Hsp70
The 70 kilodalton heat shock proteins are a family of conserved ubiquitously expressed heat shock proteins. Proteins with similar structure exist in virtually all living organisms and play crucial roles in the development of cancer, neurodegeneration, apoptosis, regulating sleep, and much more. Intracellularly localized Hsp70s are an important part of the cell's machinery for protein folding, performing chaperoning functions, and helping to protect cells from the adverse effects of physiological stresses. Additionally, membrane-bound Hsp70s have been identified as a potential target for cancer therapies and their extracellularly localized counterparts have been identified as having both membrane-bound and membrane-free structures. There is lot of potential in the Hsp70 protein as a key therapeutic target for developing new drugs for the treatment of sleep disorders, cancer, neurodegeneration, and other related pathological conditions.
Discovery
Members of the Hsp70 family are very strongly upregulated by heat stress and toxic chemicals, particularly heavy metals such as arsenic, cadmium, copper, mercury, etc. Heat shock was originally discovered by Ferruccio Ritossa in the 1960s when a lab worker accidentally boosted the incubation temperature of Drosophila. When examining the chromosomes, Ritossa found a "puffing pattern" that indicated the elevated gene transcription of an unknown protein. This was later described as the "Heat Shock Response" and the proteins were termed the "Heat Shock Proteins".Structure
The Hsp70 proteins have three major functional domains:- N-terminal ATPase domain – binds ATP and hydrolyzes it to ADP. The NBD consists of two lobes with a deep cleft between them, at the bottom of which nucleotide binds. The exchange of ATP and ADP leads to conformational changes in the other two domains.
- Substrate binding domain – is composed of a 15 kDa β sheet subdomain and a 10 kDa helical subdomain. The β sheet subdomain consists of stranded β sheets with upward protruding loops, which enclose the peptide backbone of the substrate. SBD contains a groove with an affinity for neutral, hydrophobic amino acid residues. The groove is long enough to interact with peptides up to seven residues in length.
- C-terminal domain – rich in alpha helical structure acts as a 'lid' for the substrate binding domain. The helical subdomain consists of five helices, with two helices packed against two sides of the β sheet subdomain, stabilizing the inner structure. In addition, one of the helix forms a salt bridge and several hydrogen bonds to the outer Loops, thereby closing the substrate-binding pocket like a lid. Three helices in this domain form another hydrophobic core which may be stabilization of the "lid". When an Hsp70 protein is ATP bound, the lid is open and peptides bind and release relatively rapidly. When Hsp70 proteins are ADP bound, the lid is closed, and peptides are tightly bound to the substrate binding domain.
Function
The Hsp70 system interacts with extended peptide segments of proteins as well as partially folded proteins to cause aggregation of proteins in key pathways to downregulate activity. When not interacting with a substrate peptide, Hsp70 is usually in an ATP bound state. Hsp70 by itself is characterized by a very weak ATPase activity, such that spontaneous hydrolysis will not occur for many minutes. As newly synthesized proteins emerge from the ribosomes, the substrate binding domain of Hsp70 recognizes sequences of hydrophobic amino acid residues, and interacts with them. This spontaneous interaction is reversible, and in the ATP bound state Hsp70 may relatively freely bind and release peptides. However, the presence of a peptide in the binding domain stimulates the ATPase activity of Hsp70, increasing its normally slow rate of ATP hydrolysis. When ATP is hydrolyzed to ADP the binding pocket of Hsp70 closes, tightly binding the now-trapped peptide chain. Further speeding ATP hydrolysis are the so-called J-domain cochaperones: primarily Hsp40 in eukaryotes, and DnaJ in prokaryotes. These cochaperones dramatically increase the ATPase activity of Hsp70 in the presence of interacting peptides.By binding tightly to partially synthesized peptide sequences, Hsp70 prevents them from aggregating and being rendered nonfunctional. Once the entire protein is synthesized, a nucleotide exchange factor stimulates the release of ADP and binding of fresh ATP, opening the binding pocket. The protein is then free to fold on its own, or to be transferred to other chaperones for further processing. HOP can bind to both Hsp70 and Hsp90 at the same time, and mediates the transfer of peptides from Hsp70 to Hsp90.
Hsp70 also aids in transmembrane transport of proteins, by stabilizing them in a partially folded state. It is also known to be phosphorylated which regulates several of its functions.
Hsp70 proteins can act to protect cells from thermal or oxidative stress. These stresses normally act to damage proteins, causing partial unfolding and possible aggregation. By temporarily binding to hydrophobic residues exposed by stress, Hsp70 prevents these partially denatured proteins from aggregating, and inhibits them from refolding. Low ATP is characteristic of heat shock and sustained binding is seen as aggregation suppression, while recovery from heat shock involves substrate binding and nucleotide cycling. In a thermophile anaerobe the Hsp70 demonstrates redox sensitive binding to model peptides, suggesting a second mode of binding regulation based on oxidative stress.
Hsp70 seems to be able to participate in disposal of damaged or defective proteins. Interaction with CHIP –an E3 ubiquitin ligase–allows Hsp70 to pass proteins to the cell's ubiquitination and proteolysis pathways.
Finally, in addition to improving overall protein integrity, Hsp70 directly inhibits apoptosis. One hallmark of apoptosis is the release of cytochrome c, which then recruits Apaf-1 and dATP/ATP into an apoptosome complex. This complex then cleaves procaspase-9, activating caspase-9 and eventually inducing apoptosis via caspase 3 activation. Hsp70 inhibits this process by blocking the recruitment of procaspase-9 to the Apaf-1/dATP/cytochrome c apoptosome complex. It does not bind directly to the procaspase-9 binding site, but likely induces a conformational change that renders procaspase-9 binding less favorable. Hsp70 is shown to interact with Endoplasmic reticulum stress sensor protein IRE1alpha thereby protecting the cells from ER stress - induced apoptosis. This interaction prolonged the splicing of XBP-1 mRNA thereby inducing transcriptional upregulation of targets of spliced XBP-1 like EDEM1, ERdj4 and P58IPK rescuing the cells from apoptosis. Other studies suggest that Hsp70 may play an anti-apoptotic role at other steps, but is not involved in Fas-ligand-mediated apoptosis. Therefore, Hsp70 not only saves important components of the cell but also directly saves the cell as a whole. Considering that stress-response proteins evolved before apoptotic machinery, Hsp70's direct role in inhibiting apoptosis provides an interesting evolutionary picture of how more recent machinery accommodated previous machinery, thus aligning the improved integrity of a cell's proteins with the improved chances of that particular cell's survival.
In mice, exogenous recombinant human Hsp70, delivered intranasally, increases lifespan. Although the maximum lifespan increased only moderately, the overall mortality rate in treated animals was much lower compared with the control group. Also this eHsp70-treatment improves learning and memory of mice in old age, increases their curiosity.
Cancer
Hsp70 is overexpressed in melanoma and underexpressed in renal cell cancer.In breast cancer cell line has been found that not only Hsp90 interacted with estrogen receptor alpha but also Hsp70-1 and Hsc70 interacted with ERα too.
Given the role of heat shock proteins as an ancient defense system for stabilizing cells and eliminating old and damaged cells, this system has been co-opted by cancer cells to promote their growth. Increased Hsp70 in particular has been shown to inhibit apoptosis of cancer cells, and increased Hsp70 has been shown to be associated with or directly induce endometrial, lung, colon, prostate, and breast cancer, as well as leukemia. Hsp70 in cancer cells may be responsible for tumorigenesis and tumor progression by providing resistance to chemotherapy. Inhibition of Hsp70 has been shown to reduce the size of tumors and can cause their complete regression. Hsp70/Hsp90 is a particularly attractive target for therapeutics, because it is regulated by the inhibition of its ATPase activity, while other HSPs are regulated by nucleotides. Several inhibitors have been designed for Hsp70 that are currently in clinical trials, though as of now HSP90 inhibitors have been more successful. In addition, Hsp70 has been shown to be a regulator of the immune system, activating the immune system as an antigen. Thus, tumor-derived Hsp70 has been suggested as a potential vaccine or avenue to target for immunotherapy. Given the increased expression of Hsp70 in cancer, it has been suggested as a biomarker for cancer prognostics, with high levels portending poor prognosis. An oncogenic mechanism illustrates how extracellular vesicles expressing HSP70 are produced by proliferative Acute Lymphoblastic Leukemia cells and can target and compromise a healthy hematopoiesis system during leukemia development.