Protein-tyrosine phosphatase 1B substrates and metabolic regulation

Metabolic homeostasis requires integration of complex signaling networks which, when deregulated, contribute to metabolic syndrome and related disorders. Protein-tyrosine phosphatase 1B (PTP1B) has emerged as a key regulator of signaling networks that are implicated in metabolic diseases such as obesity and type 2 diabetes. In this review, we examine mechanisms that regulate PTP1B-substrate interaction, enzymatic activity and experimental approaches to identify PTP1B substrates. We then highlight findings that implicate PTP1B in metabolic regulation. In particular, insulin and leptin signaling are discussed as well as recently identified PTP1B substrates that are involved in endoplasmic reticulum stress response, cell-cell communication, energy balance and vesicle trafficking. In summary, PTP1B exhibits exquisite substrate specificity and is an outstanding pharmaceutical target for obesity and type 2 diabetes

Coenzyme Q10 in human blood: native levels and determinants of oxidation during processing and storage.

Coenzyme Q10 (Q10) is present in the circulation mainly in its reduced form (ubiquinol-10; UL10), but oxidizes quickly ex vivo to ubiquinone-10 (UN10). Therefore, native UL10:UN10 ratios, used as markers of redox status and disease risk, are difficult to measure. We established an RP-(U)HPLC method with coulometric detection to measure natively circulating UL10 and UN10 concentrations by adding a ubiquinol/ubiquinone mixture as an internal standard immediately after plasma preparation. This allowed adjustment for unavoidable artificial UL10 oxidation as well as for total losses (or gains) of analytes during sample storage, processing, and analysis because the internal standards exactly paralleled the chemical behavior of Q10. This technique applied to blood (n = 13) revealed Q10 levels of 680-3300 nM with a mean UL10:UN10 ratio of 95:5, which was inversely associated with total Q10 (r=-0.69; p=0.004). The oxidation of UL10 to UN10 was equimolar, increased by O(2), and decreased by lower temperatures or various degassing methods. Although UL10 was stable in blood or when pure in organic solvents at 22 degrees C, its oxidation was catalyzed dose dependently by alpha-tocopherol and butylated hydroxytoluene, particularly when present in combination. Key structural features for the catalytic pro-oxidant properties of phenolic antioxidants included two substituents vicinal to the phenolic hydroxyl group

Regulation of the SNARE-interacting protein Munc18c tyrosine phosphorylation in adipocytes by protein-tyrosine phosphatase 1B

Abstract Background Protein-tyrosine phosphatase 1B (PTP1B) is a physiological regulator of insulin signaling and adiposity and is a drug target for the treatment of obesity and diabetes. The molecular mechanisms underlying PTP1B metabolic actions require additional investigation. Results Herein, we identify Munc18c as a novel PTP1B substrate in adipocytes and in vivo. We demonstrate nutritional regulation of Munc18c in adipose tissue revealing decreased expression upon high fat feeding. In addition, PTP1B deficiency leads to elevated Munc18c tyrosine phosphorylation and dissociation from syntaxin4. At the molecular level, we identify Munc18c Tyr218/219 and Tyr521 as key residues that mediate Munc18c interaction with PTP1B. Further, we uncover an essential role of Munc18c total tyrosine phosphorylation in general, and Tyr218/219 and Tyr521 in particular, in regulating its interactions and glucose uptake in adipocytes. Conclusion In conclusion, our findings identify PTP1B as the first known tyrosine phosphatase for Munc18c and a regulator of its phosphorylation and function in adipocytes

Genome-wide CRISPR screen reveals PSMA6 to be an essential gene in pancreatic cancer cells

Abstract Background Despite its relatively low incidence, pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer deaths because of the aggressive growth/metastasis of the tumor, the lack of early symptoms, and the poor treatment options. Basic research to identify potential therapeutic targets for PDAC is greatly needed. Methods We used a negative-selection genome-wide CRISPR screen to identify essential genes in the PANC-1 human pancreatic carcinoma cell line. We validated the top hits with follow-up siRNA screens, using the HPNE, HPAF-II, AsPC-1, and Mia PaCa-2 cell lines. Results The PSMA6 gene was an identified candidate hit after the CRISPR screen, siRNA validation screen, and siRNA deconvolution screen. Spheroid formation assays and flow cytometry analysis showed that PSMA6 is critical for survival in many pancreatic ductal carcinoma cell models. Lastly, as PSMA6 protein is a proteosomal subunit of the 20S core complex, we showed that bortezomib, a proteasome inhibitor, was especially toxic in PANC-1 cells. Conclusions Further study of PSMA6 and the proteasome subunit that it encodes, along with other hits identified in our CRISPR screens, may provide valuable insights into potential therapeutic targets for PDAC