| Severe burn will cause hypoxia-ischemia of tissue and organs such as heart and gastrointestinaltract. The occurrence and development of hypoxia-ischemia seriously affects the traumatic conditionand prognosis. Microtubule, the main constituents of cytoskeleton, acting as celluar frame, isassociated with mitochondrion functionally. Hypoxia leads to microtubules depolymerization andmitochondrial permeabilization, followed by energy metabolism dysfunction, and apoptosis as a result.Dynein connects the microtubule and mitochondrion like a bridge, serving many celluar progressesbesides cargo transport. Previous study disclosed that dyneinlight chain Tctex-type1(DYNLT1)hasaprotein-proteininteraction with voltage-dependent anion-selective channel(VDAC)as well as aco-localization, displaying a new mitochondrion modulating role. Knockdown of DYNLT1willincrease the sensibility of mitochondrion to hypoxia injury although the underlying mechanismremains illusive. We have found out that hypoxia will activate p38/MAPK signaling pathway andincrease microtubule associated protein phosphorylation.On the other hand, it was reported thatphosphorylation of DYNLT1at serine82has an impact on the composing of dynein complex so as toaffects the delivery of cargoes. Accordingly, we hypothesize that hypoxia may arousephosphorylation of DYNLT1, and then play a role in regulating the microtubules and mitochondria.。This study aimed to explore the effect of DYNLT1phosphorylation on microtubules andmitochondria in hypoxia. S82residue mutant recombinant adenoviruseswere constructedandtransfected objected cells to mimic phosphorylation and dephosphorylation state in order toobservetheinfluenceonmicrotubules’polymericandmonomericbalance,mitochondrialpermeabilitytransition,and celluar energy metabolism. Hence, we provide newinsights into dynein’s structure andfunction, and propose a potential therapeutic target in hypoxia cytoprotectivestudiesI. Materials and methods1. To test the DYNLT1phosphorylation change in hypoxia and establish the cellmodel of S82mutant. By immunoprecipitation (IP) and Western blot assay, we examine the serine phosphorylation of DYNLT1in H9c2and HeLa cell lines in hypoxia. Subsequently, ananti-phospho-S82antibody was constructured to specially test the S82phosphorylationchange in different hypoxic condition. Using site-directed mutagenesis technique, weconstructed recombinant adenoviruses containing various DYNLT1sequences at S82togenerateputative glutamic acid (E) and alanine (A) mutants (S82E andS82A, respectively).H9c2/HeLa cells were infected with adenoviruses to express the phosphomimic anddephosphomimic for the sake of following experiments.2. To examine the impact of DYNLT1phosphorylation on microtubules network, weemployed Western blot analysis and immunofluorescence microscopy morphologic assay.Polymeric and monomeric tubulin fractions were isolated and quantified in hypoxia first.After Ad-S82E/S82A transfection, we compared the microutubules deploymerization ofdifferent phosphorylation mutant under hypoxic condition.3. Establishing cell models as before, we measure the mitochondrial permeabilitytransition change after transfection by immunofluorescence microscopy observing.Themitochondrial permeability transition was measured by Calcein-AM/CoCl2staining.MMPwas assessed using tetramethyl rhodamine methyl ester(TMRE), a lipophilic cationicfluorescent probe.Cytochrome c release from mitochondrion was tested by western blotanalysis.4. Establishing hypoxic H9c2/HeLa cell model after the transfectionas describedbefore, we probed the impact of different S82mutagenesis on cellular energy metabolism.Cell viability was determined using CCK-8way. Two assays were employed to measure theATP content and synthesis rate respectively.5. The involvement of p38/MAPK signal pathway was explored on both sides ofnormoxic and hypoxic condition. First, we examined that whether hypoxia willactivatep38/MAPK signal pathway. Then, we used SB203580, a p38/MAPK inhibitor, toobserve the possible decreased expression of pS82in hypoxia. Simultaneously, weoverexpressed MKK6, a constitutively activated p38kinase activator, by using recombinantadenoviruses, to measure the possible pS82overexpression in normoxia.II. Results and conclusions1. Hypoxia will induce DYNLT1phosphorylation. HeLa and H9c2cells were exposedto hypoxicconditions for0.5,1,3and6h. Results showed that the tendency of DYNLT1 serineexpression increases first, reaches a peak plateau, before declining in both cell groups.The apparent high elevation occurs at1hwhich remains to3h. The tendency of S82phosphorylation is same as that of serine.To mimic S82phosphorylation and dephosphorylation, we constructedrecombinant adenoviruses containing various DYNLT1sequences atS82using site-directed mutagenesis to generateputative glutamic acid (E) and alanine (A)mutants (S82E andS82A, respectively).2. Results showed a dynamic depolymerization of tubulin with the increase offreetubulin. S82E mutagenesis accelerates MT depolymerization, causing a promotion ofmicrotubules disassembly, looser distribution and striking decrease in fluorescence intensity.S82A plays a potentiallyprotective effectinduced by hypoxia as it attenuated themicrotubules breakage.3. Hypoxia induces the mitochondrial permeabilization and dysfunctionconsequently.S82E is responsible for aggravatedMMP collapse, mPTP open and Cyt c release. Thesehypoxia-induced damages are suppressed in S82A groups.4. Mitochondrial dysfunction leads to cellular metabolic disorders.The total ATPcontent in hypoxiawas significantly decreased although the different S82mutants showedno different impact here. A further study was employed to detect the ATPsynthesis rate andresults showed that mitochondrial ATP production was impairedin the S82E groups whileS82A had no evident help.5. Hypoxia does activate p38/MAPK signal pathway. Inhibition of p38/MAPKdown-regulates the expression of pS82in hypoxia. Overexpression ofMKK6, whichactivates p38/MAPK, causes increased pS82expression in normoxia. These indicated thatp38/MAPK is involved in the phosphorylation regulation of DYNLT1at S82. |
PDF Full Text Request