Liver X receptor-Ξ± (LXR-Ξ±) which belongs to the nuclear receptor superfamily, is a ligand-activated transcription factor. Best known for its ability to regulate lipid metabolism and transport, LXRs ha Show more
Liver X receptor-Ξ± (LXR-Ξ±) which belongs to the nuclear receptor superfamily, is a ligand-activated transcription factor. Best known for its ability to regulate lipid metabolism and transport, LXRs have recently also been implicated in regulation of inflammatory response. The aim of this study was to investigate the preventive effects of synthetic LXR-Ξ± agonist T0901317 on LPS-induced mastitis in mice. The mouse model of mastitis was induced by injection of LPS through the duct of mammary gland. T0901317 was injected 1h before and 12h after induction of LPS intraperitoneally. The results showed that T0901317 significantly attenuated the infiltration of neutrophilic granulocytes, and the activation of myeloperoxidase (MPO); down-regulated the level of pro-inflammatory mediators including TNF-Ξ±, IL-1Ξ², IL-6, COX-2 and PEG2; inhibited the phosphorylation of IΞΊB-Ξ± and NF-ΞΊB p65, caused by LPS. Moreover, we report for the first time that LXR-Ξ± activation impaired LPS-induced mastitis. Taken together, these data indicated that T0901317 had protective effect on mastitis and the anti-inflammatory mechanism of T0901317 on LPS induced mastitis in mice may be due to its ability to inhibit NF-ΞΊB signaling pathway. LXR-Ξ± activation can be used as a therapeutic approach to treat mastitis. Show less
The nuclear receptor liver X receptor (LXR) has two isoforms: LXRΞ± and LXRΞ². LXR activation promotes cholesterol efflux in macrophages, but the relative importance of each LXR isoform in mediating cho Show more
The nuclear receptor liver X receptor (LXR) has two isoforms: LXRΞ± and LXRΞ². LXR activation promotes cholesterol efflux in macrophages, but the relative importance of each LXR isoform in mediating cholesterol efflux remains elusive. We evaluated the ability of different doses of LXRs agonist T0901317 to affect cholesterol efflux in human macrophages and its relationship with mRNA and protein levels of several well-characterized proteins involved in cholesterol efflux, including ABCA1, ABCG1, SR-BI, LXRΞ² and LXRΞ±, using quantitative real-time PCR, Western blotting, and siRNA techniques. Here we show that LXRΞ± rather than LXRΞ² sustains baseline cholesterol efflux in human blood-derived macrophages. Treatment of human macrophages with a non-isoform-specific LXR agonist T0901317 substantially increased HDL- and apoA-I-mediated cholesterol efflux, which was associated with increased mRNA and protein expression levels of ABCA1, ABCG1, SR-BI, LXRΞ± and LXRΞ². The siRNA- mediated silencing of LXRΞ±, but not LXRΞ² significantly reduced the protein levels of ABCA1,ABCG1, and SR-BI as wellas HDL- and ApoA1-mediated cholesterol in human macrophages. These findings imply that LXRΞ±- rather than LXRΞ²- specific agonists may promote reverse cholesterol transport in humans. Show less
Liver X receptors (LXRs) have been recognized as a promising therapeutic target for atherosclerosis; however, their role in insulin sensitivity is controversial. Adiponectin plays a unique role in mai Show more
Liver X receptors (LXRs) have been recognized as a promising therapeutic target for atherosclerosis; however, their role in insulin sensitivity is controversial. Adiponectin plays a unique role in maintaining insulin sensitivity. Currently, no systematic experiments elucidating the role of LXR activation in insulin function based on adiponectin signaling have been reported. Here, we investigated the role of LXR activation in insulin resistance based on adiponectin signaling, and possible mechanisms. C57BL/6 mice maintained on a regular chow received the LXR agonist, T0901317 (30 mg/kg.d) for 3 weeks by intraperitoneal injection, and differentiated 3T3-L1 adipocytes were treated with T0901317 or GW3965. T0901317 treatment induced significant insulin resistance in C57BL/6 mice. It decreased adiponectin gene transcription in epididymal fat, as well as serum adiponectin levels. Activity of AMPK, a key mediator of adiponectin signaling, was also decreased, resulting in decreased Glut-4 membrane translocation in epididymal fat. In contrast, adiponectin activity was not changed in the liver of T0901317 treated mice. In vitro, both T0901317 and GW3965 decreased adiponectin expression in adipocytes in a dose-dependent manner, an effect which was diminished by LXRΞ± silencing. ChIP-qPCR studies demonstrated that T0901317 decreased the binding of PPARΞ³ to the PPAR-responsive element (PPRE) of the adiponectin promoter in a dose-dependent manner. Furthermore, T0901317 exerted an antagonistic effect on the expression of adiponectin in adipocytes co-treated with 3 Β΅M Pioglitazone. In luciferase reporter gene assays, T0901317 dose-dependently inhibited PPRE-Luc activity in HEK293 cells co-transfected with LXRΞ± and PPARΞ³. These results suggest that LXR activation induces insulin resistance with decreased adiponectin signaling in epididymal fat, probably due to negative regulation of PPARΞ³ signaling. These findings indicate that the potential of LXR activation as a therapeutic target for atherosclerosis may be limited by the possibility of exacerbating insulin resistance-related disease. Show less
Dietary phytosterols have been successfully used for lowering cholesterol levels, which correlates with the fact that some phytosterols are able to act as liver X receptor (LXR) agonists. Sargassum fu Show more
Dietary phytosterols have been successfully used for lowering cholesterol levels, which correlates with the fact that some phytosterols are able to act as liver X receptor (LXR) agonists. Sargassum fusiforme is an edible marine seaweed well-known for its antiatherosclerotic function in traditional Chinese medicine. In this study, seven phytosterols including fucosterol (1), saringosterol (2), 24-hydroperoxy-24-vinyl-cholesterol (3), 29-hydroperoxy-stigmasta-5,24(28)-dien-3Ξ²-ol (4), 24-methylene-cholesterol (5), 24-keto-cholesterol (6), and 5Ξ±,8Ξ±-epidioxyergosta-6,22-dien-3Ξ²-ol (7) were purified and evaluated for their actions on LXR-mediated transcription using a reporter assay. Among these phytosterols, 2 was the most potent compound in stimulating the transcriptional activities of LXRΞ± by (3.81Β±0.15)-fold and LXRΞ² by (14.40Β±1.10)-fold, respectively. Two epimers of 2, 24(S)-saringosterol (2a) and 24(R)-saringosterol (2b), were subsequently separated by semipreparative high-performance liquid chromatography. Interestingly, 2a was more potent than 2b in LXRΞ²-mediated transactivation ((3.50Β±0.17)-fold vs (1.63Β±0.12)-fold) compared with control. Consistently, 2a induced higher expression levels of LXR target genes including key players in reverse cholesterol transport in six cell lines. These data along with molecular modeling suggested that 2a acts as a selective LXRΞ² agonist and is a potent natural cholesterol-lowering agent. This study also demonstrated that phytosterols in S. fusiforme contributed to the well-known antiatherosclerotic function. Show less
Cyanidin-3-O-Ξ²-glucoside (C3G), a typical anthocyanin pigment that exists in the human diet, has been reported to have anti-inflammatory properties. The aim of this study was to detect the effect of C Show more
Cyanidin-3-O-Ξ²-glucoside (C3G), a typical anthocyanin pigment that exists in the human diet, has been reported to have anti-inflammatory properties. The aim of this study was to detect the effect of C3G on LPS-induced acute lung injury and to investigate the molecular mechanisms. Acute lung injury was induced by intratracheal administration of LPS in mice. Alveolar macrophages from mice were stimulated with LPS and were treated with C3G. Our results showed that C3G attenuated lung histopathologic changes, myeloperoxidase (MPO) activity, TNF-Ξ±, IL-1Ξ² and IL-6 production in LPS-induced acute lung injury model. In vitro, C3G dose-dependently inhibited TNF-Ξ±, IL-1Ξ², IL-6, IL-10 and IFN-Ξ² production, as well as NF-ΞΊB and IRF3 activation in LPS-stimulated alveolar macrophages. Furthermore, C3G disrupted the formation of lipid rafts by depleting cholesterol and inhibited TLR4 translocation into lipid rafts. Moreover, C3G activated LXRΞ±-ABCG1-dependent cholesterol efflux. Knockout of LXRΞ± abrogated the anti-inflammatory effects of C3G. In conclusion, C3G has a protective effect on LPS-induced acute lung injury. The promising anti-inflammatory mechanisms of C3G is associated with up-regulation of the LXRΞ±-ABCG1 pathway which result in disrupting lipid rafts by depleting cholesterol and reducing translocation of TLR4 to lipid rafts, thereby suppressing TLR4 mediated inflammatory response. Show less
FGF21, a member of the fibroblast growth factor superfamily, is an important endogenous regulator of systemic glucose and lipid metabolism. Elevated serum FGF21 levels have been reported in subjects w Show more
FGF21, a member of the fibroblast growth factor superfamily, is an important endogenous regulator of systemic glucose and lipid metabolism. Elevated serum FGF21 levels have been reported in subjects with coronary heart disease and carotid artery plaques. However, whether FGF21 is associated with atherosclerotic diseases remains unclear. In this study, the effects of FGF21 on cholesterol efflux in THP1 macrophage-derived foam cells and the underlying mechanisms were investigated. THP1 macrophage-derived foam cells were incubated with 0, 25, 50, 100, 200, and 400βng/mL of FGF21 for varying time periods (0, 6, 12, and 24βh). Cholesterol efflux onto apoA-1 was assessed by high-performance liquid chromatography assays, while change in ABCA1 expression was analyzed by western blot and real-time quantitative PCR. Incubation was performed with the ERK1/2-specific inhibitor PD98059, PPARΞ³-specific inhibitor GW9662, and LXRΞ± siRNA. Our results show that FGF21 promotes cholesterol efflux and ABCA1 expression in THP1 macrophage-derived foam cells in a dose- and time-dependent manner. In addition, inhibition of ERK1/2 or PPARΞ³, or knockdown of LXRΞ± attenuated FGF21-mediated promotion of ABCA1 expression and cholesterol efflux. These results demonstrate that FGF21 can promote cholesterol efflux by upregulating ABCA1 through the ERK1/2-PPARΞ³-LXRΞ± pathway in THP1 macrophage-derived foam cells. Show less
To explore the relationship between the liver X receptor Ξ± gene (LXRΞ±) rsl2221497 polymorphism and the susceptibility of coronary heart disease (CHD) and serum lipids and glucose levels. The single fl Show more
To explore the relationship between the liver X receptor Ξ± gene (LXRΞ±) rsl2221497 polymorphism and the susceptibility of coronary heart disease (CHD) and serum lipids and glucose levels. The single fluorescently labeled probes technique was used to detect the genotype of rsl2221497 in LXRΞ± gene in 240 CHD patients and 250 healthy control subjects. The difference of genotype distribution between the two groups was analyzed using of Chi-square test. The serum lipids and glucose levels between the different genotypes were also compared. The risk of CHD in carriers with (AAβ+βGA) genotype was 1.76 times as that in the GG genotype carriers (ORβ=β1.76, 95% CI: 1.18-2.87, P <0.05), and the risk of CHD in carriers with A allele increased 0.88 times compared to that in G allele carriers (ORβ=β1.88, 95% CI:1.21-3.43, P <0.01). Logistic regression analysis showed that after adjusting for other confounding factors, A allele was an independent risk for CHD. However, there were no differences in serum lipids and glucose levels between each genotype. The rsl2221497 polymorphism in LXRΞ± gene was associated with susceptibility of CHD in Han population. Show less
LXR (liver X receptor) is a ligand-activated transcription factor and plays an important role in regulation of lipid homoeostasis and inflammation. Several studies indicate that LXR inhibits IFN-Ξ³ (in Show more
LXR (liver X receptor) is a ligand-activated transcription factor and plays an important role in regulation of lipid homoeostasis and inflammation. Several studies indicate that LXR inhibits IFN-Ξ³ (interferon Ξ³)-induced biological responses; however, the influence of LXR on IFN-Ξ³ expression has not been fully elucidated. In the present study, we investigated the effects of LXR activation on IFN-Ξ³ expression at different levels. At the molecular level, we surprisingly observed that LXR ligand (T0901317) induced macrophage and T-cell IFN-Ξ³ protein expression which was associated with increased mRNA and secreted protein levels in culture medium. In contrast, selective inhibition of LXRΞ± and/or LXRΞ² expression by siRNA reduced IFN-Ξ³ expression. Promoter analysis defined the multiple LXREs (LXR-responsive elements) in the proximal region of the IFN-Ξ³ promoter. EMSAs and ChIP indicated that LXR activation enhanced the binding of LXR protein to these LXREs. InΒ vivo, T0901317 increased wild-type mouse serum IFN-Ξ³ levels and IFN-Ξ³ expression in the lung and lymph nodes. Functionally, we observed that administration of T0901317 to wild-type mice increased rates of survival and being tumour-free, and inhibited tumour growth when the animals were inoculated with LLC1 carcinoma. In contrast, these protective effects were substantially attenuated in IFN-Ξ³-knockout (IFN-Ξ³-/-) mice, suggesting that the induction of IFN-Ξ³ production plays a critical role in T0901317-inhibited tumour growth. Taken together, the results of the present study show that IFN-Ξ³ is another molecular target of LXR activation, and it suggests a new mechanism by which LXR inhibits tumour growth. Show less
Liver X receptors (LXRs) including LXRΞ± and LXRΞ² are members of the nuclear hormone receptor superfamily of ligand activated transcription factors, which serve as lipid sensors to regulate expression Show more
Liver X receptors (LXRs) including LXRΞ± and LXRΞ² are members of the nuclear hormone receptor superfamily of ligand activated transcription factors, which serve as lipid sensors to regulate expression of genes controlling many aspects of cholesterol and fatty acid metabolism. The liver is the central organ in controlling lipid metabolism. In the present study, we aimed at elucidating the role of LXR activation in hepatic fatty acid homeostasis. We treated C57BL/6 mice with a synthetic non-selective LXR agonist TO901317. Fatty acid profile of lipid esters in the livers was analyzed by gas-liquid chromatography. Real-time polymerase chain reaction (PCR) and western blot were used to determine the expression of SREBP1c and SCD1 in TO901317-treated livers and HepG2 cells. Oral administration of TO901317 resulted in increased fatty acid desaturation in the liver, with concomitant increase in hepatic stearoyl CoA desaturase-1 (SCD1) expression. TO901317-induced SCD1 expression was observed in LXRΞ²-/- mice, but not in LXRΞ±-/- mice. Furthermore, TO901317 significantly increased expression of sterol regulatory element-binding protein 1c (SREBP1c), the deficiency of which almost completely abolished the induction of SCD1 by TO901317. This drug induced both SREBP1c and SCD1 expression in HepG2 cells. Overexpression of SREBP1c resulted in a significant increase in SCD1 promoter activity and expression. Taken together, the present studies demonstrate that pan-LXR activation increases hepatic fatty acid desaturation via the induction of SCD1 expression in an LXRΞ±-dependent and SREBP1c-mediated manner. Show less
Liver X receptors (LXRs), including LXRΞ± and LXRΞ² isoforms, have important roles in the metabolic regulation of glucose, cholesterol and lipid. Moreover, activation of LXRs also represses the expressi Show more
Liver X receptors (LXRs), including LXRΞ± and LXRΞ² isoforms, have important roles in the metabolic regulation of glucose, cholesterol and lipid. Moreover, activation of LXRs also represses the expression of cyclin D1 and cyclin B1, and thus suppresses the proliferation of multiple cancer cells, but the relevant mechanism is not well known. Forkhead box M1 (FOXM1) is a proliferation-specific member of forkhead box family, which is highly expressed in proliferating normal cells and numerous cancer cells. FOXM1 directly activates transcription of cyclin D1 and cyclin B1, resulting in the enhancement of cell cycle progression and cell proliferation. However, it is unclear whether LXRs are involved in the regulation of FOXM1. In this study, we demonstrated that specific LXRs agonists downregulated expression of FOXM1, cyclin D1 and cyclin B1 in hepatocellular carcinoma (HCC) cells, which led to cell cycle and cell proliferation arrest. Knockdown of FOXM1 significantly alleviated LXRs activation-mediated cell cycle arrest and cell growth suppression. Reporter assays showed that the activation of LXRs significantly reduced the transcriptional activity of FOXM1 promoter. Electrophoretic mobility shift assay and chromatin immunoprecipitation assays demonstrated that LXRΞ± but not LXRΞ² could bind to an inverted repeat IR2 (-52CCGTCAcgTGACCT-39) in the promoter region of FOXM1 gene. Moreover, the xenograft tumor growth and the corresponding FOXM1 expression in nude mice were dramatically repressed by LXRs agonists. Taken together, we conclude that LXRΞ± but not LXRΞ² functions as a transcriptional repressor for FOXM1 expression. The pathway 'LXRΞ±-FOXM1-cyclin D1/cyclin B1' is a novel mechanism by which LXRs suppress the proliferation of HCC cells, suggesting that the pathway may be a novel target for HCC treatment. Show less
To evaluate the preventive and therapeutic effects of Lactobacilluscasei Zhang on impaired glucose tolerance (IGT) by using fructose-induced hyperinsulinemia rats. Rats were fed 25 % fructose solution Show more
To evaluate the preventive and therapeutic effects of Lactobacilluscasei Zhang on impaired glucose tolerance (IGT) by using fructose-induced hyperinsulinemia rats. Rats were fed 25 % fructose solution for hyperinsulinemia with L.casei Zhang for prevention or therapy. Serum levels of insulin, glucagon-like peptide-2 (GLP-2), osteocalcin, malondialdehyde (MDA), total intestinal bile acids and hepatic glycogen contents were determined by assay kits. The major bacteria from feces and liver expression of adiponectin receptor 2 (AdipoR2), liver X receptor-Ξ± (LXR-Ξ±), peroxisome proliferator-activated receptor gamma (PPAR-Ξ³) and vitamin K epoxide reductase complex subunit 1 mRNA were assessed by RT-PCR. Pancreas injury was evaluated by histological analysis. Lactobacilluscasei Zhang significantly increased numbers of Lactobacillus and Bifidobacterium and decreased Clostridium in the intestine (p < 0.01). Meanwhile, liver glycogen contents were significantly decreased (p < 0.05). In preventive group, accompanied by significantly lower insulin and GLP-2 levels (p < 0.05), L.casei Zhang prevented rats from an increase in oral glucose tolerance area under curve (AUC) which was significant in hyperinsulinemia group (p < 0.05). In therapeutic group, L.casei Zhang administration possessed improved glucose tolerance (p < 0.05), which were associated with increased osteocalcin level (p < 0.01), improved intestinal bile acids secretion (p = 0.060), decreased serum MDA levels (p < 0.05) and upregulation of LXR-Ξ±, PPAR-Ξ³ and AdipoR2 gene expression, as well as an increase in Bacteroides fragilis (p < 0.05). Lactobacilluscasei Zhang administration exert both preventive and ameliorative effect on oral glucose tolerance AUC in IGT rats but may be via different mechanisms. L.casei Zhang could prevent rats from increased AUC through GLP-2 lowering, while the ameliorative effect in high-fructose-fed post-adolescent rats may be via B. fragilis enriched vitamin K2-dependent osteocalcin mechanism in which AdipoR2, LXR-Ξ± and PPAR-Ξ³ signaling were involved. Show less
Youjun Li, Zhiyi Wei, Yan Yan+3 more Β· 2014 Β· Proceedings of the National Academy of Sciences of the United States of America Β· National Academy of Sciences Β· added 2026-04-24
The Crumbs (Crb) complex, formed by Crb, PALS1, and PATJ, is evolutionarily conserved in metazoans and acts as a master cell-growth and -polarity regulator at the apical membranes in polarized epithel Show more
The Crumbs (Crb) complex, formed by Crb, PALS1, and PATJ, is evolutionarily conserved in metazoans and acts as a master cell-growth and -polarity regulator at the apical membranes in polarized epithelia. Crb intracellular functions, including its direct binding to PALS1, are mediated by Crb's highly conserved 37-residue cytoplasmic tail. However, the mechanistic basis governing the highly specific Crb-PALS1 complex formation is unclear, as reported interaction between the Crb tail (Crb-CT) and PALS1 PSD-95/DLG/ZO-1 (PDZ) domain is weak and promiscuous. Here we have discovered that the PDZ-Src homolgy 3 (SH3)-Guanylate kinase (GK) tandem of PALS1 binds to Crb-CT with a dissociation constant of 70 nM, which is βΌ 100-fold stronger than the PALS1 PDZ-Crb-CT interaction. The crystal structure of the PALS1 PDZ-SH3-GK-Crb-CT complex reveals that PDZ-SH3-GK forms a structural supramodule with all three domains contributing to the tight binding to Crb. Mutations disrupting the tertiary interactions of the PDZ-SH3-GK supramodule weaken the PALS1-Crb interaction and compromise PALS1-mediated polarity establishment in Madin-Darby canine kidney (MDCK) cysts. We further show that specific target binding of other members of membrane-associated guanylate kinases (MAGUKs) (e.g., CASK binding to neurexin) also requires the presence of their PDZ-SH3-GK tandems. Show less
PDZ (PSD-95/Discs-large/ZO1) domains are interaction modules that typically bind to specific C-terminal sequences of partner proteins and assemble signaling complexes in multicellular organisms. We ha Show more
PDZ (PSD-95/Discs-large/ZO1) domains are interaction modules that typically bind to specific C-terminal sequences of partner proteins and assemble signaling complexes in multicellular organisms. We have analyzed the existing database of PDZ domain structures in the context of a specificity tree based on binding specificities defined by peptide-phage binding selections. We have identified 16 structures of PDZ domains in complex with high-affinity ligands and have elucidated four additional structures to assemble a structural database that covers most of the branches of the PDZ specificity tree. A detailed comparison of the structures reveals features that are responsible for the diverse specificities across the PDZ domain family. Specificity differences can be explained by differences in PDZ residues that are in contact with the peptide ligands, but these contacts involve both side-chain and main-chain interactions. Most PDZ domains bind peptides in a canonical conformation in which the ligand main chain adopts an extended Ξ²-strand conformation by interacting in an antiparallel fashion with a PDZ Ξ²-strand. However, a subset of PDZ domains bind peptides with a bent main-chain conformation and the specificities of these non-canonical domains could not be explained based on canonical structures. Our analysis provides a structural portrait of the PDZ domain family, which serves as a guide in understanding the structural basis for the diverse specificities across the family. Show less
Autophagy, a "self-eating" cellular process, has dual roles in promoting and suppressing tumor growth, depending on cellular context. PTP4A3/PRL-3, a plasma membrane and endosomal phosphatase, promote Show more
Autophagy, a "self-eating" cellular process, has dual roles in promoting and suppressing tumor growth, depending on cellular context. PTP4A3/PRL-3, a plasma membrane and endosomal phosphatase, promotes multiple oncogenic processes including cell proliferation, invasion, and cancer metastasis. In this study, we demonstrate that PTP4A3 accumulates in autophagosomes upon inhibition of autophagic degradation. Expression of PTP4A3 enhances PIK3C3-BECN1-dependent autophagosome formation and accelerates LC3-I to LC3-II conversion in an ATG5-dependent manner. PTP4A3 overexpression also enhances the degradation of SQSTM1, a key autophagy substrate. These functions of PTP4A3 are dependent on its catalytic activity and prenylation-dependent membrane association. These results suggest that PTP4A3 functions to promote canonical autophagy flux. Unexpectedly, following autophagy activation, PTP4A3 serves as a novel autophagic substrate, thereby establishing a negative feedback-loop that may be required to fine-tune autophagy activity. Functionally, PTP4A3 utilizes the autophagy pathway to promote cell growth, concomitant with the activation of AKT. Clinically, from the largest ovarian cancer data set (GSE 9899, n = 285) available in GEO, high levels of expression of both PTP4A3 and autophagy genes significantly predict poor prognosis of ovarian cancer patients. These studies reveal a critical role of autophagy in PTP4A3-driven cancer progression, suggesting that autophagy could be a potential Achilles heel to block PTP4A3-mediated tumor progression in stratified patients with high expression of both PTP4A3 and autophagy genes. Show less
The miR-183/-96/-182 cluster is a conserved polycistronic microRNA (miRNA) cluster which is highly expressed in most breast cancers. Although there are some sporadic reports which demonstrate the impo Show more
The miR-183/-96/-182 cluster is a conserved polycistronic microRNA (miRNA) cluster which is highly expressed in most breast cancers. Although there are some sporadic reports which demonstrate the importance of each miRNA in this cluster in breast cancer, the biological roles of this cluster as a whole and its regulation mechanisms in breast cancer are still unclear. We compared the expression of this cluster in different cancer types, analyzed the regulation mechanism of this cluster, identified new target genes, and examined the impact of this cluster on breast cancer cells. The miRNA level was detected by LNA-based northern blot and Real-time PCR, and was also analyzed from TCGA dataset. Bioinformatics research and luciferase assay were applied to find the promoter regions and transcription factors. To investigate the biological effects of the miR-183/-96 /-182 cluster in breast cancer, we generated miR-96, miR-182 and miR-183 overexpression stable cell lines to check the overdose effects; we also used miR-Downβ’ antagomir for each miRNA as well as miR-183/-96 /-182 cluster sponge lentivirus to check the knockdown effects. Growth, migration, cell cycle profile and survival of these cells was then monitored by colony formation assay, MTT assay, cell wound healing assay, flow cytometry and microscopy. The target gene was validated by Real-time PCR, luciferase assay, Western blot and Phalloidin/DAPI counterstaining. The miR-183/-96/-182 cluster was highly expressed in most breast cancers, and its transcription is disordered in breast cancer. The miR-183/-96/-182 cluster was transcribed in the same pri-miRNA and its transcription was regulated by ZEB1 and HSF2. It increased breast cell growth by promoting more rapid completion of mitosis, promoted cell migration and was essential for cell survival. MiR-183 targeted the RAB21 mRNA directly in breast cancer. The miR-183/-96/-182 cluster is up-regulated in most breast cancer. It functions as an oncogene in breast cancer as it increases cell proliferation and migration. Show less
MYB-type transcription factors (TFs) play essential roles in plant growth, development and respond to environmental stresses. Role of MYB-related TFs of rice in drought stress tolerance is not well do Show more
MYB-type transcription factors (TFs) play essential roles in plant growth, development and respond to environmental stresses. Role of MYB-related TFs of rice in drought stress tolerance is not well documented. Here, we report the isolation and characterization of a novel MYB-related TF, OsMYB48-1, of rice. Expression of OsMYB48-1 was strongly induced by polyethylene glycol (PEG), abscisic acid (ABA), H2O2, and dehydration, while being slightly induced by high salinity and cold treatment. The OsMYB48-1 protein was localized in the nucleus with transactivation activity at the C terminus. Overexpression of OsMYB48-1 in rice significantly improved tolerance to simulated drought and salinity stresses caused by mannitol, PEG, and NaCl, respectively, and drought stress was caused by drying the soil. In contrast to wild type plants, the overexpression lines exhibited reduced rate of water loss, lower malondialdehyde (MDA) content and higher proline content under stress conditions. Moreover, overexpression plants were hypersensitive to ABA at both germination and post-germination stages and accumulated more endogenous ABA under drought stress conditions. Further studies demonstrated that overexpression of OsMYB48-1 could regulate the expression of some ABA biosynthesis genes (OsNCED4, OsNCED5), early signaling genes (OsPP2C68, OSRK1) and late responsive genes (RAB21, OsLEA3, RAB16C and RAB16D) under drought stress conditions. Collectively, these results suggested that OsMYB48-1 functions as a novel MYB-related TF which plays a positive role in drought and salinity tolerance by regulating stress-induced ABA synthesis. Show less
To examine the impact of single nucleotide polymorphisms in obesity-related genes on risk of obesity and metabolic disorder in childhood. A total of 3 503 Chinese children aged 6 to 18 years participa Show more
To examine the impact of single nucleotide polymorphisms in obesity-related genes on risk of obesity and metabolic disorder in childhood. A total of 3 503 Chinese children aged 6 to 18 years participated in the study, including 1 229 obese, 655 overweight and 1 619 normal weight children (diagnosed by the Chinese age- and sex- specific BMI cutoffs). Body size parameters were assessed and venipuncture blood samples were collected after a 12-hour overnight fast. Plasma glucose, insulin and serum lipid profiles were measured.Genomic DNA was isolated from peripheral blood white cells using the salt fractionation method. A total of 11 single nucleotide polymorphisms were genotyped by TaqMan allelic discrimination assays with the GeneAmp 7900 sequence detection system (Applied Biosystems, Foster City, CA, USA) (FTO rs9939609, MC4R rs17782313, GNPDA2 rs10938397, FAIM2 rs7138803, BDNF rs6265, NPC1 rs1805081, PCSK1 rs6235, KCTD15 rs29941, BAT2 rs2844479, SEC16B rs10913469 and SH2B1 rs4788102). Multiple factor analysis was performed to estimate the association between the variant and obesity-related traits. The false discovery rate (FDR) approach was used to correct for multiple comparisons. After sex, age and pubertal stage adjustment and correction for multiple testing, the rs9939609-A, rs17782313-C, rs10938397-G, and rs7138803-A alleles were associated with higher BMI (Ξ² = 0.352-0.747), fat mass percentage(Ξ² = 0.568-1.113), waist circumference (Ξ² = 0.885-1.649) and waist-to-height ratio(Ξ² = 0.005-0.010) (all P values < 0.01) in Chinese children. The rs6265-G allele increased BMI(Ξ² = 0.251, P = 0.020). The rs9939609-A, rs17782313-C, and rs10938397-G and rs6265-G alleles were also associated with risk of obesity (OR = 1.386, 95%CI:1.171-1.642; OR = 1.367, 95%CI:1.196-1.563; OR = 1.242, 95%CI:1.102-1.400; OR = 1.156, 95%CI:1.031-1.296).Rs7138803 was associated with risk of obesity only in boys (OR = 1.234, 95%CI:1.043-1.460). GNPDA2 rs10938397-G allele was associated with risk of insulin resistance(OR = 1.205, 95%CI:1.069-1.359), but there was no significance after adjusting for BMI. The association of FTO rs9939609-A, MC4R rs17782313-C, GNPDA2 rs10938397-G, and FAIM2 rs7138803-A with higher BMI, fat mass percentage, waist circumference, and waist-to height ratio and risk of obesity, and BDNF rs6265-G allele may increase BMI and obesity risk in Chinese children. GNPDA2 rs10938397-G may increase the risk of childhood insulin resistance depending on BMI. Show less
Obesity is a well-known risk factor for type 2 diabetes. Genome-wide association studies have identified a number of genetic loci associated with obesity. The aim of this study is to examine the contr Show more
Obesity is a well-known risk factor for type 2 diabetes. Genome-wide association studies have identified a number of genetic loci associated with obesity. The aim of this study is to examine the contribution of obesity-related genomic loci to type 2 diabetes in a Chinese population. We successfully genotyped 18 obesity-related single nucleotide polymorphisms among 5338 type 2 diabetic patients and 4663 controls. Both individual and joint effects of these single nucleotide polymorphisms on type 2 diabetes and quantitative glycemic traits (assessing Ξ²-cell function and insulin resistance) were analyzed using logistic and linear regression models, respectively. Two single nucleotide polymorphisms near MC4R and GNPDA2 genes were significantly associated with type 2 diabetes before adjusting for body mass index and waist circumference (OR (95% CI)β=β1.14 (1.06, 1.22) for the A allele of rs12970134, Pβ=β4.75Γ10(-4); OR (95% CI)β=β1.10 (1.03, 1.17) for the G allele of rs10938397, Pβ=β4.54Γ10(-3)). When body mass index and waist circumference were further adjusted, the association of MC4R with type 2 diabetes remained significant (Pβ=β1.81Γ10(-2)) and that of GNPDA2 was attenuated (Pβ=β1.26Γ10(-1)), suggesting the effect of the locus including GNPDA2 on type 2 diabetes may be mediated through obesity. Single nucleotide polymorphism rs2260000 within BAT2 was significantly associated with type 2 diabetes after adjusting for body mass index and waist circumference (Pβ=β1.04Γ10(-2)). In addition, four single nucleotide polymorphisms (near or within SEC16B, BDNF, MAF and PRL genes) showed significant associations with quantitative glycemic traits in controls even after adjusting for body mass index and waist circumference (all P values<0.05). This study indicates that obesity-related genomic loci were associated with type 2 diabetes and glycemic traits in the Han Chinese population. Show less
Poly(A)-binding protein-interacting protein 1 (Paip1) stimulates translational initiation by inducing the circularization of mRNA. However, the mechanisms underlying Paip1 regulation, particularly its Show more
Poly(A)-binding protein-interacting protein 1 (Paip1) stimulates translational initiation by inducing the circularization of mRNA. However, the mechanisms underlying Paip1 regulation, particularly its protein stability, are still unclear. Here, we show that the E6AP carboxyl terminus (HECT)-type ubiquitin ligase WW domain-containing protein 2 (WWP2), a homolog of the HECT-type ubiquitin ligase WWP1, interacts with and targets Paip1 for ubiquitination and proteasomal degradation. Mapping of the region including the WW domain of WWP2 revealed the interaction between WWP2 and the PABP-binding motif 2 (PAM2) of Paip1. The two consecutive PXXY motifs in PAM2 are required for WWP2-mediated ubiquitination and degradation. Furthermore, ectopic expression of WWP2 decreases translational stimulatory activity with the degradation of Paip1. We therefore provide evidence that the stability of Paip1 can be regulated by ubiquitin-mediated degradation, thus highlighting the importance of WWP2 as a suppressor of translation. Show less
Sox2 is a key factor for maintaining embryonic stem cell (ESS) pluripotency, but little is known about its posttranslational regulation. Here we present evidence that the precise level of Sox2 protein Show more
Sox2 is a key factor for maintaining embryonic stem cell (ESS) pluripotency, but little is known about its posttranslational regulation. Here we present evidence that the precise level of Sox2 proteins in ESCs is regulated by a balanced methylation andΒ phosphorylation switch. Set7 monomethylates Sox2 at K119, which inhibits Sox2 transcriptional activity and induces Sox2 ubiquitination and degradation. The E3 ligase WWP2 specifically interacts with K119-methylated Sox2 through its HECT domain to promote Sox2 ubiquitination. In contrast, AKT1 phosphorylates Sox2 at T118 and stabilizes Sox2 by antagonizing K119me by Set7 and vice versa. In mouse ESCs, AKT1 activity toward Sox2 is greater than that of Set7, leading to Sox2 stabilization and ESC maintenance. In early development, increased Set7 expression correlates with Sox2 downregulation and appropriate differentiation. Our study highlights the importance of a Sox2 methylation-phosphorylation switch in determining ESC fate. Show less
SRG3 plays essential roles both in early mouse embryogenesis and in extra-embryonic vascular development. As one of the core components of the SWI/SNF-like BAF complex, SRG3 serves as the scaffold pro Show more
SRG3 plays essential roles both in early mouse embryogenesis and in extra-embryonic vascular development. As one of the core components of the SWI/SNF-like BAF complex, SRG3 serves as the scaffold protein and its protein level controls the stability of the BAF complex, which controls diverse physiological processes through transcriptional regulation. However, little is known about how the protein level of SRG3 is regulated in mammalian cells. Previously, we identified a murine ubiquitin ligase (Wwp2) and demonstrated that it interacts with pluripotency-associated key transcription factor Oct4 and RNA polymerase II large subunit Rpb1, promoting their ubiquitination and degradation. Here, we report that Wwp2 acts as a ubiquitin ligase of SRG3. Our results show that Wwp2 and SRG3 form protein complexes and co-localize in the nucleus in mammalian cells. The interaction is mediated through the WW domain of Wwp2 and the PPPY motif of SRG3, respectively. Importantly, Wwp2 promotes ubiquitination and degradation of SRG3 through the ubiquitin-proteasome system. The expression of a catalytically inactive mutant of Wwp2 abolishes SRG3 ubiquitination. Collectively, our study opens up a new avenue to understand how the protein level of SRG3 is regulated in mammalian cells. Show less
Metabolic syndrome (MetS) is an aberration associated with increased risk for cancer and inflammation. Adiponectin, an adipocyte-produced abundant protein hormone, has countering effect on the diabeto Show more
Metabolic syndrome (MetS) is an aberration associated with increased risk for cancer and inflammation. Adiponectin, an adipocyte-produced abundant protein hormone, has countering effect on the diabetogenic and atherogenic components of MetS. Plasma levels of adiponectin are negatively correlated with onset of cancer and cancer patient mortality. We previously performed microsatellite linkage analyses using adiponectin as a surrogate marker and revealed two QTLs on chr5 (5p14) and chr14 (14q13). Using individuals from 85 extended families that contributed to the linkage and who were measured for 42 clinical and biologic MetS phenotypes, we tested QTL-based SNP associations, peripheral white blood cell (PWBC) gene expression, and the effects of cis-acting SNPs on gene expression to discover genomic elements that could affect the pathophysiology and complications of MetS. Adiponectin levels were found to be highly intercorrelated phenotypically with the majority of MetS traits. QTL-specific haplotype-tagging SNPs associated with MetS phenotypes were annotated to 14 genes whose function could influence MetS biology as well as oncogenesis or inflammation. These were mechanistically categorized into four groups: cell-cell adhesion and mobility, signal transduction, transcription and protein sorting. Four genes were highly prioritized: cadherin 18 (CDH18), myosin X (MYO10), anchor protein 6 of AMPK (AKAP6), and neuronal PAS domain protein 3 (NPAS3). PWBC expression was detectable only for the following genes with multi-organ or with multi-function properties: NPAS3, MARCH6, MYO10 and FBXL7. Strong evidence of cis-effects on the expression of MYO10 in PWBC was found with SNPs clustered near the gene's transcription start site. MYO10 expression in PWBC was marginally correlated with body composition (p = 0.065) and adipokine levels in the periphery (p = 0.064). Variants of genes AKAP6, NPAS3, MARCH6 and FBXL7 have been previously reported to be associated with insulin resistance, inflammatory markers or adiposity studies using genome-wide approaches whereas associations of CDH18 and MYO10 with MetS traits have not been reported before. Adiponectin QTLs-based SNP association and mRNA expression identified genes that could mediate the association between MetS and cancer or inflammation. Show less
High altitude pulmonary edema (HAPE), a life-threatening disease, has no biological markers used for the routine prevention, diagnosis and treatment. The aim of this study was to identify serum protei Show more
High altitude pulmonary edema (HAPE), a life-threatening disease, has no biological markers used for the routine prevention, diagnosis and treatment. The aim of this study was to identify serum proteins differentially expressed in patients with HAPE for discovering essential biomarkers. A complete serum proteomic analysis was performed on 10 HAPE patients and on 10 high altitude and 11 sea level healthy people as control using two-dimensional gel electrophoresis, followed by matrix-assisted laser desorption/ionization mass spectrometry and peptide mass fingerprinting. Finally, two most significantly changed proteins were validated by enzyme-linked immunosorbent assay (ELISA). Eight protein spots stained with differential intensity, respresenting 5 distinct proteins were identified in patients compared with healthy controls through analysis of these composite gels. Among them, four proteins, namely alpha 1-antitrypsin(alpha1-AT), Haptoglobin(Hp), apolipoprotein A-1 (apoA-1) and Complement C3 increased remarkably, while one protein, apolipoprotein A-IV (apoA-IV) decreased significantly. The variation of alpha1-AT and Haptoglobin, as detected by ELISA, was consistent with the results from proteomic analysis. It is well known that Hp, alpha1-AT and complement C3 are associated with inflammation and apoA-1 and apoA-IV play important roles in lipid absorption, transport and metabolism. Therefore, the significant expression changes of Hp, alpha1-AT and complement C3 and apoA-1 and apoA-IV between HAPE patients and their corresponding healthy controls highlight the role of inflammatory response system and lipid metabolism system in the pathophysiology of HAPE. Show less
Type 2 diabetes and its chronic complications have become a worldwide epidemic nowadays. However, its molecular mechanism is still unknown. We have previously identified a novel variant rs12742393 of Show more
Type 2 diabetes and its chronic complications have become a worldwide epidemic nowadays. However, its molecular mechanism is still unknown. We have previously identified a novel variant rs12742393 of NOS1AP for type 2 diabetes susceptibility in the Chinese population. In this study, we analyzed the total serum profiling among three genotypes of rs12742393 to discover potential crosstalk under the variant and the disease through proteomic analyses for the first time. We used OFFGEL peptide fractionation, LC-MS/MS analysis, and label-free quantification to profile the fasting human serum samples of the genotypes in rs12742393 (n = 4, for CC, AC, and AA, resp.). Four proteins were identified, including apoA4, alpha1-ACT, HABP2, and keratin 10, with blood levels changed significantly between CC and AA homozygotes of rs12742393. Compared with AA group, the levels of apoA4 increased (P = 0.000265), whereas the concentration of alpha1-ACT, HABP2, and keratin 10 decreased in CC group (P = 0.011116, 0.021175, and 0.015661, resp.). Then we selected additional fasting serum samples for ELISA and western blot validation. However, no significant differences were identified by neither ELISA nor western blot (P > 0.05). The protein profiling changes between the genotypes of rs12742393 indicated that this SNP might play a role in the development of type 2 diabetes. Show less
Apolipoprotein A5 (APOA5) and C3 (APOC3) genes are involved in the PPAR lipid metabolism pathway and thus associated with elevated triglyceride levels. However, whether APOA5 and APOC3 genetic polymor Show more
Apolipoprotein A5 (APOA5) and C3 (APOC3) genes are involved in the PPAR lipid metabolism pathway and thus associated with elevated triglyceride levels. However, whether APOA5 and APOC3 genetic polymorphisms affect intramuscular fat deposition and other meat quality traits remains unknown in pigs. One hundred and seventy-one Kele pigs were sampled to investigate genetic variants in the APOA5 and APOC3 genes and their association with seven pork quality traits. We identified 5 single nucleotide polymorphisms (SNPs) in the promoter region of the APOA5 gene and 17 SNPs in the APOC3 gene. Linkage disequilibrium analysis revealed 5 complete linkage disequilibria among these 22 SNPs. We found that 10 SNPs were significantly correlated with meat quality traits, including the mutation A5/-769 in the APOA5 gene, which was significantly associated with cooked weight percentage, and 9 SNPs in the APOC3 gene that were significantly associated with drip loss rate, meat color value of longissimus dorsi muscle and shear force. Therefore, these SNP markers will be useful for marker-assisted selection for improved pork quality. Show less
The goal of our study is to investigate the combined contribution of 10 genetic variants to diabetes susceptibility. Bibliographic databases were searched from 1970 to Dec 2012 for studies that report Show more
The goal of our study is to investigate the combined contribution of 10 genetic variants to diabetes susceptibility. Bibliographic databases were searched from 1970 to Dec 2012 for studies that reported on genetic association study of diabetes. After a comprehensive filtering procedure, 10 candidate gene variants with informative genotype information were collected for the current meta-anlayses. Using the REVMAN software, odds ratios (ORs) with 95% confidence intervals (CIs) were calculated to evaluate the combined contribution of the selected genetic variants to diabetes. A total of 37 articles among 37,033 cases and 54,716 controls were involved in the present meta-analyses of 10 genetic variants. Three variants were found to be significantly associated with type 1 diabetes (T1D): NLRP1 rs12150220 (ORβ=β0.71, 95% CIβ=β0.55-0.92, Pβ=β0.01), IL2RA rs11594656 (ORβ=β0.86, 95% CIβ=β0.82-0.91, P<0.00001), and CLEC16A rs725613 (ORβ=β0.71, 95% CIβ=β0.55-0.92, Pβ=β0.01). APOA5 -1131T/C polymorphism was shown to be significantly associated with of type 2 diabetes (T2D, ORβ=β1.27, 95% CIβ=β1.03-1.57, Pβ=β0.03). No association with diabetes was showed in the meta-analyses of other six genetic variants, including SLC2A10 rs2335491, ATF6 rs2070150, KLF11 rs35927125, CASQ1 rs2275703, GNB3 C825T, and IL12B 1188A/C. Our results demonstrated that IL2RA rs11594656 and CLEC16A rs725613 are protective factors of T1D, while NLRP1 rs12150220 and APOA5 -1131T/C are risky factors of T1D and T2D, respectively. Show less
This study aims to examine the possible associations between high density lipoprotein (HDL) subclass distribution and APOA5-1131T>C polymorphism in hypertriglyceridemia. The distribution of HDL subcla Show more
This study aims to examine the possible associations between high density lipoprotein (HDL) subclass distribution and APOA5-1131T>C polymorphism in hypertriglyceridemia. The distribution of HDL subclasses was quantified by 2-dimensional electrophoresis in conjunction with immunodetection method. The APOA5-1131T>C polymorphism was identified in 95 hypertriglyceridemic (HTG) patients and 102 healthy subjects by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). The APOA5-1131C (C) allele frequency was higher in the HTG group than in the control group. Plasma triglycerides (TG) were significantly higher and apoA5 was significantly lower in patients with the C allele when compared to patients with the APOA5-1131T (T) allele, even more dramatically so in the APOA5-1131CC homozygote. In both the HTG group and the control group, the frequency of the C allele was positively correlated with levels of TG, total cholesterol (TC), low density lipoprotein cholesterol (LDL-C) and apolipoprotein B100 (apoB100), and negatively correlated with levels of high density lipoprotein cholesterol (HDL-C), apolipoprotein A1 (apoA1) and apolipoprotein A5 (apoA5) (P<0.001). In all subjects, the frequency of the C allele was positively correlated with the level of small-sized HDL (preΞ²(1)-HDL and HDL(3a)), and negatively correlated with levels of HDL(2a) and HDL(2b). Changes in HDL subclass distributions in HTG may be related to the APOA5-1131T>C polymorphism. This polymorphism leads to a general shift towards smaller-sized HDL. Show less
The apolipoprotein A5 (APOA5) gene -1131T>C (rs662799) has been suggested to be involved in the pathway of lipid homeostasis and the development of metabolic syndrome (MetS). However, the findings are Show more
The apolipoprotein A5 (APOA5) gene -1131T>C (rs662799) has been suggested to be involved in the pathway of lipid homeostasis and the development of metabolic syndrome (MetS). However, the findings are not consistent. To systematically evaluate the associations between -1131T>C polymorphism and fasting lipid parameters and the risk of MetS, we conducted a case-control study in a Chinese population and a meta-analysis. The findings from 1840 Chinese participants indicated that the C allele carriers had significantly higher fasting total cholesterol (TC), triglycerides (TG) and lower HDL-cholesterol (HDL-C) than the TT homozygotes carriers. The -1131C allele was also found to be significantly associated with increased risk of MetS (OR β=β 1.40, 95% confidence interval (CI) β=β 1.15, 1.69) compared to the TT homozygotes. In the meta-analysis of 51,868 participants from 46 East Asian studies, 26 European studies and 19 studies of other ethnic groups, the -1131C allele was associated with higher fasting TC (weighted mean difference (WMD) β=β 0.08 mmol/L, 95% CI β=β 0.05, 0.10, Pβ=β1.74Γ10(-9)), TG (WMD β=β 0.30 mmol/L, 95% CI β=β 0.26, 0.33, Pβ=β 1.87Γ10(-55)), LDL-cholesterol (LDL-C) (WMD β=β 0.04 mmol/L, 95% CI β=β 0.02, 0.07, Pβ=β0.002), and lower HDL-C (WMD β=β -0.05 mmol/L, 95% CI β=β -0.06,-0.04, Pβ=β1.88Γ10(-21)), respectively. Based on 12 studies with 5,573 MetS cases and 8,290 controls from 5 East Asian studies, 5 European studies and 2 studies of other ethnic groups, the -1131C allele was associated with increased risk of MetS with an OR (95% CI) β=β 1.33 (1.16, 1.53) in the overall population, 1.43 (1.29, 1.58) in East Asian and 1.30 (0.94, 1.78) in European populations. In conclusion, the -1131C allele may be associated with elevated levels of fasting TG, TC, LDL-C and decreased HDL-C, and increased risk of MetS, especially in East Asians. Show less
Elevated levels of circulating triglycerides and increased arterial stiffness are associated with cardiovascular disease. Numerous studies have reported an association between levels of circulating tr Show more
Elevated levels of circulating triglycerides and increased arterial stiffness are associated with cardiovascular disease. Numerous studies have reported an association between levels of circulating triglycerides and arterial stiffness. We used Mendelian randomization to test whether this association is causal. We investigated the association between circulating triglyceride levels, the apolipoprotein A-V (ApoA5) -1131T>C single nucleotide polymorphism and brachial-ankle pulse wave velocity (baPWV) by examining data from 4421 subjects aged 18-74 years who were recruited from the Chinese population. baPWV was significantly associated with the levels of circulating triglycerides after adjusting for age, sex, body mass index (BMI), systolic blood pressure, heart rate, waist-to-hip ratio, antihypertensive treatment and diabetes mellitus status. The -1131C allele was associated with a 5% (95% confidence interval 3-8%) increase in circulating triglycerides (adjusted for age, sex, BMI, waist-to-hip ratio, diabetes mellitus and antihypertensive treatment). Instrumental variable analysis showed that genetically elevated levels of circulating triglycerides were not associated with increased baPWV. These results do not support the hypothesis that levels of circulating triglycerides have a causal role in the development of arterial stiffness. Show less
Glycosylation is one of the most important post-translational modifications to mammalian proteins. Distribution of different glycoisoforms of certain proteins may reflect disease conditions and, there Show more
Glycosylation is one of the most important post-translational modifications to mammalian proteins. Distribution of different glycoisoforms of certain proteins may reflect disease conditions and, therefore, can potentially be utilized as biomarkers. Apolipoprotein C3 (ApoC3) is one of the many plasma glycoproteins extensively studied for association with disease states. ApoC3 exists in three main glycoisoforms, including ApoC3-1 and ApoC3-2, which contain an O-linked carbohydrate moiety consisting of three and four monosaccharide residues, respectively, and ApoC3-0 that lacks the entire glycosylation chain. Changes in the ratio of different glycoisoforms of ApoC3 have been observed in pathological conditions such as kidney disease, liver disease, and diabetes. They may provide important information for diagnosis, prognosis, and evaluation of therapeutic response for metabolic conditions. In this current work, a liquid chromatography (LC)-high-resolution (HR) time-of-flight (TOF) mass spectrometry (MS) method was developed for relative quantitation of different glycoisoforms of intact ApoC3 in human plasma. The samples were processed using a solid-phase extraction (SPE) method and then subjected to LC-full scan HRMS analysis. Isotope peaks for each targeted glycoisoform at two charge states were extracted using a window of 50 mDa and integrated into a chromatographic peak. The peak area ratios of ApoC3-1/ApoC3-0 and ApoC3-2/ApoC3-0 were calculated and evaluated for assay performance. The results indicated that the ratio can be determined with excellent reproducibility in multiple subjects. It has also been observed that the ratios remained constant in plasma exposed to room temperature, freeze-thaw cycles, and long-term frozen storage. The method was applied in preliminary biomarker research of diabetes by analyzing plasma samples collected from normal, prediabetic, and diabetic subjects. Significant differences were revealed in the ApoC3-1/ApoC3-0 ratio and in the ApoC3-2/ApoC3-0 ratio among the three groups. The workflow of intact protein analysis using full scan HRMS established in this current work can be potentially extended to relative quantitation of other glycosylated proteins. To our best knowledge, this is the first time that a systematic approach of relative quantitation of targeted intact protein glycoisoforms using LC-MS has been established and utilized in biomarker research. Show less