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Elucidating the role of disulfide bond on amyloid formation and fibril reversibility of somatostatin-14: relevance to its storage and secretion.

ABSTRACT: The storage of protein/peptide hormones within subcellular compartments and subsequent release are crucial for their native function, and hence these processes are intricately regulated in mammalian systems. Several peptide hormones were recently suggested to be stored as amyloids within endocrine secretory granules. This leads to an apparent paradox where storage requires formation of aggregates, and their function requires a supply of non-aggregated peptides on demand. The precise mechanism behind amyloid formation by these hormones and their subsequent release remain an open question. To address this, we examined aggregation and fibril reversibility of a cyclic peptide hormone somatostatin (SST)-14 using various techniques. After proving that SST gets stored as amyloid in vivo, we investigated the role of native structure in modulating its conformational dynamics and self-association by disrupting the disulfide bridge (Cys(3)-Cys(14)) in SST. Using two-dimensional NMR, we resolved the initial structure of somatostatin-14 leading to aggregation and further probed its conformational dynamics in silico. The perturbation in native structure (S-S cleavage) led to a significant increase in conformational flexibility and resulted in rapid amyloid formation. The fibrils formed by disulfide-reduced noncyclic SST possess greater resistance to denaturing conditions with decreased monomer releasing potency. MD simulations reveal marked differences in the intermolecular interactions in SST and noncyclic SST providing plausible explanation for differential aggregation and fibril reversibility observed experimentally in these structural variants. Our findings thus emphasize that subtle changes in the native structure of peptide hormone(s) could alter its conformational dynamics and amyloid formation, which might have significant implications on their reversible storage and secretion.

SUBMITTER: Anoop A

PROVIDER: S-EPMC4059132 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Elucidating the role of disulfide bond on amyloid formation and fibril reversibility of somatostatin-14: relevance to its storage and secretion.

Anoop Arunagiri A Ranganathan Srivastav S Das Dhaked Bhagwan B Jha Narendra Nath NN Pratihar Supriya S Ghosh Saikat S Sahay Shruti S Kumar Santosh S Das Subhadeep S Kombrabail Mamata M Agarwal Kumud K Jacob Reeba S RS Singru Praful P Bhaumik Prasenjit P Padinhateeri Ranjith R Kumar Ashutosh A Maji Samir K SK

The Journal of biological chemistry 20140429 24

The storage of protein/peptide hormones within subcellular compartments and subsequent release are crucial for their native function, and hence these processes are intricately regulated in mammalian systems. Several peptide hormones were recently suggested to be stored as amyloids within endocrine secretory granules. This leads to an apparent paradox where storage requires formation of aggregates, and their function requires a supply of non-aggregated peptides on demand. The precise mechanism be ...[more]

PMID: 24782311

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Project description:The protein secretory pathway must maintain homoeostasis while producing a wide assortment of proteins in different conditions. It is also used extensively to produce many useful proteins in biotechnology. As such, secretory pathway dysfunction can be highly detrimental to the cell, resulting in the molecular basis for many human diseases, and can drastically inhibit product titers in biochemical production. Because the secretory pathway is a highly-integrated, multi-organelle system, dysfunction can happen at many levels and dissecting the root cause can be challenging. To better understand some of these dysfunctions, we measured multiple systems-level states of the cell (physiology, transcriptome, metabolism) while secreting a small protein (insulin precursor) or a large protein (?-amylase). This was carried out in the presence and absence of HAC1, a key transcription factor in maintaining secretory homeostasis. Clear trends in cellular stress were apparent across multiple data resulting from our perturbations. In particular, processes involving (1) degradation of protein / recycling amino acids, (2) overall transcription/translation repression, and (3) oxidative stress. Apparent runaway oxidative radical production was explained by a thermodynamic model that we put forward for disulfide formation in the endoplasmic reticulum. This model predicts that balancing the relative rates of protein folding and disulfide bond formation are key to easing oxidative stress. These predictions have direct implications in how to engineer a broad range of recombinant proteins for secretion and provide potential hypotheses for the root causes of several secretory-associated diseases. Yeast strains were constructed that produce and secrete (a) IP or (b) ?-amylase and were compared to yeast strains containing (c) an empty vector in both wild-type and HAC1 deletion backgrounds. These strains are named WN (WT with empty vector), WI (WT secreting IP), WA (WT secreting ?-amylase), dN (?hac1 with empty vector), dI (?hac1 secreting IP), and dA (?hac1 secreting ?-amylase). Strains were characterized in batch fermentation and samples were taken in mid-exponential phase. Triplicate fermentations were carried out for each strain.

Dataset Information

Elucidating the role of disulfide bond on amyloid formation and fibril reversibility of somatostatin-14: relevance to its storage and secretion.

Publications

Elucidating the role of disulfide bond on amyloid formation and fibril reversibility of somatostatin-14: relevance to its storage and secretion.

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