The Study Of Conserved Function And Molecular Mechanism Of HID-1 During Secretory Granules Biogenesis

Peptide hormones and neuropeptides are important signal materials for endocrineregulation and neuron signal transmission in organisms.These materials are packaged andstored in specialized intracellular organelles named dense core vesicles (DCV,also knownas secretory granules,SG).These vesicles generate at trans-golgi network (TGN).Theyare sorted in the golgi apparatus and then become pre-release mature DCV.The molecularmechanism in this maturation process is so far largely unknown.Our study focused on agolgi-locolized highly conserved protein HID-1 in C.elegans and mammal.This proteinoffered us a window to reveal the microscopical molecular basis in this process.hid-1 gene was first identified in a screen for mutant with high-temperature-induceddauer (HID) formation phenotype.In addition to the Hid phenotype,hid-1 mutants alsoshow pleiotropic phenotypes such as mildly uncoordinated movement (Unc) and moderatedefecation defect,which indicate an involvement of HID-1 protein in regulating vesicularexocytosis,hid-1 gene encodes a highly conserved putative protein with a single homologin Drosophila melanogaster,mouse and human.Bioinformatic analysis of HID-1 suggestsno known functional domain but predicts many transmembrane domains for the protein.The exact cellular function of this protein remains unclear.Since hid-1 gene is highly conserved in evolution,we checked its function during thematurity of DCV in C.elegans and mammalian cells.Our results indicate that ablation ofHID-1 reduced not only the number of readily releasable DCV but also the releasedbioactive peptides.By fusion fluorescent imaging and immunofluorescent imaging,weshowed that HID-1 specificly localized at medial- to trans-Golgi network.Our densitygradient separation result indicated that HID-1 functions in the sorting of membraneproteins to DCV and the transmission electron microscope (TEM) further confirmed that HID-1 affected the integrity and function of DCV.In the absence of HID-1,theSG-specific membrane proteins are reduced,which causes mislocalization of SGs andinsufficient processing of their luminal cargos.These functional presumptions wereverified by immunofluorescence and biochemistry assay both in C.elegans andmammalian cells.Our discovery of HID-1 protein as an essential component that coordinates thesorting of membrane proteins to DCV should now greatly facilitate the understanding ofthe long-standing mystery of SG biogenesis.It is clear that in the next step,we need toreveal the exact molecular role of this protein.This depends on the structure resolutionand the interacting identification of HID-1.The fulllength and domains of human HID-1 protein were expressed and purifiedfrom prokaryotic system and an initial protein crystal generation condition screen wascompleted.These are preparations to the screen of HID-1 crystal and structure resolutionby X-ray diffraction.We also set up a Tandem affinity purification system and purifiedrecombinant human HID-1 from constructed stable expression INS-1 cell line.Thediversity proteins were detected by LC-MS/MS.By further verification experiments,wewill finally reveal the interaction network of HID-1 protein and understand the molecularmechanism of the function of HID-1 during dense core vesicle biogenesis.