Protein Recognition for Remodeling and Degradation by Bacterial AAA+ ATPases

描述（由申请人提供）： AAA+蛋白酶负责所有生命领域的细胞内蛋白质降解，受控的ATP依赖性蛋白质降解和受调控的基因表达是定义蛋白质组的伙伴。此外，用于降解的相同酶机制对于分解蛋白质复合物和对抗蛋白质聚集至关重要。由于降解和分解都是固有的破坏性过程，并且底物分解的效率通常是在底物选择的水平上进行的，因此必须了解这些ATP驱动的蛋白质破坏机器指导底物选择的分子原理。该提议集中于细菌AAA+蛋白酶ClpXP、ClpAP和Lon的底物选择的三个领域。目的1集中于阐明两个特定类别的ClpX底物识别信号所使用的设计原理和分子接触。作为这一目标的一部分，我们测试了一些信号的“设计”，使它们优先识别的多蛋白复合物的背景下的hypothesis。我们还建议解决新发现的ClpX N-结构域相互作用基序与N-结构域结合的结构，以进一步了解ClpX识别的机制基础。目的2研究Lon和ClpAP蛋白酶与大肠杆菌的相互作用。coli sHsps（IbpA和IbpB）相互作用，测试这些相互作用如何影响蛋白质质量控制途径的模型。我们很高兴能探索这个新发现的蛋白质质量控制网络的聚集预防（sHsp）和蛋白质破坏（蛋白酶）臂之间的交叉点的功能后果。最终的目标开始解决机制的控制蛋白质降解反应氧化应激和氧化损伤。选择两种蛋白质进行初步研究：B。枯草杆菌过氧化物敏感转录因子PerR和E.大肠杆菌微铁蛋白（Dps.初步数据表明，PerR是特别受到加速降解的LonA蛋白酶时，其组氨酸活性中心是不可逆的氧化。与PerR相反，Dps降解被过氧化物抑制。我们建议隔离的因素负责这些例子的环境控制的监管。这些目标的成功完成将为蛋白酶识别提供实质性的新见解，并可能揭示蛋白质组控制的新范式。 公共卫生相关性： AAA+蛋白酶是许多细菌（包括主要的人类病原体）中的毒力因子。阐明底物识别的规则将是识别不稳定的毒力相关蛋白和理解其稳定性如何导致发病机制的关键。分析蛋白酶和sHsps（抗聚集的蛋白质）之间的联系，并阐明蛋白酶如何识别氧化损伤的蛋白质，有望揭示用于对抗年龄和活性氧相关蛋白毒性的新细胞机制。

DESCRIPTION (provided by applicant): AAA+ proteases are responsible for intracellular protein degradation in all domains of life and controlled ATP-dependent protein degradation and regulated gene expression are partners in defining the proteome. Furthermore, the same enzymatic machinery used for degradation is critical for disassembling protein complexes and antagonizing protein aggregation. As both degradation and disassembly are inherently destructive processes and the efficiency of substrate breakdown is often made at the level of substrate choice, it is imperative to understand the molecular principles guiding substrate selection by these ATP-driven, protein-destruction machines. This proposal focuses on three areas of substrate selection of the bacterial AAA+ proteases ClpXP, ClpAP and Lon. Aim 1 concentrates on elucidating the design principles and molecular contacts used by two specific classes of ClpX substrate-recognition signals. As part of this aim, we test the hyphothesis that some signals are "designed" such that they are preferentially recognized in the context of multi-protein complexes. We also propose to solve the structure of newly-discovered ClpX N-domain-interacting motifs bound to the N-domain to further understand the mechanistic basis of ClpX recognition. Aim 2 dissects how Lon and ClpAP proteases and the E. coli sHsps (IbpA and IbpB) interact, tests models for how these interactions impact protein quality-control pathways. We are excited to explore the functional consequences of this newly-discovered intersection between the aggregation-prevention (sHsp) and protein-destruction (protease) arms of the protein quality-control network. The final aim begins to address mechanisms for control of protein degradation in response to oxidative stress and oxidative damage. Two proteins are chosen for initial studies: the B. subtilis peroxide-sensing transcription factor, PerR and the E. coli mini-ferritin, Dps. Preliminary data indicate that PerR is specifically subject to accelerated degradation by LonA protease when its histidine active center is irreversibly oxidized. In contrast to PerR, Dps degradation is inhibited by peroxide. We propose to isolate factors responsible for these examples of environmentally-controlled regulation. Successful completion of these aims will provide substainal new insights into protease recognition and may uncover new paradigms for contol of the proteome. PUBLIC HEALTH RELEVANCE: AAA+ proteases are virulence factors in many bacteria including major human pathogens. Elucidating rules of substrate recognition will be key to identifying unstable virulence-associated proteins and understanding how their stabilization leads to pathogenesis. Analysis of the connection between proteases and the sHsps (which are proteins that fight aggregation), and elucidating how proteases recognize oxidatively damaged proteins, holds promise for uncovering new cellular mechanisms used to fight age- and reactive oxygen-associated protein toxicity.