Deficiency in Kelch protein Klhl31 causes congenital myopathy in mice
James B. Papizan, … , Ning Liu, Eric N. Olson
James B. Papizan, … , Ning Liu, Eric N. Olson
Published September 5, 2017
Citation Information: J Clin Invest. 2017;127(10):3730-3740. https://doi.org/10.1172/JCI93445.
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Research Article Muscle biology

Deficiency in Kelch protein Klhl31 causes congenital myopathy in mice

Abstract

Maintenance of muscle structure and function depends on the precise organization of contractile proteins into sarcomeres and coupling of the contractile apparatus to the sarcoplasmic reticulum (SR), which serves as the reservoir for calcium required for contraction. Several members of the Kelch superfamily of proteins, which modulate protein stability as substrate-specific adaptors for ubiquitination, have been implicated in sarcomere formation. The Kelch protein Klhl31 is expressed in a muscle-specific manner under control of the transcription factor MEF2. To explore its functions in vivo, we created a mouse model of Klhl31 loss of function using the CRISPR-Cas9 system. Mice lacking Klhl31 exhibited stunted postnatal skeletal muscle growth, centronuclear myopathy, central cores, Z-disc streaming, and SR dilation. We used proteomics to identify several candidate Klhl31 substrates, including Filamin-C (FlnC). In the Klhl31-knockout mice, FlnC protein levels were highly upregulated with no change in transcription, and we further demonstrated that Klhl31 targets FlnC for ubiquitination and degradation. These findings highlight a role for Klhl31 in the maintenance of skeletal muscle structure and provide insight into the mechanisms underlying congenital myopathies.

Authors

James B. Papizan, Glynnis A. Garry, Svetlana Brezprozvannaya, John R. McAnally, Rhonda Bassel-Duby, Ning Liu, Eric N. Olson

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Figure 6

Klhl31 targets FlnC for ubiquitination.

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Klhl31 targets FlnC for ubiquitination. (A) Coimmunoprecipitation assay ...
(A) Coimmunoprecipitation assay showing Klhl31 interacts with FlnC. (B) Transfected COS-7 cells were immunoprecipitated with anti-Flag antibody, followed by Western blotting with anti-HA antibodies. Polyubiquitination of FlnC is reduced in the presence of a K48R mutant Ub or in the absence of Klhl31. FlnC protein levels were reduced by overexpression of Klhl31, Cul3, and Ub. This reduction is rescued by the addition of the K48R mutant Ub or in the absence of Klhl31. Black arrowhead indicates increased ubiquitinated FlnC with a reduction in polyubiquitination in the presence of the K48R mutant Ub. White arrowhead indicates a nonspecific band. (C) Immunohistochemistry of WT and Klhl31-KO quadriceps with α-actinin and FlnC antibodies shows FlnC aggregates in Klhl31-KO mice. Scale bar: 20 μm. (D) Western blot analysis of WT and Klhl31-KO quadriceps at P10 using FlnC, Slmap, and Usmg5 antibodies.