Heat shock proteins: A review of the molecular chaperones☆☆☆★

The emergence of sophisticated molecular biology methods has resulted in major advantages in the basic science knowledge of vascular diseases. However, this new knowledge often is poorly understood by our readers because the methods and mechanisms of molecular and cellular biology are complex and most developments have been made since their formal training was complete. In publishing invited reviews of some of the key issues in basic science, we aim to provide vascular surgeons with basic background information that is pertinent to the understanding of the underlying basic science.

Anton Sidawy, MD K. Wayne Johnston, MD Robert B. Rutherford, MD

Heat shock proteins are ubiquitous proteins found in the cells of all studied organisms. Many types of stress, including heat, induce expression of a family of genes known as the heat shock protein genes. Heat shock proteins originally were discovered when it was observed that heat shock produced chromosomal puffs in the salivary glands of fruit flies (Drosophilia). The DNA sequence that makes up this family of genes is highly conserved across species. This family of genes originally was named because of their expression after exposure to heat. However, the genes are now known to be induced by a wide variety of environmental or metabolic stresses that include the following: anoxia, ischemia, heavy metal ions, ethanol, nicotine, surgical stress, and viral agents. Thus, the term “heat shock protein” is a misnomer because many agents other than heat induce the expression of the heat shock protein gene. Consequently, “stress protein” is the preferred term. Stress proteins are critically important because they appear to be necessary in the critical step of three-dimensional folding of some newly formed proteins within the cell. In fact, they ensure that newly formed polypeptides proceed correctly through folding and unfolding to eventually achieve a functional shape (Fig. 1).

Stress proteins also assist in the repair of denatured proteins or promote their degradation after stress or injury. They have been referred to as “molecular chaperones” because of this function.

It is thought that stress proteins are produced in response to nonlethal stress to protect organisms from subsequent severe stress that would otherwise be lethal. In the case of exposure to heat, this phenomenon has been called “thermotolerance” and has launched many experiments in which an association has been found between the heat shock response and protection against other stresses, such as hypoxia or ischemia. The addition of one type of stress may provide protection against other types of insults, which results in cross-tolerance. As examples, stress protein induction by hyperthermia may provide protection during a subsequent arterial injury or exposure to a heavy metal may provide subsequent protection against heat or ischemic injury. This thermotolerance treatment strategy has proved successful in experimental models of cardiac ischemia, arterial injury, endotoxic shock, renal and hepatic ischemia, ethanol-induced gastric ulcerations, and skeletal muscle ischemia-reperfusion.

Stress proteins belong to a multigene family and range in molecular size from 8 to 150 kd (Table I).

The nomenclature of these proteins and genes can be confusing because different nomenclature has been used in publications on this topic. The stress proteins typically are named according to their molecular size. The 70-kd protein is referred to as Hsp70, and the gene coding for that protein would be hsp70. Many of the stress proteins are present continuously (constitutive expression), and expression of other proteins is increased by stress (stress inducible). Stress proteins are rapidly induced through transcription (messenger RNA production from DNA occurs in minutes) and translation (protein production from messenger RNA) mechanisms. Gene transcription is controlled by heat shock transcription factors. Different members of the heat shock transcription factor family may be activated by specific stresses. Inactive heat shock factors exist as monomers. However, once activated, they trimerize into an active form that is capable of binding to the promotor site of the stress protein gene and initiating transcription and translation (Fig. 2).

Abnormal levels of stress proteins have been found in a number of disorders, including atherosclerosis, congestive heart failure, fever, infection, aging, Alzheimer's disease, malignant diseases, and autoimmune disorders. There is a growing body of evidence that some stress proteins may be associated with atherosclerosis. Experimentally, arteriosclerotic lesions can be induced by immunization with Hsp60/65. Hsp60/65 is found in high concentrations in human arteriosclerotic lesions, and there is a correlation between anti-Hsp60/65 antibodies and atherosclerosis. In addition to being involved in specific disease processes, the stress proteins may play a key regulatory role in cell death pathways (apoptosis) that involve DNA and protein synthesis. These proteins now are being implicated in the aging process. It appears that there is decreased expression of stress protein genes and decreased activity of HSF-1 during aging. These factors may make aging tissues more susceptible to oxidative stress injury.

Members of the Hsp70 family are the most extensively studied group of stress proteins to date. Some members of the Hsp70 family are expressed constitutively, and others are strictly stress inducible. The constitutively expressed protein shares about 95% sequence homology (identity of the DNA sequence) with the inducible form of Hsp70. However, little is known about its function within the cell. Upregulation of the inducible form of Hsp70 has been most closely associated with the development of thermotolerance.

The 90-kd (Hsp90) family of proteins represents one of the most abundant proteins in mammalian cells, yet its synthesis still increases after stress. It appears that Hsp90, in conjunction with Hsp70 and Hsp56, binds, stabilizes, and maintains the estrogen receptor complex in an active confirmation. Hsp90 appears to interact with multiple intracellular proteins and signal transduction pathways. Hsp90 serves a regulatory role by binding to and either inhibiting or stimulating the activity of its target protein. Thus, Hsp70 and Hsp90 are ubiquitous in all tissues, but some of the smaller stress proteins may have a more specialized role in the vascular system.

Hemeoxygenase (Hsp32), a rate-limiting enzyme in the degradation of Heme, is stress induced and is abundant in myocardial cells. Hsp32 is induced by sheer stress and may mediate nitric oxide–dependent platelet inhibition and vasodilatation. There is no direct evidence that Hsp32 functions as a chaperone, but its overexpression during stress events indicates that it may function in this fashion. Hsp25/27 influences the cell cytoskeleton (actin polymerization) and may be involved in cell migration. Physiologic stress increases the phosphorylation of Hsp27. Phosphorylation of Hsp25 occurs via the mitogen-activated protein kinases. Mitogen-activated protein kinases are involved in the intracellular signaling cascade and are activated during ischemia-reperfusion. Many of the small stress proteins that are present in the cytosolic compartment may be important in cardiovascular biology. The 20-kd protein, Hsp20, found in vascular smooth muscle, is a substrate for protein kinase and probably has a role in the maintenance of vascular tone and vessel wall remodeling. Ubiquitin is a small 8-kd stress protein that may facilitate targeting and removal of other proteins denatured during the stress event.

Stress proteins play a critical role in the maintenance of normal cellular homeostasis. These proteins almost certainly have a pivotal role in cell cycle progression and cell death (apoptosis) and are involved in many disease processes, including cardiovascular disease. Currently, the manipulation of stress proteins remains cumbersome because hyperthermia and pharmocologic manipulations are relatively nonspecific. Eventually, as we gain more insight into the exact role and function of these fascinating molecules, the clinical manipulation of the stress proteins will almost certainly prove beneficial.