Skeletal Muscle-Specific Methyltransferase METTL21C Trimethylates p97 and Regulates Autophagy-Associated Protein Breakdown

Summary: Protein aggregates and cytoplasmic vacuolization are major hallmarks of multisystem proteinopathies (MSPs) that lead to muscle weakness. Here, we identify METTL21C as a skeletal muscle-specific lysine methyltransferase. Insertion of a β-galactosidase cassette into the Mettl21c mouse locus revealed that METTL21C is specifically expressed in MYH7-positive skeletal muscle fibers. Ablation of the Mettl21c gene reduced endurance capacity and led to age-dependent accumulation of autophagic vacuoles in skeletal muscle. Denervation-induced muscle atrophy highlighted further impairments of autophagy-related proteins, including LC3, p62, and cathepsins, in Mettl21c−/− muscles. In addition, we demonstrate that METTL21C interacts with the ATPase p97 (VCP), which is mutated in various human MSP conditions. We reveal that METTL21C trimethylates p97 on the Lys315 residue and found that loss of this modification reduced p97 hexamer formation and ATPase activity in vivo. We conclude that the methyltransferase METTL21C is an important modulator of protein degradation in skeletal muscle under both normal and enhanced protein breakdown conditions. : Wiederstein et al. describe the skeletal muscle methyltransferase Mettl21c. They found that ablation of Mettl21c in mice results in muscle weakness and disturbance of the protein degradation machinery. Those changes are hallmarks of multisystem proteinopathies. They demonstrate that Mettl21c modulates p97 activity, which is frequently mutated in human patients with muscle weakness. Keywords: methyltransferases, skeletal muscle, p97, atrophy, autophag

Modulation of Titin-Based Stiffness in Hypertrophic Cardiomyopathy via Protein Kinase D

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The arthritis-associated HLA-B*27:05 allele forms more cell surface B27 dimer and free heavy chain ligands for KIR3DL2 than HLA-B*27:09

Objectives. HLA-B*27:05 is associated with AS whereas HLA-B*27:09 is not associated. We hypothesized that different interactions with KIR immune receptors could contribute to the difference in disease association between HLA-B*27:05 and HLAB*27:09. Thus, the objective of this study was to compare the formation of β2m-free heavy chain (FHC) including B27 dimers (B272) by HLA-B*27:05 and HLA-B*27:09 and their binding to KIR immunoreceptors. Methods. We studied the formation of HLA-B*27:05 and HLA-B*27:09 heterotrimers and FHC forms including dimers in vitro and in transfected cells. We investigated HLA-B*27:05 and HLA-B*27:09 binding to KIR3DL1, KIR3DL2 and LILRB2 by FACS staining with class I tetramers and by quantifying interactions with KIR3DL2CD3ε-reporter cells and KIR3DL2-expressing NK cells. We also measured KIR expression on peripheral blood NK and CD4 T cells from 18 HLA-B*27:05 AS patients, 8 HLA-B27 negative and 12 HLA-B*27:05+ and HLA-B*27:09+ healthy controls by FACS staining. Results. HLA-B*27:09 formed less B272 and FHC than HLA-B*27:05. HLA-B*27:05-expressing cells stimulated KIR3DL2CD3ε-reporter T cells more effectively. Cells expressing HLA-B*27:05 promoted KIR3DL2+ NK cell survival more strongly than HLA-B*27:09. HLA-B*27:05 and HLA-B*27:09 dimer tetramers stained KIR3DL1, KIR3DL2 and LILRB2 equivalently. Increased proportions of NK and CD4 T cells expressed KIR3DL2 in HLA-B*27:05+ AS patients compared with HLA-B*27:05+, HLA-B*27:09+ and HLA-B27− healthy controls. Conclusion. Differences in the formation of FHC ligands for KIR3DL2 by HLA-B*27:05 and HLA-B*27:09 could contribute to the differential association of these alleles with A

SUMO signaling by hypoxic inactivation of SUMO-specific isopeptidases

Post-translational modification of proteins with ubiquitin-like SUMO modifiers is a tightly regulated and highly dynamic process. The SENP family of SUMO-specific isopeptidases comprises six cysteine proteases. They are instrumental in counterbalancing SUMO conjugation, but their regulation is not well understood. We demonstrate that in hypoxic cell extracts, the catalytic activity of SENP family members, in particular SENP1 and SENP3, is inhibited in a rapid and fully reversible process. Comparative mass spectrometry from normoxic and hypoxic cells defines a subset of hypoxia-induced SUMO1 targets, including SUMO ligases RanBP2 and PIAS2, glucose transporter 1, and transcriptional regulators. Among the most strongly induced targets, we identified the transcriptional co-repressor BHLHE40, which controls hypoxic gene expression programs. We provide evidence that SUMOylation of BHLHE40 is reversed by SENP1 and contributes to transcriptional repression of the metabolic master regulator gene PGC-1α. We propose a pathway that connects oxygen-controlled SENP activity to hypoxic reprogramming of metabolism

Characterization of Cavin4b/Murcb deficient zebrafish.

<p>A. Representative lateral views of <i>murcb</i>^{<i>s983/s983</i>} and <i>murcb</i>^{<i>s983/+</i>} sibling zebrafish at 10 wpf. Fish in the top panels are females while those in the bottom panels are males. B. Dot plots of body mass and length of 10 wpf wt, <i>murcb</i>^{<i>s983/+</i>}, and <i>murcb</i>^{<i>s983/s983</i>} zebrafish. Length measurements were made from the anterior point to the caudal fin/trunk boundary. Approximately equal numbers of male and female fish were used. The red line represents the mean. ***p<0.0005 C. H&E stain of 10 μm sections prepared from the trunk of 10 wpf <i>murcb</i>^{<i>s983/+</i>} and <i>murcb</i>^{<i>s983/s983</i>} zebrafish. Two examples of <i>murcb</i>^{<i>s983/s983</i>} fish of different sizes are shown. D. Phalloidin staining of 10 μm transverse sections prepared from the trunk of 10 wpf <i>murcb</i>^{<i>s983/+</i>} and <i>murcb</i>^{<i>s983/s983</i>} zebrafish. E. Trichrome staining of 10 μm transverse sections prepared from the trunk of 12 mpf <i>murcb</i>^{<i>s983/+</i>} and <i>murcb</i>^{<i>s983/s983</i>} zebrafish. F. Representative lateral views of <i>murcb</i>^{<i>s983/+</i>} and <i>murcb</i>^{<i>s983/s983</i>} zebrafish at 5 dpf.</p

Filamentous actin analysis of Cavin4b/Murcb deficient zebrafish.

<p>Representative lateral views of confocal projections of phalloidin stained <i>murcb</i>^{<i>s983/+</i>} and <i>murcb</i>^{<i>s983/s983</i>} zebrafish at 3 dpf and 6 dpf. Skeletal muscle fibers are smaller and less consistent in size in <i>murcb</i> mutant animals compared to wild-type siblings.</p

Transcriptional profiling of Cavin4b/Murcb deficient zebrafish.

<p>Inset: PANTHER Database protein class statistical overrepresentation test on microarray mRNA expression profiling from <i>murcb</i>^{<i>s983/s983</i>} compared to <i>murcb</i>^{<i>s983/+</i>} 72 hpf larvae. 1309 genes with greater than 1.5 fold or less than 0.7 fold expression difference in Cavin4b/Murcb deficient larvae compared to their heterozygous siblings were recognized in the database with 25708 genes comprising the <i>Danio rerio</i> reference list. Protein classes shown had a <i>p</i> value <0.05. The log₂ ratio of <i>murcb</i>^{<i>s983/s983</i>}/<i>murcb</i>^{<i>s983/+</i>} normalized microarray values from genes in the voltage-gated ion channel PANTHER protein class show that 26 members of the voltage-gated ion channel class were misregulated where 10 would be expected. Error bars represent SEM.</p

TALEN design and generation of a <i>murcb</i> mutant allele.

<p>A. RT-PCR analysis of mRNA expression of <i>murca and murcb</i> during zebrafish development. <i>actb1</i> is shown as a loading control. B. RT-PCR analysis of mRNA expression of <i>murca</i> and <i>murcb</i> in adult zebrafish tissues. <i>myl1</i> and <i>myog</i> are shown as controls for skeletal muscle contamination of other tissue samples. <i>actb1</i> is shown as a loading control. C. <i>Whole mount in situ</i> hybridization of <i>murcb</i> mRNA in zebrafish embryos at 48 hpf. Top: lateral view. Bottom left: magnified lateral view. Bottom right: magnified dorsal view. D. TALEN construct for <i>murcb</i> mutagenesis. Sequencing results for the <i>murcb</i>^<i>s983</i> allele. Schematic of wild-type and predicted mutant proteins. The <i>murcb</i>^<i>s983</i> lesion leads to a premature stop codon after 83 missense amino acids starting at amino acid 28. Helical region (HR) domains are indicated in blue. The red arrowhead points to the TALEN target site. The red bar indicates the region of the mutant protein that is out of frame. The green bar indicates the antigen used to generate the antibody used in this work. E. Anti-Cavin4/Murc immunofluorescence (IF) micrographs of 10 μm transverse sections of skeletal muscle prepared from 10 wpf sibling and <i>murcb</i>^{<i>s983/s983</i>} fish. No 1° ab indicates control samples incubated only with secondary antibodies.</p