A Newly Uncovered Group of Distantly Related Lysine Methyltransferases Preferentially Interact with Molecular Chaperones to Regulate Their Activity

Methylation is a post-translational modification that can affect numerous features of proteins, notably cellular localization, turnover, activity, and molecular interactions. Recent genome-wide analyses have considerably extended the list of human genes encoding putative methyltransferases. Studies on protein methyltransferases have revealed that the regulatory function of methylation is not limited to epigenetics, with many non-histone substrates now being discovered. We present here our findings on a novel family of distantly related putative methyltransferases. Affinity purification coupled to mass spectrometry shows a marked preference for these proteins to associate with various chaperones. Based on the spectral data, we were able to identify methylation sites in substrates, notably trimethylation of K135 of KIN/Kin17, K561 of HSPA8/Hsc70 as well as corresponding lysine residues in other Hsp70 isoforms, and K315 of VCP/p97. All modification sites were subsequently confirmed in vitro. In the case of VCP, methylation by METTL21D was stimulated by the addition of the UBX cofactor ASPSCR1, which we show directly interacts with the methyltransferase. This stimulatory effect was lost when we used VCP mutants (R155H, R159G, and R191Q) known to cause Inclusion Body Myopathy with Paget's disease of bone and Fronto-temporal Dementia (IBMPFD) and/or familial Amyotrophic Lateral Sclerosis (ALS). Lysine 315 falls in proximity to the Walker B motif of VCP's first ATPase/D1 domain. Our results indicate that methylation of this site negatively impacts its ATPase activity. Overall, this report uncovers a new role for protein methylation as a regulatory pathway for molecular chaperones and defines a novel regulatory mechanism for the chaperone VCP, whose deregulation is causative of degenerative neuromuscular diseases.

甲基化（methylation）是一种翻译后修饰（post-translational modification），可影响蛋白质的诸多特性，尤其包括细胞定位、周转、活性与分子相互作用。近年来的全基因组分析（genome-wide analyses）已大幅扩充了编码推定甲基转移酶（putative methyltransferases）的人类基因列表。针对蛋白质甲基转移酶（protein methyltransferases）的研究表明，甲基化的调控功能并不局限于表观遗传学（epigenetics），目前已发现众多非组蛋白底物（non-histone substrates）。本研究报道了一类全新的远缘相关推定甲基转移酶家族的研究发现。亲和纯化联合质谱分析（affinity purification coupled to mass spectrometry）结果显示，这类蛋白对各类分子伴侣（chaperones）具有显著的结合偏好性。基于质谱谱图数据，我们成功鉴定出底物中的甲基化位点，尤其是KIN/Kin17的K135三甲基化（trimethylation）、HSPA8/Hsc70的K561三甲基化，以及其他Hsp70同工型（isoforms）中的对应赖氨酸残基，还有VCP/p97的K315。所有修饰位点均在体外实验中得到验证。针对VCP的研究发现，METTL21D介导的甲基化可被UBX辅因子ASPSCR1增强，而我们证实ASPSCR1可直接与该甲基转移酶相互作用。当使用已知可引发骨佩吉特病伴包涵体肌病和额颞痴呆（Inclusion Body Myopathy with Paget's disease of bone and Fronto-temporal Dementia，IBMPFD）及/或家族性肌萎缩侧索硬化（familial Amyotrophic Lateral Sclerosis，ALS）的VCP突变体（R155H、R159G和R191Q）时，这种促进效应会消失。K315位于VCP首个ATP酶（ATPase）/D1结构域的Walker B基序（Walker B motif）附近。我们的研究结果表明，该位点的甲基化会负向调控其ATP酶活性。综上，本研究揭示了蛋白质甲基化作为分子伴侣（molecular chaperones）调控通路的全新功能，并阐明了分子伴侣VCP的新型调控机制——而VCP的失调正是退行性神经肌肉疾病（degenerative neuromuscular diseases）的致病诱因之一。