Exceptions in protein superfamilies involved in exocytosis: Computational and biochemical studies

Abstract

Life is uncertain: Eat desert first. (inscription on a bakery ).;Regulated exocytosis occurs upon stimulation with external stimulus, after which the membrane of a secretory vesicle fuses with the plasma membrane and the vesicle content is released to the extracellular space. While a number of proteins are involved in this process, recent research has implicated a phosphoglucoprotein parafusin (PFUS), a member of phosphoglucomutase (PGM) super family. PFUS undergoes two covalent modifications: (phosphorylation/phosphoglycosylation) and might be involved in regulation of calcium levels in the cell after exocytosis. To further understand the function of PFUS, we have used high-speed microscopy, electrophysiology and differential inhibitor studies to correlate the biochemical cycle of PFUS with the secretory vesicle cycle in Paramecium tertaurelia cells.;Out hypothesis is that the removal of phosphate after exocytosis is actually a de-phosphoglycosylation event, followed by phosphorylation and biosynthesis of new secretory vesicles. Later, PFUS is phosphoglycosylated and re-attached to the vesicle membrane. Together with phosphoglucomutase-related protein and sarcoplasmic reticulum phosphoglucomutase, PFUS is a functionally divergent member of the PGM protein super family. In general, it appears that PGM super family members have a number of functionally divergent (possessing activity unrelated to PGM) members such as yeast PGM1 that is involved in maintenance of calcium levels, or even human PGM1 allele that is known to cause birth defects.;We have developed a machine learning approach to detect and evaluate functional exceptions in protein superfamilies, using implicit redundancy of sequence-structure relationship within protein superfamilies. We represent a multiple sequence alignment of the super family as an itemset where each protein is an item in the itemset and a residue position is a set-valued attribute. Using this representation, we use relative conservation of each residue position to evaluate any substitutions or deletions and use minimization of Kolomogorov complexity to separate a set of exceptional proteins. We also use exception-driven transformation rules to predict protein post-translational modification that is key in establishment of functional divergence of protein.;To examine structural basis for diversity of the PGM super family, we constructed homology models of all divergent members based on the crystal structure of rabbit muscle PGM. (Abstract shortened by UMI.).