Regulation Of Mitochondrial Homeostasis By Phosphates SHP2 And Its Role In Inflammation-associated Diseases

A growing number of studies have shown that there are close links mitochondrial dysfunction and diseases,including inflammatory diaseses,cancer,metabolic diseases and neurodegenerative diseases.Mitochondrion is a major organelle required for numerous functions within the cell and is essential for energy production,metabolism and cell survival.Consistant mitochondrial damage and delayed mitochondrial removement lead to the development of many human diseases.SHP2 is tyrosine phosphtase which is encoded by protein-tyrosine phosphatase,nonreceptor-type 11,PTPN11.It has been reported that SHP2 plays important roles in physiological process and diseases.Our study on SHP2 discovered that it played important role in mitochondrial function,which in turn regulated NLRP3 inflammasome activation and mitophagy.Activation of NLRP3 inflmmasome is a natural immune pattern-recognition receptors induced exogenous pathogen-associated molecular patterns and endogenous danger-associated molecular patterns clearance process and inflammation at the same time.Acute inflammation usually results in short-term immune cells infiltration,danger signal removing and eventually tissue repair,while mitophagy results in damaged mitochondrial clearance;persistent and dysregulated inflammation will become a major stimulator in the development of many human diseases including autoimmune diseases,metabolitic diseasea et.al,nonresolving inflammation may also malignant transformation,promoting and eventually developmenting tumour.We generated conditional SHP2 knockout in macrophages or dopamine neurons mice to further determine the role of SHP2 in diseases.In the first chapter,we summarized the advances in the study of NLRP3 inflammasome and mitophagy and their role in diseases,reviewed the available therapeutic tool against these diseases and also the advances of study in SHP2 and its role in different disease.This overview provided some foreshadowing for the following chapters.In the second chapter,we determined the positive role of SHP2 in the regulation of mitophagy.While knockdown of SHP2 in SH-SY5Y cells abolished cccp induced mitophagy,overexpression of SHP2 did the opposite.Further experiments showed that SHP2 affected translocation of Parkin from cytosol to mitochondrion and in turn the ubiquitination and degradation of MOM protein Tom20.Besides,SHP2 regulated mitophagy in a phosphates-dependent manner.During the activation of mitophagy,we observed interaction of Parkin and SHP2 and upregulation of p-Ser in Parkin while p-Tyr in parkin was downregulated,suggesting a role of p-Tyr in the regulation of Parkin activity apart from the reported p-Ser regulated Parkin activity.Then we determined six conserved Tyr residues by comparing amino acid sequence in Parkin from different species.Six plasmids encoding Parkin with Tyr mutation were constructed in order to determine which site of Tyr could influence the activity of Parkin.Finally,we discovered that SHP2 reversed Parkin C431S mutation induced mitophagy abolishment and the underlying mechanism still left to be digging out.In order to determine the role of SHP2 in NLRP3 inflammasome and diseases,we generated conditional-macrophage-SHP2-knockout mice(cSHP2-KO)and CASP1-knockout-conditional-SHP2-knockout mice(DKO).CSHP2-KO mice were obtained by copulated lyz-cre(expressed in lyz+macrophage cells)mouse with SHP-2f/f mouse and DKO mice were obtained by copulated cSHP2-KO mouse and CASP1-KO mouse.Furthermore,we constructed SHP2-knockdown THP-1 cells by lentivirus shRNA.So we can carry out our research based on our platform.In the third chapter,we demonstrated that LPS and ATP treatment in THP1 cells activated Src homology-2 domain containing protein tyrosine phosphatase-2(SHP2),which translocates from cytosol to mitochondria accompanying with NLRP3 inflammasome activation.SHP2 sliencing significantly aggravated mitochondrial membrane potential collapse,mtROS production and mtDNA release,while SHP2 overexpression did in the opposite.The interacting protein of SHP2 in mitochondria was discovered and proved to be adenine nucleotide translocase 1(ANT1)in GST-pulldown,co-IP and immunoflourence assays.The mutation of RRWFH motif as mitochondria target sequence in SHP2 diminished both mitochondrial translocation and interaction of SHP2 with ANT1.Furthermore,loss of SHP2 in macrophage resulted in an aberrant activation of NLRP3 inflammasome in vitro and in vivo.As the target protein of SHP2 in mitochondria,ANT1 linked mitochondrial function to NLRP3 inflammasome activation,in which silence of ANT1 significantly reduced ATP-induced collapse of mitochondrial membrane potential and secretions of IL-1?and IL-18.Finally,we confirmed that SHP2-mediated ANT1 dephosphorylation at Tyr 191 is crucial to the maintenance of mitochondrial functions and resolution of NLRP3 inflammasome.Collectively,in the second charpter,our data highlight that SHP2 constitutes a mitochondria homeostasis to limit NLRP3 inflammasome activation by inhibiting a key molecule ANT1.SHP2 in macrophages acts as a negative regulator of EAE and peritonitis,while oleanolic acid amoralities peritonitis by targeting SHP2.Next we determined the role of cSHP2-KO in high-fat diet induced fat and insulin resistance due to the close link between NLRP3 inflammsome and metabolism disease.To our surprise,we observed the resistance to high-fat diet induced bodyweight gain and insulin resistance in cSHP2-KO mice although cSHP2-KO hyperactivated NLRP3 inflammsome both in high-fat diet-mice and LPS+PA/LPS+ceramide treated macrophage cells.We observed a significantly improvement in high-fat diet induced fat liver in cSHP2-KO mice.Moreover,both CASP1-KO and anti-IL-18 treatment reversed amoralition of insulin resistance in cSHP2-KO mice.In vitro study showed that,IL-18 opposed effect of IL-? and improved insulin signaling and lipid metabolism.Targeting SHP2 may be a treatment for insulin resistance and fat liver as SHP2 inhibitor PHPS1 did so.While depletion of NLRP3 inflammsome contributes to improvement of insulin sensitivity,sufficiency of IL-18 produced by NLRP3 inflammsome activation may also inhibits high fat diet induced obesity and improve insulin sensitivity,which indicating the bidirection of NLRP3 inflammasome activation in obesity and insulin resistance.In summary,our work discovered and confirmed the predominant role of SHP2 in NLRP3 inflammasome activation and mitophagy via regulating mitochondrial function.Our discovery provided a new clue and measure for modulation of mitochondrial function and enriched the regulation network of NLRP3 inflammasome and mitophagy and a new therapeutic target for treatment for mitochondrial dysfunction associated diseases.