Effect of down-regulation of let-7c/g on triggering a double-negative feedback loop and promoting restenosis

Background:Excessive proliferation and migration of vascular smooth muscle cells(VSMCs)are the main causes of restenosis(RS)in diabetic lower extremity arterial disease(LEAD).However,the relevant pathogenic mechanisms are poorly understood.Methods:In this study,we introduced a“two-step injury protocol”rat RS model,which started with the induction of atherosclerosis(AS)and was followed by percutaneous transluminal angioplasty(PTA).Hematoxylin-eosin(HE)staining and immunohistochemistry staining were used to verify the form of RS.Two-step transfection was performed,with the first transfection of Lin28a followed by a second transfection of let-7c and let-7g,to explore the possible mechanism by which Lin28a exerted effects.5-ethynyl-2΄-deoxyuridine(EdU)and Transwell assay were performed to evaluate the ability of proliferation and migration of VSMCs.Western blotting and quantitative real-time polymerase chain reaction(qRT-PCR)were performed to detect the expression of Lin28a protein and let-7 family members.Results:Using a combination of in vitro and in vivo experiments,we discovered that let-7c,let-7g,and microRNA98(miR98)were downstream targets of Lin28a.More importantly,decreased expression of let-7c/let-7g increased Lin28a,leading to further inhibition of let-7c/let-7g.We also found an increased level of let-7d in the RS pathological condition,suggesting that it may function as a protective regulator of the Lin28a/let-7 loop by inhibiting the proliferation and migration of VSMCs.Conclusion:These findings indicated the presence of a double-negative feedback loop consisting of Lin28a and let-7c/let-7g,which may be responsible for the vicious behavior of VSMCs in RS.

Subfunctionalization of a monolignol to a phytoalexin glucosyltransferase is accompanied by substrate inhibition

Uridine diphosphate-dependent glycosyltransferases(UGTs)mediate the glycosylation of plant metabolites,thereby altering their physicochemical properties and bioactivities.Plants possess numerous UGT genes,with the encoded enzymes often glycosylating multiple substrates and some exhibiting substrate inhibition kinetics,but the biological function and molecular basis of these phenomena are not fully understood.The promiscuous monolignol/phytoalexin glycosyltransferase NbUGT72AY1 exhibits substrate inhibition(Ki)at 4 mM scopoletin,whereas the highly homologous monolignol StUGT72AY2 is inhibited at 190 mM.We therefore used hydrogen/deuterium exchange mass spectrometry and structure-based mutational analyses of both proteins and introduced NbUGT72AY1 residues into StUGT72AY2 and vice versa to study promiscuity and substrate inhibition of UGTs.A single F87I and chimeric mutant of NbUGT72AY1 showed significantly reducedscopoletin substrate inhibition,whereas its monolignolgly cosylation activity was almost unaffected.Reverse mutations in StUGT72AY2 resulted in increased scopoletin glycosylation,leading to enhanced promiscuity,which was accompanied by substrate inhibition.Studies of 3D structures identified open and closed UGT conformers,allowing visualization of the dynamics of conformational changes that occur during catalysis.Previously postulated substrate access tunnels likely serve as drainage channels.The results suggest a two-site model in which the second substrate molecule binds near the catalytic site and blocks product release.Mutational studies showed that minor changes in amino acid sequence can enhance the promiscuity of the enzyme and add new capabilities such as substrate inhibition without affecting existing functions.The proposed subfunctionalization mechanism of expanded promiscuity may play a role in enzyme evolution and highlights the importance of promiscuous enzymes in providing new functions.