Dataset Information

Mutational analysis of predicted interactions between the catalytic and P domains of prohormone convertase 3 (PC3/PC1).

ABSTRACT: The subtilisin-like prohormone convertases (PCs) contain an essential downstream domain (P domain), which has been predicted to have a beta-barrel structure that interacts with and stabilizes the catalytic domain (CAT). To assess possible sites of hydrophobic interaction, a series of mutant PC3-enhanced GFP constructs were prepared in which selected nonpolar residues on the surface of CAT were substituted by the corresponding polar residues in subtilisin Carlsberg. To investigate the folding potential of the isolated P domain, signal peptide-P domain-enhanced GFP constructs with mutated andor truncated P domains were also made. All mutants were expressed in betaTC3 cells, and their subcellular localization and secretion were determined. The mutants fell into three main groups: (i) Golgisecreted, (ii) ERnonsecreted, and (iii) apoptosis inducing. The destabilizing CAT mutations indicate that the side chains of V292, T328, L351, Q408, H409, V412, and F441 and nonpolar fragments of the side chains of R405 and W413 form a hydrophobic patch on CAT that interacts with the P domain. We also have found that the P domain can fold independently, as indicated by its secretion. Interestingly, T594, which is near the P domain C terminus, was not essential for P domain secretion but is crucial for the stability of intact PC3. T594V produced a stable enzyme, but T594D did not, which suggests that T594 participates in important hydrophobic interactions within PC3. These findings support our conclusion that the catalytic and P domains contribute to the folding and thermodynamic stability of the convertases through reciprocal hydrophobic interactions.

SUBMITTER: Ueda K

PROVIDER: S-EPMC156251 | biostudies-literature | 2003 May

REPOSITORIES: biostudies-literature

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Mutational analysis of predicted interactions between the catalytic and P domains of prohormone convertase 3 (PC3/PC1).

Ueda Kazuya K Lipkind Gregory M GM Zhou An A Zhu Xiaorong X Kuznetsov Andrey A Philipson Louis L Gardner Paul P Zhang Chunling C Steiner Donald F DF

Proceedings of the National Academy of Sciences of the United States of America 20030429 10

The subtilisin-like prohormone convertases (PCs) contain an essential downstream domain (P domain), which has been predicted to have a beta-barrel structure that interacts with and stabilizes the catalytic domain (CAT). To assess possible sites of hydrophobic interaction, a series of mutant PC3-enhanced GFP constructs were prepared in which selected nonpolar residues on the surface of CAT were substituted by the corresponding polar residues in subtilisin Carlsberg. To investigate the folding pot ...[more]

PMID: 12721373

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Project description:Transcription factor cAMP responsive element-binding protein 3 like 1 (Creb3l1) is a non-classical endoplasmic reticulum stress molecule that is emerging as an important component for cellular homeostasis, particularly within cell types with high peptide secretory capabilities. We have previously shown that Creb3l1 serves an important role in body fluid homeostasis through its transcriptional control of the gene coding for antidiuretic hormone arginine vasopressin in the neuropeptide-rich magnocellular neurones of the supraoptic nucleus. In response to osmotic stimuli such as dehydration, vasopressin magnocellular neurones undergo remarkable transcriptome changes, including increased Creb3l1 expression, to ensure that the supply of vasopressin meets demand. To determine where else Creb3l1 fits into the secretory cell supply chain, we performed RNA-sequencing of Creb3l1 knockdown anterior pituitary mouse corticotroph cell line AtT20. The target chosen for further investigation was Pcsk1, which encodes proprotein convertase enzyme 1 (PC1/3). PC1/3 is crucial for processing of neuropeptides and peptide hormones such as pro-opiomelanocortin (POMC), proinsulin, proglucagon, vasopressin and oxytocin. Viral manipulations in supraoptic nuclei by over-expression of Creb3l1 increased Pcsk1, whereas Creb3l1 knockdown decreased Pcsk1 expression. In vitro promoter activity and binding studies showed that Creb3l1 was a transcription factor of the Pcsk1 gene binding directly to a G-box motif in the promoter. In the dehydrated rat anterior pituitary, Creb3l1 and Pcsk1 expression decreased in parallel compared to control, supporting our findings from manipulations in AtT20 cells and the supraoptic nucleus. No relationship was observed between Creb3l1 and Pcsk1 expression in the neurointermediate lobe of the pituitary, indicating a different mechanism of PC1/3 synthesis by these POMC-synthesising cells. Therefore, Creb3l1, by regulating the expression of Pcsk1, does not control the processing of POMC peptides in the intermediate lobe.

Dataset Information

Mutational analysis of predicted interactions between the catalytic and P domains of prohormone convertase 3 (PC3/PC1).

Publications

Mutational analysis of predicted interactions between the catalytic and P domains of prohormone convertase 3 (PC3/PC1).

Shared Molecules

Only show the datasets with similarity scores above: 0.5

Threshold

0.5

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