The last enzyme of the de novo purine synthesis pathway 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC) plays a central role in insulin signaling and the Golgi/endosomes protein network.

Insulin is internalized with its cognate receptor into the endosomal apparatus rapidly after binding to hepatocytes. We performed a bioinformatic screen of Golgi/endosome hepatic protein fractions and found that ATIC, which is a rate-limiting enzyme in the de novo purine biosynthesis pathway, and PTPLAD1 are associated with insulin receptor (IR) internalization. The IR interactome (IRGEN) connects ATIC to AMPK within the Golgi/endosome protein network (GEN). Forty-five percent of the IR Golgi/endosome protein network have common heritable variants associated with type 2 diabetes, including ATIC and AMPK. We show that PTPLAD1 and AMPK are rapidly compartmentalized within the plasma membrane (PM) and Golgi/endosome fractions after insulin stimulation and that ATIC later accumulates in the Golgi/endosome fraction. Using an in vitro reconstitution system and siRNA-mediated partial knockdown of ATIC and PTPLAD1 in HEK293 cells, we show that both ATIC and PTPLAD1 affect IR tyrosine phosphorylation and endocytosis. We further show that insulin stimulation and ATIC knockdown readily increase the level of AMPK-Thr172 phosphorylation in IR complexes. We observed that IR internalization was markedly decreased after AMPKα2 knockdown, and treatment with the ATIC substrate AICAR, which is an allosteric activator of AMPK, increased IR endocytosis in cultured cells and in the liver. These results suggest the presence of a signaling mechanism that senses adenylate synthesis, ATP levels, and IR activation states and that acts in regulating IR autophosphorylation and endocytosis.

A type 2 diabetes disease module with a high collective influence for Cdk2 and PTPLAD1 is localized in endosomes.

Despite the identification of many susceptibility genes our knowledge of the underlying mechanisms responsible for complex disease remains limited. Here, we identified a type 2 diabetes disease module in endosomes, and validate it for functional relevance on selected nodes. Using hepatic Golgi/endosomes fractions, we established a proteome of insulin receptor-containing endosomes that allowed the study of physical protein interaction networks on a type 2 diabetes background. The resulting collated network is formed by 313 nodes and 1147 edges with a topology organized around a few major hubs with Cdk2 displaying the highest collective influence. Overall, 88% of the nodes are associated with the type 2 diabetes genetic risk, including 101 new candidates. The Type 2 diabetes module is enriched with cytoskeleton and luminal acidification-dependent processes that are shared with secretion-related mechanisms. We identified new signaling pathways driven by Cdk2 and PTPLAD1 whose expression affects the association of the insulin receptor with TUBA, TUBB, the actin component ACTB and the endosomal sorting markers Rab5c and Rab11a. Therefore, the interactome of internalized insulin receptors reveals the presence of a type 2 diabetes disease module enriched in new layers of feedback loops required for insulin signaling, clearance and islet biology.

Insulin-dependent phosphorylation of DPP IV in liver. Evidence for a role of compartmentalized c-Src.

Dipeptidyl peptidase IV (DPP IV, CD26, EC 3.4.14.5) serves as a model aimed at elucidating protein sorting signals. We identify here, by MS, several tyrosine-phosphorylated proteins in a rat liver Golgi/endosome (G/E) fraction including DPP IV. We show that a pool of DPP IV is tyrosine-phosphorylated. Maximal phosphorylation was observed after 2 min following intravenous insulin injection. DPP IV coimmunoprecipitated with the cellular tyrosine kinase Src (c-Src) with maximal association also observed after 2 min following insulin injection. DPP IV was found phosphorylated after incubation of nonsolubilized G/E membranes with [gamma-32P]ATP. The c-Src inhibitor PP2 inhibited DPP IV phosphorylation. Oriented proteolysis experiments indicate that a large pool of c-Src is protected in G/E fractions. Following injection of the protein-tyrosine phosphatase inhibitor bpV(phen), DPP IV levels markedly decreased by 40% both in plasma membrane and G/E fractions. In the fraction designated Lh, DPP IV levels decreased by 50% 15 min following insulin injection. Therefore, a pool of DPP IV is tyrosine-phosphorylated in an insulin-dependent manner. The results suggest the presence of a yet to be characterized signalling mechanism whereby DPP IV has access to c-Src-containing signalling platforms.

Use of electron density critical points as chemical function-based reduced representations of pharmacological ligands.

In this paper, we propose a reduced representation of molecules of pharmacological interest based on their chemical functions. The proposed representations of the molecules are obtained by a topological analysis of their electron density maps at medium resolution, leading to graphs of critical points. The distribution of the different types of critical points are compared at various levels of resolution for a training set of 22 molecules in order to define the optimal resolution level leading to the best representation of the various chemical functions. The reduced representations can in the future be used for molecular similarity research and pharmacophore proposals.