Lysine methyltransferase Smyd2 regulates Hsp90-mediated protection of the sarcomeric titin springs and cardiac function.

Protein lysine methylation controls gene expression and repair of deoxyribonucleic acid in the nucleus but also occurs in the cytoplasm, where the role of this posttranslational modification is less understood. Members of the Smyd protein family of lysine methyltransferases are particularly abundant in the cytoplasm, with Smyd1 and Smyd2 being most highly expressed in the heart and in skeletal muscles. Smyd1 is a crucial myogenic regulator with histone methyltransferase activity but also associates with myosin, which promotes sarcomere assembly. Smyd2 methylates histones and non-histone proteins, such as the tumor suppressors, p53 and retinoblastoma protein, RB. Smyd2 has an intriguing function in the cytoplasm of skeletal myocytes, where it methylates the chaperone Hsp90, thus promoting the interaction of a Smyd2-methyl-Hsp90 complex with the N2A-domain of titin. This complex protects the sarcomeric I-band region and myocyte organization. We briefly summarize some novel functions of Smyd family members, with a focus on Smyd2, and highlight their role in striated muscles and cytoplasmic actions. We then provide experimental evidence that Smyd2 is also important for cardiac function. In the cytoplasm of cardiomyocytes, Smyd2 was found to associate with the sarcomeric I-band region at the titin N2A-domain. Binding to N2A occurred in vitro and in yeast via N-terminal and extreme C-terminal regions of Smyd2. Smyd2-knockdown in zebrafish using an antisense oligonucleotide morpholino approach strongly impaired cardiac performance. We conclude that Smyd2 and presumably several other Smyd family members are lysine methyltransferases which have, next to their nuclear activity, specific regulatory functions in the cytoplasm of heart and skeletal muscle cells. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.

Smyd2 controls cytoplasmic lysine methylation of Hsp90 and myofilament organization.

Protein lysine methylation is one of the most widespread post-translational modifications in the nuclei of eukaryotic cells. Methylated lysines on histones and nonhistone proteins promote the formation of protein complexes that control gene expression and DNA replication and repair. In the cytoplasm, however, the role of lysine methylation in protein complex formation is not well established. Here we report that the cytoplasmic protein chaperone Hsp90 is methylated by the lysine methyltransferase Smyd2 in various cell types. In muscle, Hsp90 methylation contributes to the formation of a protein complex containing Smyd2, Hsp90, and the sarcomeric protein titin. Deficiency in Smyd2 results in the loss of Hsp90 methylation, impaired titin stability, and altered muscle function. Collectively, our data reveal a cytoplasmic protein network that employs lysine methylation for the maintenance and function of skeletal muscle.

Expression of CD133 and other putative stem cell markers in uveal melanoma.

'Cancer stem cells' (CSCs) are tumor cells with stem cell properties hypothesized to be responsible for tumorigenesis, metastatis, and resistance to treatment, and have been identified in different tumors including cutaneous melanoma, using stem cell markers such as CD133. This study explored expression of CD133 and other putative stem cell markers in uveal melanoma. Eight uveal melanoma cell lines were subjected to flow-cytometric (fluorescence-activated cell sorting) analysis of CD133 and other stem cell markers. Eight paraffin-embedded tumors were analyzed by immunohistochemistry for CD133, Pax6, Musashi, nestin, Sox2, ABCB5, and CD68 expressions. Ocular, uveal melanoma, and hematopoietic stem cell distributions of C-terminal and N-terminal CD133 mRNA splice variants were compared by reverse-transcription PCR. Fluorescence-activated cell sorting analysis revealed a population of CD133-positive/nestin-positive cells in cell lines Mel270, OMM 2.3, and OMM2.5. All cell lines studied were positive for nestin, CXCR-4, CD44, and c-kit. Immunohistochemistry identified cells positive for CD133, Pax6, Musashi, nestin, Sox2, ABCB5, and CD68 predominantly at the invading tumor front. C-terminal primers interacting with CD133 splice variant s2 detected a novel variant lacking exon 27. Differential expression of CD133 splice variants was found in iris, ciliary body, retina, and retinal pigment epithelium/choroid as well as in uveal melanoma cell lines. mRNA for nestin, Sox2, and Musashi was present in all studied cell lines. Uveal melanoma such as cutaneous melanoma may therefore contain CSCs. Further experiments are needed to isolate stem cell marker-positive cells, to evaluate their functional properties and to explore therapeutical approaches to these putative CSCs in uveal melanoma.

Conformation-regulated mechanosensory control via titin domains in cardiac muscle.

The giant filamentous protein titin is ideally positioned in the muscle sarcomere to sense mechanical stimuli and transform them into biochemical signals, such as those triggering cardiac hypertrophy. In this review, we ponder the evidence for signaling hotspots along the titin filament involved in mechanosensory control mechanisms. On the way, we distinguish between stress and strain as triggers of mechanical signaling events at the cardiac sarcomere. Whereas the Z-disk and M-band regions of titin may be prominently involved in sensing mechanical stress, signaling hotspots within the elastic I-band titin segment may respond primarily to mechanical strain. Common to both stress and strain sensor elements is their regulation by conformational changes in protein domains.