AdoMet-dependent tRNA methyl transferases

 描述(申请人提供)：外排引起的多重耐药是治疗细菌感染最紧迫的问题之一。抗生素是从细胞中挤出的，不能 达到足够高的细胞内浓度以发挥治疗效果。虽然解决这一问题的努力集中在一次针对一个外排泵，但耐药性突变可能会迅速发展。我们建议以TrmD为靶点，同时减少多个泵的外排，以加速杀菌作用。TrmD是一种细菌特异性的S-腺苷-蛋氨酸(ADO-Met)依赖的tRNA甲基转移酶，它控制阅读框架上蛋白质合成的准确性。TrmD的缺失会导致+1移码(+1FS)错误的积累，从而导致蛋白质合成的提前终止。我们最近发现，在大肠杆菌和其他革兰氏(-)细菌中，多个外排基因在阅读框架的Aug起始密码子附近含有TrmD依赖的密码子。因此，我们假设靶向TrmD可以使所有这些基因的蛋白表达失活。通过一次减少多个泵的药物外流，我们认为靶向TrmD为未得到满足的医疗需求提供了一种新的解决方案。成功的靶向TrmD需要了解其ADOMet结构域及其产品m1G37-tRNA维持的质量控制机制。以E.ColiTrmD(EcTrmD)为模型，我们在以下三个目标中提供了这些先决条件。在目标1中，我们将从分子水平上了解TrmD的ADOMet结构域。这个结构域具有非同寻常的能力，即使在低水平的ADOMet下也能保持甲基化活性。我们将测试 一种假说认为，这种能力是由结构域的不寻常的拓扑蛋白质结折叠所赋予的，该结构域在结合时使甲基供体“弯曲”。使用动力学和细胞分析，我们将确定蛋白质结折叠如何利用Adobe Met结合来促进tRNA结合和促进甲基转移。我们假设，Adobe Met结合和催化活性之间的这种轻松协调是该结构域独特生物学的根源。在目标2中，我们将从机制水平上了解TrmD的m1G37-tRNA产物如何提高核糖体上蛋白质合成的准确性。准确性将取决于m1G37-tRNA在光滑的mRNA序列上减少+1FS错误的能力，以及减少cmo5U34的解码错误的能力，cmo5U34是一种摆动修饰，在天然tRNA中经常伴随着m1G37。在目标3中，我们将在细胞水平上了解TrmD的失活如何减少外排蛋白的合成。我们假设这种外流蛋白的减少将增加传统抗生素在细胞内的积累，导致更快的杀菌肌动蛋白。我们还将使用核糖体图谱来定义TrmD活性的范围，以确定其蛋白表达在TrmD缺乏症中受阻的基因。这将为抗生素靶向的新战略和抗生素发现的新范式提供基础。

 DESCRIPTION (provided by applicant): Multi-drug resistance due to efflux is one of the most pressing issues in treating bacterial infections. Antibiotics are extruded from the cell and cannot reach high enough intracellular concentrations to exert a therapeutic effect. While efforts to address this problem have focused on targeting one efflux pump at a time, resistance mutations can quickly develop. We propose to target TrmD to reduce efflux at multiple pumps simultaneously so as to accelerate bactericidal action. TrmD is a bacterial-specific S-adenosyl-methionine (AdoMet)-dependent tRNA methyl transferase that controls the accuracy of protein synthesis on the reading frame. Loss of TrmD leads to accumulation of +1 frameshift (+1FS) errors, which cause pre-mature termination of protein synthesis. We recently discovered that multiple efflux genes in E. coli and in other Gram (-) bacteria contain TrmD-dependent codons near the AUG start codon of the reading frame. We therefore hypothesize that targeting TrmD can inactivate protein expression of all of these genes. By reducing drug efflux of multiple pumps at once, we propose that targeting TrmD offers a novel solution to an unmet medical need. Successful targeting TrmD requires an understanding of its AdoMet domain and the quality control mechanism maintained by its product m1G37-tRNA. Using E. coli TrmD (EcTrmD) as a model, we provide these prerequisites in the following three aims. In Aim 1, we will develop a molecular-level understanding of the AdoMet domain of TrmD. This domain has the unusual ability to maintain the methylation activity even at low levels of AdoMet. We will test the hypothesis that this ability is conferred by the unusual topological protein knot-fold of the domain that "bends" the methyl donor upon binding. Using both kinetic and cellular assays, we will determine how the protein knot-fold uses AdoMet binding to facilitate tRNA binding and to promote methyl transfer. We hypothesize that this facile coordination between AdoMet binding and catalytic activity is at the root of the unique biology of the domain. In Aim 2, we will develo a mechanistic-level understanding of how the m1G37-tRNA product of TrmD improves the accuracy of protein synthesis on the ribosome. Accuracy will be determined by the ability of m1G37-tRNA to reduce +1FS errors at slippery mRNA sequences and to reduce decoding errors of cmo5U34, a wobble modification that frequently accompanies m1G37 in natural tRNAs. In Aim 3, we will develop a cellular-level understanding of how inactivation of TrmD reduces synthesis of efflux proteins. We hypothesize that this reduction of efflux proteins will increase intracellular accumulation of traditional antibiotics, leading to faster bactericidal actin. We will also define the scope of TrmD activity using ribosome profiling to identify genes whose protein expression is arrested in TrmD deficiency. This will provide the basis for new strategies of antibiotic targeting and new paradigms for antibiotic discovery.