Study On The Localization Mechanism Of Melanocytes In Aspergillus Fumigatus

Objective:Aspergillus fumigatus is an important filamentous pathogenic fungus.It is one of the most important pathogenic fungi that causes fungal pneumonia.Its pathogenicity is mainly determined by its specific virulence factors.The virulence factor is synthesized and released to the outside of the cell(conidia)to form a polymelanin layer.Animal experiments show that melanin is the most important virulence factor that determines the virulence.Genes,destroying the synthesis of black or the formation of the final product,their ability to infect animals becomes very weak.Therefore,the process of melanin synthesis and the gene regulation of melanin synthase and spatial positioning of cells are important for studying the pathogenicity of Aspergillus fumigatus It is very important to control and find drug targets.It is not clear whether the synthesis of melanin is synthesized in the cell in a free state or in a "closed" small space.Therefore,this paper will study the cell positioning of melanin synthase to explore Cellular localization of melanin synthesis.Early experimental data and literature indicate that the spatial localization of melanin synthase is in "closed" cell particlesIn these,these particles are related to the early lysosomal marker protein Rab5,indicating that these melanin synthases are localized in the early lysosomes of the cells and complete the early synthesis of melanin.For melanin synthases with "typical" cytoplasmic protein characteristics The mechanism of recognition and sorting into early lysosomes for localization is not clear.In this paper,the specific sorting mechanism of melanin synthase will be explored.Methods:First,melanin synthase was labeled with fluorescent protein.It was observed that melanin synthase was localized in the early lysosome and co-localized with the early lysosomal marker protein Rab5.Second,we used immunoprecipitation to find the interaction with the synthase.The protein,mainly through the early synthase labeled with melanin(FLAG / GFP labeling),is subjected to the next affinity chromatography to obtain the protein that interacts with the labeled synthase,and is identified by LC-MS / MS Interacting proteins,analyzing proteins that may be involved in protein modification,sorting,positioning,and transportation,and verifying whether these proteins are actually involved in the cell localization of melanin synthase and affecting the final synthesis of melanin through gene knockout.Finally,through preliminary work,We know that protein palmitoylation is an important mechanism for protein localization.We want to know whether melanin synthase interacts withpalmitoyl transferase and is palmitoylated.Therefore,we will apply palmitoyl protein purification to extract palmitoyl Protein and liquid chromatography-mass spectrometry(LC-MS / MS)were used to identify the palmitoylated protein in Aspergillus fumigatus.Finally,bioinformaticsAnalyze the functions of the proteins identified in the proteome and perform functional verification.By inhibiting the activity of palmitoyl transferase,observe the cell localization of melanin synthase and whether melanin synthesis is affected.In future experiments to further verify,we through gene knockout Overexpression followed by phenotypic observation and measurement is a direct method to verify gene function.The effect of palmitoyl modification on melanin synthase cell localization will be verified from two aspects,one is after analysis and determination of palmitoyl modification sites Site-directed mutations make the modification impossible to verify the function and cell location of melanin synthase;in addition,after directly knocking out palmitoyl transferase,it is verified whether melanin synthase can still normal cell positioning and complete melanin synthesis.Result:According to the subcellular localization of DHN-melanin synthase,we can infer that melanin is synthesized in the secretory granules(early endosomes)and then secreted to the cell surface through the endosomes.The enzyme(Abr1 / Abr2)completes further catalysis and polymerization to form the final melanin,which is distributed on the cell surface.Melanin completes the initial synthesis in secretory granules,and early synthase Alb1 / Ayg1 / Arp1 / Arp2 co-localizes in secretion In the endosome,the precursor of melanin synthesis can be efficiently completed in the granular vesicles,and then this precursor is secreted to the cell surface through exocytosis.In this paper,we further confirmed the subsynthesis of early synthase Cell localization-in lysosomes,late synthases are localized on the cells;in addition,we initially purified and mass spectrometry identified the palmitoyl proteome during the conidial stage,and the mass spectrometry results confirmed that the four melanin early synthases are all in the protein The group was identified,and further Western blot analysis(Western Blotting)confirmed that melanin early synthase was detected in the palmitoylated protein group.The activity of transferase or the occurrence of palmitoylation modification can interfere with the correct positioning of palmitoyl synthase by inhibiting the melanin synthase from being palmitoylated.Conclusion:The early synthase is located on the endosome to complete the early synthesis of melanin,and then is secreted to the cell surface.The late melanin synthase located on the cell wall is further catalyzed and polymerized to form the final melanin.If the Mvp1 protein is missing,it is located in the endosome The recovery of the early synthase in the body is blocked,so that the newly synthesized melanin intermediatecan not be transported by the endosome to the cell surface through exocytosis.Through bioinformatics analysis and cell location prediction,it can be seen that the early melanin synthase is a typical The "cytoplasmic" protein.How this cytoplasmic protein is recognized and sorted into endosomes,our preliminary experiments have confirmed that palmitoylation modification is a very important step.If the activity of palmitoyl transferase is inhibited,melanin The localization of early synthase in endosomes will be prevented.