Fibrillation of Human Calcitonin and Its Analogs: Effects of Phosphorylation and Disulfide Reduction

Renawala, Harshil K.; Chandrababu, Karthik Balakrishna; Topp, Elizabeth M.

doi:10.1016/j.bpj.2020.11.009

Cited by 15 publications

(29 citation statements)

References 67 publications

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“… 392 Sabareesan and Udgaonkar 393 tracked the aggregation of the wild-type prion protein (PrP) to the N-terminal region of the protein, consistent with positions of mutations known to increase aggregation. Finally, Renawala et al 394 studied calcitonin, an aggregation-prone therapeutic peptide hormone, to determine the effects of disulfide reduction on its aggregation kinetics. Interestingly, they found that reduced calcitonin aggregation involves different residues than wild-type ( Figure 33 ), demonstrating the utility of pulsed HDX approaches to therapeutic protein development.…”

Section: Current Uses Of Hdx-msmentioning

confidence: 99%

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Advances in Hydrogen/Deuterium Exchange Mass Spectrometry and the Pursuit of Challenging Biological Systems

et al. 2021

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Solution-phase hydrogen/deuterium exchange (HDX) coupled to mass spectrometry (MS) is a widespread tool for structural analysis across academia and the biopharmaceutical industry. By monitoring the exchangeability of backbone amide protons, HDX-MS can reveal information about higher-order structure and dynamics throughout a protein, can track protein folding pathways, map interaction sites, and assess conformational states of protein samples. The combination of the versatility of the hydrogen/deuterium exchange reaction with the sensitivity of mass spectrometry has enabled the study of extremely challenging protein systems, some of which cannot be suitably studied using other techniques. Improvements over the past three decades have continually increased throughput, robustness, and expanded the limits of what is feasible for HDX-MS investigations. To provide an overview for researchers seeking to utilize and derive the most from HDX-MS for protein structural analysis, we summarize the fundamental principles, basic methodology, strengths and weaknesses, and the established applications of HDX-MS while highlighting new developments and applications.

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Section: Current Uses Of Hdx-msmentioning

confidence: 99%

Section: Current Uses Of Hdx-msmentioning

confidence: 99%

Advances in Hydrogen/Deuterium Exchange Mass Spectrometry and the Pursuit of Challenging Biological Systems

et al. 2021

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“…Mechanistic insights into the fibrillization of hCT is essential for the rational design of aggregation-resistant hCT analogues. The fibrillization tendency of hCT is strong both in vivo and in vitro. , The fibrillization kinetics of hCT features a typical sigmoidal curve as other pathological amyloid proteins (e.g., Aβ, hIAPP, and α-synuclein). ,, A two-step reaction mechanism was proposed, isolated hCT monomers first nucleated β-sheet-rich protofibrils via helical accumulation along with a series of complex conformation conversion, followed by fibril growth of forming mature fibrils. ,,, As revealed by numerous studies of other disease-related amyloid peptides, soluble low-molecular-weight, β-sheet-rich oligomers formed in the early nucleation stages are likely the major cytotoxic species of hCT aggregation. − All the calcitonin family proteins contain a highly conserved intramolecular disulfide bond formed by residues Cys-1 and Cys-7 at the N-terminus. , Reducing the disulfide bond of sCT causes the loss of helical conformation, indicating that the disulfide bond may stabilize the helices of calcitonin. , The hCT monomer also mainly adopts α-helix, and the helical structure in hCT is more flexible than sCT. ,,, Residues from 15 to 19 of hCT, DFNKF, are known as the amyloidogenic core of hCT. ,, A recent study has shown that the nitration of tyrosine Y12 of hCT would significantly decrease the aggregation and cytotoxicity of hCT, indicating a critical role of this particular aromatic residue in the amyloid aggregation of hCT . Two single-point mutants of F16L and F19L hCT aggregated much slower than the wild type but faster than a multi-point mutant TL-hCT with Y12L, F16L, and F19L substitutions .…”

Section: Introductionmentioning

confidence: 97%

“…5,9 Reducing the disulfide bond of sCT causes the loss of helical conformation, indicating that the disulfide bond may stabilize the helices of calcitonin. 29,32 The hCT monomer also mainly adopts α-helix, and the helical structure in hCT is more flexible than sCT. 2,17,33,34 Residues from 15 to 19 of hCT, 15 DFNKF, 19 are known as the amyloidogenic core of hCT.…”

Section: ■ Introductionmentioning

confidence: 99%

Structural Perturbation of Monomers Determines the Amyloid Aggregation Propensity of Calcitonin Variants

Liu

Wang

Zou

et al. 2022

J. Chem. Inf. Model.

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Human calcitonin (hCT) is a polypeptide hormone that participates in calcium-phosphorus metabolism. Irreversible aggregation of 32amino acid hCT into β-sheet-rich amyloid fibrils impairs physiological activity and increases the risk of medullary carcinoma of the thyroid. Amyloid-resistant hCT derivatives substituting critical amyloidogenic residues are of particular interest for clinical applications as therapeutic drugs against bone-related diseases. Uncovering the aggregation mechanism of hCT at the molecular level, therefore, is important for the design of amyloid-resistant hCT analogues. Here, we investigated the aggregation dynamics of hCT, non-amyloidogenic salmon calcitonin (sCT), and two hCT analogues with reduced aggregation tendency� TL-hCT and phCT�using long timescale discrete molecular dynamics simulations. Our results showed that hCT monomers mainly adopted unstructured conformations with dynamically formed helices around the central region. hCT self-assembled into helix-rich oligomers first, followed by a conformational conversion into βsheet-rich oligomers with β-sheets formed by residues 10−30 and stabilized by aromatic and hydrophobic interactions. Our simulations confirmed that TL-hCT and phCT oligomers featured more helices and fewer β-sheets than hCT. Substitution of central aromatic residues with leucine in TL-hCT and replacing C-terminal hydrophobic residue with hydrophilic amino acid in phCT only locally suppressed β-sheet propensities in the central region and C-terminus, respectively. Having mutations in both central and Cterminal regions, sCT monomers and dynamically formed oligomers predominantly adopted helices, confirming that both central aromatic and C-terminal hydrophobic residues played important roles in the fibrillization of hCT. We also observed the formation of β-barrel intermediates, postulated as the toxic oligomers in amyloidosis, for hCT but not for sCT. Our computational study depicts a complete picture of the aggregation dynamics of hCT and the effects of mutations. The design of next-generation amyloid-resistant hCT analogues should consider the impact on both amyloidogenic regions and also take into account the amplification of transient β-sheet population in monomers upon aggregation.

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“…Moreover, although the murine CAIA model affects joints symmetrically similar to human RA, its transient nature is different from the chronic disease progression in affected patients. Despite these limitations, we believe that our findings are especially important from a clinical point of view, as novel water soluble CT prodrugs have recently been introduced for the treatment of osteoporosis and related musculoskeletal disorders like RA (Renawala et al, 2021).…”

Section: Limitations Of the Studymentioning

confidence: 98%

The calcitonin receptor protects against bone loss and excessive inflammation in collagen antibody-induced arthritis

et al. 2022

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Fibrillation of Human Calcitonin and Its Analogs: Effects of Phosphorylation and Disulfide Reduction

Cited by 15 publications

References 67 publications

Advances in Hydrogen/Deuterium Exchange Mass Spectrometry and the Pursuit of Challenging Biological Systems

Advances in Hydrogen/Deuterium Exchange Mass Spectrometry and the Pursuit of Challenging Biological Systems

Structural Perturbation of Monomers Determines the Amyloid Aggregation Propensity of Calcitonin Variants

The calcitonin receptor protects against bone loss and excessive inflammation in collagen antibody-induced arthritis

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