Study On Protein Interaction Between Helicobacter Pylori Vacuolating Cytotoxin And Mitochondrial Adenine Nucleotide Translocator

Helicobacter pylori (Hp) is most common pathogenic bacteria, which infection presently affects approximately one-half of the world's population and seven hundred million people in our country, and the health people are very susceptible to it.Hp leads to chronic gastritis, the most frequent chronic inflammation worldwide.It is also aetiologically associated with gastric and duodenal ulcer, mucosa associated lymphoid tissue (MALT) gastric lymphoma and gastric cancer (GC). Since then, data from several epidemiological, interventional and experimental studies have been gathered, confirming the causal link between Hp and GC. Ecological studies mostly confirm the geographical association between the prevalence of Hp and prevalence of GC.It has been evident for over 20 years that Hp is involved in the development of gastric cancer; in 1994, the WHO concluded that Hp is a definite or class I carcinogen in humans.Prospective studies reveal that the risk for development of gastric cancer is much greater in Hp-infected populations than in uninfected populations. The development of GC is a multistep process that is multi-factorial. The most important risk factor is the infection with Hp. The pathogenesis of GC includes a sequence of events that begins with Hp-induced chronic superficial gastritis, progressing towards atrophic gastritis, intestinal metaplasia, dysplasia and eventually GC. The"driving force"of GC is a chronic gastric inflammation, whose intensity and localization depending on the bacteria infection. The mechanisms by which chronic inflammation lead to epithelial and pre-cancerous lesions include induction of oxidative stress, perturbation of the epithelial cells proliferation/apoptosis ratio, and cytokine secretion. The end-stage of the multistep process of GC corresponds to accumulation of molecular alterations involving either the suppressor pathway (defect in tumour suppressor genes) or the mutator pathway (defect in DNA mismatch repair genes).The abnormality of gastric epithelial cells proliferation or apotosis is playing the most important roles in development to GC which caused by Hp infections.While the mechanisms of Hp associated cells apotosis are still relatively poorly defined, the elucidation may provide opportunities to develop effective strategies for GC prevention and therapy. Chronic gastritis induced by Hp is a strong risk factor for the development of distal gastric adenocarcinoma. A specific host response to Hp that may contribute to gastric carcinogenesis is epithelial cell apoptosis. VacA is one of the most important virulence factors of Hp that is extracellular and causes damage to the gastric epithelial layer as a result of vacuolation of late endosomal and lysosomal compartments of these cells. VacA-producing Hp strains are found in the vast majority of patients with GC, powerful evidences were discovered that it has important contributions to the development of GC.VacA has also been shown to be directly involved in mucosal damage in GC. This damage might be attributed to the vacuolating as well as to the apoptosis-inducing activity of VacA. Therefore, elucidation of VacA as an apoptosis-inducing factor might offer a better understanding of the pathogenesis of Hp-related diseases such as GC. The VacA is a unique proteinous cytotoxin producted by Hp that showed no striking primary sequence homology with other known bacterial toxins.The cytotoxin is an important factor in the pathogenesis of Hp, which induces vacuolating of epithelial cells and plays an important role in gastric epithelial apotosis. Hp induces cell death by apoptosis. However, the mechanism of apoptosis-inducing factor is still unknown.The virulence factor VacA is a potential candidate.Cellular vacuolation and mitochondrial cytochrome c release are independent outcomes of VacA cytotoxin activity that are each dependent on membrane channel formation. Hp VacA which causes vacuolation of gastric epithelial cells and other types of cultured cells,is known to enter mammalian cells,localizes to the mitochondrial, induced an inactivation of energy metabolism followed by mitochondrial damage,leading to impairment of the cell cycle in gastric epithelial cells,and modulates mitochondrial membrane permeability resulting in reduction of mitochondrial transmembrane potential by a mechanism dependent on toxin channel activity ultimately resulting in cytochrome c release,stimulate apoptosis via a mitochondrial- dependent pathway.Mitochondrial apoptosis pathway is endogenous cell death pathway. Mitochondria are important organelles for energy production, Ca2+ homeostasis, and cell death. In recent years, the role of the mitochondria in apoptotic has received much attention.In apoptotic, an increase of mitochondrial membrane permeability is considered to be one of the key events, although the detailed mechanism remains to be elucidated. The mitochondrial membrane permeability transition (MPT) is increase in the permeability of the mitochondrialmembrane that leads to loss ofΔψm,mitochondrial swelling, and rupture of the outer mitochondrial membrane.The MPT is thought to occur after the opening of a channel, which is termed the permeability transition pore (MPTP) and core constitution consists of the voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane (OM), the adenine nucleotide translocator (ANT) in the innermembrane (IM) and cyclophilin D (Cyp D) in the mitochondrial matix. Mitochondrial membrane permeabilization can be a rate limiting step of apoptotic as well as necrotic cell death.Permeabilization of the OM and/or IM is, at least in part, mediated by the permeability transition pore complex (PTPC). The PTPC is formed in the IM/OM contact site and contains the two most abundant IM and OM proteins, ANT and VDAC,the matrix protein cyclophilin D,which can interact with ANT, as well as apoptosis-regulatory proteins from the Bax/Bcl-2 family.The MPTP is a non-specific pore, permeant to any molecule of < 1.5 kDa, that opens in the inner mitochondrial membrane under conditions of elevated matrix [Ca2+], especially when this is accompanied by oxidative stress and depleted adenine nucleotides. Opening of the MPTP causes massive swelling of mitochondria, rupture of the outer membrane and release of intermembrane components that induce apoptosis. In addition mitochondria become depolarised causing inhibition of oxidative phosphorylation and stimulation of ATP hydrolysis. Thus, in many cases, the PT appears to be the mastermind that orchestrates apoptosis. Alteration of mitochondrial membrane permeability is a central mechanism leading invariably to cell death. Indeed, extended PTPC opening is sufficient to trigger an increase in mitochondrial membrane permeability and apoptosis. Among the various PTPC components, the ANT appears to act as a bi-functional protein which, on the one hand, contributes to a crucial step of aerobic energy metabolism, the ADP/ATP translocation, and on the other hand, can be converted into a pro-apoptotic pore under the control of onco- and anti-oncoproteins from the Bax/Bcl-2 family.Additionally,VacA likes as an A-B type cytotoxin, binds to cells primarily via amino acid sequences in its B subunit—p58,and it is necessary for enzymatic activity of A subunit—p37.Meanwhile, research indicated that the residues are situated in 418 aa to 799 aa of p58 fragment madiate the development of gastric diseases through cell cycle arrest in the G1 phase and induction of apoptosis is associated with up-regulation of apoptosis related factors.To summarized,we speculated that VacA and ANT formation the complex, then changed the channel activity of mitochondrial membrane, leaded to MPT eventually. In present study, apply to Yeast Two-Hybrid and Co-immunoprecipitation to explored the hypothesis that the protein interaction between VacA and ANT isoforms,VacA and ANT formation the complex induced the mitochondria membrane transition.Expected to clarify the mechanism of VacA induce cell apotosis,for the purpose of supply new clues of molecular mechanism of Hp infections evocated GC by mitochondrial apoptosis pathway.1. Construction and identification of the"bait"plasmid of Yeast Two-Hybrid: Extracted the genome DNA from Helicobacter pylori standard strain NCTC11637, the gene encoding Vacuolating cytotoxin partial sequences was amplified by PCR and then cloned into pGBKT7 to construct the pGBKT7-VacA bait plasmid. Insert-contained plasmid was confirmed by restriction endonuclease analysis and DNA sequencing. And then the bait plasmid were transformed into the host S.cerevisiae AH109,the results of recombinant protein detection by Western Blot indicated that the protein translated correctly; meanwhile, the bait plasmid also have no cytotoxin, no leakage and no auto-activated activities. This work was important for the further verification of the protein interaction with VacA.2. Construction and identification of the"prey"plasmids of Yeast Two-Hybrid: Each open reading frame (ORF) of the ANT isoforms were amplified by RT-PCR and then cloned into the pGADT7 respectively, to construct the prey plasmids pGADT7-ANT1, pGADT7-ANT2 and pGADT7-ANT3. After proved by restrict endonuclease digesting and DNA sequencing, the prey plasmids were transformed into S.cerevisiae Y187 separately, then the expression of the prey proteins were analyzed by Western Blot severally. A series of cytotoxin and activity performances of the prey plasmids were detected, the results indicated that all of these plasmids have natural activity and have no toxincity, leakage and auto-activated activities.These achievements provided the foundation for inspect the combination partner of ANT in the Yeast Two-Hybrid System.3. Yeast Two-Hybrid: Transform AH109 with pGBKT7-VaA and transform Y187 with pGADT7-ANT1 or pGADT7-ANT2 or pGADT7-ANT3.The two types of cells were co-cultured in liquid SD/-Trp/-Leu medium.After mating,the plasmids were introduced into the same host cells,centrifuged cells and plate culture on Low-stringency,Medium-stringency, High-stringency plates sequentially.Verify the protein interaction afterβ-galactosidase assays. None of all experimental groups activated the reporter genes transcription, there were means that have no protein interactions between each other in our test system.4. Construction and Identification of the pCMV-Myc-VacA expression vector: The pGBKT7-VacA plasmid DNA as template, amplified VacA fragment by PCR, and cloned into plasmid pCMV-Myc. After identificated by restriction endonuclease digested and then transient transfected into AGS cells by using lipofectamine 2000.Detected the recombinant protein by Western Blot analysis. The results demonstrated that the recombinant protein translated correctly.This study offers the basis for further research of co-immunoprecipitation protein of VacA.5. Construction and Identification of the pCMV-HA-ANT expression vectores: Set up the Yeast Two-hybrid prey plasmids as the templates.Amplified the ANT fragments by PCR respectively.And then cloned into the HA-tag expression vector pCMV-HA. Identificated by restriction endonuclease digested and transient transfected into AGS cells by using lipofectamine 2000 separately. All the expression plasmids were proofed that they were translated exactly by Western Blot assays. 6. Co-Immunoprecipitation: The Myc-tag plasmid pCMV-Myc-VacA and the HA-tag plasmids pCMV-HA-ANT1 or pCMV-HA-ANT2, pCMV-HA-ANT3 were co-transfected into AGS cells with optimal combination. The cells were homogenized in lysis buffer, and then added the first primary antibody into the supernatant, after 4 hours incubation, added the Protein A beads in it and incubated overnight at low velocity rotary conditions. After that, the Protein A beads were pelleted, then Western Blot were performed by using another primary antibody, to verified the protein interactions at physiological circumstance.Detected the protein interactions by Co-immunoprecipitation approach. No matter how VacA immunoprec- ipitation ANT or ANT immunoprecipitation VacA, there were no phenomenons had been detected that revealed where the co-immunoprecipitation happened between each other.