Biomedical subjects

Peter L Pedersen

Publications and source records attributed to Peter L Pedersen.

17 recordsLinked to original sources

PubMed2006

Mitochondrial ATP synthase. Crystal structure of the catalytic F1 unit in a vanadate-induced transition-like state and implications for mechanism.

ATP synthesis from ADP, P(i), and Mg2+ takes place in mitochondria on the catalytic F1 unit (alpha3beta3gammedeltaepsilon) of the ATP synthase complex (F0F1), a remarkable nanomachine that interconverts electrochemical and mechanical energy, producing the high energy terminal bond of ATP. In currently available structural models of F1, the P-loop (amino acid residues 156GGAGVGKT163) contributes to substrate binding at the subunit catalytic sites. Here, we report the first transition state-like structure of F1 (ADP.V(i).Mg.F1) from rat liver that was crystallized with the phosphate (P(i)) analog vanadate (VO(3-)4 or V(i)). Compared with earlier "ground state" structures, this new F1 structure reveals that the active site region has undergone significant remodeling. P-loop residue alanine 158 is located much closer to V(i) than it is to P(i) in a previous structural model. No significant movements of P-loop residues of the subunit were observed at its analogous but noncatalytic sites. Under physiological conditions, such active site remodeling involving the small hydrophobic alanine residue may promote ATP synthesis by lowering the local dielectric constant, thus facilitating the dehydration of ADP and P(i). This new crystallographic study provides strong support for the catalytic mechanism of ATP synthesis deduced from earlier biochemical studies of liver F1 conducted in the presence of V(i) (Ko, Y. H., Bianchet, M., Amzel, L. M., and Pedersen, P. L. (1997) J. Biol. Chem. 272, 18875-18881; Ko, Y. H., Hong, S., and Pedersen, P. L. (1999) J. Biol. Chem. 274, 28853-28856).

Adenosine Diphosphate↗

PubMed2005

Transport ATPases: structure, motors, mechanism and medicine: a brief overview.

Today we know there are four different types of ATPases that operate within biological membranes with the purpose of moving many different types of ions or molecules across these membranes. Some of these ions or molecules are transported into cells, some out of cells, and some in or out of organelles within cells. These ATPases span the biological world from bacteria to eukaryotic cells and have become most simply and commonly known as "transport ATPases." The price that each cell type pays for transport work is counted in molecules of hydrolyzed ATP, a metabolic currency that is itself regenerated by a transport ATPase working in reverse, i.e., the ATP synthase. Four major classes of transport ATPases, the P, V, F, and ABC types are now known. In addition to being involved in many different types of biological/physiological processes, mutations in these proteins also account for a large number of diseases. The purpose of this introductory article to a mini-review series on transport ATPases is to provide the reader with a very brief and focused look at this important area of research that has an interesting history and bears significance to cell physiology, biochemistry, immunology, nanotechnology, and medicine, including drug discovery. The latter involves potential applications to a whole host of diseases ranging from cancer to those that affect bones (osteoporosis), ears (hearing), eyes (macromolecular degeneration), the heart (hypercholesterolemia/cardiac arrest,), immune system (immune deficiency disease), kidney (nephrotoxicity), lungs (cystic fibrosis), pancreas (diabetes and cystic fibrosis), skin (Darier disease), and stomach (ulcers).

ATP-Binding Cassette Transporters↗

PubMed2004

Advanced cancers: eradication in all cases using 3-bromopyruvate therapy to deplete ATP.

A common feature of many advanced cancers is their enhanced capacity to metabolize glucose to lactic acid. In a challenging study designed to assess whether such cancers can be debilitated, we seeded hepatocellular carcinoma cells expressing the highly glycolytic phenotype into two different locations of young rats. Advanced cancers (2-3cm) developed and were treated with the alkylating agent 3-bromopyruvate, a lactate/pyruvate analog shown here to selectively deplete ATP and induce cell death. In all 19 treated animals advanced cancers were eradicated without apparent toxicity or recurrence. These findings attest to the feasibility of completely destroying advanced, highly glycolytic cancers.

Adenosine Triphosphate↗

PubMed2004

Mitochondrial ATP synthasome: three-dimensional structure by electron microscopy of the ATP synthase in complex formation with carriers for Pi and ADP/ATP.

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.

Animals↗

PubMed2004

Mitochondrial ATP synthase: a bioinformatic approach reveals new insights about the roles of supernumerary subunits g and A6L.

The mitochondrial ATP synthase is a membrane protein complex which couples the proton gradient across the mitochondrial inner membrane to the synthesis of ATP from ADP+Pi. The complex is composed of essential subunits for its motor functions and supernumerary subunits, the roles of which remain to be elucidated. Subunits g and A6L are supernumerary subunits, and the specific roles of these subunits are still matters of debate. To gain insight into the functions of these two subunits, we carried out the alignment and the homolog search of the protein sequences of the subunits and found the following features: Subunit g appears to have isoforms in animals, and the transmembrane domain of the animal subunit g contains a completely conserved acidic residue in the middle of a helix on the conserved side of the transmembrane helix. This finding implicates the conserved acidic residue as important for the function of subunit g. The alignment of A6L protein sequences shows a conserved aromatic residue at the N-terminal domain with which the N-terminal MPQL sequence comprises a unique MPQLX4Ar motif that can signify the protein A6L. The conserved aromatic residue may also be important for the function of A6L.

Amino Acid Motifs↗

PubMed2004

Recently elucidated energy catabolism pathways provide opportunities for novel treatments in hepatocellular carcinoma.

It has long been known that tumors depend on energy production pathways that are different from those of normal cells. These unique pathways require the expression and function of tumor-specific enzymes. Some of these glycolytic enzymes, as well as other modulators of tumor behavior, have recently been elucidated. In theory, inhibiting such enzymes or appropriately affecting such modulators should deprive tumors of energy, while leaving nontransformed cells unaffected. These factors include certain hexokinases that catalyze glycolysis in tumors and can be inhibited by 3-bromopyruvate. 2-deoxyglucose is another modulator that depletes hexokinase stores and cannot undergo further catabolism, thus depriving tumors of their energy source. Other enzymes or modulators are under scrutiny and have shown promise. Preliminary experiments on animals with hepatocellular carcinoma have indeed shown very encouraging results. It appears that modulating the energy production pathways of tumors is poised to become a substantial research area for cancer treatment. This review will focus on the energy production pathways of transformed cells, highlight the differences between transformed and normal cells in this regard and summarize recent experiments that take advantage of these disparities in cancer treatment.

Animals↗

PubMed2003

Glucose metabolism in cancer: importance of transcription factor-DNA interactions within a short segment of the proximal region og the type II hexokinase promoter.

A common signature of many cancers is a high glucose catabolic rate frequently dependent on the overexpression of Type II hexokinase (HKII), a mitochondrial bound enzyme that also suppresses cell death. As the tumor HKII promoter plays a significant role in HKII overexpression, studies reported here were undertaken to identify both the major regions and transcription factors involved under tumor-like conditions. Reporter gene assays following transfection of hepatoma cells with decreasing segments of the HKII promoter traced its known strength to the proximal region (-281 to -35). Mutational analyses showed that in this short region GC boxes 1, 2, 5, and 6, a CCAAT box, an inverted CCAAT box, and CRE are involved in promoter activation. Other studies demonstrated binding of transcription factors Sp1, Sp2, and Sp3 to GC boxes 1 and 6, Sp1 and Sp2 to GC boxes 2 and 5, NF-Y to CCAAT boxes, and CREB, ATF1, and CREM to CRE. In addition, transfection studies involving Sp1, Sp2, Sp3, CREB, and NFY (dominant negative form) provided evidence that these proteins are promoter activators. Finally, alignment of available HK proximal promoters showed strong conservation only among HKII sequences. These findings implicate signaling pathways directed to a short segment of the proximal region of the HKII promoter as major contributors to HKII overexpression in many cancers.

Animals↗

PubMed2003

Subunit E of mitochondrial ATP synthase: a bioinformatic analysis reveals a phosphopeptide binding motif supporting a multifunctional regulatory role and identifies a related human brain protein with the same motif.

The mitochondrial adenosine triphosphate (ATP) synthase is located in the inner membrane and consists of at least 16 subunit types in animals, one of which is subunit e, the function of which is not clearly defined. A highly homologous protein is located in the nucleus and named progesterone receptor binding protein (RBF), to designate its role in this organelle. In addition, the expression level of subunit e in mammalian cells fluctuates greatly and is induced by certain carcinogens and elevated in liver cancers. Because these previous observations suggested to us that subunit e may play multifunctional regulatory roles, we employed a bioinformatic approach to test this view. First, from sequence alignment studies, secondary structure analyses, and basic local alignment search tool (BLAST) searches, we concluded that mitochondrial subunit e and the homologous nuclear protein RBF are most likely the same protein. Second, we examined the known sequence and structure of one of the most common multifunctional cell regulatory proteins, the 14-3-3 protein, involved in phosphopeptide binding, and deduced that it has an apparent binding motif (-KX(6)R---RY-). Third, from careful examination of the conserved residues within all subunit e sequences in the database, we discovered that this protein has a comparable binding motif (-RY---KX(6)R-). Finally, in a BLAST search for additional homologs of subunit e, we found a human brain protein, KIAA1578, the C-terminal 30 amino acids of which are identical to those of human subunit e. This protein also contains a potential phosphopeptide binding motif. In summary, these studies provide support for the view that subunit e is a multifunctional cell regulator involved in cell signaling, and implicate the involvement of the KIAA1578 protein in cell signaling as well. These studies suggest also that, while functioning as a subunit of mitochondrial ATP synthases, subunit e may help regulate these complexes by binding to phosphopeptides within one or more of the other subunit types.

14-3-3 Proteins↗

PubMed2003

Cystic fibrosis transmembrane conductance regulator: the NBF1+R (nucleotide-binding fold 1 and regulatory domain) segment acting alone catalyses a Co2+/Mn2+/Mg2+-ATPase activity markedly inhibited by both Cd2+ and the transition-state analogue orthovanadate.

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.

Adenosine Triphosphate↗

PubMed2003

Glucose metabolism in cancer. Evidence that demethylation events play a role in activating type II hexokinase gene expression.

One of the "signature" phenotypes of highly malignant, poorly differentiated tumors, including hepatomas, is their remarkable propensity to utilize glucose at a much higher rate than normal cells, a property frequently dependent on the marked overexpression of type II hexokinase (HKII). As the expression of the gene for this enzyme is nearly silent in liver tissue, we tested the possibility that DNA methylation/demethylation events may be involved in its regulation. Initial studies employing methylation restriction endonuclease analysis provided evidence for differential methylation patterns for the HKII gene in normal hepatocytes and hepatoma cells, the latter represented by a highly glycolytic model cell line (AS-30D). Subsequently, sequencing following sodium bisulfite treatment revealed 18 methylated CpG sites within a CpG island (-350 to +781 bp) in the hepatocyte gene but none in that of the hepatoma. In addition, treatment of a hepatocyte cell line with the DNA methyltransferase inhibitors, 5'-azacytidine and 5'-aza-2'-deoxycytidine, activated basal expression levels of HKII mRNA and protein. Finally, stably transfecting the hepatocyte cell line with DNA demethylase also resulted in activating the basal expression levels of HKII mRNA and protein. These novel observations indicate that one of the initial events in activating the HKII gene during either transformation or tumor progression may reside at the epigenetic level.

Animals↗

PubMed2003

Mitochondrial ATP synthasome. Cristae-enriched membranes and a multiwell detergent screening assay yield dispersed single complexes containing the ATP synthase and carriers for Pi and ADP/ATP.

The terminal step of ATP synthesis in intact mitochondria is catalyzed by the ATP synthase (F(0)F(1)) that works in close synchrony with the P(i) and ADP/ATP carriers. Each carrier consists of only a single polypeptide chain in dimeric form, while the ATP synthase is highly complex consisting in animals of 17 known subunit types and more than 30 total subunits. Although structures at high resolution have been obtained for the water-soluble F(1) part of the ATP synthase consisting of only five subunit types, such structures have not been obtained for either the complete ATP synthase or the P(i) and ADP/ATP carriers. Here, we report that all three proteins are localized in highly purified cristae-like vesicles obtained by extensive subfractionation of the mitochondrial inner membrane. Moreover, using a multiwell detergent screening assay, 4 nonionic detergents out of 80 tested were found to disperse these cristae-like vesicles into single soluble complexes or "ATP synthasomes" that contain the ATP synthase in association with the P(i) and ADP/ATP carriers. These studies offer new mechanistic insights into the terminal steps of oxidative phosphorylation in mitochondria and set the stage for future structural efforts designed to visualize in atomic detail the entire complex involved. They also provide evidence that the cristae are a subcompartment of the inner membrane.

Animals↗

PubMed2003

ATP synthases: insights into their motor functions from sequence and structural analyses.

ATP synthases are motor complexes comprised of F0 and F1 parts that couple the proton gradient across the membrane to the synthesis of ATP by rotary catalysis. Although a great deal of information has been accumulated regarding the structure and function of ATP synthases, their motor functions are not fully understood. For this reason, we performed the alignments and analyses of the protein sequences comprising the core of the ATP synthase motor complex, and examined carefully the locations of the conserved residues in the subunit structures of ATP synthases. A summary of the findings from this bioinformatic study is as follows. First, we found that four conserved regions in the sequence of gamma subunit are clustered into three patches in its structure. The interactions of these conserved patches with the alpha and beta subunits are likely to be critical for energy coupling and catalytic activity of the ATP synthase. Second, we located a four-residue cluster at the N-terminal domain of mitochondrial OSCP or bacterial (or chloroplast) delta subunit which may be critical for the binding of these subunits to F1. Third, from the localizations of conserved residues in the subunits comprising the rotors of ATP synthases, we suggest that the conserved interaction site at the interface of subunit c and delta (mitochondria) or epsilon (bacteria and chloroplasts) may be important for connecting the rotor of F1 to the rotor of F0. Finally, we found the sequence of mitochondrial subunit b to be highly conserved, significantly longer than bacterial subunit b, and to contain a shorter dimerization domain than that of the bacterial protein. It is suggested that the different properties of mitochondrial subunit b may be necessary for interaction with other proteins, e.g., the supernumerary subunits.

Amino Acid Sequence↗

PubMed2002

Mitochondrial bound type II hexokinase: a key player in the growth and survival of many cancers and an ideal prospect for therapeutic intervention.

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.

Cell Survival↗

PubMed2002

ATP synthase of yeast: structural insight into the different inhibitory potencies of two regulatory peptides and identification of a new potential regulator.

Mitochondrial ATP synthases, the major producers of ATP in higher eukaryotic cells, are known to be regulated by a peptide designated IF(1). In contrast, in yeast three such peptides have been identified, IF(1) and STF(1), which inhibit the reverse ATPase reaction, and STF(2), a modulator of the action of these inhibitors. Despite significant homology to IF(1), STF(1) exhibits less than half ( approximately 40%) its inhibitory potency. The two-fold purpose of this bioinformatic study was to gain structural insight into the different inhibitory potencies of IF(1) and STF(1) and to determine to what extent yeast are unique in employing multiple peptides to regulate the ATP synthase. Sequence and secondary structural analyses and comparison with the known structure of bovine IF(1) predicted a dimeric structure for yeast STF(1) in which the C-terminal regions form a coiled-coil. Moreover, sequence comparisons showed that within this C-terminal region a conserved acidic residue (Asp 59) in yeast IF(1) is replaced by Asn in STF(1). In the known structure of bovine IF(1), predicted to be very similar to that of yeast IF(1), the residue Glu 68 corresponding to Asp 59 participates in the formation of a four-residue conserved acidic cluster in the middle of the coiled-coil in the C-terminal region. It is deduced here that this acidic cluster is likely to be important in the regulation of IF(1)'s inhibitory capacity and that replacement of conserved Asp 59 by Asn in STF(1) may reduce its potency. Although other homologs to the inhibitors IF(1) and STF(1) were not found in searches of available eukaryotic genomes, including human, a new homolog, named STF(3), with 65% identity to the modulator STF(2), was discovered within the yeast genome and identified to be expressed by searching the yeast EST database. Thus, yeast appears unique in regulating the ATP synthase by involving multiple peptides (IF(1), STF(1), STF(2), and perhaps STF(3)).

Amino Acid Sequence↗

PubMed2002

Novel therapy for liver cancer: direct intraarterial injection of a potent inhibitor of ATP production.

Most types of cancer are difficult to eradicate and some, like liver carcinomas, are almost always fatal. Significantly, we report here that direct intraarterial delivery of 3-bromopyruvate (3-BrPA), a potent inhibitorof cell ATP production, to liver-implanted rabbit tumors, inflicts a rapid, lethal blow to most cancer cells therein. Moreover, systemic delivery of 3-BrPA suppresses "metastatic" tumors that arise in the lungs. In both cases, there is no apparent harm to other organs or to the animals. Thus, intraarterial delivery of agents like 3-BrPA directly to the site of the primary tumor, followed by systemic delivery only when necessary, may represent a powerful new strategy for arresting the growth of liver and other cancers while minimizing toxic side effects.

Adenosine Triphosphate↗

PubMed2002

Signal transduction to mitochondrial ATP synthase: evidence that PDGF-dependent phosphorylation of the delta-subunit occurs in several cell lines, involves tyrosine, and is modulated by lysophosphatidic acid.

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.

Journal Article↗

PubMed2002

Transport ATPases in biological systems and relationship to human disease: a brief overview.

Interest in the field of transport ATPases has grown dramatically during the past 20 years and gained considerable visibility for several reasons. First, it was shown that most transport ATPases can be lumped into only a few categories designated simply as P, V, F, and ABC types, the latter consisting of a large superfamily. Second, it has been shown that many transport ATPases have a clear relevance to human disease. Third, the field of transport ATPases has become rather advanced in the study of the reaction mechanisms and structure-function relationships associated with several of these enzymes. Finally, the Nobel committee recently recognized major accomplishments in this field of research. Here, the author provides a brief discussion of transport ATPases that are present in biological systems and their relevance or possible relevance to human disease.

Adenosine Triphosphatases↗