Utilizing small molecule probes to study the regulation of proteins by specific phospholipids in membrane bilayers

Biochemistry, Drug Discovery, Membrane Biology

Sparks, Robert Pleasants

关键词: Biochemistry, Drug Discovery, Membrane Biology;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/106173/SPARKS-DISSERTATION-2019.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

The segregation and upkeep of distinct compartmental organization within the cell requires both organelle specific SNAREs as well as sorting receptors to allow for continual rounds of productive fusion bringing in appropriate cargo into designated organelles.Generally, sorting receptors function by providing unique signal sequences in the c-terminal tail region of the receptor that bind to specific sorting proteins allowing cargo to be transmitted to appropriate organelles.However, the yeast Vps10 vacuolar sorting protein functions by an as yet undefined signal sequence segregating proteins to either the secretory pathway or to the vacuole.Additionally, we have found that the mammalian Vps-10 orthologue, sortilin binds to PI(3,4,5)tri-phosphate (PIP3) and this may have relevance to the eventual sorting functions in this pathway.Furthermore, this pathway is defined by its requirement for the protein Vps-34, the only class III phosphatidyl-inositol-3-kinase that makes PI3P in both yeast and mammalian cells.The relationship between Vps-34 or PI3P production and phosphatidic acid (PA) levels is a fairly unexplored subject.However, our group identified the PA phosphatase Pah1 as being the sole PA phosphatase, which when knocked out showed any effect on yeast vacuole fusion.Furthermore, in Pah1 knockouts, there is no Vps34 at the yeast vacuole and levels of PI3P at the yeast vacuole are almost completely abolished.We have shown that PA levels at the vacuole affect fusion via regulation at the priming step of fusion.Priming is the unraveling of cis-SNAREs resulting from trans-SNARE complexes remaining from a prior fusion event. The unique job of priming all cellular SNAREs is the responsibility of AAA+ ATPase NSF or Sec18, which transmits the energy released from ATP hydrolysis to mechanically unwind SNAREs.In order to investigate Vps10 dependent sorting and SNARE priming, small molecules were developed to probe the lipid binding pockets of both yeast Vps-10 and Sec18.These molecules have been shown to affect levels of priming as well as targets of sorting protein Vps-10.It is proposed that computational drug discovery in companion with molecular dynamics (MD) simulations can be coupled to biochemical techniques to further elucidate mechanisms of protein fusion and sorting involving specific lipid-protein interactions to further understand the mechanisms by which pathways such as the Vps-10 protein sorting pathway and the yeast fusion priming pathway operate.