Molecular mechanism of the Hsp70 Chaperone network

70 kDa热休克蛋白（Hsp 70）家族的分子伴侣是细胞蛋白质质量监测网络的核心组成部分。没有其他类型的伴侣蛋白的功能多样性已被归因于可比。Hsp 70通过其蛋白质折叠功能影响许多调节信号传导过程和细胞控制回路，影响细胞稳态、增殖、分化和程序性细胞死亡。因此，Hsp 70参与许多病理生理过程，如癌症、神经变性、炎症和许多类型病原体的感染。这种广泛功能的基础是Hsp 70 s底物结合结构域与客户蛋白中短的变性氨基酸序列基序的镊子样相互作用，其由Hsp 70 s核苷酸结合结构域的复杂变构机制调节。Hsp 70是由JDP家族的辅伴侣蛋白（cochaperones of J-domain protein，JDP）靶向其客户，Hsp 70-客户复合物的寿命受核苷酸交换因子（nucleotide exchange factors，NEFs）的调节。本研究的主要目标是进一步了解Hsp 70的分子机制，不仅是单个Hsp 70、单个JDP和单个NEF的三方核心机制，但它处于寡聚状态，并且是由多个Hsp 70、JDP和NEF组成的复杂网络，这些Hsp 70、JDP和NEF可能相互合作或竞争。阐明细胞Hsp 70系统及其调控的复杂性将为开发增强或抑制这种机制活性的药物奠定更好的基础，可能以更具体的方式对抗癌症和神经变性等疾病，以及潜在的感染。具体目标是：（1）开发一种用于高通量检测Hsp 70与氨基酸序列基序的JDP靶向相互作用的传感器;（2）阐明Hsp 70系统内寡聚化的功能意义;（3）绘制人类细胞核质区室的Hsp 70-JDP网络的互连性。为了实现这些目标，我们将采用遗传，生物化学和细胞生物学方法，包括诱变，基于发光的相互作用屏幕，荧光共振能量转移，荧光寿命成像。

The molecular chaperones of the 70 kDa heat shock protein (Hsp70) family are central components of the cellular protein quality surveillance network. To no other class of chaperones a comparable diversity of functions have been attributed. Through their protein folding functions Hsp70s influence many regulatory signaling processes and cellular control circuits, impacting cellular homeostasis, proliferation, differentiation and programmed cell death. Thereby Hsp70s are involved in many pathophysiological processes like cancer, neurodegeneration, inflammation and infections with many types of pathogens. The basis for this wide variety of functions is the tweezer-like interaction of Hsp70s’ substrate binding domain with short degenerative amino acid sequence motifs in client proteins that is regulated by an intricate allosteric mechanism by Hsp70s’ nucleotide binding domain. Hsp70s are targeted to their clients by cochaperones of the J-domain protein (JDP) family and the life-time of the Hsp70-client complex is regulated by nucleotide exchange factors (NEFs).The overarching goal of this research projects is to further our molecular understanding of the Hsp70 machinery, not only as a tripartite core machine of a single Hsp70, a single JDP and a single NEF, but in its oligomeric states and as a complex network of multiple Hsp70s, JDPs and NEFs that might cooperate or compete with each other. The elucidation of the intricacies of the cellular Hsp70 system and its regulation will lay a better foundation for developing drugs that enhance or inhibit the activity of this machinery possibly in a more specific way to fight diseases like cancer and neurodegeneration and, potentially, infections. The specific aims are (1) to develop a sensor for high throughput detection of JDP-targeted interaction of Hsp70s with amino acid sequence motifs; (2) to elucidate the functional implications of the oligomerization within the Hsp70 system; and (3) to map the interconnectivity of the Hsp70-JDP network of the nuclear-cytoplasmic compartment of human cells. To achieve these goals we will employ genetic, biochemical, and cell biological methods, including mutagenesis, luminescence-based interaction screens, fluorescence resonance energy transfer, and fluorescence life-time imaging.