Frontiers in Structural Biology

The HSP70 family of molecular chaperones plays a significant role in cancer. Notably, the inducible protein Hsp72 becomes expressed in many cancers, often to high levels and underlies escape of tumor cells from senescence and increased tumor initiation and metastasis. Examination of database suggests that mutation within the ORFs of HSP70 genes in cancer is relatively rare suggesting a requirement for intact function. At the molecular level, Hsp72 is thought to chaperone key proteins in tumorigenesis and permit their accumulation in the malignant cell. In addition, an important role for Hsp72 in RNA metabolism is emerging, indicating mechanisms potentially involving the RNA binding protein HuR. The existence of multiple HSP70 pseudogenes may also be important for future studies of long non-coding RNA (lncRNA) regulation through this family of chaperones. As the significance of this family of chaperones in cancer emerges, small molecule inhibitors have been developed as future potential cancer pharmaceuticals. We discuss the targeting of individual HSP70 families at key functional domains in the proteins.

The 70 kDalton (Hsp70) chaperones perform a variety of functions in living cells, the most crucial being assisting correct protein folding, refolding misfolded proteins, and participating in iron-sulfur cluster biogenesis. The chaperones consist of the nucleotide-binding domain which, upon transitions between the ADP- to ATPbound state, undergoes slight conformational changes, which trigger major conformational changes in the conformation of the whole molecule, ultimately leading to substrate binding or release. This chapter summarizes our work on the simulations of the chaperone cycle by means of all-atom and coarse-grained molecular dynamics, and on modeling the structure and interactions of two complexes that are formed during the process of iron-sulfur biogenesis: the binary complex composed of the Iron-sulfur protein 1 and the Jac1 Hsp40 cochaperone from yeast, and the ternary complex composed of the Iron-sulfur protein 1, the Jac1 Hsp40 cochaperone, and the Stressseventy subfamily Q protein 1 Hsp70 chaperone from yeast.

High-resolution techniques, such as X-ray crystallography and nuclear magnetic resonance, are not always capable of providing insight into the quaternary structure of full-length forms of eukaryotic chaperones. This has somewhat limited the field’s ability to understand the mechanisms by which chaperones regulate and specify their functions. To fill this information gap, small angle X-ray scattering (SAXS) has been used to gain insight into the quaternary structure of chaperones and co-chaperones in the absence and in the presence of their ligands. This chapter will review selected structural biology publications of the Hsp70 system, in which SAXS was used to investigate the quaternary structure of these molecular chaperones, and will examine how analytical ultracentrifugation can be used as an important tool to validate SAXS data.

Nature employs multiple repeat protein scaffolds in order to promote protein-protein interactions. In this sense, TPR proteins participate in different natural pathways, especially in diverse processes of eukaryotic cells. An important aspect for cellular homeostasis is the maintenance of the folding of recently synthesized peptides as well as all mature proteins such as SHRs. Since, an aberrant protein folding drives loss of function, this effect induce the expression or modulate the function of molecular chaperones. Hsp90 and Hsp70 with the cooperation of cochaperones are involved in the stabilization of several proteins implicated in signaling, and in the tumor phenotype of various cancers. Therefore, cochaperones are essential component of the cytosolic Hsp90 folding pathway, since their function comprises targeting clients to Hsp90, modulating their conformational changes or Hsp90 ATPase activity. The scientific knowledge in the properties and structure of chaperones and the searching of compounds that can modulate their function on different cellular mechanism has became remarkably important in the treatment of diverse diseases specially those in which a protein mechanism is involved. A description of diverse structural aspects of Hsp90-TPR cochaperones interaction in the context of SHR, as well as a structural comparison of different isoforms of Hsp90 is presented in this chapter. Besides, the primary and new biotechnological approaches inhibiting Hsp90 interactions are also discussed, since Hsp90 and its interactions have become the main targets for inhibiting the growth of specific tumor types.

Molecular chaperones and especially chaperonins are primarily known as special members of the family of heat shock proteins which participate in folding, assembly, transmembrane transport and degradation of a wide variety of cellular proteins both in prokaryotes and eukaryotes. The multifunctional character of chaperones underlies their involvement in many diseases. One of their functions is binding to polypeptides lacking a rigid tertiary structure, i.e., to those with many exposed hydrophobic amino acids. The current review is focused on interaction of polypeptides of this type with GroEL, the best-studied Escherichia coli chaperonin. The literature data on driving forces of their interaction, localization of substrate polypeptides on the GroEL surface, and the effect of GroEL ligands on its interaction with substrate polypeptides are considered. Some biotechnological applications of this event are also discussed.

Protein homeostasis depends on the ability of molecular chaperones to assist proteins with complex folding patterns to achieve their native state. An important molecular chaperone found in the eukaryotic cytosol is the chaperonin containing tailless complex polypeptide 1 (CCT, also called TRiC). CCT is composed of eight homologous subunits, each with ATPase activity, that form a double ring complex with protein folding cavities in the center of each ring. CCT folds primarily nascent polypeptides that have yet to achieve their native state by binding the unfolded protein in the folding cavity of the open, nucleotide-free conformation of CCT. Upon binding and hydrolysis of ATP, CCT undergoes a conformational change that simultaneously creates a lid over the folding cavity and releases the nascent protein into the cavity. The protein is then allowed to fold in this confined space isolated from other proteins in the cell. Dissociation of phosphate and ADP allows CCT to relax back into its open conformation and release the protein if it has achieved its native conformation. Recent studies suggest that CCT initially extends and unfolds at least some substrate proteins by binding them at sites on opposite sides of the folding cavity. Interestingly, subunits on one side of the CCT ring bind and hydrolyze ATP more effectively than subunits on the other side, suggesting a sequential release mechanism first from one side and then the other, effectively dictating the folding trajectory of the protein. The CCT folding process is facilitated by co-chaperones that deliver substrates for folding, stabilize folding intermediates or promote substrate release. In this manner, CCT and its cochaperones accommodate the folding of many protein substrates with diverse folding patterns.

The Hsp70 chaperone system is central to the proteostasis network in the cytosol of human cells. Hsp70 has roles in protein folding, the prevention and clearance of aggregates, and various other biological processes. A range of co-chaperone proteins regulate Hsp70, among these the Hsp40/DNAJ proteins are important activators of Hsp70 function. Stress inducible Hsp70 and its non-inducible form Hsc70 are ubiquitously expressed, and the most abundant DNAJs are DNAJA1 (Hdj2), DNAJA2 and DNAJB1 (Hsp40, Hdj1). Here, the mechanisms of Hsc70/Hsp70 with the DNAJs is reviewed. An overview of biological functions of the major DNAJs is then presented, particularly in the context of protein misfolding diseases.