Regulation of PC1/3 and the role of proSAAS in secretory protein aggregation

Abstract

Prohormone convertase 1/3 (PC1/3), a eukaryotic serine protease related to the bacterial subtilase family, is critically involved in the maturation of neuropeptides and peptide hormones, such as proopiomelanocortin and proinsulin. Recently, mutations and SNPs have been identified in the Pcsk1 gene (encoding PC1/3) associating PC1/3 with obesity. In the regulated secretory pathway of neuroendocrine and endocrine tissues, PC1/3 cleaves after dibasic residues. PC1/3 is initially synthesized as a zymogen, and undergoes autocatalytic processing at the N-terminus to yield active 87 kDa PC1/3. This is followed by sequential cleavage at the C-terminus resulting in an intermediate 74 kDa form, and the final 66 kDa PC1/3 species. In addition, PC1/3 activity is regulated by its endogenous binding partner, proSAAS, which has been shown to inhibit PC1/3. PC1/3 is a notoriously slow enzyme; in fact, the 87 kDa form is 100 times less active than other family members, such as PC2 and furin. Therefore, additional regulatory mechanisms beyond autocatalytic cleavage and proSAAS binding may contribute to PC1/3 activity. Using a variety of biochemical techniques, I demonstrated that PC1/3 is also regulated via self-association and substrate binding. I found that both secreted and intracellular PC1/3 exist as multiple ionic and high molecular weight forms and that PC1/3 oligomers are reversible and exhibit increased enzymatic activity upon dissociation. Furthermore, PC1/3 dimers are activated upon preincubation with short KR-containing peptides. Since proSAAS is found in PC1/3-expressing cells, I hypothesized that proSAAS could dissociate and activate PC1/3. However, under the conditions tested, proSAAS was not able to dissociate PC1/3 in vitro, nor could it affect PC1/3 oligomerization/aggregation within neuroendocrine cells. Interestingly, proSAAS is more broadly expressed than PC1/3; therefore proSAAS may have additional functions unrelated to PC1/3 regulation. Uncontrolled protein aggregation is deleterious to neuronal function; for example, aggregation of Aβ<subscript>1-42 and α-synuclein has been implicated in Alzheimer's and Parkinson's disease, respectively. ProSAAS efficiently blocked the fibrillation of both of these proteins and protected cells from Aβ<subscript>1-42-induced cytotoxicity. In summary, the data in this thesis clearly demonstrate that PC1/3 is regulated by self-association and substrate binding, and that proSAAS has an anti-aggregant function for neurodegeneration-associated proteins.