Loss Of USP46 Facilitates K63-linked Polyubiquitination And HDAC6-mediated Aggregation Of NLRP3 In Podocytes

Part 1.HDAC6 inhibition with CAY10603 ameliorates podocyte injury in diabetic mouse by suppressing NLRP3 inflammasome activationObjectives:To observe the regulation of HDAC6 on NLRP3 inflammasome activation in podocyte injury of diabetic nephropathy.Methods:(1)The 8-week-old male CD-1 mice were selected to construct the mouse model of diabetes nephropathy.After the model was established,the mice were intraperitoneally injected with HDAC6 inhibitor CAY10603(1,2 and 5mg/kg/d).The therapeutic effect was evaluated by proteinuria,serum creatinine and renal pathology;(2)The inhibitory effects of CAY10603 on NLRP3 inflammasome was evaluated by IHC staining and WB in vivo and in vitro podocytes,respectively.Results:(1)5mg/kg/d CAY10603 exhibited the most significant effect on the reduction of proteinuria and serum creatinine and restoration of renal pathology;(2)CAY10603 significantly inhibited the activation of NLRP3 inflammasome in podocytes in vivo and in vitro.Conclusion:HDAC6 inhibition with CAY10603 ameliorates podocyte injury in diabetic mouse by suppressing NLRP3 inflammasome activationPart 2.Loss of USP46 facilitates K63-linked polyubiquitination and HDAC6mediated aggregation of NLRP3 in podocytesObjectives:NLRP3 inflammasome activation contributes to podocyte injury in DN,and ubiquitination and deubiquitination regulate NLRP3 inflammasome assembly and activation.To investigate whether USP46 regulates the activation of NLRP3 inflammasome in podocytes.Methods:(1)Soluble and insoluble proteins were isolated from podocytes treated with bortezomib,proteasome function,aggresome formation,NLRP3 expression and its ubiquitin modification were detected;(2)Short interfering RNAs(siRNAs)were used to suppress endogenous USP46 expression in cultured human podocytes,proteasome function,aggresome formation,NLRP3 expression and its ubiquitin modification were detected;(3)The USP46-NLRP3 interaction was delineated by CoIP,the polyubiquitination sites in NLRP3 affected by USP46 was identified by mass spectrometry(MS);(4)HDAC6-dependent NLRP3 aggregation affected by polyubiquitination site in K930 was determined;(5)Mice with podocyte-specific deletion of USP46(USP46PKO)were generated,urine albumin and creatinine were tested to access kidney function.Kidney histology and transmission electron microscopy(TEM)were carried out.Aggresome pathway and inflammasome activation were detected in mice;(6)IF staining were performed to determine the expression pattern of USP46 in human kidney tissues;Association of glomerular mRNA expression of USP46 with proteinuria,eGFR and NLRP3 activation in biopsied samples,as determined by Spearman’s R test.Results:(1)In human podocytes,bortezomib treatment caused proteasome function impairment and the formation of aggresome,in which,aggregated NLRP3 underwent K63-linked polyubiquitination;(2)Loss of USP46 expression leads to protein aggregates and aggresome formation in podocytes,impaired proteasome function and promoted NLRP3 aggregation,which was undergoing K63-linked polyubiquitination;(3)Both the NACHT and LRR domains of NLRP3 were crucial for the USP46NLRP3 interaction,USP46 directly deubiquitinated the K63-linked polyubiquitinated chain on NLRP3 K930;(4)K63-linked polyubiquitination of NLRP3 at site K930 is essential for HDAC6-mediated aggresomal assembly and activation of the NLRP3 inflammasome;(5)Compared to the USP46flox/flox mice,the USP46PKO mice developed proteinuria 12 weeks after birth.Kidney histological examination with PAS staining revealed that USP46PKO mice developed glomerulosclerosis and thickening of the glomerular basement membrane(GBM).TEM imaging revealed that the USP46PKO mice exhibited mild podocyte foot process effacement and obvious thickening of the GBM.IHC staining revealed downregulation of WT1 and NPHS2 expression in USP46PKO mice.Importantly,induction and activation of HDAC6,NLRP3 and Caspase-1 were evident in podocyte of USP46PKO mice.Conclusion:USP46 deficiency facilitates the K63-linked polyubiquitination of NLRP3 at site K930,in turn promoting the NLRP3-HDAC6 interaction and HDAC6dependent aggresome formation.Part 3.Virtual Screening identify Acarbose as a potential agonist of USP46 Objectives:To screen a potential agonist of USP46.Methods:(1)Using the molecular docking operation between small molecule compounds and USP46 protein,targeting the USP46-WDR20 and USP46-WDR48 binding interface,the potential agonists of usp46 were virtually screened from MCE bioactive compound library plus(containing 10.8k compounds);(2)The activation of USP46 by subsequent small molecule agonist and the protective effect were verified cultured human podocytes in vitro;(3)The 8-week-old male CD-1 mice were selected to construct the mouse model of diabetes nephropathy.After the model was established,the diets conteined 1000 ppm acarbose were daily given for 4 weeks.Results:(1)The top 200 small molecule compounds targeting USP46-WDR20 and USP46-WDR48 binding interfaces were virtually screened from MCE bioactive compound library plus.Among the top 10 small molecule compounds approved by FDA,acarbose is the only one small molecule drug targeting both binding interfaces at the same time;(2)In vitro cultured human podocytes,acarbose activated the expression of USP46 dose dependently;(3)Acarbose reduced the proteinuria and imporved renal pathology in diabetic CD-1 mice,IHC staining also confirmed the suppressed activation of HDAC6 in podocytes of diabetic CD-1 mice.Conclusion:Acarbose is a potential agonist of USP46 through suppressing aggresome pathway.