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Mitochondrial Biology Lab

Dept of Biochemistry, Indian Institute of Science, Bangalore.

Protein Translocation Across Mitochondrial Inner Membrane.

Mitochondria are the 'power plants' of the cell, are essential not just for energy generation but also as a site for a variety of metabolic reactions. 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 (1) characterize the human mitochondrial import motor and translocon components, (2) Dynamic interaction studies of translocon components and (3) Mechanism and regulation of import motor component's interactions with TIM23 channel in order to understand fundamental aspects of the mitochondrial protein transport process with respect to mitochondrial biogenesis in higher organisms.

Folding in Cell: Mechanism and Regulation.

The proper folding of nascent and/or denatured polypeptide chains into their biologically active conformations require the assistance of other pre-existing proteins known as molecular chaperones. Despite considerable progress in the biochemical and biophysical analyses of such chaperone proteins, very little is known about the actual mechanism of protein folding under cellular conditions. In vitro experiments are unable to reflect the precise physicochemical conditions and other complex regulatory mechanisms that exist within the cell. It is known that successful folding of polypeptide chains and prevention of aggregation is mediated by highly organized chaperone families like the Hsp70, Hsp40/J-proteins and chaperonins like TRic. The Hsp70 and J-protein genes have proliferated during the course of evolution and their products exist in virtually every cellular compartment, including cytosol, nucleus, ER and mitochondrial matrix. Yet, owing to their critical function in cell survival, it is found that these genes show a high extent of evolutionary conservation.

The Saccharomyces cerevisiae genome encodes for 12 Hsp70s and 22 J-proteins, while human genome analysis has revealed 13 Hsp70s and 41 J-protein members. As, a long term goal we will probe the exact mechanism regulating Hsp70 action in the cell and dissect the intricate functional network between Hsp70 and J-proteins at different cellular locations. Our lab will also investigate how these 'chaperone machineries' prevent aggregation of proteins and aid proper folding, using yeast and mammalian model systems.

Iron-Sulphur Clusters (Fe/S centers) Biogenesis.

Iron-Sulphur clusters act as essential electron carriers and enzyme cofactors in many proteins. They play a vital role in a wide range of cellular processes including electron transfer in oxidative phosphorylation, control of oxidative stress inside the cell, DNA repair, iron homeostasis and ribosome biogenesis. These moieties are indispensable for the activity of certain critical enzymes involved in metabolic pathways of biomolecules including carbohydrates, fatty acids and nucleotides. Any defects in Fe/S cluster biogenesis can lead to various biochemical and genetic disorders such as Friedreich ataxia, X-linked sideroblastic anemia, cerebellar ataxia, Xeroderma pigmentosum etc.

From experimental studies, it has been found that mitochondria perform a central role in synthesis and assembly of Fe/S centers in vast majority of proteins that are targeted into different cellular locations. Biogenesis of Fe/S clusters can be divided into two major events. Initially the Fe/S cluster is assembled on a scaffold protein that provides a transient platform for this process. The second major step of biogenesis involves the release of the scaffold-bound Fe/S cluster and its transfer to apoprotein by coordination with specific aminoacid ligands. This step is specifically assisted by a chaperone system comprised of the Hsp70 family member, the J-proteins and the nucleotide exchange factor. Our goal is to understand the complex chaperone network involved in Fe/S cluster biogenesis in mammalian mitochondria.

Reactive oxygen species.

Mapping ROS signaling networks in eukaryotic systems: 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 (OH°). It is generated primarily as a byproduct of cellular metabolism through leakage of electrons by the electron transport chain (ETC) in mitochondria. A basal level of ROS is essential as a signaling molecule for multiple cellular functions. Cellular redox homeostasis is critically maintained by equilibrium between ROS production and its removal through the involvement of well-defined antioxidant machinery. Any alteration in redox balance generates severe oxidative stress leading to multiple cellular damages. The association of ROS is well known in several pathological conditions, including neurodegeneration, cancer progression, type 2 diabetes mellitus and atherosclerosis. Therefore, our long term goal is to identify novel ROS regulators and elucidate their intricate signaling pathways in different pathophysiological conditions.

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.