•A peptide is designed to inhibit the activity of PI3K, a critical molecule for cell growth.•The peptide is able reduce the proliferation of gastric cancer cells.•The peptide can block tumor growth of gastric cancer cells in a mouse model.•The peptide also inhibits the function of a hotspot mutation of PI3K found in many human cancers.Activation of class I Phosphoinositide 3-kinases (PI3Ks) by mutation or overexpression closely correlates with the development of various human cancers. Class I PI3Ks are heterodimers composed of p110 catalytic subunits and regulatory subunits represented by p85. PAQR3 has been found to inhibit p110α activity by blocking its interaction with p85. In this study, we identified the N-terminal 6–55 amino acid residues of PAQR3 being sufficient for its interaction with p110α. A synthetic peptide, P6-55, that contains the N-terminus of PAQR3 could disrupt the interactions of p110α with both PAQR3 and p85. The activity of PI3K was also inhibited by P6-55, accompanied by significant inhibition of cancer cell proliferation. In a xenograft mouse model, P6-55 was able to reduce tumor growth in vivo. Furthermore, P6-55 was capable of inhibiting the elevated basal PI3K activity of H1047R, a hotspot mutation found in many types of human cancers. The cell proliferation and migration of cancer cells bearing H1047R mutation were also reduced by P6-55. In conclusion, our study provides a proof of concept that blocking the interaction of p110α with p85 by a peptide can serve as a new strategy to inhibit the oncogenic activity of PI3K in cancer therapy.

•DNA damage repair is modulated by changing subcellular distribution of RAD23B-XPC complex during global genome NER•The subcellular compartmentation of RAD23B is regulated by PAQR3, a Golgi-localized protein•PAQR3 reduces the protein level of XPC with accelerated degradation/polyubiquitination of XPC•DNA damage repair in response to chemotherapy drugs is regulated by PAQR3The RAD23B-XPC complex in the nucleus plays a key role in the initial damage recognition during global genome nucleotide excision repair (NER). Within the complex, XPC, a product of Xeroderma pigmentosum C, recognizes and interacts with the unpaired bases in the undamaged DNA strand, while RAD23B stabilizes XPC. However, how RAD23B is regulated by other factors is not well known. We report here a mode of spatial regulation of RAD23B that controls XPC stability and DNA damage repair. We first identified that RAD23B was able to directly associate with PAQR3, a newly-discovered tumor suppressor implicated in many types of human cancers. PAQR3 reduced the protein level of XPC, together with accelerated degradation and enhanced polyubiquitination of XPC. Mechanistically, PAQR3 reduces nucleic distribution of RAD23B by tethering it to the Golgi apparatus, thus diminishing the amount of RAD23B proteins available to interact with XPC in the nucleus. The viability of gastric cancer cells upon treatment with chemotherapy drugs including etoposide, cisplatin and doxorubicin was reduced by PAQR3 overexpression, but enhanced by PAQR3 knockdown. The degree of DNA damage induced by these drugs, as measured by immunoblotting with γ-H2AX, was elevated by PAQR3 overexpression and lessened by PAQR3 knockdown. Furthermore, a synthetic peptide comprising the N-terminus of PAQR3 was able to recapitulate the activity of PAQR3 in reducing XPC stability and enhancing chemotherapy drug-induced DNA damage. In conclusion, our study reveals that RAD23B is controlled by subcellular compartmentation, thus affecting XPC-mediated DNA damage repair in cancer cells.

Ras plays a pivotal role in many cellular activities, and its subcellular compartmentalization provides spatial and temporal selectivity. Here we report a mode of spatial regulation of Ras signaling in the Golgi apparatus by two highly homologous proteins PAQR10 and PAQR11 of the progestin and AdipoQ receptors family. PAQR10 and PAQR11 are exclusively localized in the Golgi apparatus. Overexpression of PAQR10/PAQR11 stimulates basal and EGF-induced ERK phosphorylation and increases the expression of ERK target genes in a dose-dependent manner. Overexpression of PAQR10/PAQR11 markedly elevates Golgi localization of HRas, NRas and KRas4A, but not KRas4B. PAQR10 and PAQR11 can also interact with HRas, NRas and KRas4A, but not KRas4B. The increased Ras protein at the Golgi apparatus by overexpression of PAQR10/PAQR11 is in an active state. Consistently, knockdown of PAQR10 and PAQR11 reduces EGF-stimulated ERK phosphorylation and Ras activation at the Golgi apparatus. Intriguingly, PAQR10 and PAQR11 are able to interact with RasGRP1, a guanine nucleotide exchange protein of Ras, and increase Golgi localization of RasGRP1. The C1 domain of RasGRP1 is both necessary and sufficient for the interaction of RasGRP1 with PAQR10/PAQR11. The simulation of ERK phosphorylation by overexpressed PAQR10/PAQR11 is abrogated by downregulation of RasGRP1. Furthermore, differentiation of PC12 cells is significantly enhanced by overexpression of PAQR10/PAQR11. Collectively, this study uncovers a new paradigm of spatial regulation of Ras signaling in the Golgi apparatus by PAQR10 and PAQR11.

Vascular endothelial growth factors (VEGFs) are crucial regulators of angiogenesis and vasculogenesis. The autocrine VEGF signaling is required for maintaining the homeostasis of vasculature. Dysregulation of angiogenesis is implicated in the development of many human cancers, especially in clear-cell renal cell carcinoma (ccRCC), a highly vascularized tumor. Meanwhile, antiangiogenesis has become a mainstay in the treatment of human cancers. In this study, we analyzed the functional roles of RKTG (Raf Kinase Trapping to Golgi), a negative regulator of mitogen-activated protein kinase (Raf/MEK/ERK) signaling, by sequestration of Raf kinase to the Golgi apparatus, in angiogenesis and ccRCC. Through a series of in vitro and in vivo experiments, we found that RKTG has a negative effect on cell proliferation, migration, sprouting and angiogenesis of endothelial cells. RKTG, by suppressing mitogen-activated protein kinase signaling, negatively regulates the transactivation activity of hypoxia-inducible factor 1α (HIF-1α) by inhibiting formation of HIF-1α/p300 complex and suppressing VEGF transcription, thereby reducing hypoxia-induced VEGF production. The expression level of RKTG is significantly downregulated in clinical ccRCC tumor samples, with an inverse correlation with VEGF expression level. These results highlight the functional roles of RKTG and its regulated Raf/ERK/MEK signaling cascade in angiogenesis and autocrine VEGF signaling. In addition, this study indicates that RKTG is likely implicated in the development of ccRCC through its regulation on angiogenesis.

Both genetic variations and diet-disrupted gut microbiota can predispose animals to metabolic syndromes (MS). This study assessed the relative contributions of host genetics and diet in shaping the gut microbiota and modulating MS-relevant phenotypes in mice. Together with its wild-type (Wt) counterpart, the Apoa-I knockout mouse, which has impaired glucose tolerance (IGT) and increased body fat, was fed a high-fat diet (HFD) or normal chow (NC) diet for 25 weeks. DNA fingerprinting and bar-coded pyrosequencing of 16S rRNA genes were used to profile gut microbiota structures and to identify the key population changes relevant to MS development by Partial Least Square Discriminate Analysis. Diet changes explained 57% of the total structural variation in gut microbiota, whereas genetic mutation accounted for no more than 12%. All three groups with IGT had significantly different gut microbiota relative to healthy Wt/NC-fed animals. In all, 65 species-level phylotypes were identified as key members with differential responses to changes in diet, genotype and MS phenotype. Most notably, gut barrier-protecting Bifidobacterium spp. were nearly absent in all animals on HFD, regardless of genotype. Sulphate-reducing, endotoxin-producing bacteria of the family, Desulfovibrionaceae, were enhanced in all animals with IGT, most significantly in the Wt/HFD group, which had the highest calorie intake and the most serious MS phenotypes. Thus, diet has a dominating role in shaping gut microbiota and changes of some key populations may transform the gut microbiota of Wt animals into a pathogen-like entity relevant to development of MS, despite a complete host genome.

In eukaryotic cells, receptor endocytosis is a key event regulating signaling transduction. Adiponectin receptors belong to a new receptor family that is distinct from G-protein-coupled receptors and has critical roles in the pathogenesis of diabetes and metabolic syndrome. Here, we analyzed the endocytosis of adiponectin and adiponectin receptor 1 (AdipoR1) and found that they are both internalized into transferrin-positive compartments that follow similar traffic routes. Blocking clathrin-mediated endocytosis by expressing Eps15 mutants or depleting K+ trapped AdipoR1 at the plasma membrane, and K+ depletion abolished adiponectin internalization, indicating that the endocytosis of AdipoR1 and adiponectin is clathrin-dependent. Depletion of K+ and overexpression of Eps15 mutants enhance adiponectin-stimulated AMP-activated protein kinase phosphorylation, suggesting that the endocytosis of AdipoR1 might downregulate adiponectin signaling. In addition, AdipoR1 colocalizes with the small GTPase Rab5, and a dominant negative Rab5 abrogates AdipoR1 endocytosis. These data indicate that AdipoR1 is internalized through a clathrin- and Rab5-dependent pathway and that endocytosis may play a role in the regulation of adiponectin signaling.

Herbert et al reported association with obesity of a common DNA variant rs7566605 at 10 kb upstream of the INSIG2 gene. We analyzed rs7566605 polymorphism in 3125 Chinese in a cross-sectional study. We found no significant association of rs7566605 polymorphism with body mass index (BMI) and waist circumference among all participants (P=0.52). However, if geographic location is considered, the C/C genotype of rs7566605 was marginally associated with increased levels of BMI and risk of obesity among individuals living in Shanghai (P=0.06), indicating that the C/C genotype may contribute to obesity in certain subpopulation among Chinese under certain environmental settings.

Subcellular compartmentalization has become an important theme in cell signaling such as spatial regulation of Ras by RasGRP1 and MEK/ERK by Sef. Here, we report spatial regulation of Raf kinase by RKTG (Raf kinase trapping to Golgi). RKTG is a seven-transmembrane protein localized at the Golgi apparatus. RKTG expression inhibits EGF-stimulated ERK and RSK phosphorylation, blocks NGF-mediated PC12 cell differentiation, and antagonizes Ras- and Raf-1-stimulated Elk-1 transactivation. Through interaction with Raf-1, RKTG changes the localization of Raf-1 from cytoplasm to the Golgi apparatus, blocks EGF-stimulated Raf-1 membrane translocation, and reduces the interaction of Raf-1 with Ras and MEK1. In RKTG-null mice, the basal ERK phosphorylation level is increased in the brain and liver. In RKTG-deleted mouse embryonic fibroblasts, EGF-induced ERK phosphorylation is enhanced. Collectively, our results reveal a paradigm of spatial regulation of Raf kinase by RKTG via sequestrating Raf-1 to the Golgi apparatus and thereby inhibiting the ERK signaling pathway.

Germline mutations of the serine/threonine kinase LKB1 (also known as STK11) lead to Peutz–Jeghers syndrome (PJS) that is associated with increased incidence of malignant cancers. However, the tumor suppressor function of LKB1 has not been fully elucidated. We applied yeast two-hybrid screening and identified that a novel WD-repeat protein WDR6 was able to interact with LKB1. Immunofluorescence staining revealed that WDR6 was localized in cytoplasm, similar to the localization of LKB1. Expression of LKB1 was able to inhibit colony formation of Hela cells. Interestingly, coexpression of WDR6 with LKB1 enhanced the inhibitory effect of LKB1 on Hela cell proliferation. Consistently, WDR6 was able to synergize with LKB1 in cell cycle G1 arrest in Hela cells. Coexpression of WDR6 and LKB1 was able to induce a cyclin-dependent kinase (CDK) inhibitor p27Kip1. Furthermore, the stimulatory effect of LKB1 on p27Kip1 promoter activity was significantly elevated by coexpression with WDR6. Collectively, these results provided initial evidence that WDR6 is implicated in the cell growth inhibitory pathway of LKB1 via regulation of p27Kip1.

•A new mode of regulation of mTOR signaling in response to amino acids is proposed.•PAQR3 inhibits mTOR signaling by disrupting the integrity of mTORC1 formation.•Amino acid deprivation-induced autophagy is modulated by PAQR3.Amino acids are the key activators of the mTOR complex 1 (mTORC1, mainly composed of mTOR, Raptor and mLST8) required for cell growth and proliferation. On the other hand, deprivation of amino acids induces autophagy via inhibition of mTORC1 signaling. We report here that amino acid-induced mTORC1 activity and amino acid deprivation-induced autophagy are regulated by PAQR3, a newly found tumor suppressor. At the cellular level, PAQR3 negatively regulates amino acid-induced activation of mTORC1. The N-terminal end of PAQR3 interacts with the WD domains of Raptor and mLST8 directly. PAQR3 reduces the interaction of mTOR with Raptor and mLST8, thus disrupts formation of intact mTORC1 complex. PAQR3 modulates leucine-induced alteration in cell size. In addition, PAQR3 knockdown reduces amino acid deprivation-induced autophagy. The inhibitory effect of PAQR3 knockdown on autophagy is abrogated by rapamycin treatment, indicating that PAQR3 modulates autophagy via its regulation on mTORC1 signaling. In conclusion, our finding reveals a new mode of regulation of mTORC1 signaling and autophagy by PAQR3 in response to alterations of amino acids.

For thousands of years, cinnamon has been used as a traditional treatment in China. However, there are no studies to date that investigate whether cinnamon supplements are able to aid in the treatment of type 2 diabetes in Chinese subjects. We hypothesized cinnamon should be effective in improving blood glucose control in Chinese patients with type 2 diabetes. To address this hypothesis, we performed a randomized, double-blinded clinical study to analyze the effect of cinnamon extract on glycosylated hemoglobin A1c and fasting blood glucose levels in Chinese patients with type 2 diabetes. A total of 66 patients with type 2 diabetes were recruited and randomly divided into 3 groups: placebo and low-dose and high-dose supplementation with cinnamon extract at 120 and 360 mg/d, respectively. Patients in all 3 groups took gliclazide during the entire 3 months of the study. Both hemoglobin A1c and fasting blood glucose levels were significantly reduced in patients in the low- and high-dose groups, whereas they were not changed in the placebo group. The blood triglyceride levels were also significantly reduced in the low-dose group. The blood levels of total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and liver transaminase remained unchanged in the 3 groups. In conclusion, our study indicates that cinnamon supplementation is able to significantly improve blood glucose control in Chinese patients with type 2 diabetes.

•Reduced glycogen deposition reduces high-fat diet induced obesity in mice.•Increasing glycogen synthesis elevates lipid accumulation in adipocytes.•As a glycogen-targeting subunit of PP1, PPP1R3G regulates metabolism in vivo.Glycogen and triglyceride are two major forms of energy storage in the body and provide the fuel during different phases of food deprivation. However, how glycogen metabolism is linked to fat deposition in adipose tissue has not been clearly characterized. We generated a mouse model with whole-body deletion of PPP1R3G, a glycogen-targeting subunit of protein phosphatase-1 required for glycogen synthesis. Upon feeding with high-fat diet, the body weight and fat composition are significantly reduced in the PPP1R3G−/- mice compared to the wild type controls. The metabolic rate of the mice as measured by O2 consumption and CO2 production is accelerated by PPP1R3G deletion. The high-fat diet-induced liver steatosis is also slightly relieved by PPP1R3G deletion. The glycogen level in adipose tissue is reduced by PPP1R3G deletion. In 3T3L1 cells, overexpression of PPP1R3G leads to increases of both glycogen and triglyceride levels. In conclusion, our study indicates that glycogen is actively involved in fat accumulation in adipose tissue and obesity development upon high-fat diet. Our study also suggests that PPP1R3G is an important player that links glycogen metabolism to lipid metabolism in vivo.

Adiponectin is an adipose-derived hormone that has anti-diabetic and anti-atherogenic effects through interaction with adiponectin receptors AdipoR1 and AdipoR2. We analyzed the transcriptional regulation of AdipoR1 by insulin. Insulin repressed the promoter activity of AdipoR1 in C2C12 myoblasts via PI3K and Foxo1. Deletion studies demonstrated the presence of a putative insulin-responsive region which is composed of a nuclear inhibitory protein (NIP) binding element. Mutation of the NIP element abrogated the negative regulation of AdipoR1 promoter by insulin. Insulin treatment could induce formation of a protein complex that bound the NIP element. Collectively, our data suggest that a repressive NIP element is involved in the negative regulation of AdipoR1 promoter by insulin.