Differential Lysine Acetylation And Succinylation In Hypertrophic Scars Based On Quantitative Modification Omics

Hypertrophic scar(HS)is the most common co mplication of skin surgery,burns,and trauma.It is a pathological scar caused by abnormal tissue rep air after skin in jury.Its essence is the proliferation and enhanced activity of fibroblasts,resulting in excessive collagen production,including a large amount of ex tracellu lar matrix components such as type I and type III collagen deposited in the tissue,A pathological condition that is difficult to be absorbed or reshaped by the body.The clinical manifestations of HS include local harden ing and thick ening,purplish red or flushing on the surface,accompan ied by v arying degrees of itch ing and tingling.In the early stage,blisters can easily form,leading to scar ulcers and pigmentation.This not only affects th e appearance,but in severe cases,it can even cause scar contracture deformities,greatly affecting the quality of life and physical and mental health of patien ts.However,its specific molecular bio logical level and pathophysio logical mechanism are still unclear,which has become a difficult point in clinical treatment.As the execu tor of life functions,proteins play a pivotal ro le in the o ccurren ce and development of diseases.Proteomics is an emerging discip line th at comprehen-sively studies the structure and function of life,physiological functions,and laws of life activities.It has b ecome an important content of research in the post-genome era,and has receiv ed widespread attention in many fie lds.As an importan t bran ch of proteomics research,post translational modification of proteins plays an important role in in creasing protein div ersity and condu cting complex physiological regulation processes,which can better reveal the essence and laws of life phenomena.Lysine acety lation(KAc)is a common post translational modification that regulates a variety of cellular processes and is associated with many diseases.Histone acety lation can activ ate type VI collagen and promote th e migration of fibroblasts in lung cancer.Type I collagen promotes th e migration of preadipocytes through acetylation,and excessive migration of preadipocytes can lead to abnormal d evelop ment and fibrosis-related diseases,such as hypertrophic scars.In addition,lysine succinylation(KSuc)is a n ewly discovered post translational modification.Compared to acety lation of lysine,succinylation of lysine can cause more changes in protein properties by ch anging th e ch arge of lysine from+1 to-1 and introducing groups with larger structures.Succinylation can regulate protease activity and gen e expression,and participate in various life activ ities such as glucose metabolism,amino acid metabo lism,fatty acid metabolism,ketone body synthesis,and active oxygen scavenging.The abnormal lev el of succinylation modification is closely related to the occurrence and developmen t of various d iseases,in cluding tumors,card iac metabolic diseases,liver metabolic diseases,and neurological diseases.This project intends to study th e changes in proteo mics and modification histology of hypertrophic scars,and discover th e pathological changes and changes in protein modification after translation,which will help promo te the dev elopment of drugs,provide new eviden ce for the molecular mechan ism of the occurrence and development of hypertrophic scars,as well as the discovery of biomark ers for diagnosis and treatment,and will h elp us find breakthroughs in the diagnosis and treatment of hypertrophic scars.This topic consists of the following four parts.Chapter 1 Preliminary study on the differential modification of lysine acetylation in hypertrophic scar and normal skin.Objective: To explore the differential modif ication of lysine acetylation in hypertrophic scar and normal skin.Methods: The expression profile of acetylated proteins in HS was studied using protein modification proteomics techniques.Results: KAc was found in both HS and normal skin,and the level of KAc in HS was higher than that in normal skin.A total of 556 KAc modification sites were identified on 259 proteins,of which 88 KAc proteins and 175 KAc sites were quantifiable.Comp ared with normal skin,50 KAc proteins and 92 KAc sites were upregulated in HS,while 10 KAc proteins and 13 KAc sites were downregulated.14 differentially acetylated proteins in HS related signaling pathways form a PPI network,including DCN(TGF-β/ Smad signal pathway),tenascin-X(TNXB,PI3k-AKT signal pathway),collagen α-3(VI)chain(COL6A3,PI3k-AKT signaling pathway),COL6A2(PI3k-AKT signaling pathway),heat shock protein β-1(HSPB1,MAPK signaling pathway),fatty acid synthase(FASN,MAPK signaling pathway),TF(MAPK signaling pathway),COL1A1(Relaxin signaling pathway),GAPDH(HIF-1 signaling pathway),diphosphate aldolase a(ALDOA,PI3k-AKT signaling pathway,HIF-1 signaling pathway),α Enolase(ENO1,HIF-1 signaling p athway),S100A9(IL-17 signaling pathway),14-3-3 protein sig ma(SFN,p53 signaling pathway).Conclusion: KAc is differentially modified in HS and normal skin,and is associated with 14 differentially acetylated proteins in HS-related signaling pathways,which will provide a new way on the study of the mechanism of KAc action in HSChapter 2 Preliminary Study of Lysine Succinylation in Hypertrophic Scar and Normal SkinObjective: To explore the differential modification of Lysine Succinylation in Hypertrophic Scar and Normal Skin.Methods: The expression profile of succinylated proteins in HS was studied by protein modification technology,and th e differen tially modified proteins were verified by PRM.Results: A total of 79 KSuc modified proteins and 159 KSuc modified sites were identified.We an alyzed differen tially modified pro teins and sites between HS and normal skin,and qu antified a to tal of 33 KSuc proteins and 53 KSuc sites.Comp ared with normal skin,29 KSuc proteins and 38 KSuc sites in HS were upregulated by more than 1.5 times,but there was no downregulation of KSuc proteins and sites.These proteins modified by KSuc are main ly located in mito chondria,ex tracellu lar components,cytoplasm,and nucleus,which may be related to en ergy metabolism or collag en synthesis.Select TGF-β/ The PRM validation of KSuc was p erformed on 8 proteins(4 differential amber modified proteins(COL6A2,COL1A1,DCN,and TF) and 4 amber modified proteins of interest(TGFB1,ACTA2,HSPB1,and COL6A3)from the three classical signal p athways of Smad,PI3k-AKT,and MAPK.The results confirmed th at the PRM results of the 4 differential amb er modified proteins were consistent with the proteomic d ata,and the 4 amber modified proteins of in terest met expectations.Conclusion: It is the first time to confirm the differential modification of KSuc in HS and normal skin.TGF-β/KSuc occurs in eight proteins(TGFB1,ACTA2,DCN,HSPB1,TF,COL6A3,COL6A2,and COL3A1)of the three classical signaling pathways Smad,PI3k-AKT,and MAPK,which lays a theoretical foundation for the study of the functional role of KSuc in HS.Chapter 3 Histological Analysis of Co-modification of Lysine Acetylation and Succinylation in Hypertrophic ScarsObjective: Crosstalk Analysis of Lysine Acetylation and Succinylation in Hypertrophic Scars.Methods: Bioinformatics methods were used to analyze the co-modification of acetylation and succinylation in hypertrophic scars.Results: In HS,48 proteins and 52 sites were found to be modified by KAc and KSuc simultaneously.These overlapping modification proteins and proteins with overlapping modification sites are mainly located in mitochondria,ex tracellu lar components,cytoplasm,and nucleus.HS-related TGF-β/ In signal pathways such as Smad,PI3k-AKT,MAPK,relaxin,HIF-1,AGE-RAGE,and IL-17,we found th at DCN,COL6A2,TF,COL1A1,GAPDH,COL3A1,S100A9,and S100A8 simultaneously undergo lysine acetylation and succinylation.Conclusion: The crosstalk analysis of Kac and Ksuc in HS revealed overlapping modified proteins and overlapping modified sites,deepening th e understanding and understanding of the mech anism of PTMs in HS,and providing a new idea for subsequent research on HS.Chapter 4 the function of lysine acetylation and succinylation modification in human skin fibroblastsObjective: To investigate the function of KAc and KSuc in human fibroblasts in vitro.Methods: After human skin fibroblasts were treated with sodium acetate or disodium succinate,CC K8,Western Blot and q RT-PCR were used to detect cell proliferation,KAc,KSuc and α-SMA,COL1,COL3 of human skin fibroblasts.Results: Both sodium acetate and disodium succinate could significantly promote the proliferation of human skin fibroblasts and increase the levels of KAc and KSuc of human skin fibroblasts.In human skin fibroblasts,sodium acetate significan tly in creased th e m RNA lev els of α-SMA,COL1 and COL3,while disodium succinate did not affect the m RNA lev els of these genes.Sodium acetate and disodium succinate significan tly up-regulated the protein lev els of α-SMA,COL1 and COL3.Conclusion: KAc and KSuc can promote the proliferation of human fibroblasts,which provides experimental basis for the further study of the function of KAc and KSuc in HS.