Enzyme-engineering Approaches to Dissect Protein Methylation Profiles

描述(由申请人提供) 摘要：表观遗传调控涉及许多生物过程，这些过程中的错误已被认为与许多疾病有关，如癌症。在协调表观遗传学的关键酶中，有60多种人类蛋白质甲基转移酶。越来越多的证据表明，表观遗传多样性需要蛋白质甲基转移酶作用于组蛋白和非组蛋白靶标。然而，由于我们不能明确地描述体内60多种人类蛋白甲基转移酶的靶标，阐明这些过程的生理和病理作用受到了显著的阻碍。为了应对这一巨大的挑战，从而垂直推进表观遗传学研究，我们建议开发一种高度创新的技术，我们称之为蛋白质甲基化的生物正交谱(BPPM)，并将该方法应用于癌症相关蛋白质甲基转移酶的靶向谱分析。结合我们支持性的初步结果，我们设想可以合理地设计蛋白质甲基转移酶以利用S-腺苷-L-蛋氨酸类似物作为替代辅助因子，从而将其靶标以不同的化学基团。不同的修改可以有选择地丰富，并与BPPM的各自记者明确地描述。我们还计划通过采用一种创造性的方法来原位生产SAM类似物，从而利用这种方法在体内进行BPPM。关于预期结果，拟议的工作旨在为蛋白质甲基转移酶的靶标分析提供变革性的技术进步和普遍适用的试剂。与传统方法相比，我们的BPPM具有以下特点：在细胞蛋白质组中使用完整的甲基转移酶具有高度的完整性，通过将特定细胞类型的甲基化特征与指定的甲基转移酶联系起来具有高度的特异性，通过选择性地从蛋白质组中富集修饰的靶标具有高的敏感性。这些结果有望揭示不同蛋白质甲基转移酶之间的甲基化特征如何不同，在正常与病理阶段，或在非侵袭性疾病与侵袭性疾病中。 公共卫生相关性：未受管制的蛋白质甲基化可导致发育异常、神经紊乱和癌症。这一提议的成功完成有望为理解这些疾病过程中涉及的分子机制提供宝贵的工具。由此产生的知识有望加强我们对这些疾病中异常甲基化途径的理解，并可能导致识别用于疾病诊断的新生物标记物或药物干预的靶标。此外，这项研究将有助于对干细胞分化的更广泛的理解，对于干细胞分化，蛋白质甲基化涉及的表观遗传学调节尚未被很好地理解。

DESCRIPTION (Provided by the applicant) Abstract: Epigenetic regulations are involved in numerous biological processes and errors in these processes have been implicated in many diseases such as cancer. Among the key enzymes that orchestrate epigenetics are over 60 human protein methyltransferases. Accumulated evidence showed that epigenetic diversity requires protein methyltransferases to act on histone and nonhistone targets. Nonetheless, elucidating the physiological and pathological roles of these processes has been significantly hindered by our inability to unambiguously profile the targets of the over 60 human protein methyltransferases in vivo. To address this formidable challenge and thus vertically advance the epigenetic research, we propose to develop a highly innovative technology, which we termed Bioorthogonal Profiling of Protein Methylation (BPPM) and, as a paradigm, apply the approach for target profiling of cancer-relevant protein methyltransferases. In conjunction with our supportive preliminary results, we envisioned that protein methyltransferases can be rationally engineered to exploit S-adenosyl-L-methionine analogues as alternate cofactors and thus label their targets with distinct chemical groups. The distinct modifications can be selectively enriched and unambiguously characterized with respective reporters for BPPM. We further plan to leverage the approach for in vivo BPPM by incorporating a creative approach for in situ production of SAM analogues. With regard to expected outcomes, the proposed work is aimed at providing transformative technological advancement and generally applicable reagents for target profiling of protein methyltransferases. Compared with conventional approaches, our BPPM is featured as the high integrity by using intact methyltransferases in context of cellular proteome, the high specificity by linking cell-type-specific methylation profiles to designated methyltransferases, and the high sensitivity by selectively enriching modified targets from proteome. The results are expected to reveal how the methylation profiles differ between diverse protein methyltransferases, in normal versus pathological stages or in non-aggressive versus aggressive diseases. Public Health Relevance: Deregulated protein methylation can cause developmental abnormalities, neurological disorders and cancer. The successful completion of this proposal is expected to provide the valuable tools to understand the molecular mechanisms involved in these disease processes. The resultant knowledge is expected to enhance our understanding of aberrant methylation pathways in these diseases and likely lead to the identification of novel biomarkers for disease diagnosis or targets for pharmacological intervention. Additionally, this research will contribute to a broader understanding of stem cell differentiation, for which protein-methylationinvolved epigenetic regulations have not been well understood.