Gade, M. | Search bioRxiv

bioRxiv2022

Nucleoside-driven specificity of DNA Methyltransferase

We have studied adenosine binding specificities of two bacterial DNA methyltransferase Taq methyltransferase (M.TaqI), and HhaI methyltransferase (M.HhaI). While these DNA methyltransferases have similar cofactor binding pocket interactions, experimental data showed different specificity for novel cofactors ((SNM) (S-guanosyl-L-methionine (SGM), S-cytidyl-L-methionine (SCM), S-uridyl-L-methionine (SUM)). Protein dynamics corroborate the experimental data on the cofactor specificities. For M.TaqI the specificity for S-adenosyl-L-methionine (SAM) is governed by the tight binding on the nucleoside part of the cofactor, while for M.HhaI the degree of freedom of the nucleoside chain allows the acceptance of other bases. The experimental data proves a catalytically productive methylation by M.HhaI binding pocket for all the SNM. Our results suggest a new route for successful design of unnatural SNM analogues for methyltransferases as a tool for cofactor engineering. Table of Content O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/513954v2_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@2562aforg.highwire.dtl.DTLVardef@71de9forg.highwire.dtl.DTLVardef@1cead77org.highwire.dtl.DTLVardef@1de8682_HPS_FORMAT_FIGEXP M_FIG TOC: Methylation by DNA methyltransferase is nucleobase dependent. While M.Taq1 is specific for SAM, M.HhaI is promiscuous for other SAM analogs C_FIG

biochemistry↗

bioRxiv2022

Transcriptomic analysis of aging mouse sciatic nerve reveals early pathways leading to sarcopenia

BackgroundSarcopenia, the age-associated decline in skeletal muscle mass and strength, has long been considered a disease of muscle only, but accumulating evidence suggests that sarcopenia could originate from the neural components controlling muscles. To identify early molecular changes in the efferent nerves that may drive sarcopenia initiation, we performed a longitudinal transcriptomic analysis of the sciatic nerve in aging mice. MethodsSciatic nerve and gastrocnemius muscle were obtained from young adult, middleaged, old, and sarcopenic (5,18, 21 and 24 months old, respectively) C57BL/6J female mice (n=6 per age group). Sciatic nerve RNA was extracted and subjected to RNA sequencing (RNA-seq), with real-time quantitative reverse transcription PCR (qRT-PCR) validation of differentially expressed genes (DEGs). Functional enrichment analysis of clusters of genes associated with patterns of gene expression across age groups was performed. Sarcopenia was confirmed with qRT-PCR of previously established markers of sarcopenia onset in gastrocnemius muscle. ResultsWe detected 33 significant DEGs in sciatic nerve of 18-month-old mice compared to 5-month-old mice (absolute value of fold change > 2; false discovery rate [FDR] < 0.05) which we validated with qRT-PCR of the three top up- and down-regulated genes. Up-regulated genes were associated with circadian rhythm and the AMPK signaling pathway, while down-regulated genes were associated with biosynthesis and metabolic pathways and circadian rhythm. Strikingly, we detected a significant increase in Myog expression (log2 fold change = 18.93, FDR q-value = 1.54x10-12) in sciatic nerve of 18-month-old mice, before up-regulation in muscle was observed. We identified seven clusters of genes with similar expression patterns across groups. Functional enrichment analysis of these clusters revealed biological processes that may be implicated in sarcopenia initiation including extracellular matrix organization and circadian regulation of gene expression. ConclusionsGene expression changes in mouse peripheral nerve can be detected prior to overt clinical onset of sarcopenia. These early molecular changes we report shed a new light on biological processes that may be implicated in sarcopenia initiation and pathogenesis. Future studies will validate which of the key changes we reported have disease modifying and/or biomarker potential.

neuroscience↗