The Mechanism Of DENN-Domain Protein FAM45A In Regulating Membrane Trafficking

The MADD/DENN(Differentially Expressed in Normal cells and Neoplasia)domain is an evolutionarily ancient and highly conserved protein module.It has its own activity of guanine nucleo tide exchange factors(GEF).Research has shown that proteins with DENN domain often function as GEF of Rab proteins.It is responsible for mediating the binding of specific Rab proteins to GTP,which activiates them and allows them participate in downstream reactions.Rabs is the largest known small GTPase family and,together with their effector proteins,plays an important role in regulating cellular vesicle transport,organelle function and many other aspects.FAM45A is a non-classical DENN domain Protein.Thro?gh bioinformatics analysis,it is shown that the higher order protein structure is very similar to the classical denn domain protein,s?ggesting that it may have the function of Rab protein GEF and participate in intracellular vesicle transport.It is found in many eukaryotes such as amoeba,sea urchin,a number of insects and most vertebrates.It is highly conserved and may have important biological functions,but the function and mechanism of FAM45A protein are still elusive.Our previous studies showed that FAM45A is a regulator of late endosomes/multivesicular bodies,which regulates the positioning,maturation and secretion of late endosomes,but the specific mechanism of action is still unclear.In this study,we constructed a variety of Rab protein GTP/GDP locked mutants,using immunoprecipitation and functional complementation experiments,and found that FAM45A interacts with many Rabs in vitro and the knockdown phenotype of FAM45A can be rescued by Rab27a/b,suggesting that FAM45A is upstream of Rab27a/b.Secondly,by immunoprecipitation coupled to mass spectrometry,we identified an interaction between FAM45A and another vesicle transport regulator protein complex COMMD/CCDC22/CCDC93(CCC).Finally,by constructing a double knockdown cell line,we explored the functional similarity and interaction between FAM45A and another DENN family protein MADD.Objective:To study the mechanism of FAM45A protein in regulating vesicular vesicle transportMethods:?.Identification of downstream Rab for FAM45A protein1.Construct the expression vector of 3xFlag tagged FAM45 A and a panel of Citrine tagged Rab proteins,examine the interaction between FAM45A and Rab protein by co-immunoprecipitation;Construct GTP-binding locked mutants and GDP-binding mutants of various Rab proteins tagged with Citrine,co-immunoprecipitation to detect the interaction strength of GTP-binding locked mutants and GDP-binding locked mutants with FAM45A.2.Construct FAM45A shRNA stable knockdown cell line and observe the location of Cit-Rab protein.Using the localization of CD63(late endosome)as the assaying phenotype and test the ability of Rab GTP binding locking protein to compensate for the FAM45A knockdown phenotype;Construct mCherry-FAM45A and co-transfected into Hela cells together with Citrine-Rab protein,and their co-localization was observed by fluorescence microscopy.?.Potential interacting proteins of FAM45A1.Immunoprecipitation/mass spectrometry to identify FAM45A interacting proteins2.Co-immunoprecipitation and fluorescence microscopy to verify mass spectrometry results?.Explore the relationship between FAM45A and another DENN family protein MADD1.Establish FAM45A and MADD double-knock cell lines2.The effects of FAM45A and MADD gene knockdown on CD63 localization and exosome secretionResult:?.Downstream Rab identification of FAM45A protein1.All Rab proteins used in this experiment interact to some extent with FAM45A,indicating that FAM45A has the ability to bind Rab protein,but its specificity is either promiscuous or cannot be differentiated in in vitro experiments;2.Compared with GDP-binding locked mutants,FAM45A has a higher affinity for GTP-binding mutants of Rab27a/b;3.Successfully established the FAM45A shRNA stable knockdown cell line,with phenotypes consistent with previous reports;4.Cit-Rab27a/b protein clusters to the perinuclear region in FAM45A knockdown cell line;5.Cit-Rab27a/b GTP locked mutant can reverse FAM45A knockdown phenotype;6.Cit-Rab27a/b co-located with mCherry-FAM45A.?.Potential interacting proteins of FAM45A1.We found a CCC protein complex(consisting of CCDC22,CCDC93,and any of 10 COMMD proteins)that interact with FAM45A;2.Through co-immunoprecipitation and fluorescence microscopy,we confirmed that FAM45A interacts and colocalizes with CCDC22.III.Explore the relationship between FAM45A and another DENN family protein MADD1.Similar to FAM45A,the knowdown of MADD also causes CD63 clusterin in the perinuclear region,and also leads to defective exosome release;2.MADD knockdown causes FAM45A mRNA to increase,but FAM45A knockdown does not cause significant changes in MADDConclusion:1.Rab27a/b and FAM45A have a nucleotide-dependent interaction,and can reverse the phenotype of FAM45A after knockdown;2.FAM45A interacts with CCC complex;3.Another DENN family protein MADD has similar,possibly redundant,function with FAM45A.