Protein methyltransferases mediate posttranslational modifications of both histone and nonhistone proteins. Whereas histone methylation is well-known to regulate gene expression, the biological significance of nonhistone methylation is poorly understood. Methyltransferase-like 21c (Mettl21c) is a newly classified nonhistone lysine methyltransferase whose in vivo function has remained elusive. Using a Mettl21c LacZ knockin mouse model, we show here that Mettl21c expression is absent during myogenesis and restricted to mature type I (slow) myofibers in the muscle. Using co-immunoprecipitation, MS, and methylation assays, we demonstrate that Mettl21c trimethylates heat shock protein 8 (Hspa8) at Lys-561 to enhance its stability. As such, Mettl21c knockout reduced Hspa8 trimethylation and protein levels in slow muscles, and Mettl21c overexpression in myoblasts increased Hspa8 trimethylation and protein levels. We further show that Mettl21c-mediated stabilization of Hspa8 enhances its function in chaperone-mediated autophagy, leading to degradation of client proteins such as the transcription factors myocyte enhancer factor 2A (Mef2A) and Mef2D. In contrast, Mettl21c knockout increased Mef2 protein levels in slow muscles. These results identify Hspa8 as a Mettl21c substrate and reveal that nonhistone methylation has a physiological function in protein stabilization.

The outer membranes (OM) of Gram-negative bacteria contain a class of proteins (TBDTs) that require energy for the import of nutrients and to serve as receptors for phages and protein toxins. Energy is derived from the proton motif force (pmf) of the cytoplasmic membrane (CM) through the action of three proteins, namely, TonB, ExbB, and ExbD, which are located in the CM and extend into the periplasm.

Skeletal muscles contain heterogeneous myofibers that are different in size and contractile speed, with type IIb myofiber being the largest and fastest. Here, we identify methyltransferase‐like 21e (Mettl21e), a member of newly classified nonhistone methyltransferases, as a gene enriched in type IIb myofibers. The expression of Mettl21e was strikingly up‐regulated in hypertrophic muscles and during myogenic differentiation in vitro and in vivo. Knockdown (KD) of Mettl21e led to atrophy of cultured myotubes, and targeted mutation of Mettl21e in mice reduced the size of IIb myofibers without affecting the composition of myofiber types. Mass spectrometry and methyltransferase assay revealed that Mettl21e methylated valosin‐containing protein (Vcp/p97), a key component of the ubiquitin‐proteasome system. KD or knockout of Mettl21e resulted in elevated 26S proteasome activity, and inhibition of proteasome activity prevented atrophy of Mettl21e KD myotubes. These results demonstrate that Mettl21e functions to maintain myofiber size through inhibiting proteasome‐mediated protein degradation.—Wang, C., Zhang, B., Ratliff, A. C., Arrington, J., Chen, J., Xiong, Y., Yue, F., Nie, Y., Hu, K., Jin, W., Tao, W. A., Hrycyna, C. A., Sun, X., Kuang, S. Methyltransferase‐like 21e inhibits 26S proteasome activity to facilitate hypertrophy of type IIb myofibers. FASEB J. 33, 9672–9684 (2019). http://www.fasebj.org

Isoprenylcysteine carboxyl methyltransferase (Icmt) is an integral membrane protein localized to the endoplasmic reticulum that is responsible for the post‐translational α‐carboxyl methylesterification of the C‐terminus in CaaX proteins. CaaX proteins (where C is Cys, a is generally an aliphatic amino acid and X is one of several amino acids) include a variety of regulatory proteins, most notably Ras. The Icmt from S. cerevisiae, Ste14, is comprised of six transmembrane (TM) domains in which TM1 contains a putative dimerization motif, G31XXXG35XXXG39, where G is a glycine amino acid residue and X is a subset of hydrophobic amino acids. Ste14 has been shown biochemically to form functional homodimers or higher order oligomers, yet further studies must be performed to better understand this mechanism. To explore these elements as possible sequence and/or structural determinants for dimerization, we used cysteine‐scanning mutagenesis to generate a library of single cysteine mutants for each residue in TM1 (amino acids 25–42). These TM1 cysteine mutants were characterized with immunoblot analyses to assess protein expression, activity, stability, and the ability to form dimers. We determined that residues S27, Y28, L30, G31, G35, and G39 are critical for both methyltransferase activity and the structural stability of the protein. Interestingly, several of the TM1 cysteine mutants that are proposed to lie on the same face of an α‐helix in a helical wheel diagram formed dimers upon the addition of sulfhydryl specific cross‐linkers. We further validated the homodimerization of Ste14 by size exclusion chromatography, multi‐angle light scattering and small‐angle X‐ray scattering. Together, these data suggest that the active form of the Ste14 Icmt is, in fact, a homodimer and these data will ultimately be useful as we further explore the mechanism of action of Ste14.Support or Funding Information1. Purdue Research Foundation2. National Institutes of Health/NIGMS (Grant #: 5R01GM106082‐05)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

No abstract