Andrew PedenSALEM DAHISH ALAMRI2022-05-302022-05-30https://drepo.sdl.edu.sa/handle/20.500.14154/53074SNAP29, a Synaptosomal-associated protein 29, plays a key role on intracellular membrane trafficking and membrane fusion. Here, we studied SNAP29 extensively using overexpression and knockout techniques in the evaluation of its roles in the endocytic pathways and cellular activities. The conservation of NPF motif (asparagine-proline-phenylalanine) of SNAP29 and the phosphorylation process are confirmatory to its functionality in its interactivity with other proteins such as the EHD1 and SNAREs. The split-GFP system in HeLa cells transfection with SNAP29 constructs demonstrated the abnormal accumulation of endosomal markers (EEA1, EHD1, Rab8, and MICAL1) and the perturbation of Rab8 and MICAL-L1 tubules (recycling tubules) in response to the overexpression of SNAP29, which also lead to the reduction of transferrin utilization in strongly and weakly transfected cells (abnormal endocytic pathways). The mutation of the NPF motif of SNAP29 using CRISPR/Cas9 in stabilized split-HeLa cells did not significantly alert the expression of genes (endocytic and autophagy genes) in the microarray. The negative response of the genes in the absence of SNAP29 suggests the contributions of other proteins and the deficiency of SNAP29 is not compensated, and thus, the need for further investigation in the future such as the immunofluorescence staining of autophagy and other SNAREs proteins in SNAP29 mutated cells. Ultimately, our results highlight the importance of SNAP29 in post-Golgi trafficking and its effect in the recycling of ligands, and consequently, may contribute to the understanding of CEDNIK syndrome pathogenesis.en