GroEL is a protein which belongs to the chaperonin family of molecular chaperones, and is found in many bacteria. It is required for the proper folding of many proteins. To function properly, GroEL requires the lid-like cochaperonin protein complex GroES. In eukaryotes the organellar proteins Hsp60 and Hsp10 are structurally and functionally nearly identical to GroEL and GroES, respectively, due to their endosymbiotic origin.
HSP60 is implicated in mitochondrial protein import and macromolecular assembly. It may facilitate the correct folding of imported proteins, and may also prevent misfolding and promote the refolding and proper assembly of unfolded polypeptides generated under stress conditions in the mitochondrial matrix. HSP60 interacts with HRAS and with HBV protein X and HTLV-1 protein p40tax. HSP60 belongs to the chaperonin (HSP60) family. Note: This description may include information from UniProtKB.
Alternate Names: 60 kDa chaperonin, Chaperonin 60, CPN60, Heat shock protein 60, HSP-60, HuCHA60, Mitochondrial matrix protein P1, P60 lymphocyte protein, HSPD1
Heat shock protein 60 (HSP60) is a mitochondrial chaperonin that is typically held responsible for the transportation and refolding of proteins from the cytoplasm into the mitochondrial matrix. In addition to its role as a heat shock protein, HSP60 functions as a chaperonin to assist in folding linear amino acid chains into their respective three-dimensional structure. Through the extensive study of groEL, HSP60’s bacterial homolog, HSP60 has been deemed essential in the synthesis and transportation of essential mitochondrial proteins from the cell's cytoplasm into the mitochondrial matrix. Further studies have linked HSP60 to diabetes, stress response, cancer and certain types of immunological disorders.
Discovery
Mammalian HSP60 was first reported as a mitochondrial P1 protein. It was subsequently cloned and sequenced by Radhey Gupta and coworkers. The amino acid sequence showed a strong homology to GroEL. It was initially believed that HSP60 functioned only in the mitochondria and that there was no equivalent protein located in the cytoplasm. Recent discoveries have discredited this claim and have suggested that there is a recognizable difference between HSP60 in the mitochondria and in the cytoplasm. A similar protein structure exists in the chloroplast of certain plants. This protein presence provides evidence for the evolutionary relationship of the development of the mitochondria and the chloroplast by means of endosymbiosis.
Structure Under normal physiological conditions, HSP60 is a 60 kilodalton oligomer composed of monomers that form a complex arranged as two stacked heptameric rings. This double ring structure forms a large central cavity in which the unfolded protein binds via hydrophobic interactions. This structure is typically in equilibrium with each of its individual components: monomers, heptamers, and tetradecamers. Recent studies have begun to suggest that in addition to its typical location in the mitochondria, HSP60 can also be found in the cytoplasm under normal physiological conditions.
Each subunit of HSP60 has three domains: the apical domain, the equatorial domain, and the intermediate domain. The equatorial domain contains the binding site for ATP and for the other heptameric ring. The intermediate domain binds the equatorial domain and the apical domain together. The intermediate domain induces a conformational change when ATP is bound allowing for an alternation between the hydrophilic and hydrophobic substrate binding sites. In its inactive state, the protein is in a hydrophobic state. When activated by ATP, the intermediate domain undergoes a conformational change that exposes the hydrophilic region. This insures fidelity in protein binding. Chaperonin 10 aids HSP60 in folding by acting as a dome-like cover on the ATP active form of HSP60. This causes the central cavity to enlarge and aids in protein folding. See the above figure for further detail on the structure.
The mitochondrial HSP60 sequence contains a series of G repeats at the C-terminal. The structure and function of this sequence is not quite known. The N-terminal contains a pre-sequence of hydroxylated amino acids, namely arginine, lysine, serine, and threonine, which serve as directors for the importation of the protein into the mitochondria.
The predicted structure of HSP60 includes several vertical sine waves, alpha helices, beta sheets, and 90 degree turns. There are regions of hydrophobicity where the protein presumably spans the membrane. There are also three N-linked glycosylation sites at positions 104, 230, 436. The sequence and secondary structure for the mitochondrial protein are illustrated in the above image obtained from the Protein Data Bank.
Newer information has begun to suggest that the HSP60 found in the mitochondria differs from that of the cytoplasm. With respect to the amino acid sequence, the cytoplasmic HSP60 has an N-terminal sequence not found in the mitochondrial protein. In gel electrophoresis analysis, significant differences were found in the migration of cytoplasmic and mitochondrial HSP60. The cytoplasmic HSP60 contains a signal sequence of 26 amino acids on the N terminus. This sequence is highly degenerate and is capable of folding into amphiphilic helix. Antibodies against HSP60 targeted both the mitochondrial and cytoplasmic form. Nonetheless, antibodies against the signal sequence targeted only the cytoplasmic form. Under normal physiological condition, both are found in relatively equal concentrations. In times of stress or high need of HSP60 in either the cytoplasm or the mitochondria, the cell is capable for compensating by increasing the presence of HSP60 in one compartment and decreasing its concentration in the opposite compartment.
Function
Common Heat shock proteins are amongst the most evolutionarily conserved of proteins. The significant function, structural, and sequential homology between HSP60 and its prokaryotic homolog, groEL, demonstrates this level of conservation. Moreover, HSP60’s amino acid sequence bears a similarity to its homolog in plants, bacteria, and humans. Heat shock proteins are primarily responsible for maintaining the integrity of cellular proteins particularly in response to environmental changes. Stresses such as temperature, concentration imbalance, pH change, and toxins can all induce heat shock proteins to maintain the conformation of the cell’s proteins. HSP60 aids in the folding and conformation maintenance of approximately 15-30% of all cellular proteins. In addition to HSP60’s typical role as a heat shock protein, studies have shown that HSP60 plays an important role in the transport and maintenance of mitochondrial proteins as well as the transmission and replication of mitochondrial DNA.
Mitochondrial protein transport HSP60 possesses two main responsibilities with respect to mitochondrial protein transport. It functions to catalyze the folding of proteins destined for the matrix and maintains protein in an unfolded state for transport across the inner membrane of the mitochondria. Many proteins are targeted for processing in the matrix of the mitochondria but then are quickly exported to other parts of the cell. The hydrophobic portion HSP60 is responsible for maintaining the unfolded conformation of the protein for transmembrane transport. Studies have shown how HSP60 binds to incoming proteins and induces conformational and structural changes. Subsequent changes in ATP concentrations hydrolyze the bonds between the protein and HSP60 which signals the protein to exit the mitochondria. HSP60 is also capable of distinguishing between proteins designated for export and proteins destined to remain in the mitochondrial matrix by looking for an amphiphilic alpha-helix of 15-20 residues. The existence of this sequence signals that the protein is to be exported while the absence signals that the protein is to remain in the mitochondria. The precise mechanism is not yet entirely understood.
DNA metabolism In addition to its critical role in protein folding, HSP60 is involved in the replication and transmission of mitochondrial DNA. In extensive studies of HSP60 activity in Saccharomyces cerevisiae, scientists have proposed that HSP60 binds preferentially to the single stranded template DNA strand in a tetradecamer like complex Kaufman, BA. Studies on mitochondria DNA nucleoids in Saccharomyces cerevisiae: identification of bifunctional proteins. In Genetics and Development, UT Southwestern Medical Center at Dallas, Dallas, TX. 241pp. This tetradecamer complex interacts with other transcriptional elements to serve as a regulatory mechanism for the replication and transmission of mitochondrial DNA. Mutagenic studies have further supported HSP60 regulatory involvement in the replication and transmission of mitochondrial DNA.Mutations in HSP60 increase the levels of mitochondrial DNA and result in subsequent transmission defects.
Cytoplasmic vs mitochondrial HSP60 In addition to the already illustrated structural differences between cytoplasmic and mitochondrial HSP60, there are marked functional differences. Studies have suggested that HSP60 plays a key role in preventing apoptosis in the cytoplasm. The cytoplasmic HSP60 forms a complex with proteins responsible for apoptosis and regulates the activity of these proteins. The cytoplasmic version is also involved in immune response and cancer. These two aspects will be elaborated on later. Extremely recent investigations have begun to suggest a regulatory correlation between HSP60 and the glycolytic enzyme, 6-phosphofructokinase-1. Although not much information is available, cytoplasmic HSP60 concentrations have influenced the expression of 6-phosphofructokinase in glycolysis. Despite these marked differences between the cytoplasmic and mitochondrial form, experimental analysis has shown that the cell is quickly capable of moving cytoplasmic HSP60 into the mitochondria if environmental conditions demand a higher presence of mitochondrial HSP60.
Synthesis and assembly HSP60 is typically found in the mitochondria and has been found in organelles of endosymbiotic origin. HSP60 monomers form two heptameric rings that bind to the surface of linear proteins and catalyze their folding in an ATP dependent process. HSP60 subunits are encoded by nuclear genes and translated into the cytosol. These subunits then move into the mitochondria where they are processed by other HSP60 molecules. Several studies have shown how HSP60 proteins must be present in the mitochondria for the synthesis and assembly of additional HSP60 components. There is a direct positive correlation between the presence of HSP60 proteins in the mitochondria and the production of additional HSP60 protein complexes.
The kinetics of assembly of HSP60 subunits into the 2-heptameric rings takes two minutes. The subsequent protease-resistant HSP60 is formed in a half-time of 5–10 minutes. This rapid synthesis indicates that there is an ATP-dependent interaction where the formed HSP60 complex stabilizes the intermediate of the HSP60 assembly complex, effectively serving as a catalyst. The necessity of preexisting HSP60 in order to synthesize additional HSP60 molecules supports the endosymbiotic theory of the origin of mitochondria. There must have been a rudimentary prokaryotic homologous protein that was capable of similar self-assembly.
Immunological role As discussed above, HSP60 has generally been known as a chaperonin which assists in protein folding in mitochondria. However, some new research has indicated that HSP60 possibly plays a role in a “danger signal cascade” immune response. There is also mounting evidence that it plays a role in autoimmune disease.