Young Hee Ko’s research while affiliated with Johns Hopkins University and other places

The mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channel plays a central role in protection of cardiac and neuronal cells against ischemia and apoptosis, but its molecular structure is unknown. Succinate dehydrogenase (SDH) is inhibited by mitoK(ATP) activators, fueling the contrary view that SDH, rather than mitoK(ATP), is the target of cardioprotective drugs. Here, we report that SDH forms part of mitoK(ATP) functionally and structurally. Four mitochondrial proteins [mitochondrial ATP-binding cassette protein 1 (mABC1), phosphate carrier, adenine nucleotide translocator, and ATP synthase] associate with SDH. A purified IM fraction containing these proteins was reconstituted into proteoliposomes and lipid bilayers and shown to confer mitoK(ATP) channel activity. This channel activity is sensitive not only to mitoK(ATP) activators and blockers but also to SDH inhibitors. These results reconcile the controversy over the basis of ischemic preconditioning by demonstrating that SDH is a component of mitoK(ATP) as part of a macromolecular supercomplex. The findings also provide a tangible clue as to the structural basis of mitoK(ATP) channels.

The terminal steps involved in making ATP in mitochondria require an ATP synthase (F(0)F(1)) comprised of two motors, a phosphate carrier (PIC), and an adenine nucleotide carrier (ANC). Under mild conditions, these entities sub-fractionate as an ATP synthase/PIC/ANC complex or "ATP synthasome" (Ko, Y.H., Delannoy, M, Hullihen, J., Chiu, W., and Pedersen, P.L. (2003) J. Biol. Chem. 278, 12305-12309). As a first step toward obtaining three-dimensional information about this large complex or "metabolon" and the locations of PIC and ANC therein, we dispersed ATP synthasomes into single complexes and visualized negatively stained images by electron microscopy (EM) that showed clearly the classical headpiece, central stalk, and basepiece. Parallel immuno-EM studies revealed the presence of PIC and ANC located non-centrally in the basepiece, and other studies implicated an ATP synthase/PIC/ANC stoichiometry near 1:1:1. Single ATP synthasome images (7506) were boxed, and, using EMAN software, a three-dimensional model was obtained at a resolution of 23 A. Significantly, the basepiece is oblong and contains two domains, the larger of which connects to the central stalk, whereas the smaller appears as an extension. Docking studies with known structures together with the immuno-EM studies suggest that PIC or ANC may be located in the smaller domain, whereas the other transporter resides nearby in the larger domain. Collectively, these finding support a mechanism in which the entry of the substrates ADP and P(i) into mitochondria, the synthesis of ATP on F(1), and the release and exit of ATP are very localized and highly coordinated events.

The terminal steps involved in making ATP in mitochondria require an ATP synthase (F0F1) comprised of two motors, a phosphate carrier (PIC), and an adenine nucleotide carrier (ANC). Under mild conditions, these entities sub-fractionate as an ATP synthase/PIC/ANC complex or “ATP synthasome” (Ko, Y.H., Delannoy, M, Hullihen, J., Chiu, W., and Pedersen, P.L. (2003) J. Biol. Chem. 278, 12305–12309). As a first step toward obtaining three-dimensional information about this large complex or “metabolon” and the locations of PIC and ANC therein, we dispersed ATP synthasomes into single complexes and visualized negatively stained images by electron microscopy (EM) that showed clearly the classical headpiece, central stalk, and basepiece. Parallel immuno-EM studies revealed the presence of PIC and ANC located non-centrally in the basepiece, and other studies implicated an ATP synthase/PIC/ANC stoichiometry near 1:1:1. Single ATP synthasome images (7506) were boxed, and, using EMAN software, a three-dimensional model was obtained at a resolution of 23 Å. Significantly, the basepiece is oblong and contains two domains, the larger of which connects to the central stalk, whereas the smaller appears as an extension. Docking studies with known structures together with the immuno-EM studies suggest that PIC or ANC may be located in the smaller domain, whereas the other transporter resides nearby in the larger domain. Collectively, these finding support a mechanism in which the entry of the substrates ADP and Pi into mitochondria, the synthesis of ATP on F1, and the release and exit of ATP are very localized and highly coordinated events.

Cystic fibrosis (CF) is caused by mutations in the gene encoding CFTR (cystic fibrosis transmembrane conductance regulator), a regulated anion channel and member of the ATP-binding-cassette transporter (ABC transporter) superfamily. Of CFTR's five domains, the first nucleotide-binding fold (NBF1) has been of greatest interest both because it is the major 'hotspot' for mutations that cause CF, and because it is connected to a unique regulatory domain (R). However, attempts have failed to obtain a catalytically active NBF1+R protein in the absence of a fusion partner. Here, we report that such a protein can be obtained following its overexpression in bacteria. The pure NBF1+R protein exhibits significant ATPase activity [catalytic-centre activity (turnover number) 6.7 min(-1)] and an apparent affinity for ATP ( K (m), 8.7 microM) higher than reported previously for CFTR or segments thereof. As predicted, the ATPase activity is inhibited by mutations in the Walker A motif. It is also inhibited by vanadate, a transition-state analogue. Surprisingly, however, the best divalent metal activator is Co(2+), followed by Mn(2+) and Mg(2+). In contrast, Ca(2+) is ineffective and Cd(2+) is a potent inhibitor. These novel studies, while demonstrating clearly that CFTR's NBF1+R segment can act independently as an active, vanadate-sensitive ATPase, also identify its unique cation activators and a new inhibitor, thus providing insight into the nature of its active site.

Despite more than 75 years of research by some of the greatest scientists in the world to conquer cancer, the clear winner is still cancer. This is reflected particularly by liver cancer that worldwide ranks fourth in terms of mortality with survival rates of no more than 3-5%. Significantly, one of the earliest discovered hallmarks of cancer had its roots in Bioenergetics as many tumors were found in the 1920s to exhibit a high glycolytic phenotype. Although research directed at unraveling the underlying basis and significance of this phenotype comprised the focus of cancer research for almost 50 years, these efforts declined greatly from 1970 to 1990 as research into the molecular and cell biology of this disease gained center stage. Certainly, this change was necessary as the new knowledge obtained about oncogenes, gene regulation, and programmed cell death once again placed Bioenergetics in the limelight of cancer research. Thus, we now have a much better molecular understanding of the high glycolytic phenotype of many cancers, the pivotal roles that Type II hexokinase-mitochondrial interactions play in this process to promote tumor cell growth and survival, and how this new knowledge can lead to improved therapies that may ultimately turn the tide on our losing war on cancer.

Although signal transduction mechanisms originating from receptors on the plasma membrane and targeted to metabolic and other enzymes/proteins localized in the cytoplasm or the nucleus have been extensively studied in animal cells, few such studies have focused on the mitochondrial energy producing machinery, i.e. the electron transport chain and ATP synthase complex (F0F1). Significantly, it was shown in an earlier collaborative study that platelet-derived growth factor (PDGF), which is linked in signal transduction pathways to tyrosine kinase-dependent phosphorylations, regulates the phosphorylation of the mitochondrial ATP synthase delta subunit in cortical neurons (Zhang et. al., 1995. J. Neurochem. 65, 2812-2815). This is a particularly intriguing finding in light of more recent reports demonstrating that ATP synthases are nanomotors with a central rotor, one component of which is the delta subunit. In this report, evidence is provided that the PDGF-dependent phosphorylation of the ATP synthase delta subunit is not confined to neuronal cells but can be demonstrated also in studies with PDGF-treated NIH3T3 and kidney cells. Evidence is provided also that phosphorylation of the ATP synthase delta subunit may involve its single tyrosine residue, and that this phosphorylation is modulated when the cell based assay includes lysophosphatidic acid (LPA), a phospholipid signaling molecules. Finally, results are presented of an analysis which revealed a number of potential tyrosine phosphorylation sites on three other subunits (alpha, beta, and gamma) of the F1 (catalytic) moiety of the mitochondrial ATP synthase, thus making this important complex a most attractive target for future signal transduction studies.

The disease Cystic Fibrosis (CF) is caused by mutations in the protein called CFTR, cystic fibrosis transmembrane conductance regulator, an ABC-transporter-like protein found in the plasma membrane of animal cells. CFTR is believed to function primarily as a Cl- channel, but evidence is mounting that this protein has other roles as well. Structurally, CFTR consists of a single polypeptide chain (1480 amino acids) that folds into 5 distinct domains. These include 2 transmembrane domains that are involved in channel formation; 2 nucleotide-binding domains (NBF1 and NBF2), the first of which clearly binds and hydrolyzes ATP; and 1 regulatory domain (R) that is phosphorylated in a cAMP-dependent process. Currently, the 3D structure of neither CFTR nor its domains has been elucidated, although both nucleotide domains have been modeled in 3D, and solution structures in 3D have been obtained for peptide segments of NBF1. The most common mutation causing CF is the deletion (delta) of a single phenylalanine (F) in position 508 within a putative helix located in NBF1. CF patients bearing this deltaF508 mutation frequently experience chronic lung infections, particularly by Pseudomonas aeruginosa, and have a life span that rarely exceeds the age of 30. Since the CFTR gene was cloned and sequenced in 1989, there has been over a decade of research focused on understanding the molecular basis of CF caused by the deltaF508 mutation, with the ultimate objective of using the knowledge gained to carry out additional research designed to correct the underlying defect. In general, this pioneering or "ground roots" research has succeeded according to plan. This brief review summarizes some of the highlights with a focus on those studies conducted in the authors' laboratory. For us, this research has been both exciting and rewarding mainly because the results obtained, despite very limited funding, have provided considerable insight, not only into the chemical, molecular, and pathogenic basis of CF, but have made it possible for us and others to now develop novel, chemically rational, and "cost effective" strategies to identify agents that correct the structural defect in the deltaF508 CFTR protein causing most cases of CF.

The rabbit VX2 tumor when implanted in the liver has proven convenient as a model for studying hepatocellular carcinomas. However, its metabolic properties have not been well studied. Significantly, studies described here show that the VX2 tumor exhibits a high glycolytic/high hexokinase phenotype that is retained following implantation and growth in rabbit liver. In addition, results of a limited screen show that the glycolytic rate is inhibited best by 2-deoxyglucose (2DOG) and 3-bromopyruvate (3BrPA), the former compound of which is phosphorylated by hexokinase but not further metabolized, while the latter directly inhibits hexokinase. Finally, when tested on hepatoma cells in culture both inhibitors facilitated cell death. These studies underscore the usefulness of the VX2 tumor model for the study of advanced liver cancer and for selecting anti-hepatoma agents.

Recent research indicates that ATP synthases (F(0)F(1)) contain two distinct nanomotors, one an electrochemically driven proton motor contained within F(0) that drives an ATP hydrolysis-driven motor (F(1)) in reverse during ATP synthesis. This is depicted in recent models as involving a series of events in which each of the three alphabeta pairs comprising F(1) is induced via a centrally rotating subunit (gamma) to undergo the sequential binding changes necessary to synthesize ATP (binding change mechanism). Stabilization of this rotary process (i.e., to minimize "wobble" of F(1)) is provided in current models by a peripheral stalk or "stator" that has recently been shown to extend from near the bottom of the ATP synthase molecule to the very top of F(1). Although quite elegant, these models envision the stator as fixed during ATP synthesis, i.e., bound to only a single alphabeta pair. This is despite the fact that the binding change mechanism views each alphabeta pair as going through the same sequential order of conformational changes which demonstrate a chemical equivalency among them. For this reason, we propose here two different dynamic models for stator function during ATP synthesis. Both models have been designed to maintain chemical equivalency among the three alphabeta pairs during ATP synthesis and both have been animated.