Understanding how proteins accurately fold into three-dimensional structures in the cell is a central problem in modern biology. After ribosomal synthesis, the nascent polypeptides must actively fold into their native conformations to function properly within the cell. Protein misfolding leads to cellular accumulation of incorrectly folded proteins, which is the cause of many diseases including Alzheimer's, Huntington, Prion diseases, and Cancer. Ordered protein folding in the cellular cramped chaos is only possible under the supervision of specialized proteins, collectively referred as "Molecular Chaperones" which are belongs to Hsp70 and Hsp40/J-class. These highly conserved group of proteins recognize and stabilize partially folded intermediates of proteins during cellular processes such as protein translation, transport across organellar membranes, folding and degradation, as well as facilitating the refolding of proteins damaged after exposure of cells to stress. The long-term goal of our research is to understand the intricate functional network of Hsp70 and Hsp40/J-class in the cytosol and different cell organelles. By utilizing yeast and mammalian model systems, my lab will investigate the cellular functions of molecular chaperones using a combination of experimental tools from genetics and cell biology with biochemistry and biophysics. We will address the role(s) of molecular chaperones in following different physiological functions.
1. Protein Translocation Across Mitochondrial Inner Membrane.
Mitochondria are essential organelles and the 'power plants' of the cell. The biogenesis of mitochondria requires the import and folding of hundreds of proteins that are synthesized on cytosolic ribosomes. Utilizing the 'yeast' model system it has been demonstrated that transport of proteins across the inner mitochondrial membrane 'translocon/import channel' is driven by an import motor, whose components have been highly conserved in evolution. Yeast 'import motor' consists of five essential sub-units (mtHsp70, Tim44, Mge1, Pam18 and Pam16) and one non-essential subunit called Pam17. A critical core component of this machine is the major mitochondrial 70 kDa heat shock protein (mtHsp70), tethered to the import channel via its interaction with an essential peripheral membrane component, Tim44. Recently, two additional heat shock components of the import motor, a J-protein (Pam18 /Tim14) and J-like protein (Pam16/Tim16) have been identified. Pam18 and Pam16 form a stable heterodimer and regulate the import process by modulating the activity of the 'import motor'. However, the mechanism of regulation of import motor is poorly understood. The orthologs of yeast 'import motor' components are reported in human mitochondria comprising the part of 'human import motor'. However, the human import system differs in composition due to the presence of additional components and the architecture of the import machinery itself. The regulation of human import motor activity is critical for the proper functioning and maintaining normal mitochondrial physiology. The altered regulation of 'import motor' leads to severe mitochondrial genetic disorders including neuro-degenerative diseases, malignancy, aging and heart failure. Therefore, maintaining an efficient protein transport system in mitochondria is critical for cell function, thus preventing pathophysiology associated with mitochondrial diseases. The long-term goal of this project is to characterize the components of 'human import motor' and its mechanism of regulation in order to understand fundamental aspects of the mitochondrial protein transport process in higher organisms.
2. Protein Folding in Cell: Mechanism and Regulation.
Despite considerable progress in the biochemical and biophysical analysis of molecular chaperones, surprisingly little is known about how protein folding occurs in cellular conditions. Successful folding of newly-translated proteins is mediated by a highly organized chaperone machinery namely the Hsp70, Hsp40/J-proteins and the chaperonin, TRiC which prevents aggregation and assists in proper folding to their native conformations. Hsp70 and J-protein genes have proliferated during evolution and exist in virtually every cellular compartment, including the cytosol, nucleus, ER and mitochondrial matrix. Saccharomyces cerevisiae genome encodes for 12 Hsp70s and 22 J-proteins, while human genome analysis has revealed 13 Hsp70s and 41 J-protein members. The functional relationship amongst the members of these two groups of proteins is not clearly understood. As a long-term goal, we will dissect the intricate functional network between Hsp70 and J-proteins present at different cellular locations in humans. Our lab will also investigate how these 'chaperone machines' prevent aggregation of proteins and aids in proper folding under stress conditions using mammalian model systems.
3. Biogenesis of Iron-Sulphur Clusters (Fe/S centers) in Proteins
Fe/S clusters are vital moiety of proteins involved in diverse cellular process. A vital function of mitochondria is the biogenesis of Fe/S clusters, as Fe/S cluster-containing proteins perform critical roles in cells, including electron transfer in oxidative phophorylation. Mitochondrial molecular chaperones are responsible for the synthesis and assembling the Fe/S centers in vast majority of proteins that are targeted into different cellular locations. We will investigate the molecular mechanisms of Fe/S cluster formation and iron homeostasis in mammalian mitochondria.
4. Role of Heat Shock Proteins in Health and Diseases.
The stress-protective heat-shock proteins are often overexpressed in cells of various cancers and have been suggested to be contributing factors in tumorigenesis. The overexpression of molecular chaperones has also been shown to protect cells against apoptotic cell death. Heat-shock proteins with dual roles as regulators of protein conformation and stress sensors may, therefore have intriguing roles in both cell proliferation and apoptosis. The function of molecular chaperones is also vital for aging process, autoimmunity and the replication of many viruses. The involvement of chaperones, therefore, in such diverse roles suggests novel therapeutic approaches by targeting heat-shock protein function for a broad spectrum of tumor types, various pathogenic disease states, and protein conformational diseases.
5. Reactive oxygen species (ROS) are the chemical species formed by the incomplete reduction of oxygen. ROS includes Superoxide anion (O2-), hydrogen peroxide (H2O2), and hydroxyl radicals (OHo) (r). It is generated primarily as a byproduct of cellular metabolism through leakage of electrons by the electron transport chain (ETC) in mitochondria. Production of reactive oxygen species is a natural phenomenon. An appropriate level of ROS is necessary for the proper signaling in cell. This optimum level is maintained by equilibrium between ROS production and its removal through the involvement of antioxidants. Oxidative stress is generated when there is an alteration in this equilibrium. The contribution to oxidative stress does not solely belong to reactive oxygen species; reactive nitrogen species (RNS) also play an important role. RNS are produced from L-arginine by enzyme nitric oxide synthase. But reactive oxygen species plays the major role in augmentation of oxidative stress. Being a signaling molecule, ROS can regulate many physiological processes. The association of ROS with assorted pathological conditions such as neurodegeneration, type 2 diabetes mellitus, atherosclerosis
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