereas Olfm2 overexpression promoted the SM marker expression. TGF-β induced Olfm2 nuclear accumulation, suggesting that Olfm2 may be involved in transcriptional activation of SM markers. Indeed, Olfm2 regulated SM marker expression and promoter activity in a serum response factor (SRF)/CArG box-dependent manner. Olfm2 physically interacted with SRF without affecting SRF-myocardin interaction. Olfm2-SRF interaction promoted the dissociation of SRF from HERP1, a transcriptional repressor. Olfm2 also inhibited HERP1 expression. Moreover, blockade of Olfm2 expression inhibited TGF-β-induced SRF binding to SM gene promoters in a chromatin setting, whereas overexpression of Olfm2 dose dependently enhanced SRF binding. These results demonstrate that Olfm2 mediates TGF-β-induced SM gene transcription by empowering SRF binding to CArG box in SM gene promoters. Show less
Phenotypic switching of vascular smooth muscle cells (VSMCs) plays an initial role in neointimal hyperplasia, the main cause of many occlusive vascular diseases. The aim of this study was to measure t Show more
Phenotypic switching of vascular smooth muscle cells (VSMCs) plays an initial role in neointimal hyperplasia, the main cause of many occlusive vascular diseases. The aim of this study was to measure the effects of resveratrol (RSV) on the phenotypic transformation of VSMCs and to investigate its mechanism of action. Cultured VSMCs isolated from rat thoracic aorta were prepared with serum starvation for 72 hours followed by RSV treatment (50-200 μmol/L) and 10% serum stimulation. Male Sprague-Dawley rats, subjected to carotid arteries injury from a balloon catheter, were exposed to intraperitoneal injection of RSV (1 mg/kg) or saline and were killed after 7 or 28 days. Compared with cells in the serum-induced group, VSMCs in the RSV or N-[N-(3, 5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) treatment group exhibited significant decreases of proliferation and migration. The total and cytoplasmic Notch-1 levels were declined by RSV, accompanied by a significant increase in smooth muscle α-actin and smooth muscle myosin heavy chain protein. The expression of Notch-1, Jagged-1, Hey-1, and Hey-2 mRNA in balloon-injured arteries at 7 days was decreased by RSV treatment. Arteries from RSV-treated rats showed less neointimal hyperplasia, lower collagen content, and a lower rate of cells positive for proliferating cell nuclear antigen 28 days after injury, compared with saline controls. The results indicate that RSV can attenuate phenotypic switching of VSMCs after arterial injury through inhibition of the Notch pathway. Show less
Neointimal formation after vessel injury is a complex process involving multiple cellular and molecular processes. Inhibition of intimal hyperplasia plays an important role in preventing proliferative Show more
Neointimal formation after vessel injury is a complex process involving multiple cellular and molecular processes. Inhibition of intimal hyperplasia plays an important role in preventing proliferative vascular diseases, such as restenosis. In this study, we intended to identify whether sodium ferulate could inhibit neointimal formation and further explore potential mechanisms involved. Cultured vascular smooth muscle cells (VSMCs) isolated from rat thoracic aorta were pre-treated with 200 µmol/L sodium ferulate for 1 hour and then stimulated with 1 µmol/L angiotensin II (Ang II) for 1 hour or 10% serum for 48 hours. Male Sprague-Dawley rats subjected to balloon catheter insertion were administrated with 200 mg/kg sodium ferulate (or saline) for 7 days before sacrificed. In presence of sodium ferulate, VSMCs exhibited decreased proliferation and migration, suppressed intracellular reactive oxidative species production and NADPH oxidase activity, increased SOD activation and down-regulated p38 phosphorylation compared to Ang II-stimulated alone. Meanwhile, VSMCs treated with sodium ferulate showed significantly increased protein expression of smooth muscle α-actin and smooth muscle myosin heavy chain protein. The components of Notch pathway, including nuclear Notch-1 protein, Jagged-1, Hey-1 and Hey-2 mRNA, as well as total β-catenin protein and Cyclin D1 mRNA of Wnt signaling, were all significantly decreased by sodium ferulate in cells under serum stimulation. The levels of serum 8-iso-PGF2α and arterial collagen formation in vessel wall were decreased, while the expression of contractile markers was increased in sodium ferulate treated rats. A decline of neointimal area, as well as lower ratio of intimal to medial area was observed in sodium ferulate group. Sodium ferulate attenuated neointimal hyperplasia through suppressing oxidative stress and phenotypic switching of VSMCs. Show less
Chih-Hsiang Huang, Yue-Ru Chu, Yihong Ye+1 more · 2014 · The Journal of biological chemistry · American Society for Biochemistry and Molecular Biology · added 2026-04-24
Misfolded proteins of the endoplasmic reticulum (ER) are retrotranslocated to the cytosol and degraded by the proteasome via a process termed ER-associated degradation (ERAD). The precise mechanism of Show more
Misfolded proteins of the endoplasmic reticulum (ER) are retrotranslocated to the cytosol and degraded by the proteasome via a process termed ER-associated degradation (ERAD). The precise mechanism of retrotranslocation is unclear. Here, we use several lumenal ERAD substrates targeted for degradation by the ubiquitin ligase HRD1 including SHH (sonic hedgehog) and NHK (null Hong Kong α1-antitrypsin) to study the geometry, organization, and regulation of the HRD1-containing ERAD machinery. We report a new HRD1-associated membrane protein named HERP2, which is homologous to the previously identified HRD1 partner HERP1. Despite sequence homology, HERP2 is constitutively expressed in cells, whereas HERP1 is highly induced by ER stress. We find that these proteins are required for efficient degradation of both glycosylated and nonglycosylated SHH proteins as well as NHK. In cells depleted of HERPs, SHH proteins are largely trapped inside the ER with a fraction of the stabilized SHH protein bound to the HRD1-SEL1L ligase complex. Ubiquitination of SHH is significantly attenuated in the absence of HERPs, suggesting a defect in retrotranslocation. Both HERP proteins interact with HRD1 through a region located in the cytosol. However, unlike its homolog in Saccharomyces cerevisiae, HERPs do not regulate HRD1 stability or oligomerization status. Instead, they help recruit DERL2 to the HRD1-SEL1L complex. Additionally, the UBL domain of HERP1 also seems to have a function independent of DERL2 recruitment in ERAD. Our studies have revealed a critical scaffolding function for mammalian HERP proteins that is required for forming an active retrotranslocation complex containing HRD1, SEL1L, and DERL2. Show less
Patrick J Antonellis, Lana M Pollock, Shih-Wei Chou+7 more · 2014 · The Journal of neuroscience : the official journal of the Society for Neuroscience · Society for Neuroscience · added 2026-04-24
The precise morphology of the mechanosensitive hair bundle requires seamless integration of actin and microtubule networks. Here, we identify Acf7a (actin crosslinking family protein 7a) as a protein Show more
The precise morphology of the mechanosensitive hair bundle requires seamless integration of actin and microtubule networks. Here, we identify Acf7a (actin crosslinking family protein 7a) as a protein positioned to bridge these distinct cytoskeletal networks in hair cells. By imaging Acf7a-Citrine fusion protein in zebrafish and immunolabeling of vestibular and cochlear mouse hair cells, we show that Acf7a and ACF7 circumscribe, underlie, and are interwoven into the cuticular plate (CP), and they also encircle the basal body of the kinocilium. In cochlear hair cells, ACF7 localization is graded, with the highest concentration near each fonticulus--an area free of F-actin in the region of the CP that contains the basal body. During hair-cell development and regeneration, Acf7a precedes formation of the hair bundle and CP. Finally, electron tomography demonstrates that the ends of microtubules insert into the CP and are decorated with filamentous linkers connecting microtubules to the CP. These observations are consistent with ACF7 being a linker protein, which may shape the cytoskeleton of the hair cell early during hair-bundle genesis. Show less