Study Of T2D Differential Proteomic Analysis In White Adipose Tissues And The Key Protein SSAO's Interactome

SSAO,on one hand,belongs to amine oxidases,and on the other hand,is an adhesion molecule,which is also named as VAP-1.It distribute widely in kinds of tissues of human body especially in vasculature,endothelial and smooth muscle.SSAO mainly locate in cytoplasm membrane and membrane structure of organelles.Besides catalyzing oxidation of amines,it can also mediate adhesion and extravasation of leukocyte and granulocyte,simulate insulin effects,take part in growth and maturation of specific cells,maintain and deposit extracellular matrix in smooth muscle,lipid transportation in adipocyte,and bind drugs.It is involved in a series of pathological processes,like diabetes,atherosclerosis,hypertension,inflammation,cardiac failure,Alzheimer's disease,severe depression,chronic nephrosis,liver dysfunction and tumor.Plasma SSAO activity was increased in a large amount of chronic diseases,which might be related to its cell adhesion and catalyzing amine metabolism functions.The SSAO activity showed positive correlation with the content of glycosylated hemoglobin.SSAO substrate administration could relieve chronic hyperglycemia,and through inhibiting SSAO activity by antibody or inhibitor could suppress inflammation.More attention was paid to SSAO as potential drug target,however,but the mechanism is still not determined so far.The dissertation studied the SSAO as mainline and conducted multi-aspect researches related to SSAO.On one aspect,through applying high resolution clean native polyacrylamide gel electrophoresis?hrCN-PAGE?and immunoprecipitation method,SSAO potential interaction partners were discovered;on the other aspect,differentially expressed adipose tissue proteins were identified by ¹⁸O-labeling based proteomics approach;at last,we analyzed the SSAO function in diabetes.Here are the main research contents and results:1.The hrCN-PAGE coupled mass spectrometry method was employed for separation and identification of microsome protein complexes of rat adipocytes.The results showed:1)Rat adipocyte proteins were significantly enriched in clusters related to the regulation of the protein metabolic process,the cellular carbohydrate catabolic process,response to stimulus and wounding,macromolecular complex subunit organization,positive regulation of molecular function,regulation of programmed cell death and biomolecule transport with enrichment scores more than 1.3.This may suggest the active role of adipose tissue in these processes.2)Some SSAO potential interaction partners,among which the interaction between SSAO and ALDH2 were validated.2.For a better understanding of the mechanism linking the WAT to diabetes,a differential proteomics study was conducted to investigate alterations in epididymis fat pad comparing HFD fed T2D KKAy mice to its genetic background,C57BL/6J mice,by¹⁸O-labeling technique coupled with 2D LC-MS/MS.The results showed:1)121 proteins showed consistent changes in T2D mice as compared to controls with CV?20%?fold changes of>2 or<0.5 as threshold?.2)Proteins involved in metabolic process,oxidative stress,ion homeostasis,apoptosis and cell division were found to be significantly regulated in T2D mice compared to normal controls.The synthesis of fatty acid might be inhibited,and the?-oxidation of fatty acid might be activated in WAT as T2D develops.Glucose metabolism disorder was manifested in the increase of glycolysis enzymes and the decrease of TCA cycle enzymes.Elevated oxidative stress suggests the deterioration of cellular environment of WAT.However,this condition wasn't eliminated by apoptosis,and in contrast,the cell proliferation seems to be enhanced causing an ever-increasing fat mass in epididymis fat tissue.3)The down-regulation of SSAO indicated crippled ability of eliminating amines of WAT,but yet reduces the production of aldehydes.It may also relate to elevated SSAO in blood that revealed by other previous studies.3.Immunoprecipitation method was applied to explore SSAO potential interaction partner,the results of which were then combined with differentially expressed proteins in T2D for analyzation.1)A total of 171 proteins were identified as SSAO potential interaction partners.2)Clusters related to motor activity in molecular function?MF?,adhesion in cellular component?CC?,calcium ion transport in biological process?BP?,and carbon metabolism,pyruvate metabolism,insulin secretion and cholinergic synapse in KEGG pathway were found to have enrichment score more than 1.3,which showed the importance of SSAO in these function or processes.3)Thirty five SSAO potential interaction proteins were found to be altered in T2D,which suggested the motor function and adhesion ability of adipocyte might be weakened.In summary,the results from hrCN-PAGE method were more accurate,whereas immunoprecipitation method could test a wider range of SSAO potential interaction proteins.The differential proteomics study displayed the whole proteome changes of WAT in T2D.The present research results revealed SSAO interactome and their changes in T2D,which provided a lot of information as reference of follow-up researches for understanding SSAO's role in T2D.