Heat Shock Proteins

Cellular proteins are exposed to various kinds of stresses such as changes in temperature, pH and metal ions concentrations which induces protein misfolding and aggregation. The intracellular accumulation of protein aggregates adversely affects cellular viability and is the underlying basis of various human diseases. Thus each cell has evolved set of proteins known as heat shock proteins that prevents protein aggregation by interacting with exposed hydrophobic patches of aggregation prone proteins, and thereby protects cells from the deleterious effects of protein aggregates. Heat shock proteins are ubiquitously present in all organisms, and highly conserved across different species from bacteria to humans. In addition to their role in preventing protein aggregation,Hsps are also involved in protein synthesis, protein trafficking, assembly of multi-protein complexes and the protein degradation. Based upon the approximate molecular weight,Hsps are categorizedinto different families such as Hsp100, Hsp90, Hsp70, Hsp60 or Hsp40 family. Many of the Hsp families possess multiple members which are highly homolougous yet perform both redundant as well as non-redundant functions. As the primary role of Hsps is to maintain protein homeostasis, these have been extensively explored as therapeutic targets against number of protein misfolding disorders such asneurodegenerative disorders and various forms of cancer. The Hsps are also major therapeutic targets against malaria and various types of bacterial infections. Considerable efforts have been made over the past two decades to identify modulators of manyHsps and many of these modulators are currently being evaluated for their efficacy in different phases of clinical trials.

Heat Shock Protein 100 (Hsp100)
Hsp100 family members belong to AAA+ superfamily of ATPases that are involved in diverse cellular activities including protein disaggregation, intracellular trafficking and DNA replication. Based upon the number of nucleotide binding domains, the family of proteins are divided into two classes; Class I and Class II containing two or one nucleotide binding domain respectively. Each nucleotide binding domain contains Walker A and Walker B motifs which are critical for nucleotide binding and hydrolysis. The members of Hsp100 family that have disaggregation function belong to ClpB/Hsp104 subfamily. The ClpB and Hsp104 subfamily of proteins are highly conserved in bacteria, and lower eukaryotes however not yet found in animals and humans. The absence of ClpB/Hsp104 members in metazoans cytosol makes them attractive therapeutic targets against number of infectious pathogens. The yeast Hsp104 and bacterial ClpB uses energy from ATP hydrolysis to disassemble larger protein aggregates and thus confer thermotolerance to host organism. ClpA, ClpX and ClpY are some of other members of Hsp100 family that binds to unfolded protein substrates and direct their delivery to associated peptidase subunits ClpP or ClpQ for further degradation.

Heat Shock Protein 90
Hsp90 is ubiquitously expressed in many organisms and highly conserved across different species. Most eukaryotes including yeast and humans contain two highly homologous cytosolic Hsp90 isoforms one of which is constitutively present whereas other is stress inducible. Though not essential in prokaryotes, the presence of at least one of the Hsp90 isoforms is critical for cellular viability in eukaryotes. Each monomer of dimeric Hsp90 consists of an N-terminal ATP binding domain, middle domain and a C-terminal domain. Similar to Hsp70, Hsp90 activity is regulated by various co-chaperones with which it interacts. Hsp90 is involved in the maturation of kinases, steroid hormone receptors and nonsignal transduction clients. As many kinases require Hsp90 for its maturation, the chaperone has been extensively targeted against various forms of cancers.

Hsp70 and Hsp40 chaperones
Hsp70s are ubiquitously present from bacteria to humans and are highly conserved. Eukaryotes contain multiple highly homologues members of cystosolic Hsp70s which perform similar as well as distinction functions. Some of Hsp70 members are constitutively expressed whereas others are stress inducible. In addition to cytosolic Hsp70s, members of the family are also present in different cellular organelles such as endoplasmic reticulum (Bip or Grp78 in humans), mitochondria (mtHsp70 or GRP75 in humans) and lysosomes. The family members are involved in variety of functions such as protein folding, cellular signalling, protein trafficking and protein degradation. Structurally, Hsp70 consists for three domains, a nucleotide binding ATPase domain (NBD), substrate binding domain (SBD) and C-terminal domain (CTD). Hsp70s have low intrinsic ATPase activity which is further stimulated by various co-chaperones such as Hsp40s. The ATP bound Hsp70 has low binding affinity for the substrate. The binding of substrate at SBD followed by Hsp40 stimulated ATP hydrolysis induces conformational changes in Hsp70s that traps substrate by closing of C-terminal lid on SBD. Nucleotide exchange factors then exchanges ADP with ATP leading to opening of the C-terminal lid and beginning of new reaction cycle.
Hsp40s are the largest and the most diverse family among heat shock proteins. Hsp40s are required not only to stimulate Hsp70 ATPase activity but also for its functional specificity. In addition, Hsp40s are also known to deliver various substrates to Hsp70s. Some members of Hsp40 family also show chaperoning activity independent of Hsp70s. The Hsp40s are characterized by the presence of a conserved J-domain which mediates their interaction with NBD of Hsp70. Similar to Hsp70s, multiple Hsp40 members exists in each organism and based on the presence/absence of Gly/Phe rich region and cys repeats, are divided into three groups; type I, II or III. Hsp70 alone or Hsp70-Hsp40 interactions has been explored extensively as therapeutic targets against various protein misfolding disorders.

Figure: Schematic representation of Hsp70 cycle

Hsp60
Hsp60 family of proteins are abundantly expressed in bacterial cytosol, and different organelles of plant cells (chloroplast) and other eukaryote cells (primarily mitochondria). Some of Hsp60 members as constitutively expressed whereas other are stress inducible. Based upon the requirement of co-chaperones, Hsp60 members are classified into two major groups, group I and II. Group I requires the presence of co-chaperones and are present in bacterial cytosol or membrane bound organelles such as mitochondria or cholorplast. The bacterial GroEL is one of the extensively studied Hsp60 members of group I that along with GroES (Hsp10) constitutes major chaperoning complex that promotes ATP dependent folding of most the aggregation prone partially unfolded protein substrates in bacteria. GroEL exists as a double-ring complex with each ring composed of 7 monomers. GroES also forms a heptameric ring and acts as a lid to the cavity formed within GroEL. Group II members of Hsp60 such as chaperonin containing TCP-1 complex (CCT), possess an intrinsic lid like structure and thus does not require additional co-factor for their in vivo function. The crucial role of Hsp60 in maintenance of protein homeostasis primarily of mitochondrial proteins is evident from findings that multiple mutations that adversely affect the protein refolding activity of the chaperone is associated with various human diseases.