Sweeny Elizabeth A, DeSantis Morgan E, Shorter James
Department of Biochemistry and Biophysics, University of Pennsylvania, USA.
J Vis Exp. 2011 Sep 30(55):3190. doi: 10.3791/3190.
Hsp104 is a hexameric AAA+ protein(1) from yeast, which couples ATP hydrolysis to protein disaggregation (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure2). This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils. Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease as well as amyloid forms of PrP. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease and Huntington's disease. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors. To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102 kDa protein with a pI of -5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His(6)-tag removal compared to a previous purification method from E. coli. Moreover, our protocol is more facile and convenient than two more recent protocols.
热休克蛋白104（Hsp104）是一种来自酵母的六聚体AAA +蛋白（1），它将ATP水解与蛋白质解聚耦合起来（图1）。这种活性赋予了两个关键的选择优势。首先，Hsp104使无序聚集体复性，使酵母在各种蛋白质错误折叠应激（包括热休克）后能够存活。其次，Hsp104对交叉β淀粉样纤维进行重塑，使酵母能够利用无数朊病毒（传染性淀粉样蛋白）作为有益和可遗传表型变异的储存库。值得注意的是，Hsp104直接重塑淀粉样前体寡聚体和淀粉样纤维，包括由酵母朊病毒蛋白Sup35和Ure2组成的那些。这种淀粉样重塑功能是酵母Hsp104的一个特殊方面。大肠杆菌的同源物ClpB无法重塑淀粉样前体寡聚体或淀粉样纤维。除了令人困惑的动物外，在所有生命王国中都发现了Hsp104的同源物。实际上，动物细胞是否拥有任何将蛋白质解聚与复性（而不是降解）耦合的酶系统仍然未知。因此，我们和其他人提出，Hsp104可能被开发为一种治疗剂，用于治疗与特定蛋白质错误折叠成有毒淀粉样前体寡聚体和淀粉样纤维相关的各种神经退行性疾病。目前没有直接针对与这些疾病相关的聚集物的治疗方法。然而，Hsp104可溶解由α-突触核蛋白组成的有毒寡聚体和淀粉样纤维，这些与帕金森病以及PrP的淀粉样形式有关。重要的是，Hsp104可减少蛋白质聚集，并改善帕金森病和亨廷顿舞蹈病啮齿动物模型中的神经退行性变。理想情况下，为了优化治疗并最小化副作用，Hsp104将被设计和增强，以选择性地重塑所讨论疾病核心的特定聚集体。然而，对Hsp104如何解聚如此多样的聚集结构和不相关蛋白质的结构和机制了解有限，这阻碍了这些努力。为了了解Hsp104的结构和机制，研究纯蛋白并用最少的成分重建其解聚酶活性至关重要。Hsp104是一种102 kDa的蛋白质，pI为 -5.3，在ADP或ATP存在下，或在无核苷酸时高蛋白浓度下会形成六聚体。在这里，我们描述了一种从大肠杆菌中纯化高活性、稳定Hsp104的优化方案。使用大肠杆菌可简化大规模生产，我们的方法可快速可靠地用于众多Hsp104变体。与之前从大肠杆菌中纯化的方法相比，我们的方案提高了Hsp104的纯度并简化了His(6)-标签去除。此外，我们的方案比最近的另外两种方案更简便、更方便。
