The Structural and Functional Study of the Human Mitochondrial Hsp60 Chaperonin in Neurodegenerative Diseases
Proteins are essential elements that are responsible for a variety of cellular activities within organisms, including catalyzing metabolic reactions, DNA replication, responding to stimuli, providing structure to cells and organisms, and transporting molecules. After protein synthesis in the ribosome, the unfolded protein need to fold into their unique compact structures so that they can perform their full biological functions. The biologically active structure of a protein is referred to the native-state of the protein with biological activity. The process of protein folding is one of the most important and challenging research topics of contemporary biochemistry, especially for its central role in life. The folding of polypeptide chains into their unique three-dimensional structure is of fundamental importance in biology. Chaperonins are a class of proteins that assemble into barrel-shaped chamber to renature the misfolded, non-functional proteins to the folded, functional state. The human mitochondrial heat shock protein 60 (hsp60) functions as a chaperonin, folding proteins in the human mitochondrial matrix. D3G and V72I point mutations in Hsp60 have been shown to lead to the neurodegenerative disorders MitCHAP-60 and SPG13, respectively. A D3G mutation in hsp60 leads to MitCHAP-60, an early onset neurodegenerative disease characterized by muscle weakness, limb spasticity and involuntary eye movement. Another hsp60 mutation, V72I, has been linked with SPG13, a form of hereditary spastic paraplegia, characterized by progressive weakness, spasticity in the lower limbs, impaired vision, deafness, and cognitive impairment. There are no specific treatments to prevent, slow, or reverse these two diseases. The detailed mechanism of the two diseases are also unknown. The β-subunit of the human mitochondrial ATP synthase has been co-immunoprecipitated together with the Hsp60 indicating that the β-subunit is likely being folded by the chaperonin. The biological effects of the interaction have not been studied but we hypothesize that the lack of active β-subunit is the cause of the neurological disorders. We expressed and purified recombinant wild type and mutant hsp60 complexes and analyzed them via electron microscopy, dynamic light scattering (DLS), and in-vitro protein-folding assays. We characterized the wild type hsp60 conformational changes upon nucleotide binding and postulate a likely catalytic cycle with the various conformational intermediates. Negative-stain electron microscopy along with DLS suggest that the D3G and V72I complexes fall apart when treated with ATP or ADP and are therefore unable to fold denatured substrates such as α-lactalbumin, malate dehydrogenase (MDH), and the β-subunit of ATP synthase. Our data suggests that hsp60 plays a crucial role in folding important players in aerobic respiration such as the β-subunit of the ATP synthase and MDH. D3G and V72I mutations in hsp60 impair its ability to fold these substrates leading to abnormal ATP synthesis and the MitCHAP-60 and SPG13 neurodegenerative disorders. These studies indicate that that hsp60 is important for folding members of mitochondrial energy producing pathways such as the ATP synthase β-subunit or malate dehydrogenase. Mutations that lead to inactive Hsp60 complexes indirectly lead to MitCHAP-60 and SPG13. The hydrophobic flexible C-terminal end of each subunit in the hsp60 chaperonin, protrudes inside the central cavity. The central cavity is the place where the protein folding cycle happens. We generated and characterized E. coli strains expressing Hsp60 C-terminus deletion chaperonin protein to study the hsp60 protein folding cycle. If the C-terminus deletion of hsp60 chaperonin impaired the interaction between neighboring subunits, the yeast strains will dead. Researchers have shown that the C-terminus deletion hsp60 chaperonin remained capable of the binding activity with the cochaperonin hsp10 in the nucleotide-dependent manner. By generating the C-terminus 26 amino acid deletion (CTD) hsp60, we try to explain the mechanism of the hsp60 chaperonin protein folding cycle with the unfolded substrates or the folded, unreleased substrates by the protein reconstruction analyze. We found that the protein folding activity of the hsp60-MDH is much lower than the hsp60 wildtype, even hsp60-CTD can still form the complex. We also found the hsp60-CTD binds the denatured malate dehydrogenase (MDH) by using the native gel and dynamic light scattering. Keywords: MitCHAP-60, Hereditary Spastic Paraplegia, SPG-13, Hsp60, Chaperonin, Protein folding, ATP Synthase β-Subunit, Neurodegeneration.
Wang, Jinliang, "The Structural and Functional Study of the Human Mitochondrial Hsp60 Chaperonin in Neurodegenerative Diseases" (2018). ETD Collection for University of Texas, El Paso. AAI13421724.