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Phosphoproteomic analysis of ORM2 overexpression in the liver of ob/ob mice

ABSTRACT: In order to evaluate ORM2 could activate which signaling pathway, phosphoproteomic analysis was performed. First, liver proteins were extracted from ob/ob mice injected with Ad-ORM2 and Ad-GFP using RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors. Next, the phosphorylated peptides were enriched by immobilized metal affinity chromatograph and the fractions were pre-separated by reversed phase liquid chromatograph. Electron transfer dissociation was used to effectively retain the skeletal information and phosphorylation modification information of the peptide, and accurately determine the molecular mass of proteins or peptides. Finally, the phosphoproteomic analysis was performed by tandem mass spectrometry and database retrieval.

ORGANISM(S): Mus Musculus

SUBMITTER: Yan Lu

PROVIDER: PXD035372 | iProX | Fri Jul 15 00:00:00 BST 2022

REPOSITORIES: iProX

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Project description:Matthew F. Hockin, Kevin M. Cawthern, Michael Kalafatis & Kenneth G. Mann. A model describing the inactivation of factor Va by APC: bond cleavage, fragment dissociation, and product inhibition. Biochemistry 38, 21 (1999). The inactivation of factor Va is a complex process which includes bond cleavage (at three sites) and dissociation of the A2N.A2C peptides, with intermediate activity in each species. Quantitation of the functional consequences of each step in the reaction has allowed for understanding of the presentation of disease in individuals possessing the factor V polymorphism factor VLEIDEN. APC cleavage of membrane-bound bovine factor Va (Arg306, Arg505, Arg662) leads to the dissociation of fragments of the A2 domain, residues 307-713 (A2N.A2C + A2C-peptide), leaving behind the membrane-bound A1.LC species. Evaluation of the dissociation process by light scattering yields invariant mass loss estimates as a function of APC concentration. The rate constant for A2 fragment dissociation varies with [APC], reaching a maximal value of k = 0.028 s-1, the unimolecular rate constant for A2 domain fragment dissociation. The APC binding site resides in the factor Va light chain (LC) (Kd = 7 nM), suggesting that the membrane-bound LC.A1 product would act to sequester APC. This inhibitory interaction (LC.A1.APC) is demonstrated to exist with either purified factor Va LC or the products of factor Va inactivation. Utilizing these experimental data and the reported rates of bond cleavage, binding constants, and product activity values for factor Va partial inactivation products, a model is developed which describes factor Va inactivation and accounts for the defect in factor VLEIDEN. The model accurately predicts the rates of inactivation of factor Va and factor VaLEIDEN, and the effect of product inhibition. Modeled reaction progress diagrams and activity profiles (from either factor Va or factor VaLEIDEN) are coincident with experimentally derived data, providing a mechanistic and kinetic explanation for all steps in the inactivation of normal factor Va and the pathology associated with factor VLEIDEN.

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Phosphoproteomic analysis of ORM2 overexpression in the liver of ob/ob mice

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