For many years, our laboratory has been interested primarily in unravelling the molecular mechanism behind the function of two groups of proteins. One is the sophisticated machinery that mitochondria use for translocating proteins from the cytosol. Most mitochondrial proteins are encoded in the nuclear genome, synthesized in the cytosol as preproteins and then imported into mitochondria. Uptake of these preproteins into the mitochondria is mediated by a number of oligomeric protein complexes, which are found in the cytosol, outer mitochondrial membrane, intermembrane space, inner mitochondrial membrane and in the mitochondrial matrix. The translocase of the inner membrane, TIM23 complex, with its associated proteins, is the focus of one major project in our laboratory.
The second major topic of our research is the chaperonin family of proteins. The most well-known are the GroEL and GroES proteins of E. coli. The cpn60 (or Hsp60) protein (GroEL) binds to nascent or stress-denatured proteins and facilitates their refolding with the assistance of the cpn10 (Hsp10, GroES) co-chaperonin. In mitochondria, Hsp60 mediates the folding of proteins that have been translocated from the cytosol to the matrix. The importance of these chaperonins in mitochondria is highlighted by the discovery of genetic diseases caused by mutations in these proteins, and of extramitochondrial roles, some related to cancer development. We have been studying the structure and function of mitochondrial and chloroplast chaperonins in vitro. Recently, we published the first crystal structure of the the mitochondrial chaperonin complex, which lent insight into the unique mechanism of this system.
More recently, we have become interested in the mechanism behind genetic diseases. Modern deep-sequencing technologies have facilitated the identification of mutations responsible for recessive genetic diseases. We use this information to delineate the molecular basis of the disease, taking a two-pronged approach. We utilize a yeast model to study the effects of the mutated protein in a biological system. In parallel, we produce the mutated protein and purify it in vitro, in order to investigate its properties and its interaction with other proteins.
The primary research approach that we utilize is reconstitution of a studied system from indvidual purified proteins. We then employ a wide range of biochemistry and biophysics methods to study the structural properties of the reconstituted complexes, in vitro. Finally, we use functional assays (protein refolding and import) to establish a mechanistic link between the structure and function. In parallel, we study the behavior of protein variants in a living system, using bacterial and yeast as model systems.
Innovation and Discoveries
Over the years, we have reported several discoveries that further our mechanistic understanding of the systems studied in our lab. We were the first to show that Tim44, a component of the mitochondrial import channel channel, binds the lipid cardiolipin, and succeeded in identifying the lipid-binding site on the protein. We also reconstituted the interaction between several components of the TIM23 complex, as follows. i) The ATP hydrolyzing component of the TIM23 complex, Hsp70, and the membrane anchor Tim44. ii) Two components of the TIM23 complex, Tim50 and Tim23, and their respective interactions with precursor proteins. iii) In our most recent work, we showed that the membrane-situated GXXXG motifs of Tim23 mediate vital protein-protein interactions between various components.
We have also made significant progress in understanding organellar chaperonins. The chloroplast Cpn60 and Cpn10 are each represented by a family of paralogous proteins. We were the first laboratory to clone and reconstitute a full set of chaperonin proteins from a single type of plant chloroplast in vitro. Our studies showed that these proteins combine to form a wide variety of homo and hetero-oligomers with different physical and functional properties, allowing for a great amount of flexibility, which may help these sessile organisms deal with a changing environment.
In the field of mitochondrial chaperonins, we solved the first crystal structure of the mammalian mitochondrial chaperonin (Hsp60-Hsp10) complex. Our structure demonstrates that the mitochondrial system has unique characteristics from the well-studied E. coli system.
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