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Pathogenic Mechanism of Disseminated Superficial Actinic Porokeratosis-associated Mevalonate Kinase Mutations

Author: ZhuTengFei
Tutor: XiaKun
School: Central South University
Course: Genetics
Keywords: DSAP mevalonate kinase protein half-time homodimer
CLC: R751
Type: Master's thesis
Year: 2013
Downloads: 21
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Abstract


AimMevalonate kinase(MVK) catalyze the phosphorylation of mevalonic acid into5-phosphomevalonate.There are several mutantsions which have been identified from a variety of disseminated superficial actinic porokeratosis (DSAP) predigrees and sporadic DSAP patients, but the pathogenesis remains clear. Our research on pathogenic mechanism of mevalonate kinase disseminated superficial actinic porokeratosis associated mutants will improve the understanding of DSAP.MethodTotal RNA was from lymphoid cells of patients and normal people. mRNA expression was detected by using real time PCR and RT-PCR. MVK wide type(WT) and DSAP association mutants plasmid were constructed and expressed in HEK293cell to determine protein level of MVK WT and DSAP association mutants.To determine half-life of MVK WT and DSAP associated mutants,HEK293cell stably expressing MVK WT and association mutants were treated with Chcloheximide in various time periods followed by detecting protein level of MVK using western blot.To clarify the degradation pathway of MVK WT and DSAP association mutants,HEK293cell stably expressing MVK WT and DSAP association mutants were treated with proteasomal inhibitor MG132or lysosomal inhibitor CQ followed by detecting protein level MVK using western blot.To determine subcelluar localization of MVK WT and DSAP association mutants,HEK293CELL with transient expressing GFP-target or FLAG-target MVK WT and DSAP associated mutants were immunostained with peroxisome marker catalase, mitochondrial marker TOM20,ER marker calreticulin and anti-GFP/anti-FLAG antibodies.Constructed MVK WT and DSAP associated mutant plasmids in pGEX-4T-2and induced BL21E.coli expression GST fusion protein using IPTG. Determined the ability to form homodimer of MVK WT and DSAP association mutants by using GST pull down.Co-transfect GFP-target and FLAG-target MVK WT and/or DSAP associated mutant plasmids into HEK293cells and determined the ability to form homodimer of MVK WT and DSAP association mutants by using Co-immunoprecipitation(Co-IP)ResultsLA189V mRNA expression was lower than normal.DASP associated mutants protein level was lower than wide type2.Half-time of MVK DSAP associated mutants were significantly shorter than MVK WTMVK DSAP associated mutants were degraded by the proteasome degradation pathway. 3.MVK WT and DSAP associated mutants were localized in cytoplasm.MVK WT and DSAP association mutants did not co-localized with peroxisome, mitochondrial or endplasmic reticulum4.MVK WT could form homodimer itself, MVK WT could form homodimer with MVK A189V,MVK A189V could form homodimer itself.MVK WT could not form homodimer with MVK H312R and MVK H312R could not form homodimer itself.Conclusion1. MVK DSAP associated mutants do not change subcellular localization.2. MVK DSAP associated mutants reduce protein stability, and mutants were degrade by the proteasome degradation pathway.3.MVK H312R defect in the formation of homodimer.

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