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Nowadays heat shock proteins (HSP) seem to be everywhere and can apparently do anything. But it was not always so. For many years genes were academic , curiosities apparently confined to the salivary glands of fruit flies. Their study was initiated by the discovery of a new gene expression pattern, through a happy accident involving the overheating of a salivary gland preparation on a microscope stage (). This was first reported as, “A new puffing pattern induced by temperature shock and DNP in ” in 1962 (). However, it was to take another 10–15 years before the first HSP mRNA was isolated (). Around this time (1978) the HSP “went global” and were discovered in mammalian tissue culture cells, in , in yeast, and in plants (; ; ; ; ; ). The heat shock field emerged as a major study area in experimental biology at the 1982 meeting , held at the Cold Spring Harbor Laboratory (Ashburner, 1982). At this time, however, the functions of the HSP remained mysterious and the details of regulation of gene expression were only beginning to emerge. All that was known was that the proteins appeared to possess “homeostatic activity” and were (as they are to this day) associated with resistance to heat shock and other stresses (Chapter 2). However, with the intensive international effort and the wealth of experimental systems available in the early 1980s, the concept began to emerge that the HSP belonged to a new kind of proteins which function to modify the structures of other proteins.

The heat shock or stress response has been studied mainly as a cellular response. Most of the data come from bacterial cells, eukaryotic microorganisms (yeast primarily), and cultured animal cells. Often these cultured cells are tumor cell lines, i.e., cells that are functionally eukaryotic microorganisms as a consequence of genetic changes that change their social behavior and proliferative control. These systems have provided useful information about stress protein function and their roles in the defensive cellular state of cytoprotection. However, a full understanding of stress response biology in complex multicellular organisms requires different thinking and different models. This conclusion stems from the paradigm that the basic unit of function in animals and plants is not the individual cell but the tissue. Therefore stress response biology in these complex biological systems is primarily about tissue-level protection. Ultimately we would like to know how these responses are deployed in humans and how these inducible defenses may be used to prevent tissue damage from disease and from surgical intervention.

Bacterial cells have limited abilities to modify and choose their dynamic environment. They utilize information processing systems to monitor their surroundings constantly for important changes. Among the appropriate responses to environmental changes are alterations in physiology, development, virulence, and location. In most species, highly sophisticated global regulatory networks modulate the expression of genes. These effects are mediated in large part through the activation or repression of mRNA transcript initiation by DNA-binding proteins, σ-factors, and corresponding signal transduction systems. This adaptive response is based on appropriate genetic programmes allowing them to respond rapidly and effectively to environmental changes that impair growth or even threaten their life. Cellular homeostasis is achieved by a multitude of sensors and transcriptional regulators, which are able to sense and respond to changes in temperature (heat and cold shock), external pH (alkaline and acid shock), reactive oxygen species (hydrogen peroxide and superoxide), osmolarity (hyper- and hypoosmotic shock), and nutrient availability to mention the most important ones. These changes are often called stress factors, and stresses can come at a sudden (catastrophic stress) or grow and grow (pervasive stress). Each stress factor leads to the induction of a subset of genes, the stress genes coding for stress proteins. It should be mentioned that challenge to any stress factor will not only result in induction of genes, but also in repression or even turn off of a subset of genes, but the underlying mechanisms are largely unknown. While some genes are induced by only one single stress factor, others respond to several. The former are termed specific stress genes and the latter general stress genes.

The unfolded protein response (UPR) is a multifaceted signal transduction pathway that is activated in all eukaryotic organisms in response to changes in the environment of the endoplasmic reticulum (ER) that adversely affect protein folding and assembly in the secretory pathway. The response is generally thought to protect cells from the transient alterations that can occur in the ER environment and serves to restore homeostatis in this organelle. Under extreme or prolonged stress, apoptotic pathways can be activated to destroy the cell. Recent studies reveal that in addition to protecting cells from adverse physiological conditions, the UPR plays an essential role in the normal development and functioning of some tissues and can be a major contributor to the pathology of some diseases.

Since Ritossa’s seminal discovery in 1962 that the puffing pattern changes of salivary gland polytene chromosomes can be induced by heat shock and chemical treatment (), the heat shock response (HSR) has served as an excellent model and paradigm of inducible gene expression. During the ensuing decades considerable evidence has accumulated, from diverse areas of biology, about the regulation of the stress response during development, homeostatic maintenance of organs and organisms, and pathophysiological conditions. From bacteria to man, environmental stress, cell growth, differentiation, and pathophysiological states are all known to induce the rapid and reversible synthesis of evolutionary conserved set of proteins commonly termed, heat shock proteins (HSPs). HSPs, acting as molecular chaperones, play essential roles in protein folding, trafficking, higher order assembly and degradation of proteins thereby ensuring survival under both stressful and extreme physiological conditions (; ; ).

Induction of hsp genes by heat and chemicals is strictly dependent on the presence in their non-transcribed regulatory sequences of so-called heat shock elements (abbreviated HSEs) (; ). HSEs are arrays of three or more modules of the sequence element NGAAN (or AGAAN) or variations thereof (; ). Heat shock factors (HSFs) are defined as proteins that are capable of specifically binding HSE sequences. The first attempts at identifying and/or purifying an HSF were undertaken by () and (). Whereas certain organisms appear to express a single HSF, others, including plants, avian species and mammals express multiple related but distinct factors (reviewed, e.g., in ). One of these proteins, HSF1, rapidly took center stage because it was found to be the major heat- and chemically induced HSE DNA-binding protein in mammalian cells and, therefore, likely to be the major transcription factor that activates or enhances transcription of hsp genes in response to these stresses (; ). Gene knockouts confirmed this expected role of HSF1 (; ; for , see ). As it must be capable of carrying out the same function, the single HSF present in certain organisms will also be referred to as HSF1 herein for the sake of simplicity.

The small heat shock proteins (sHsps) family comprises several members found in prokaryotes and eukaryotes with important variations in the number of members between species (e.g., ,10 in mammals, ,20 in plants) (; ). Their common feature is a central α-crystallin domain. The core structure of the α-crystallin domain, rather than its amino acid sequence, is conserved between species. This ,90 amino acid domain presents a well-preserved double β-sheet sandwich structure, which is surrounded by N- and C-terminal domains whose length and sequence can vary extensively between and within species giving rise to proteins ranging from 11 to 42 kilodaltons (kDa). These extensions have been suggested to confer functional specificities to the different sHsps. Another property of sHsps is their propensity to form large oligomers, which are in dynamic equilibrium with smaller subunits (dimers, trimers, or tetramers depending on the sHsp). From X-ray and electron microscopic data, these particles have a diameter of 10–18 nm with a hollow core. The number of subunits can vary from 12 () to 24 ( and yeast). The quaternary structure is quite variable with polydisperse complexes in the range of 400 to over 800 kDa (see ; for recent detailed reviews on the structure of sHsps).

All living organisms respond to conditions such as mild heat shock, oxidative stress, reperfusion injury, or other stressful situations by increasing the expression of specific sets of protective proteins that have been commonly referred to for more than 30 years as heat shock proteins (hsps). Most, if not all, of these proteins are also expressed in the absence of stress. Many of these highly conserved proteins function as molecular chaperones to guide changes in conformational states that are critical to the synthesis, folding, translocation, assembly, and degradation of other proteins (). Additionally, they can act to inhibit the irreversible aggregation of denatured proteins caused by protein-damaging stresses and, in some instances, assist in the refolding of denatured proteins (; ). The principal hsps of mammalian cells can be classified into several sequence-related families that are characterized by molecular size, i.e., the hsp25/hsp27 (small heat shock protein), the hsp40 (J-domain proteins), the hsp60, the hsp70, the hsp90, and the hsp110/Sse family. The regulation of hsps is coordinated by heat shock transcription factors (HSFs) that interact with heat shock elements (HSEs) in the promoters of the hsp genes (). The hsps are principally found in the cytoplasm, nucleus, and mitochondria.

Anfinsen discovered over 30 years ago that the information necessary to dictate the tertiary structure of a protein was contained in the primary amino acid sequence (). However, Afinsen’s experiments were performed in vitro with dilute solutions of a small globular protein (ribonuclease A), and these conditions are distinct from the highly crowded environment inside a cell where protein conformations vary and protein concentrations may be as high as 300 mg/mL (). In fact, most newly synthesized proteins inside a cell would fail to fold efficiently without the assistance of additional “machines.” Important components of these machines are molecular chaperones, and three of the most abundant classes of molecular chaperones are the Hsp70s, Hsp40s, and Hsp90s. In part, the Hsp70 and Hsp40 chaperones prevent protein aggregation and catalyze polypeptide folding because they bind to hydrophobic patches on unfolded or misfolded proteins. Hsp70 function can be regulated by specific Hsp40 partners and by nucleotide exchange factors (NEFs). Hsp90 chaperones are regulated by a distinct group of proteins, and although they also associate with polypeptides, Hsp90s do not bind preferentially to exposed hydrophobic amino acid patches ().

This chapter surveys evidence that Hsp40 and Hsp70 act as a molecular machine to disassemble protein complexes. Recent papers have discussed whether Hsp70 actively unfolds proteins during translocation across membranes (), whether it fragments aggregates or extracts polypeptides (), and whether it acts a “holdase” or a “foldase” (,). These are related questions because they ask whether Hsp70 exerts a force on its clients during these processes. The first two sections of the chapter discuss the structures of Hsp70 and Hsp40 with an eye toward their interactions and conformational changes, that is, the “moving parts”. Hsp40-Hsp70 chaperone machines have three levels of functional organization: (i) the articulation of Hsp70 domains, (ii) the interactions with J-domain-containing protein(s), and (iii) interactions with additional protein factors for targeting (e.g., TPR proteins) and regulation (e.g., GrpE). These additional factors are diverse and probably not fundamental to a disassembly activity, and thus they generally will not be discussed. Subsequent sections discuss the machine-like role of Hsp40-Hsp70 in several major classes of biochemical systems: the disassembly of protein complexes, protein degradation, protein translocation across membranes, and protein folding.