KBTBD13 is an actin-binding protein that modulates muscle kinetics
Josine M. de Winter, … , Nicol C. Voermans, Coen A.C. Ottenheijm
Josine M. de Winter, … , Nicol C. Voermans, Coen A.C. Ottenheijm
Published October 31, 2019
Citation Information: J Clin Invest. 2020;130(2):754-767. https://doi.org/10.1172/JCI124000.
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Research Article Muscle biology

KBTBD13 is an actin-binding protein that modulates muscle kinetics

Abstract

The mechanisms that modulate the kinetics of muscle relaxation are critically important for muscle function. A prime example of the impact of impaired relaxation kinetics is nemaline myopathy caused by mutations in KBTBD13 (NEM6). In addition to weakness, NEM6 patients have slow muscle relaxation, compromising contractility and daily life activities. The role of KBTBD13 in muscle is unknown, and the pathomechanism underlying NEM6 is undetermined. A combination of transcranial magnetic stimulation–induced muscle relaxation, muscle fiber– and sarcomere-contractility assays, low-angle x-ray diffraction, and superresolution microscopy revealed that the impaired muscle-relaxation kinetics in NEM6 patients are caused by structural changes in the thin filament, a sarcomeric microstructure. Using homology modeling and binding and contractility assays with recombinant KBTBD13, Kbtbd13-knockout and Kbtbd13R408C-knockin mouse models, and a GFP-labeled Kbtbd13-transgenic zebrafish model, we discovered that KBTBD13 binds to actin — a major constituent of the thin filament — and that mutations in KBTBD13 cause structural changes impairing muscle-relaxation kinetics. We propose that this actin-based impaired relaxation is central to NEM6 pathology.

Authors

Josine M. de Winter, Joery P. Molenaar, Michaela Yuen, Robbert van der Pijl, Shengyi Shen, Stefan Conijn, Martijn van de Locht, Menne Willigenburg, Sylvia J.P. Bogaards, Esmee S.B. van Kleef, Saskia Lassche, Malin Persson, Dilson E. Rassier, Tamar E. Sztal, Avnika A. Ruparelia, Viola Oorschot, Georg Ramm, Thomas E. Hall, Zherui Xiong, Christopher N. Johnson, Frank Li, Balazs Kiss, Noelia Lozano-Vidal, Reinier A. Boon, Manuela Marabita, Leonardo Nogara, Bert Blaauw, Richard J. Rodenburg, Benno Küsters, Jonne Doorduin, Alan H. Beggs, Henk Granzier, Ken Campbell, Weikang Ma, Thomas Irving, Edoardo Malfatti, Norma B. Romero, Robert J. Bryson-Richardson, Baziel G.M. van Engelen, Nicol C. Voermans, Coen A.C. Ottenheijm

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Figure 6

Effect of KBTBD13 on sarcomere structure and function.

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Effect of KBTBD13 on sarcomere structure and function. (A) Homology mode...
(A) Homology modeling reveals that KBTBD13 β propeller motifs (left) have striking similarities with those of the actin-binding protein scruin in limulus sperm (middle). KBTBD13 has cysteine residue (Cys367) located in nearly the exact same position as scruin (Cys837, right). (B) SDS-PAGE image after pulldown assay in a mouse muscle lysate (muscle lys.). One unique band (*) was identified, excised, and analyzed with mass spectrometry. Actin was prominently present in excised band. (C, left). Amounts of KBTBD13 (gray) and KBTBD13R408C (blue) bound to actin versus free KBTBD13 were plotted and fitted. The shared fit is shown in black. (C, right) Western blot of KBTBD13 in pellet and supernatant fraction (in presence of actin, P+, and S+, respectively) and pellet fraction in absence of actin (P–) (lanes 1-3). Lanes 6–8 show dose-dependent detection of serial dilution of recombinant KBTBD13. (D) APEX-based EM of zebrafish expressing GFP-tagged KBTBD13. Positive cells stained dark in electron micrographs. Note that in cells, myofibrils in particular show dark staining. Scale bar: 1 μm. Top right: To increase contrast between KBTBD13-positive and -negative areas, images were converted from gray scale to spectrum scale, with KBTBD13 in light blue. Below, right: high magnification of myofibrillar compartment. Scale bar: 0.3 μm. Within myofibrils, A-band and I-band show labeling, indicating that KBTBD13 binds to thin filament and thick filament. Furthermore, z-discs, where thin filaments from adjacent sarcomeres overlap, show strong labeling. Below, left: high magnification of myofibrillar compartment of negative cell. Scale bar: 0.3 μm. (E) Left: representative image of actin filaments in solution. Inset illustrates determination of parameters required to calculate persistence length (Lp) of actin filaments. Right: KBTBD13WT protein does not affect Lp, but KBTDB13R408C increases Lp of actin filaments. (F). Left: relaxation traces of 2 segments of same permeabilized muscle fiber: one incubated with recombinant KBTBD13WT, the other with recombinant KBTBD13R408C. Right: In healthy control muscle, incubation with KBTBD13R408C slows relaxation kinetics. In NEM6 muscle, incubation with KBTBD13WT does not affect relaxation kinetics. Student’s t tests or Mann-Whitney U tests were performed. For detailed information on number of samples and statistical tests and outcomes, see Supplemental Table 1.