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P L Pedersen

Publications and source records attributed to P L Pedersen.

At least 55 records · Page 3Linked to original sources
PubMed1995

Defective protein folding as a basis of human disease.

The ability of a polypeptide to fold into a unique, functional, three-dimensional structure in vivo is dependent upon its amino acid sequence and the function of molecular chaperone proteins and enzymes that catalyse folding. Intense study of the physical chemistry and cell biology of folding have greatly aided our understanding of the mechanisms normally employed. Evidence is accumulating that many disease-causing mutations and modifications exert their effects by altering protein folding. Here we discuss the pathobiology of these processes.

Amino Acid Isomerases↗
PubMed1994

ATP synthase. The machine that makes ATP.

The recently determined crystal structure of the F1 part of mitochondrial ATP synthase provides new insights into the workings of one of the most remarkable and complex biochemical machines.

Adenosine Triphosphatases↗
PubMed1994

Alteration of pyruvate metabolism in African trypanosomes during differentiation from bloodstream into insect forms.

In the presence of glucose and ample oxygen, insect form African trypanosomes release pyruvate more than 100-fold more slowly than do bloodstream forms. This rate decrease could not be accounted for simply by an increased mitochondrial pyruvate oxidation rate as inhibiting mitochondrial respiration increases pyruvate efflux to rates only 2-3% of that observed for bloodstream form trypanosomes. Alternatively, decreased pyruvate efflux from insect form trypanosomes could not be accounted for by decreased pyruvate transporter activity, which, surprisingly, was nearly as high in insect form trypanosomes as reported by us earlier for bloodstream forms (J.P. Barnard, B. Reynafarje, and P.L. Pedersen (1993) J. Biol. Chem. 268, 3654-3661). Rather, the low pyruvate efflux rate appears to be due primarily to reduced levels of the enzyme pyruvate kinase, which, in contrast to conclusions of an earlier study, is readily detected in insect form trypanosomes in the absence of added activators at an activity level about 4% of that found in bloodstream forms. Insect form pyruvate kinase seems to be located in the cytosol and exhibits kinetic profiles and constants nearly identical to those reported by us earlier for the bloodstream form enzyme (J.P. Barnard, and P.L. Pedersen (1988) Mol. Biochem. Parasitol. 31, 141-148). It is suggested that the reduced levels of pyruvate kinase, and hence the reduced pyruvate efflux rates, in insect form trypanosomes result from down regulation of the gene encoding the cytosolic enzyme.

Animals↗
PubMed1994

The cystic fibrosis transmembrane conductance regulator. Nucleotide binding to a synthetic peptide segment from the second predicted nucleotide binding fold.

Previous studies from this laboratory with a 67-amino acid synthetic peptide (P-67) demonstrated directly that the first predicted nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator (CFTR) binds ATP (Thomas, P.J, Shenbagamurthi, P., Ysern, S., and Pedersen, P.L. (1991) Science 251, 555-557). Although mutational analysis within the predicted second nucleotide binding fold indicates that this domain may be functionally important also, direct evidence for nucleotide binding is lacking. Here, we report the design, chemical synthesis, and purification of a 51-amino acid segment (P-51) of the second predicted nucleotide binding fold of CFTR and demonstrate that this peptide binds ATP. P-51 consists of amino acid residues from glutamic acid 1228 through threonine 1278 and contains a motif, GX4GKS, very similar or identical to that found in many nucleotide-binding proteins. The freshly dissolved peptide moves predominantly as a single species upon molecular sieve chromatography and readily binds ATP without eliciting its hydrolysis. P-51 also readily binds the fluorescent ATP analogs TNP-ATP (2'(3')-0-(2,4,6-trinitrophenyl)-adenosine-5'-triphosphate) and TNP-ADP but exhibits much less capacity to bind TNP-AMP. ATP displaces TNP-ATP with a Kd (ATP) of 0.46 mM. In the presence of the denaturant urea, P-51 loses most of its binding capacity indicating that structure is important for binding. Consistent with this conclusion, circular dichroism spectroscopy revealed that P-51 has significant secondary structure. Elements of such structure calculated from deconvolution of the circular dichroism spectra compare favorably with those predicted from the program of Chou, P.Y., and Fasman, G.D. (1977) J. Mol. Biol. 115, 135-175. These experiments provide the first direct evidence that the second predicted nucleotide binding fold of CFTR binds ATP and define a 51-amino acid segment within the approximately 150-amino acid fold critical for this function. They also indicate that the beta and gamma phosphate groups of ATP may be important for binding and that the 51-amino acid region studied is not sufficient to catalyze ATP hydrolysis. Finally, as seven different mutations within P-51 are known to cause cystic fibrosis, these studies will be important in future efforts to understand the molecular basis of the disease.

Adenosine Triphosphate↗
PubMed1994

Chromosomal localization of genes required for the terminal steps of oxidative metabolism: alpha and gamma subunits of ATP synthase and the phosphate carrier.

The terminal steps of oxidative phosphorylation include transport of phosphate and ADP into the mitochondrial matrix, synthesis of ATP in the matrix, and transport of the product ATP into the cytosol where it can be utilized to perform cellular work. Three nuclear genome encoded membrane proteins, namely, the phosphate carrier (PHC), the adenine nucleotide carrier (ANT), and the ATP synthase complex, consisting of at least 13 individual subunits, catalyze these reactions. The locations of the alpha and gamma subunits of the mitochondrial ATP synthase complex and the mitochondrial phosphate carrier, PHC, on human chromosomes were determined using cloned rat liver cDNA as probes. Human homologues of the alpha subunit are on chromosomes 9 and 18, the gamma subunit are on chromosomes 10 and 14, and the PHC was localized to chromosome 12.

Animals↗
PubMed1993

The cystic fibrosis transmembrane conductance regulator. Overexpression, purification, and characterization of wild type and delta F508 mutant forms of the first nucleotide binding fold in fusion with the maltose-binding protein.

The first nucleotide binding fold (NBF1) of the cystic fibrosis transmembrane conductance regulator (CFTR) and its disease-causing mutant form (delta F508,NBF1) were overexpressed in high yield in Escherichia coli in fusion with the maltose-binding protein (MBP). The rationale for producing the chimerae was to aid in domain purification, solubilization, and crystallization and to examine the effect of protein-protein interactions on the properties of the mutant NBF1. Both the purified wild type and delta F508 mutant fusion proteins fold into functional nucleotide binding domains as determined by using the fluorescent nucleotide analog TNP-ATP (2'-(3')-O-(2,4,6-trinitrophenyl)adenosine-5'-triphosphate). Moreover, the prominent secondary structural features of the two proteins as assessed by ultraviolet circular dichroism spectropolarimetry are very similar, as is the higher order structure evident in three separate protease digestion patterns. Finally, the stability of the nucleotide binding function of the two proteins is similar as assessed by sensitivity to urea. Gel filtration chromatography and electron and confocal microscopy reveal that both fusion proteins, but not MBP alone, form organized fibers, suggesting that NBF1 self-associates, thus raising the possibility that CFTR may be oligomeric in the plasma membrane. Significantly, in the presence of high salt, these fusion proteins also have a propensity to form microcrystals. Finally, the two separate domains (NBF1 and MBP) constituting the fusion proteins appear to interact quite strongly as both proteins remain associated even after cleavage of their fusion junction. The possible relevance of these novel findings to those approaches that might be taken to elucidate the three-dimensional structural differences between the wild type and delta F508 mutant forms of CFTR, as well as to ameliorate the severity of cystic fibrosis, is discussed.

ATP-Binding Cassette Transporters↗
PubMed1993

Structure/function relationships in hexokinase. Site-directed mutational analyses and characterization of overexpressed fragments implicate different functions for the N- and C-terminal halves of the enzyme.

Hexokinases are comprised of two highly homologous approximately 50-kDa halves and are product-inhibited by glucose-6-P. Four amino acid residues, Ser603, Asp657, Glu708, and Glu742, located in the C-terminal half of the tumor mitochondrial enzyme have been shown to be essential for enzyme function (Arora, K. K., Filburn, C. R., and Pedersen, P. L. (1991) J. Biol. Chem. 266, 5359-5362). Here we have assessed also the role of the N-terminal half of the same enzyme. Site-directed mutagenesis of residues predicted to interact with glucose in the N-terminal half, i.e. Ser155, Asp209, and Glu260, to Ala, have no effect on hexokinase activity. In addition, inhibition by hexose mono- and bisphosphates is unchanged for each of the mutant enzymes. Significantly, the overexpressed N-terminal polypeptide is devoid of catalytic activity but does have the capacity to bind ATP-agarose and be released with ATP and glucose-6-P. In contrast, the overexpressed C-terminal polypeptide is catalytically active and shows the same product inhibition pattern as the complete 100-kDa parent enzyme. These results emphasize that the N-terminal half of tumor hexokinase is essential neither for catalysis nor product modulation. Rather, the N-terminal half may play another role, perhaps in modulation of the ATP/glucose-6-P-dependent binding of the enzyme to tumor mitochondria or by acting as a spacer between the outer mitochondrial membrane and the C-terminal catalytic unit.

Amino Acid Sequence↗
PubMed1993

Regulation of the mitochondrial ATP synthase/ATPase complex: cDNA cloning, sequence, overexpression, and secondary structural characterization of a functional protein inhibitor.

The ATPase inhibitor protein of the rat liver mitochondrial ATP synthase/ATPase complex has been cloned from a rat liver cDNA library, and its nucleotide sequence determined. The sequence is highly homologous to both the bovine heart (approximately 70%) and the yeast inhibitor proteins (approximately 40%). The deduced protein sequence is 107 amino acids in length, and based on homology to the bovine heart protein, the first 25 N-terminal amino acids encode a putative mitochondrial targeting sequence. The "mature" protein (without the targeting sequence) fused to the maltose binding protein has been overexpressed in Escherichia coli. The maltose binding protein was used as a handle for the development of a rapid one-step purification of the fusion protein by affinity chromatography on an amylose resin. The purified fusion protein was cleaved with Factor Xa protease at the fusion junction, and the resulting ATPase inhibitor protein was purified to > 90% purity. The purified, overexpressed inhibitor protein displays normal inhibitor activity. The protein inhibits ATP hydrolysis catalyzed by the ATP synthase/ATPase complex in submitochondrial particles in a manner kinetically indistinguishable from the same protein purified from rat liver mitochondria, and exhibits a specific activity of approximately 10,000 units/mg. The secondary structure of the inhibitor protein was determined by circular dichroism spectropolarimetry. The experimentally determined structure shows a high content of alpha-helix and is in good agreement with sequence-based structural predictions. As the function of the inhibitor protein is known to exhibit a high dependence on pH, a study of the pH dependence of inhibitor secondary structure was performed. It is shown that as pH is lowered, conditions which activate inhibitory capacity, the protein loses significant alpha-helical structure. This is the first report of the overexpression in E. coli of a functional ATPase inhibitor protein. Secondary structural analysis of this protein indicates that conversion from its active to its inactive form involves a significant conformational change.

Adenosine Triphosphatases↗
PubMed1993

Glucose catabolism in African trypanosomes. Evidence that the terminal step is catalyzed by a pyruvate transporter capable of facilitating uptake of toxic analogs.

The protozoan parasite Trypanosoma brucei derives its metabolic energy exclusively from a unique type of glycolysis in which pyruvate derived from glucose catabolism is released into the host bloodstream. In this study, this terminal metabolic step has been examined in detail. Pyruvate release from trypanosomal cells supplied with glucose is very rapid, proceeding with an apparent Vmax of 214 nmol x min-1 x mg-1. Counterflow experiments with [14C]pyruvate demonstrate that this metabolic end product can be taken up by actively metabolizing cells consistent with the presence of a plasma membrane transporter. The findings that [14C] acetate exhibits a much lower capacity for cell entry and that the structural analog alpha-cyano-3-hydroxycinnamic acid inhibits pyruvate release provide additional support for the presence of a pyruvate transporter. The substrate analog and alkylating agent 3-bromopyruvate inhibits completely both cell motility and pyruvate release. Surprisingly, however, it is a poor inhibitor of pyruvate transport per se. Rather, its preferential site of action and that of iodoacetic acid were identified by radiolabeling studies and microsequence analysis as glyceraldehyde-3-phosphate dehydrogenase. In extending these studies, 3-bromopyruvate was found to be over 20 times less effective in inhibiting glyceraldehyde-3-phosphate dehydrogenase in intact erythrocytes than in trypanosomal cells. However, in sonicated preparations from both cell types, the enzyme exhibits nearly identical sensitivities to inhibition by 3-bromopyruvate. Experiments reported here provide the first direct evidence that pyruvate release in African trypanosomes is catalyzed by a specific transport system and implicate this transporter as a vehicle for delivering toxic alkylating agents into trypanosomal cells.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1993

Effects of the delta F508 mutation on the structure, function, and folding of the first nucleotide-binding domain of CFTR.

The fatal autosomal recessive disease cystic fibrosis (CF) is caused by mutations in the gene which encodes the cystic fibrosis transmembrane conductance regulator (CFTR). Many of these disease-causing mutations, including the deletion of F508 (delta F508) which accounts for approximately 70% of the disease alleles, occur in one of the two consensus nucleotide binding sequences. Peptide studies have directly demonstrated that the N-terminal nucleotide binding sequences bind adenine nucleotides. Structurally, circular dichroism spectropolarimetry indicates that this region of CFTR assumes a beta-stranded structure in solution. The delta F508 mutation causes a diminution in the amount of beta-stranded structure and a concomitant increase in the amount of random coil structure present, indicating that either the mutant peptide has a different native structure or that the conformational equilibrium is shifted toward a more disordered form. Furthermore, the mutant peptide is more sensitive to denaturation, indicating that delta F508 is a stability, or protein-folding mutant. Here we review these results and discuss their implications for interpreting the behavior of delta F508 in situ and for the rational design of new CF drugs.

Amino Acid Sequence↗
PubMed1993

Phosphate transport in mitochondria: past accomplishments, present problems, and future challenges.

The requirement of inorganic phosphate (Pi) for oxidative phosphorylation in eukaryotic cells is fulfilled through specific Pi transport systems. The mitochondrial proton/phosphate symporter (Pic) is a membrane-embedded protein which translocates Pi from the cytosol into the mitochondrial matrix. Pic is responsible for the very rapid transport of most of the Pi used in ATP synthesis. During the past five years there have been advances on several fronts. Genomic and cDNA clones for yeast, bovine, rat, and human Pic have been isolated and sequenced. Functional expression of yeast Pic in yeast strains deficient in Pi transport and expression in Escherichia coli of a chimera protein involving Pic and ATP synthase alpha subunit have been accomplished. Pic, in contrast to other members of the family of transporters involved in energy metabolism, was demonstrated to have a presequence, which optimizes the import of the precursor protein into mitochondria. Six transmembrane segments appear to be a structural feature shared between Pic and other mitochondrial anion carriers, and recent-site directed mutagenesis studies implicate structure-functional relationships to bacteriorhodopsin. These recent advances on Pic will be assessed in light of a more global interpretation of transport mechanism across the inner mitochondrial membrane.

Adenosine Triphosphate↗
PubMed1992

Altered protein folding may be the molecular basis of most cases of cystic fibrosis.

Experiments have demonstrated that the cystic fibrosis transmembrane conductance regulator protein (CFTR), containing the most common cystic fibrosis (CF)-causing mutation (delta F508), reaches the plasma membrane in reduced amounts. Studies of a peptide model of CFTR indicate that the delta F508 mutated region is more sensitive to denaturating conditions. This paper proposes that altered protein folding accounts for these findings, and, thus, most cases of CF. Significantly, the hypothesis makes specific predictions about the effect of stabilizing conditions on mutant CFTR, and, further, suggests a new class of pharmaceuticals that may prove effective in the treatment of this important genetic disease.

Cystic Fibrosis↗