John W. Hawes

Absence of γ-Interferon–inducible Lysosomal Thiol Reductase in Melanomas Disrupts T Cell Recognition of Select Immunodominant Epitopes

Long-lasting tumor immunity requires functional mobilization of CD8+ and CD4+ T lymphocytes. CD4+ T cell activation is enhanced by presentation of shed tumor antigens by professional antigen-presenting cells (APCs), coupled with display of similar antigenic epitopes by major histocompatibility complex class II on malignant cells. APCs readily processed and presented several self-antigens, yet T cell responses to these proteins were absent or reduced in the context of class II+ melanomas. T cell recognition of select exogenous and endogenous epitopes was dependent on tumor cell expression of γ-interferon–inducible lysosomal thiol reductase (GILT). The absence of GILT in melanomas altered antigen processing and the hierarchy of immunodominant epitope presentation. Mass spectral analysis also revealed GILTs ability to reduce cysteinylated epitopes. Such disparities in the profile of antigenic epitopes displayed by tumors and bystander APCs may contribute to tumor cell survival in the face of immunological defenses.

Studies on the regulation of the mitochondrial α-ketoacid dehydrogenase complexes and their kinases

Five mitochondrial protein kinases, all members of a new family of protein kinases, have now been identified, cloned, expressed as recombinant proteins, and partially characterized with respect to catalytic and regulatory properties. Four members of this unique family of eukaryotic protein kinases correspond to pyruvate dehydrogenase kinase isozymes which regulate the activity of the pyruvate dehydrogenase complex, an important regulatory enzyme at the interface between glycolysis and the citric acid cycle. The fifth member of this family corresponds to the branched-chain alpha-ketoacid dehydrogenase kinase, an enzyme responsible for phosphorylation and inactivation of the branched-chain alpha-ketoacid dehydrogenase complex, the most important regulatory enzyme in the pathway for the disposal of branched-chain amino acids. At least three long-term control mechanisms have evolved to conserve branched chain amino acids for protein synthesis during periods of dietary protein insufficiency. Increased expression of the branched-chain alpha-ketoacid dehydrogenase kinase is perhaps the most important because this leads to phosphorylation and nearly complete inactivation of the liver branched-chain alpha-ketoacid dehydrogenase complex. Decreased amounts of the liver branched-chain alpha-ketoacid dehydrogenase complex secondary to a decrease in liver mitochondria also decrease the livers capacity for branched-chain keto acid oxidation. Finally, the number of E1 subunits of the branched-chain alpha-ketoacid dehydrogenase complex is reduced to less than a full complement of 12 heterotetramers per complex in the liver of protein-starved rats. Since the E1 component is rate-limiting for activity and also the component of the complex inhibited by phosphorylation, this decrease in number further limits overall enzyme activity and makes the complex more sensitive to regulation by phosphorylation in this nutritional state. The branched-chain alpha-ketoacid dehydrogenase kinase phosphorylates serine 293 of the E1 alpha subunit of the branched-chain alpha-ketoacid dehydrogenase complex. Site-directed mutagenesis of amino acid residues surrounding serine 293 reveals that arginine 288, histidine 292 and aspartate 296 are critical to dehydrogenase activity, that histidine 292 is critical to binding the coenzyme thiamine pyrophosphate, and that serine 293 exists at or in close proximity to the active site of the dehydrogenase. Alanine scanning mutagenesis of residues in the immediate vicinity of the phosphorylation site (serine 293) indicates that only arginine 288 is required for recognition of serine 293 as a phosphorylation site by the branched-chain alpha-ketoacid dehydrogenase kinase. Phosphorylation appears to inhibit dehydrogenase activity by introducing a negative charge directly into the active site pocket of the E1 dehydrogenase component of the branched-chain alpha-ketoacid dehydrogenase complex. A model based on the X-ray crystal structure of transketolase is being used to predict residues involved in thiamine pyrophosphate binding and to help visualize how phosphorylation within the channel leading to the reactive carbon of thiamine pyrophosphate inhibits catalytic activity. The isoenzymes of pyruvate dehydrogenase kinase differ greatly in terms of their specific activities, kinetic parameters and regulatory properties. Chemically-induced diabetes in the rat induces significant changes in the pyruvate dehydrogenase kinase isoenzyme 2 in liver. Preliminary findings suggest hormonal control of the activity state of the pyruvate dehydrogenase complex may involves tissue specific induced changes in expression of the pyruvate dehydrogenase kinase isoenzymes.

A Domain with Homology to Neuronal Calcium Sensors Is Required for Calcium-dependent Activation of Diacylglycerol Kinase α

Diacylglycerol kinases (DGKs) phosphorylate diacylglycerol produced during stimulus-induced phosphoinositide turnover and attenuate protein kinase C activation. Diacylglycerol kinase α is an 82-kDa DGK isoform that is activated in vitro by Ca2+. The DGKα regulatory region includes tandem C1 protein kinase C homology domains and Ca2+-binding EF hand motifs. It also contains an N-terminal recoverin homology (RVH) domain that is related to the N termini of the recoverin family of neuronal calcium sensors. To probe the structural basis of Ca2+ regulation, we expressed a series of DGKα deletions spanning its regulatory domain in COS-1 cells. Deletion of the RVH domain resulted in loss of Ca2+-dependent activation. Further deletion of the EF hands resulted in a constitutively active enzyme, suggesting that sequences in or near the EF hands are sufficient for autoinhibition. Binding of Ca2+ to the EF hands protected sites within both the RVH domain and EF hands from trypsin cleavage and increased the phenyl-Sepharose binding of a recombinant DGKα fragment that included both the RVH domain and EF hands. These observations suggested that Ca2+ elicits a concerted conformational change of these two domains. A cationic amphiphile, octadecyltrimethylammonium chloride, also activated DGKα. As with Ca2+, this activation required the RVH domain. However, this agent did not protect the EF hands and RVH domain from trypsin cleavage. These findings indicate that the EF hands and RVH domain act as a functional unit during Ca2+-induced DGKα activation.

Structural and mechanistic similarities of 6-phosphogluconate and 3-hydroxyisobutyrate dehydrogenases reveal a new enzyme family, the 3-hydroxyacid dehydrogenases

Rat 3‐hydroxyisobutyrate dehydrogenase exhibits significant amino acid sequence homology with 6‐phosphogluconate dehydrogenase, d‐phenylserine dehydrogenase from Pseudomonas syringae, and a number of hypothetical proteins encoded by genes of microbial origin. Key residues previously proposed to have roles in substrate binding and catalysis in sheep 6‐phosphogluconate dehydrogenase are highly conserved in this entire family of enzymes. Site‐directed mutagenesis, chemical modification, and substrate specificity studies were used to compare possible mechanistic similarities of 3‐hydroxyisobutyrate dehydrogenase with 6‐phosphogluconate dehydrogenase. The data suggest that 3‐hydroxyisobutyrate and 6‐phosphogluconate dehydrogenases may comprise, in part, a previously unrecognized family of 3‐hydroxyacid dehydrogenases.

15N NMR Study of the Ionization Properties of the Influenza Virus Fusion Peptide in Zwitterionic Phospholipid Dispersions

Influenza virus hemagglutinin (HA)-mediated membrane fusion involves insertion into target membranes of a stretch of amino acids located at the N-terminus of the HA(2) subunit of HA at low pH. The pK(a) of the alpha-amino group of (1)Gly of the fusion peptide was measured using (15)N NMR. The pK(a) of this group was found to be 8.69 in the presence of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine). The high value of this pK(a) is indicative of stabilization of the protonated form of the amine group through noncovalent interactions. The shift reagent Pr(3+) had large effects on the (15)N resonance from the alpha-amino group of Gly(1) of the fusion peptide in DOPC vesicles, indicating that the terminal amino group was exposed to the bulk solvent, even at low pH. Furthermore, electron paramagnetic resonance studies on the fusion peptide region of spin-labeled derivatives of a larger HA construct are consistent with the N-terminus of this peptide being at the depth of the phosphate headgroups. We conclude that at both neutral and acidic pH, the N-terminal of the fusion peptide is close to the aqueous phase and is protonated. Thus neither a change in the state of ionization nor a significant increase in membrane insertion of this group is associated with increased fusogenicity at low pH.

Peroxisomal Metabolism of Propionic Acid and Isobutyric Acid in Plants

The subcellular sites of branched-chain amino acid metabolism in plants have been controversial, particularly with respect to valine catabolism. Potential enzymes for some steps in the valine catabolic pathway are clearly present in both mitochondria and peroxisomes, but the metabolic functions of these isoforms are not clear. The present study examined the possible function of these enzymes in metabolism of isobutyryl-CoA and propionyl-CoA, intermediates in the metabolism of valine and of odd-chain and branched-chain fatty acids. Using 13C NMR, accumulation of β-hydroxypropionate from [2-13C]propionate was observed in seedlings of Arabidopsis thaliana and a range of other plants, including both monocots and dicots. Examination of coding sequences and subcellular targeting elements indicated that the completed genome of A. thaliana likely codes for all the enzymes necessary to convert valine to propionyl-CoA in mitochondria. However, Arabidopsis mitochondria may lack some of the key enzymes for metabolism of propionyl-CoA. Known peroxisomal enzymes may convert propionyl-CoA to β-hydroxypropionate by a modified β-oxidation pathway. The chy1–3 mutation, creating a defect in a peroxisomal hydroxyacyl-CoA hydrolase, abolished the accumulation of β-hydroxyisobutyrate from exogenous isobutyrate, but not the accumulation of β-hydroxypropionate from exogenous propionate. The chy1–3 mutant also displayed a dramatically increased sensitivity to the toxic effects of excess propionate and isobutyrate but not of valine. 13C NMR analysis of Arabidopsis seedlings exposed to [U-13C]valine did not show an accumulation of β-hydroxypropionate. No evidence was observed for a modified β-oxidation of valine. 13C NMR analysis showed that valine was converted to leucine through the production of α-ketoisovalerate and isopropylmalate. These data suggest that peroxisomal enzymes for a modified β-oxidation of isobutyryl-CoA and propionyl-CoA could function for metabolism of substrates other than valine.

Profiling of differentially expressed apoptosis-related genes by cDNA arrays in human cord blood CD34 cells treated with etoposide

OBJECTIVE Understanding the molecular events that contribute to survival of and drug-induced apoptosis in hematopoietic stem and progenitor cells (HSC/P) can have impact on more rational approaches to blood cancer therapeutic design, as well as on strategies to minimize toxic side effects of chemotherapeutic drugs. Here we sought to systematically evaluate the basic molecular components and main pathways that govern and mediate cellular response initiated within human CD34(+) cells to etoposide-induced apoptosis. MATERIALS AND METHODS Human CD34(+) cells were isolated from umbilical cord blood (CB) and expanded in vitro. Expression of apoptosis-related genes in the control and etoposide treated cells was determined using cDNA array and quantitative real-time RT-PCR. RESULTS We identified a set of apoptosis-related genes expressed in highly purified normal human CB CD34(+) cells and determined how the expression of these genes changed in response to etoposide treatment. In addition, TRAIL does not induce apoptosis of normal human CD34(+) cells, and it has no cytotoxic effect on human CD34(+) cells that are undergoing apoptosis in response to growth factor withdrawal. This may be due to upregulation of cytotoxic receptors as well as the decoy receptor for TRAIL, and c-FLIP. CONCLUSION p53, c-Myc, and BAFF pathways are main pathways utilized by CD34(+) cells to arrest cell-cycle progression at multiple checkpoints, to halt proliferation, and to induce apoptosis as part of their cellular response to etoposide. Multiple known pro-survival and pro-apoptotic pathways are simultaneously activated in etoposide-treated CD34(+) cells. Also, TRAIL, used alone or in concert with chemotherapeutic drugs, may be of use as a safe blood cancer therapeutic with no or low toxicity for HSC/P.

New developments in our understanding of the β-hydroxyacid dehydrogenases

The beta-hydroxyacid dehydrogenases are a structurally conserved family of enzymes that catalyze the NAD(+) or NADP(+)-dependent oxidation of specific beta-hydroxyacid substrates like beta-hydroxyisobutyrate. These enzymes share distinct domains of amino acid sequence homology, most of which now have assigned putative functions. 6-phosphogluconate dehydrogenase and beta-hydroxyisobutyrate dehydrogenase, the most well-characterized members, both appear to be readily inactivated by chemical modifiers of lysine residues, such as 2,4,6-trinitrobenzene sulfonate (TNBS). Peptide mapping by ESI-LCMS showed that inactivation of beta-hydroxyisobutyrate dehydrogenase with TNBS occurs with the labeling of a single lysine residue, K248. This lysine residue is completely conserved in all family members and may have structural importance relating to cofactor binding. The structural framework of the beta-hydroxyacid dehydrogenase family is shared by many bacterial homologues. One such homologue from E. coli has been cloned and expressed as recombinant protein. This protein was found to have enzymatic activity characteristic of tartronate semialdehyde reductase, an enzyme required for bacterial biosynthesis of D-glycerate. A homologue from H. influenzae was also cloned and expressed as recombinant protein. This protein was active in the oxidation of D-glycerate, but showed approximately ten-fold higher activity with four carbon substrates like beta-D-hydroxybutyrate and D-threonine. This enzyme might function in H. influenzae, and other species, in the utilization of polyhydroxybutyrates, an energy storage form specific to bacteria. Cloning and characterization of these bacterial beta-hydroxyacid dehydrogenases extends our knowledge of this enzyme family.

Structural and Mechanistic Aspects of a New Family of Dehydrogenases, the β-Hydroxyacid Dehydrogenases

The catabolism of valine, unlike that of other branched-chain amino acids, occurs with the formation of a free branched-chain acid, (S)-β-hydroxyisobutyrate or HIBA, whereas other branched-chain amino acids are metabolized solely as coenzyme A thioesters. Because it exists as a free acid, HIBA can be released into the blood stream by specific tissues and is cleared by the liver where it can serve as a substrate for gluconeogenesis (Letto et al., 1986). During the past decade there has been significant interest in the metabolism and interorgan trafficking of the R- and S- enantiomers of HIBA. HIBA is oxidized in mitochondria to methylmalonate semialdehyde by a highly specific, NAD+-dependent dehydrogenase (HIBADH or 3-hydroxy-2-methyl-propionate: NAD+ oxidoreductase, EC 1.1.1.31). Previous studies of rat HIBADH tentatively placed the enzyme in the now well-established short-chain alcohol dehydrogenase family (Crabb et al., 1993; Hawes et al., 1995). This assignment was based on amino acid sequence homology, enzymatic properties such as the lack of a metal requirement for catalysis, and effects of tyrosine-specific chemical modification. However, site-directed mutagenesis studies indicated that HIBADH differs in mechanism from the short-chain dehydrogenases studied to date, such as Drosophila alcohol dehydrogenase (Hawes et al., 1995). Furthermore, the short-chain dehydrogenases mostly prefer secondary alcohols as optimal substrates whereas HIBADH is only active with primary alcohol substrates. HIBADH, therefore, is most likely not closely related to the short-chain dehydrogenases. More recent studies showed that HIBADH shares better amino acid sequence homology and enzymatic properties with a separate, previously unrecognized family of enzymes that includes D-phenylserine dehydrogenase from Pseudomonas syringae, 6-phosphogluconate dehydrogenase from numerous species, and numerous hypothetical proteins of microbial origin (Hawes et al., 1996).

[20] – Production of Recombinant E1 Component of Branched-Chain α-Keto Acid Dehydrogenase Complex

The activity state of mitochondrial branched-chain α-keto acid dehydrogenase (BCKDH) is highly regulated according to the physiological requirements for protein synthesis or degradation of excess branched-chain amino acids.1 This regulation is primarily exerted through reversible phosphorylation of the catalytic subunit of BCKDH by a specific protein kinase and a specific phosphoprotein phosphatase. Two phosphorylation sites exist on the E1α subunit although regulation of BCKDH activity results exclusively from phosphorylation of site 1, Ser-293 in rat E1α. Phosphorylation of site 2, Ser-303 in rat E1α, occurs at a significantly slower rate2 and is silent with respect to regulation of BCKDH activity.3 Thiamine pyrophosphate (TPP) and branched-chain α-keto acids (cofactor and substrates for the BCKDH-catalyzed reaction) inhibit the phosphorylation by BCKDH kinase, and it has been suggested that BCKDH kinase inactivates BCKDH E1 by placing a covalently linked phosphate directly into the active site of the dehydrogenase.4 Studies of the mechanism of inactivation of BCKDH by phosphorylation require a ready source of purified subunits of this multienzyme complex that can be reconstituted into an active BCKDH complex under various conditions. To this end we have expressed the BCKDH E1 component as a recombinant enzyme5 in a form that can be easily purified and reconstituted with the purified transacylase core (E2) of the BCKDH complex. The purified recombinant E1 subunit is enzymatically active when combined with E2, can be inactivated by treatment with the purified recombinant BCKDH kinase and ATP, and can be manipulated by site-directed mutagenesis. Materials R408 and VCS M13 are purchased from Stratagene (La Jolla, CA). Vectors pET15 and pET21 are purchased from Novagen (Madison, WI). Nickel-NTA-agarose is purchased from Qiagen (Valencia, CA). Restriction enzymes, restriction enzyme buffers, and other DNA-modifying enzymes are purchased from BRL (Gaithersburg, MD). All chemical reagents for enzyme assay are purchased from Sigma (St. Louis, MO). The site-directed mutagenesis system is purchased from Amersham (Arlington Heights, IL). Native rat liver BCKDH E2 subunit is prepared by a modification of the method of Cook et al.6 according to Shimomura et al.7 The pGroESL plasmid was a kind gift of A. Gatenby (Central Research and Development, Du Pont, Wilmington, DE).