KLHL41 stabilizes skeletal muscle sarcomeres by nonproteolytic ubiquitination
- University of Texas Southwestern Medical Center, United States
Figures
Figure 1 with 1 supplement
Muscle-specific expression of Klhl41.
(A) Distribution of Klhl41 transcript in adult mouse tissues as determined by qRT-PCR. Expression of Klhl41 transcript was normalized to 18 s rRNA. GP: Gastrocnemius plantaris, TA: Tibialis …
Figure 1—figure supplement 1
Klhl41 KO allele.
Structure of Klhl41 WT (top) and KO allele (bottom). Insertion of the LacZ cassette into intron 1 is sufficient to abolish Klhl41 expression.
Figure 2
Klhl41 KO mice display neonatal lethality.
(A) Klhl41 gene structure: the regions coding for BTB (blue), BACK (red) and Kelch repeats (green) are indicated (top). Detection of Klhl41 transcript in P0 muscle of WT and Klhl41 KO mice by …
Figure 3 with 2 supplements
Loss of KLHL41 in mice leads to nemaline myopathy.
(A) Hematoxylin and eosin staining of longitudinal sections of diaphragm muscle of WT and Klhl41 KO mice at P0. Arrows point to ragged fibers (fibers with discontinuous H&E staining) present in KO …
Figure 3—figure supplement 1
Additional histology of Klhl41 KO mice.
(A) Hematoxylin and eosin staining of transverse sections of heart ventricle (top) and quadriceps (bottom) of WT and Klhl41 KO mice at P3. Scale bar: 100 µm. (B) Sarcomeric α-actinin immunostaining …
Figure 3—figure supplement 2
Ultrastructural abnormalities in Klhl41 KO hindlimb.
EM of WT and Klhl41 KO hindlimb muscle at P3. Scale bar: 1 µm.
Figure 4 with 3 supplements
KLHL41 is required to maintain normal levels of sarcomeric proteins in vivo.
(A) Heat map of changes in protein levels identified by proteomics in WT and Klhl41 KO hindlimb muscles at P0 (n = 3 mice per genotype). A total of 389 proteins were identified by proteomics to be …
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Figure 4—source data 1
Global protein changes in WT and KO mice by quantitative proteomics.
Unbiased quantitative proteomics was performed in WT and Klhl41 KO hindlimb muscle at P0 (n = 3 per genotype). Muscle samples were labeled with tandem mass tags (TMT). TMT129-131 were used to label WT samples and TMT126-128 were used to label KO samples. Data are provided as protein intensities (blue columns): for each channel, the signal of each unique peptide was added and normalized to the 126 channel. Then, protein intensities were generated by summing all of the unique peptide intensities to the corresponding protein. Proteins significantly regulated (p<0.05 and fold change >1.3) are shown. For up-regulated proteins, fold change was calculated as KOmean signal/WTmean signal. For down-regulated proteins, fold change was calculated as -WTmean signal/KOmean signal.
- https://doi.org/10.7554/eLife.26439.013
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Figure 4—source data 2
Global mRNA changes in WT and KO mice by RNA-seq.
RNA-seq (n = 3 mice per genotype) in WT and KO hindlimb muscle at P0 was performed by the UT Southwestern Genomic and Microarray Core Facility. Data are provided as log of fold change (log(2)FC) and log2 of counts per million (log(2)CPM). For analysis of multiple groups, Holm-Sidak correction for multiple comparisons was utilized with a false discovery rate of 0.05 (FDR). Gene transcripts with significant changes (FDR < 0.05 and fold change >1.5) are included.
- https://doi.org/10.7554/eLife.26439.014
Figure 4—figure supplement 1
Pathway analysis of differentially regulated proteins in Klhl41 KO muscle by unbiased proteomics.
(A) Top 10 biological pathways enriched in down-regulated proteins in Klhl41 KO hindlimb muscle at P0 as identified by DAVID analysis. (B) Top 10 biological pathways enriched in up-regulated …
Figure 4—figure supplement 2
Global changes in mRNA expression by RNA-seq.
(A) Heat map of mRNA changes in WT and Klhl41 KO hindlimb muscle at P0 by RNA-seq (n = 3 per genotype). Data are represented as Z-score. (B) Ingenuity pathway analysis of up-regulated mRNA …
Figure 4—figure supplement 3
KLHL40 and KLHL41 show distinct partner specificity.
(A) Alignment between mouse KLHL40 and KLHL41 protein sequences. The different domains are indicated with distinct colors: BTB (blue), BACK (red) and Kelch repeats (green). Long insertions are …
Figure 5
KLHL41 association with CUL3 is mediated by its BTB domain.
(A) Co-immunoprecipitation in transfected COS-7 cells indicates that KLHL41 homodimerizes with itself and heterodimerizes with KLHL40. Western blots of co-immunoprecipitation and input with …
Figure 6 with 1 supplement
KLHL41 stabilization of NEBfrag is poly-ubiquitin-dependent.
(A) KLHL41 can stabilize NEBfrag in the presence of HA-Ub-WT but not HA-Ub-K0 mutant. Expression of NEBfrag and KLHL41 was determined by western blot analysis with the indicated antibodies in COS-7 …
Figure 6—figure supplement 1
KLHL40 stabilization of LMOD3 is independent of poly-ubiquitination.
The ability of KLHL40 to stabilize LMOD3 in the presence of HA-Ub (WT) or HA-Ub (K0) was determined by western blot analysis with the indicated antibodies. The laddered smear detected in HA input …
Figure 7 with 1 supplement
KLHL41 prevents NEBfrag aggregation in the nucleus.
(A) KLHL41 regulation of NEBfrag in the absence or presence of proteasome inhibitor (10 µM MG132 for 24 hr) was determined by western blot analysis with the indicated antibodies in COS-7 cells. Note …
Figure 7—figure supplement 1
KLHL41 prevents NEBfrag aggregation in the nucleus.
(A) Determination of NEBfrag half-life. COS-7 cells transfected with NEBfrag-myc and FLAG-KLHL41 were pulsed with cycloheximide (100 µg/ml) for the indicated times and protein levels were determined …
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.26439.020
