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Functional significance of HCM mutants of tropomyosin, V95A and D175N, studied with in vitro motility assays.

ABSTRACT: The majority of hypertrophic cardiomyopathy (HCM) is caused by mutations in sarcomere proteins. We examined tropomyosin (Tpm)'s HCM mutants in humans, V95A and D175N, with in vitro motility assay using optical tweezers to evaluate the effects of the Tpm mutations on the actomyosin interaction at the single molecular level. Thin filaments were reconstituted using these Tpm mutants, and their sliding velocity and force were measured at varying Ca2+ concentrations. Our results indicate that the sliding velocity at pCa ?8.0 was significantly increased in mutants, which is expected to cause a diastolic problem. The velocity that can be activated by Ca2+ decreased significantly in mutants causing a systolic problem. With sliding force, Ca2+ activatable force decreased in V95A and increased in D175N, which may cause a systolic problem. Our results further demonstrate that the duty ratio determined at the steady state of force generation in saturating [Ca2+] decreased in V95A and increased in D175N. The Ca2+ sensitivity and cooperativity were not significantly affected by the mutations. These results suggest that the two mutants modulate molecular processes of the actomyosin interaction differently, but to result in the same pathology known as HCM.

SUBMITTER: Ishii S

PROVIDER: S-EPMC6435021 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Functional significance of HCM mutants of tropomyosin, V95A and D175N, studied with in vitro motility assays.

Ishii Shuya S Suzuki Madoka M Ishiwata Shin'ichi S Kawai Masataka M

Biophysics and physicobiology 20190202

The majority of hypertrophic cardiomyopathy (HCM) is caused by mutations in sarcomere proteins. We examined tropomyosin (Tpm)'s HCM mutants in humans, V95A and D175N, with in vitro motility assay using optical tweezers to evaluate the effects of the Tpm mutations on the actomyosin interaction at the single molecular level. Thin filaments were reconstituted using these Tpm mutants, and their sliding velocity and force were measured at varying Ca2+ concentrations. Our results ind ...[more]

PMID: 30923661

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Project description:BACKGROUND:Novel foods may provide new protein sources for a growing world population but entail risks of unexpected food-allergic reactions. No guidance on allergenicity assessment of novel foods exists, while for genetically modified (GM) crops it includes comparison of sequence identity with known allergens, digestibility tests and IgE serum screening. OBJECTIVE:As a proof of concept, to evaluate non-/allergenic tropomyosins (TMs) regarding their potential as new calibrator proteins in functional biological in vitro assays for the semi-quantitative allergy risk assessment of novel TM-containing animal foods with mealworm TM as an example. METHODS:Purified TMs (shrimp, Penaeus monodon; chicken Gallus gallus; E coli overexpression) were compared by protein sequencing, circular dichroism analysis and in vitro digestion. IgE binding was quantified using shrimp-allergic patients' sera (ELISA). Biological activities were investigated (skin testing; titrated basophil activation tests, BAT), compared to titrated biological mediator release using humanized rat basophil leukaemia (RBL) cells. RESULTS:Shrimp and chicken TMs showed high sequence homology, both alpha-helical structures and thermal stability. Shrimp TM was stable during in vitro gastric digestion, chicken TM degraded quickly. Both TMs bound specific IgE from shrimp-allergic patients (significantly higher for shrimp TM), whereas skin reactivity was mostly positive with only shrimp TM. BAT and RBL cell assays were positive with shrimp and chicken TM, although at up to 100- to 1000-times lower allergen concentrations for shrimp than chicken TM. In RBL cell assays using both TM as calibrators, an activation of effector cells by mealworm TM similar to that by shrimp TM confirmed the already reported high allergenic potency of mealworm TM as a novel protein source. CONCLUSIONS & CLINICAL RELEVANCE:According to current GM crops' allergenicity assessment, non-allergenic chicken TM could falsely be considered an allergen on a weight-of-evidence approach. However, calibrating allergenic potency in functional BAT and RBL cell assays with clinically validated TMs allowed for semi-quantitative discrimination of novel food protein's allergenicity. With TM calibration as a proof of concept, similar systems of homologous protein might be developed to scale on an axis of allergenicity.

Project description:The in vitro motility assay (IVMA) is a technique that enables the measurement of the interaction between actin and myosin providing a relatively simple model to understand the mechanical muscle function. For actin-myosin IVMA, myosin is immobilized in a measurement chamber, where it converts chemical energy provided by ATP hydrolysis into mechanical energy. The result is the movement of fluorescently labeled actin filaments that can be recorded microscopically and analyzed quantitatively. Resulting sliding speeds and patterns help to characterize the underlying actin-myosin interaction that can be affected by different factors such as mutations or active compounds. Additionally, modulatory actions of the regulatory proteins tropomyosin and troponin in the presence of calcium on actin-myosin interaction can be studied with the IVMA. Zebrafish is considered a suitable model organism for cardiovascular and skeletal muscle research. In this context, straightforward protocols for the isolation and use of zebrafish muscle proteins in the IVMA would provide a useful tool in molecular studies. Currently, there are no protocols available for the mentioned purpose. Therefore, we developed fast and easy protocols for characterization of zebrafish proteins in the IVMA. Our protocols enable the interested researcher to (i) isolate actin from zebrafish skeletal muscle and (ii) extract functionally intact myosin from cardiac and skeletal muscle of individual adult zebrafish. Zebrafish tail muscle actin is isolated after acetone powder preparation, polymerized, and labeled with Rhodamine-Phalloidin. Myosin from ventricles of adult zebrafish is extracted directly into IVMA flow-cells. The same extraction protocol is applicable for comparably small tissue pieces as from zebrafish tail, mouse and frog muscle. After addition of the fluorescently labeled F-actin from zebrafish-or other origin-and ATP, sliding movement can be visualized using a fluorescence microscope and an intensified CCD camera. Taken together, we introduce a method for functional analysis in zebrafish cardiac and skeletal muscle research to study mutations at the molecular level of thick or thin filament proteins. Additionally, preliminary data indicate the usefulness of the presented method to perform the IVMA with myosin extracted from muscles of other animal models.

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Functional significance of HCM mutants of tropomyosin, V95A and D175N, studied with in vitro motility assays.

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Functional significance of HCM mutants of tropomyosin, V95A and D175N, studied with <i>in vitro</i> motility assays.

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