The Molecular Function Of The Interaction Between Alpha B-grystallin And ATP6V1A In Regulating Lysosomal Activity

BackgroundIn eukaryotes,lysosomes are the basic digestive organs in cells,which maintain an acidic environment of PH 5,containing more than 50 hydrolases to degrade the phagocytic substances.The mechanism of lysosomal acidification is the pathogenesis of many lysosomal storage diseases?LSDs?.The mechanism of lysosomal acidity is regulated by V-ATPase complex,which is a proton pump driven by ATP and exists in the cells and cell membranes of eukaryotes.It is mainly responsible for the establishment and maintenance of intracellular acidic PH,and the injection of H⁺into the cell cavity relying on ATP.V-ATPase is the reversible assembly of V1/V0 proton pump on lysosomal membrane,which is a multisubunit complex.The complex including the v1 complex located in the cytoplasm and the v0 complex anchored on the lysosomal membrane.V1 includes at least eight different subunits?A-H?,The three catalytic sites for ATP hydrolysis are composed of subunits A and B.The V0 region contains the subunit a,c,c',c",d and e,and is responsible for the transport of protons across the membrane.ATP6V1A is a component of V-ATPase and is an A subunit located in the V1 region of proton pump.In early experiments,we have found that Hsf4 up-regulates lysosomal activity by maintaining lysosomal acid PH.Hsf4 regulates the expression of small heat shock proteins in lens epithelial cells,such as HSP25 and CRYAB.Hsp 25 and alpha B-crystallin are key molecular chaperones to maintaining intracellular homeostasis and survival.Molecular chaperones play important roles in all aspects of protein quality control.Our preliminary data demonstrated that alpha B-crystallin interacts with ATP6V1A.However,the molecular mechanism of the interaction between alpha B-crystallin and atp6v1a in regulating lysosomal acidification has not been well identified.Through this research,we probe into this molecular mechanism in detail.AimThe purpose of this study was to explore the significance of the interaction between alphaB-crystallin and ATP6V1A and the molecular mechanism of regulating the maintenance of lysosomal acidic PH.The mouse lens epithelial cell line mlec/Hsf4b^-/-,mlec/HA-Hsf4b,mlec/HA-Hsf4b/sh-cry?B,which Knock out and overexpress Hsf4b,and Knock out alpha B-crystallin,was used as the study object.Methodes1.In the mouse lens epithelial cell line mlec/Hsf4b^-/-,mlec/HA-Hsf4b,mlec/HA-Hsf4b/sh-cry?B which Knock out and overexpress Hsf4b,and Knock out alpha B-crystallin.we detected the expression of ATP6V1A protein by Western blot and the expression of ATP6V1A mRNA by real time PCR.2.We processed mlec/Hsf4b^-/-,mlec/HA-Hsf4b,mlec/HA-Hsf4b/sh-cry?B cell line using CHX,and detected of protein stability of ATP6V1A by Western blot.3.We processed mlec/Hsf4b^-/-,mlec/HA-Hsf4b,mlec/HA-Hsf4b/sh-cry?B cell line by CHX.At the same time,mlec/HA-Hsf4b/sh-cry?B was processed using CQ and MG132,respectively.The protein degradation pathway of ATP6V1A was detected by Western blot.4.After the processing of mlec/HA-Hsf4b/sh-cry?B cells by MG132,Immunoprecipitation ATP6V1A,Western blot detects ubiquitin to clear the level of ATP6V1A ubiquitination.After processing mlec/HA-Hsf4b and mlec/HA-Hsf4b/sh-cry?B with MG132,ubiquitin pull down test,ATP6V1A was detected by western blot to study its ubiquitin level.5.In the mlec/Hsf4b^-/-and mlec/HA-Hsf4b cells,the expression of alpha B-crystallin and ATP6V1A in lysosome was detected by lysosomal separation,and the subcellular localization of alpha B-crystallin and ATP6V1A was detected by immunofluorescence.6.The interaction between alpha B-crystallin and other subunits of V-ATPase proton pump was investigated by GST Pull down.7.Four truncated bodies of alpha B-crystallin were constructed,NT?1-66aa?,CD?66-149aa?,CC?149-176aa?,CT?66-176aa?,GST pull down was used to explore the interaction between ATP6V1A and alpha B-crystallin truncated bodies.and the interaction between ATP6V1A and alpha B-crystallin.8.The three alpha B-crystallin Phosphorylation site mutant plasmid PEBG-?B-S19A?PEBG-?B-S45A and PEBG-?B-S59A were constructed.The interaction between alpha B-crystallin mutants and ATP6V1A was detected by GST Pull down.After incubation of the lysosome probe Green DND-189,we detected of lysosomal PH when alpha B-crystallin phosphorylation site mutation by multifunctional enzyme labeling instrument.9.Inhibition of mTORC1 complex by RAPA,the influence on the interaction between ATP6V1A and alpha B-crystallin was investigated by GST Pull down experiment.Detection of protein Stability of ATP6V1A after mTORC1 inhibition by Western blot was done,after treatment of mlec/HA-Hsf4b cells with CHX and RAPA.Lysosome probe Green DND-189 was used to detect lysosomal PH in mlec/HA-Hsf4b cells treated with RAPA.Results1.In lens epithelial cells,overexpression of HSF4b significantly increased the protein stability.2.Knockout alpha B-crystallin significantly reduced the protein level and protein stability of ATP6V1A.3.ATP6V1A protein was degraded mainly by ubiquitin proteasome pathway,knockout of alpha B-crystallin could significantly enhance the level of ATP6V1A ubiquitization.4.The interaction between alpha B-crystallin and ATP6V1A might occur in lysosome.5.Alpha B-crystallin only interacts with ATP6V1A.6.There was no interaction between ATP6V1A and truncated bodies of alpha B-crystallin,and the phosphorylation modification of alpha B-crystallin did not participate in this process.7.The mTOR pathway was involved in the interaction between ATP6V1A and alpha B-crystallin.Conclusion1.ATP6V1A protein degradation is mainly through the ubiquitin proteasome pathway in lens epithelial cells,Alpha B-crystallin enhanced its protein stability by interacting with ATP6V1A.2.Alpha B-crystallin does not bind to other subunits of v-atpase proton pump and only interacts with atp6v1a.The interaction between alpha b-crystallin and atp6v1a depends on its full length,and the phosphorylation modification of alpha B-crystallin does not participate in the process of interaction.3.Alpha b-crystallin is involved in the regulation of lysosomal activity by v-atpase proton pump mediated by mtor.