Molecular mechanism of the Hsp70 Chaperone network

70 kDa热休克蛋白（Hsp70）家族的分子伴侣是细胞蛋白质量监测网络的核心组成部分。没有其他种类的伴侣具有如此多样的功能。hsp70通过其蛋白折叠功能影响许多调节信号过程和细胞控制回路，影响细胞稳态、增殖、分化和细胞程序性死亡。因此，hsp70参与了许多病理生理过程，如癌症、神经变性、炎症和多种病原体的感染。这种多种功能的基础是Hsp70s底物结合域与客户蛋白中短退化氨基酸序列基序的镊子状相互作用，这种相互作用受Hsp70s核苷酸结合域复杂的变抗机制调节。hsp70是由j结构域蛋白（JDP）家族的伴侣蛋白靶向的，Hsp70-client复合体的寿命由核苷酸交换因子（nef）调节。本研究项目的总体目标是进一步加深我们对Hsp70机制的分子理解，不仅是单个Hsp70，单个JDP和单个NEF的三方核心机器，而且在其寡聚状态下，作为多个Hsp70， JDP和NEF的复杂网络，它们可能相互合作或竞争。阐明细胞Hsp70系统及其调控的复杂性将为开发药物奠定更好的基础，这些药物可能以更具体的方式增强或抑制该机制的活性，以对抗癌症、神经变性和潜在的感染等疾病。具体目标是：(1)开发一种高通量检测hsp70与jdp靶向氨基酸序列基序相互作用的传感器；(2)阐明Hsp70系统中寡聚化的功能意义；(3)绘制人类细胞核细胞质区Hsp70-JDP网络的互联性。为了实现这些目标，我们将采用遗传、生化和细胞生物学方法，包括诱变、基于发光的相互作用筛选、荧光共振能量转移和荧光终身成像。

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.