Title Page
Abstract
Contents
Ⅰ. Introduction 9
Ⅱ. Materials and Methods 11
Ⅱ-1. Construction of plasmids 11
Ⅱ-2. Cell culture and Transfection 11
Ⅱ-3. Streptavidin blot 11
Ⅱ-4. Sample preparation for MS 11
Ⅱ-5. Functional enrichment analysis 12
Ⅱ-6. Protein-protein interaction (PPI) networks and clustering 12
Ⅲ. Results 13
Ⅲ-1. Construction of plasmid to identify NPC1-interacting proteins using TurboID-mediated proximity labeling 13
Ⅲ-2. TurboID fused with NPC1 promiscuously biotinylates adjacent proteins 13
Ⅲ-3. MS data suggest candidates for NPC-interacting proteins at ER-Lysosome MCS 14
Ⅲ-4. Protein-protein network suggests NPC1-associated protein complex at ER-Lysosome MCS 15
Ⅳ. DISCUSSION 48
Ⅴ. REFERENCES 49
Ⅵ. ABSTRACT IN KOREAN 51
Table 1. Description about the function of biotinylated proteins by NPC1-TurboID mass results 32
Table 2. Gene Ontology (GO) term enrichment analysis of NPC1-TurboID (Biological Process) 38
Figure 1. TurboID system to identify NPC1-interacting proteins at ER-Lysosome Contact Sites 16
Figure 2. TurboID fused with NPC1 proteins biotinylates closely associated proteins in U2OS cells 18
Figure 3. Sampling of biotinylated proteins by NPC1-TurboID and TurboID for mass analysis 19
Figure 4. Analysis of the proteins identified in NPC1-TurboID through mass spectrometry 22
Figure 5. GO analysis of NPC1-TurboID results, covering Biological Process, Cellular Component, and Molecular Function categories 29
Figure 6. The multiple protein list obtained from the mass spectrometry results for NPC1-TurboID conducted on Homo sapiens was subjected to k-means clustering with a cluster number of 4, and the... 31