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Peptididylarginine deiminase type 2 (PADI2) catalyzes the conversion of arginine residues to citrulline residues on proteins. We demonstrate that PADI2 induces T cell activation and investigate how PADI2 promotes activated T cell autonomous death (ACAD). In activated Jurkat T cells, overexpression of PADI2 significantly increases citrullinated proteins and induces endoplasmic reticulum (ER) stress and unfolded protein response (UPR) signaling, ultimately resulting in the expression of autophagy-related proteins and autophagy. PADI2 promoted autophagy and resulted in the early degradation of p62 and the light chain 3B (LC3B)-II accumulation. In Jurkat T cells, silencing the autophagy-related gene (Atg) 12 protein inhibits PADI2-mediated autophagy and promotes ER stress and apoptosis, whereas overexpression of Atg12 decreased ER stress and prolonged autophagy to promote cell survival. Additionally, PADI2 regulates T cell activation and the production of Th17 cytokines in Jurkat T cells (interleukins 6, IL-17A, IL-17F, IL-21, and IL-22). In Jurkat T cells, silencing IL-6 promotes autophagy mediated by PADI2 and inhibits PADI2-induced apoptosis, whereas silencing Beclin-1 increases the activation and survival of Th17-like T cells while decreasing autophagy and apoptosis. PADI2 silencing alleviates ER stress caused by PADI2 and decreases cytokine expression associated with Th17-like T cell activation and ACAD. We propose that PADI2 was involved in Th17 lymphocyte ACAD via a mechanism involving ER stress and autophagy that was tightly regulated by PADI2-mediated citrullination. These findings suggest that inhibiting Th17 T cell activation and the development of severe autoimmune diseases may be possible through the use of novel antagonists that specifically target PADI2.

Antizyme (AZ) is a protein that negatively regulates ornithine decarboxylase (ODC). AZ achieves this inhibition by binding to ODC to produce AZ-ODC heterodimers, abolishing enzyme activity and targeting ODC for degradation by the 26S proteasome. In this study, we focused on the biomolecular interactions between the C-terminal domain of AZ (AZ95&ndash, 228) and ODC to identify the functional elements of AZ that are essential for binding, inhibiting and degrading ODC, and we also identified the crucial factors governing the differential binding and inhibition ability of AZ isoforms toward ODC. Based on the ODC inhibition and AZ-ODC binding studies, we demonstrated that amino acid residues reside within the &alpha, 1 helix, &beta, 5 and &beta, 6 strands, and connecting loop between &beta, 6 and &alpha, 2 (residues 142&ndash, 178), which is the posterior part of AZ95&ndash, 228, play crucial roles in ODC binding and inhibition. We also identified the essential elements determining the ODC-degradative activity of AZ, amino acid residues within the anterior part of AZ95&ndash, 228 (residues 120&ndash, 145) play crucial roles in AZ-mediated ODC degradation. Finally, we identified the crucial factors that govern the differential binding and inhibition of AZ isoforms toward ODC. Mutagenesis studies of AZ1 and AZ3 and their binding and inhibition revealed that the divergence of amino acid residues 124, 150, 166, 171, and 179 results in the differential abilities of AZ1 and AZ3 in the binding and inhibition of ODC.

Summary Human mitochondrial NAD(P)+-dependent malic enzyme (ME2) is well recognized to associate with cancer cell metabolism, and the single nucleotide variants (SNVs) of ME2 may play a role in enzyme regulation. Here we reported that the SNVs of ME2 occurring in the allosteric sites lead to inactivation or overactivation of ME2. Two ME2-SNVs, ME2_R67Q and ME2-R484W, that demonstrated inactivating or overactivating enzyme activities of ME2, respectively, have different impact toward the cells. The cells with overactivating SNV enzyme, ME2_R484W, grow more rapidly and are more resistant to cellular senescence than the cells with wild-type or inactivating SNV enzyme, ME2_R67Q. Crystal structures of these two ME2-SNVs reveal that ME2_R67Q was an inactivating “dead form,” and ME2_R484W was an overactivating “closed form” of the enzyme. The resolved ME2-SNV structures provide a molecular basis to explain the abnormal kinetic properties of these SNV enzymes., Graphical abstract, Highlights • Single nucleotide variants lead to dysregulation of the human ME2 • ME2 SNVs that occur in the regulatory sites lead to dysregulated kinetics of ME2 • The dysregulated ME2 SNVs have an impact on cell growth and cellular senescence • ME2-SNV structures provide a molecular basis to explain dysregulated kinetics of ME2, Biological Sciences; Genetics; Cell Biology; Structural Biology; Cancer

Polyamines are organic polycations essential for cell growth and differentiation; their aberrant accumulation is often associated with diseases, including many types of cancer. To maintain polyamine homeostasis, the catalytic activity and protein abundance of ornithine decarboxylase (ODC), the committed enzyme for polyamine biosynthesis, are reciprocally controlled by the regulatory proteins antizyme isoform 1 (Az1) and antizyme inhibitor (AzIN). Az1 suppresses polyamine production by inhibiting the assembly of the functional ODC homodimer and, most uniquely, by targeting ODC for ubiquitin-independent proteolytic destruction by the 26S proteasome. In contrast, AzIN positively regulates polyamine levels by competing with ODC for Az1 binding. The structural basis of the Az1-mediated regulation of polyamine homeostasis has remained elusive. Here we report crystal structures of human Az1 complexed with either ODC or AzIN. Structural analysis revealed that Az1 sterically blocks ODC homodimerization. Moreover, Az1 binding triggers ODC degradation by inducing the exposure of a cryptic proteasome-interacting surface of ODC, which illustrates how a substrate protein may be primed upon association with Az1 for ubiquitin-independent proteasome recognition. Dynamic and functional analyses further indicated that the Az1-induced binding and degradation of ODC by proteasome can be decoupled, with the intrinsically disordered C-terminal tail fragment of ODC being required only for degradation but not binding. Finally, the AzIN-Az1 structure suggests how AzIN may effectively compete with ODC for Az1 to restore polyamine production. Taken together, our findings offer structural insights into the Az-mediated regulation of polyamine homeostasis and proteasomal degradation.

Dibenzoylmethane (DB), a minor constituent of the root extract of licorice, belongs to the flavonoid family. Hydroxydibenzoylmethane (HDB) and hydroxymethyldibenzoylmethane (HMDB) have an identical structure to DB, but also possess a hydroxyl group and a hydroxyl and methyl group bonded to aromatic rings, respectively. They inhibit cellular proliferation and induce apoptosis in a variety of types of cancer cell, however, the antimetastatic effects of DB, HDB and HMDB on human breast carcinoma cells remain to be elucidated. The present study aimed to clarify the molecular mechanisms underlying the effects of DB and its analogues on phorbol‑12‑myristate 13‑acetate (PMA)‑induced MCF‑7 cell metastasis. The results revealed that DB, HDB and HMDB inhibited cell migration and invasion. In addition, PMA‑mediated MCF‑7 cell invasion was inhibited by DB, HDB and HMDB by inhibiting the expression of matrix metalloproteinase (MMP)‑9. Rottlerin, a protein kinase C (PKC)δ inhibitor and LY294002, a phosphatidylinositide 3‑kinase (PI3K) inhibitor, reduced the PMA‑mediated expression of MMP‑9 and cell invasion. Furthermore, DB, HDB and HMDB prevented the activation of PKCδ and PI3K by inhibiting their phosphorylation. The present study was the first, to the best of our knowledge, to demonstrate the antimetastatic potential of DB, HDB and HDMB, which decreased cancer cell invasion through the PI3K/PKCδ‑mediated MMP‑9 pathway.

Our recent studies of peptidylarginine deiminase 4 (PAD4) demonstrate that its non-catalytic Ca2+-binding sites play a crucial role in the assembly of the correct geometry of the enzyme. Here, we examined the folding mechanism of PAD4 and the role of Ca2+ ions in the folding pathway. Multiple mutations were introduced into the calcium-binding sites, and these mutants were termed the Ca1_site, Ca2_site, Ca3_site, Ca4_site and Ca5_site mutants. Our data indicate that during the unfolding process, the PAD4 dimer first dissociates into monomers, and the monomers then undergo a three-state denaturation process via an intermediate state formation. In addition, Ca2+ ions assist in stabilizing the folding intermediate, particularly through binding to the Ca3_site and Ca4_site to ensure the correct and active conformation of PAD4. The binding of calcium ions to the Ca1_site and Ca2_site is directly involved in the catalytic action of the enzyme. Finally, this study proposes a model for the folding of PAD4. The nascent polypeptide chains of PAD4 are first folded into monomeric intermediate states, then continue to fold into monomers, and ultimately assemble into a functional and dimeric PAD4 enzyme, and cellular Ca2+ ions may be the critical factor governing the interchange.

Our previous studies suggest that the fully active form of Peptidylarginine deiminase 4 (PAD4) should be a dimer and not a monomer. This paper provides a plausible mechanism for the control of PAD4 catalysis by molecular interplay between its dimer-interface loop (I-loop) and its substrate-binding loop (S-loop). Mutagenesis studies revealed that two hydrophobic residues, W347 and V469, are critical for substrate binding at the active site; mutating these two residues led to a severe reduction in the catalytic activity. We also identified several hydrophobic amino acid residues (L6, L279 and V283) at the dimer interface. Ultracentrifugation analysis revealed that interruption of the hydrophobicity of this region decreases dimer formation and, consequently, enzyme activity. Molecular dynamic simulations and mutagenesis studies suggested that the dimer interface and the substrate-binding site of PAD4, which consist of the I-loop and the S-loop, respectively, are responsible for substrate binding and dimer stabilization. We identified five residues with crucial roles in PAD4 catalysis and dimerization: Y435 and R441 in the I-loop, D465 and V469 in the S-loop, and W548, which stabilizes the I-loop via van der Waals interactions with C434 and Y435. The molecular interplay between the S-loop and the I-loop is crucial for PAD4 catalysis.

Dibenzoylmethane (DBM) belongs to the flavonoid family and is a minor constituent of the root extract of licorice and the β-diketone analogue of curcumin. It exhibits antimutagenic, anticancer, and chemopreventive effects. Ornithine decarboxylase (ODC), the rate-limiting enzyme of the polyamine biosynthetic pathway, plays an important role in growth, proliferation, and transformation. Our previous studies showed ODC overexpression prevented etoposide-, paclitaxel-, and cisplatin-induced apoptosis. Here, we investigated one mechanism of DBM-induced apoptosis and the antiapoptotic effects of ODC during DBM treatment. We found that DBM induced apoptosis, promoted reactive oxygen species (ROS) generation, and disrupted the mitochondrial membrane potential (Δ ψm. N-acetylcysteine, a ROS scavenger, reduced DBM-induced apoptosis, which led to the loss of Δ ψm due to reduced ROS. Overexpression of ODC in parental cells had the same effects as the ROS scavenger. The results demonstrated that DBM-induced apoptosis was a ROS-dependent pathway and ODC overexpression blocked DBM-induced apoptosis by inhibiting intracellular ROS production. © 2011 Wiley Periodicals, Inc. J Biochem Mol Toxicol 25:312–319, 2011; View this article online at wileyonlinelibrary.com. DOI 10.1002/jbt.20391

Human mitochondrial NAD(P)(+)-dependent malic enzyme (m-NAD-ME) is a malic enzyme isoform with dual cofactor specificity, ATP inhibition and substrate cooperativity. The determinant of ATP inhibition in malic enzyme isoforms has not yet been identified. Sequence alignment of nucleotide-binding sites of ME isoforms revealed that Lys346 is conserved uniquely in m-NAD-ME. In other ME isoforms, this residue is serine. As the inhibitory effect of ATP is more pronounced on m-NAD-ME than on other ME isoforms, we have examined the possible role of Lys346 by replacing it to alanine, serine or arginine. Our kinetic data indicate that the K346S mutant enzyme displays a shift in its cofactor preference from NAD(+) to NADP(+) upon increasing k(cat,NADP) and decreasing K(m,NADP). Furthermore, the cooperative binding of malate becomes less significant in human m-NAD-ME after mutation of Lys346. The h value for the wild-type is close to 2, but those of the K346 mutants are approximately 1.5. The K346 mutants can also be activated by fumarate and the cooperative effect can be abolished by fumarate, suggesting that the allosteric property is retained in these mutants. Our data strongly suggest that Lys346 in human m-NAD-ME is required for ATP inhibition. Mutation of Lys346 to Ser or Ala causes the enzyme to be much less sensitive to ATP, similar to cytosolic NADP-dependent malic enzyme. Substitution of Lys to Arg did not change the isoform-specific inhibition of the enzyme by ATP. The inhibition constants of ATP are increased for K346S and K346A, but are similar to those of the wild-type for K346R, suggesting that the positive charge rather than group specificity is required for binding affinity of ATP. Thus, ATP inhibition is proposed to be determined by the electrostatic potential involving the positive charge on the side chain of Lys346.

The human mitochondrial NAD(P)+-dependent malic enzyme (m-NAD-ME) is a malic enzyme isoform with dual cofactor specificity and substrate binding cooperativity. Previous kinetic studies have suggested that Lys362 in the pigeon cytosolic NADP+-dependent malic enzyme has remarkable effects on the binding of NADP+ to the enzyme and on the catalytic power of the enzyme (Kuo, C. C., Tsai, L. C., Chin, T. Y., Chang, G.-G., and Chou, W. Y. (2000) Biochem. Biophys. Res. Commun. 270, 821-825). In this study, we investigate the important role of Gln362 in the transformation of cofactor specificity from NAD+ to NADP+ in human m-NAD-ME. Our kinetic data clearly indicate that the Q362K mutant shifted its cofactor preference from NAD+ to NADP+. The Km(NADP) and kcat(NADP) values for this mutant were reduced by 4-6-fold and increased by 5-10-fold, respectively, compared with those for the wild-type enzyme. Furthermore, up to a 2-fold reduction in Km(NADP)/Km(NAD) and elevation of kcat(NADP)/kcat(NAD) were observed for the Q362K enzyme. Mutation of Gln362 to Ala or Asn did not shift its cofactor preference. The Km(NADP)/Km(NAD) and kcat(NADP)/kcat(NAD) values for Q362A and Q362N were comparable with those for the wild-type enzyme. The ΔG values for Q362A and Q362N with either NAD+ or NADP+ were positive, indicating that substitution of Gln with Ala or Asn at position 362 brings about unfavorable cofactor binding at the active site and thus significantly reduces the catalytic efficiency. Our data also indicate that the cooperative binding of malate became insignificant in human m-NAD-ME upon mutation of Gln362 to Lys because the sigmoidal phenomenon appearing in the wild-type enzyme was much less obvious that that in Q362K. Therefore, mutation of Gln362 to Lys in human m-NAD-ME alters its kinetic properties of cofactor preference, malate binding cooperativity, and allosteric regulation by fumarate. However, the other Gln362 mutants, Q362A and Q362N, have conserved malate binding cooperativity and NAD+ specificity. In this study, we provide clear evidence that the single mutation of Gln362 to Lys in human m-NAD-ME changes it to an NADP+-dependent enzyme, which is characteristic because it is non-allosteric, non-cooperative, and NADP+-specific.

Human mitochondrial NAD(P)+-dependent malate dehydrogenase (decarboxylating) (malic enzyme) can be specifically and allosterically activated by fumarate. X-ray crystal structures have revealed conformational changes in the enzyme in the absence and in the presence of fumarate. Previous studies have indicated that fumarate is bound to the allosteric pocket via Arg67 and Arg91. Mutation of these residues almost abolishes the activating effect of fumarate. However, these amino acid residues are conserved in some enzymes that are not activated by fumarate, suggesting that there may be additional factors controlling the activation mechanism. In the present study, we tried to delineate the detailed molecular mechanism of activation of the enzyme by fumarate. Site-directed mutagenesis was used to replace Asp102, which is one of the charged amino acids in the fumarate binding pocket and is not conserved in other decarboxylating malate dehydrogenases. In order to explore the charge effect of this residue, Asp102 was replaced by alanine, glutamate or lysine. Our experimental data clearly indicate the importance of Asp102 for activation by fumarate. Mutation of Asp102 to Ala or Lys significantly attenuated the activating effect of fumarate on the enzyme. Kinetic parameters indicate that the effect of fumarate was mainly to decrease the Km values for malate, Mg2+ and NAD+, but it did not notably elevate kcat. The apparent substrate Km values were reduced by increasing concentrations of fumarate. Furthermore, the greatest effect of fumarate activation was apparent at low malate, Mg2+ or NAD+ concentrations. The Kact values were reduced with increasing concentrations of malate, Mg2+ and NAD+. The Asp102 mutants, however, are much less sensitive to regulation by fumarate. Mutation of Asp102 leads to the desensitization of the co-operative effect between fumarate and substrates of the enzyme.

Identification and use of effective cancer chemopreventive agents have become an important issue in public health-related research. For identification of potential cancer chemopreventive constituents we have set up a battery of cell- and enzyme-based in vitro marker systems relevant for prevention of carcinogenesis in vivo. These systems include modulation of drug metabolism (inhibition of Cyp1A activity, induction of NAD(P)H:quinone reductase (QR) activity in Hepa1c1c7 murine hepatoma cell culture), determination of radical scavenging (DPPH scavenging) and antioxidant effects (scavenging of superoxide anion-, hydroxyl- and peroxyl-radicals), anti-inflammatory mechanisms (inhibition of lipopolysaccharide (LPS)-mediated nitric oxide (NO) generation by inducible NO synthase (iNOS) in Raw 264.7 murine macrophages, cyclooxygenase-1 (Cox-1) inhibition), and anti-tumor promoting activities (inhibition of phorbol ester-induced ornithine decarboxylase (ODC) activity in 308 murine keratinocytes). We have tested a series of known chemopreventive substances belonging to several structural classes as reference compounds for the identification of novel chemopreventive agents or mechanisms. These include organosulfur compounds (phenethylisothiocyanate (PEITC), diallylsulfide, diallyldisulfide), terpenes (limonene, perillyl alcohol, oleanolic acid, 18--glycyrrhetinic acid), short-chain fatty acids (sodium butyrate), indoles (indole-3-carbinol), isoflavonoids (quercetin, silymarin, genistein), catechins (( −)-epigallocatechin gallate (EGCG)), simple phenols (ellagic acid, resveratrol, piceatannol, curcumin), pharmaceutical agents (piroxicam, acetylsalicylic acid, tamoxifen), and vitamins/derivatives (ascorbic acid, Trolox). We confirmed known chemopreventive mechanisms of these compounds. Additionally, we could demonstrate the usefulness of our approach by identification of hitherto unknown mechanisms of selected agents. As an example, we detected anti-inflammatory properties of PEITC, based on NF-B-mediated inhibition of NO production. Further, PEITC inhibited phorbol ester-induced superoxide anion radical production in granulocytes, and ODC induction in the 308 cell line. These mechanisms might contribute to the chemopreventive potential of PEITC. © 2002 Elsevier Science B.V. All rights reserved.

Cutaneous melanoma is the most life-threatening neoplasm of the skin, accounting for most of the skin cancer deaths. Accumulating evidence suggests that targeting metabolism is an appealing strategy for melanoma therapy. Mitochondrial NAD(P) + –dependent malic enzyme (ME2), an oxidative decarboxylase, was evaluated for its biological significance in cutaneous melanoma progression. ME2 mRNA and protein expression significantly increased during melanoma progression, as evidenced by Gene Expression Omnibus analysis and immunohistochemistry on clinically annotated tissue microarrays, respectively. In addition, ME2 knockdown attenuated melanoma cell proliferation in vitro . ME2 ablation resulted in reduced cellular ATP levels and elevated cellular reactive oxygen species production, which activated the AMP-activated protein kinase pathway and inhibited acetyl-CoA carboxylase. Furthermore, ME2 expression was associated with cell migration and invasion. ME2 knockdown decreased anchorage-independent growth in vitro and tumor cell growth in vivo. These results suggested that ME2 might be an important factor in melanoma progression and a novel biomarker of invasion.

Ornithine decarboxylase (ODC) catalyzes the decarboxylation of ornithine to putrescine and is the rate-limiting enzyme in the polyamine biosynthesis pathway. ODC is a dimeric enzyme, and the active sites of this enzyme reside at the dimer interface. Once the enzyme dissociates, the enzyme activity is lost. In this paper, we investigated the roles of amino acid residues at the dimer interface regarding the dimerization, protein stability and/or enzyme activity of ODC. A multiple sequence alignment of ODC and its homologous protein antizyme inhibitor revealed that 5 of 9 residues (residues 165, 277, 331, 332 and 389) are divergent, whereas 4 (134, 169, 294 and 322) are conserved. Analytical ultracentrifugation analysis suggested that some dimer-interface amino acid residues contribute to formation of the dimer of ODC and that this dimerization results from the cooperativity of these interface residues. The quaternary structure of the sextuple mutant Y331S/Y389D/R277S/D332E/V322D/D134A was changed to a monomer rather than a dimer, and the K d value of the mutant was 52.8 µM, which is over 500-fold greater than that of the wild-type ODC (ODC_WT). In addition, most interface mutants showed low but detectable or negligible enzyme activity. Therefore, the protein stability of these interface mutants was measured by differential scanning calorimetry. These results indicate that these dimer-interface residues are important for dimer formation and, as a consequence, are critical for enzyme catalysis.

Peroxyacetyl nitrate (PAN), an ubiquitous air pollutant, induced apoptosis in human leukemia HL-60, human chronic myelogenous leukemia K-562, and mouse monocyte-macrophage RAW 264.7 cell lines. In the HL 60 cells, characteristic apoptosis morphology could be observed 4 h after the cells were treated with 50 μM PAN. Exposure of HL-60 cells to increasing concentrations of PAN (from 1 μM to 100 μM) confirmed the concentration dependence of apoptosis as evidenced by DNA fragmentation in HL-60 cells, chromatin condensation by acridine-orange staining, and the appearance of the DNA apoptotic peak in flow cytometry. During apoptosis in HL-60 cells, 3-nitrotyrosine and 3,5-dinitrotyrosine were detected by high-performance liquid chromatography and liquid chromatography–mass spectrometry–mass spectrometry. We hypothesized that PAN might induce cell death in human leukemia cells by releasing peroxynitrite and other reactive oxygen species (ROS) such as superoxide and hydrogen peroxide. Moreover, exogenous superoxide dismutase promoted PAN-induced apoptosis, and in contrast, a combination of superoxide dismutase and catalase suppressed this apoptosis. We also hypothesize that the generation of ROS during PAN-induced apoptosis in HL-60 cells could activate stress-activated protein kinase/jun N-terminal kinase activity. The formation of H2O2 produced from the dismutation of PAN-elicited superoxide anion contributed to the apoptotic mechanism in HL-60 cells through ROS pathways. These findings suggested that induction of apoptosis by the air pollutant PAN might occur as a result of the release of ROS. Mol. Carcinog. 25:196–206, 1999. © 1999 Wiley-Liss, Inc.

Prolinedithiocarbamate (PDTC) and diethyldithiocarbamate (DDTC) are cancer chemopreventive agents and can be biotransformed to prolinethiuramdisulfide (PTDS) and tetraethylthiuramdisulfide (disulfiram; DTDS), respectively. We found that the reactive metabolites PTDS and DTDS induced apoptosis after G1/S arrest. Phosphorylation of cyclin E, inhibition of cyclin-dependent kinase 2 activity, and degradation of cyclin E were found in human hepatoma Hep G2 cells during apoptosis. Moreover, PTDS and DTDS decreased the level of bcl-2 but increased the level of p53. In contrast, PDTC, DDTC, and ammonium dithiocarbamate (ADTC) did not induce apoptosis; rather they led to the induction of p53 and p21 followed by G1/S arrest. PDTC, DDTC, and ADTC also arrested cells in G1 phase. We then examined the effects of PTDS and DTDS on the signal transduction mechanisms leading to apoptosis. Although the transcription factors NFκB and AP-1 cooperatively decreased their DNA-binding activities to κB and 12-O-tetradecanoylphorbol-13-acetate–responsive elements, respectively, and p53 increased DNA-binding activity in the early stage but decreased it in the latter stage after treatment with PTDS, when the human Hep G2 cells were undergoing apoptosis. In summary, our results indicated that (i) PTDS and DTDS induced apoptosis and G1/S arrest mediated by p53, whereas PDTC, DDTC, and ADTC induced p53-dependent p21 expression leading to G1/S arrest; (ii) PDTC, DDTC, and ADTC induced p21/KIP1/CIP1 expression in a p53-dependent pathway leading to G1/S arrest; and (iii) NFκB, AP-1, and bcl-2 were downregulated during PTDS- and DTDS-induced apoptosis. These results suggested that PTDS and DTDS induced p53-dependent apoptosis, whereas PDTC, DDTC, and ADTC induced G1/S arrest. Apoptosis is regulated by the modulation of intracellular effectors such as NFκB, AP-1, and bcl-2 and activation of p53 in early stages. Mol. Carcinog. 22:235–246, 1998. © 1998 Wiley-Liss, Inc.

Human mitochondrial NAD(P)+-dependent malic enzyme (m-NAD(P)-ME) is allosterically activated by the four-carbon trans dicarboxylic acid, fumarate. Previous studies have suggested that the dicarboxylic acid in a trans conformation around the carbon-carbon double bond is required for the allosteric activation of the enzyme. In this paper, the allosteric effects of fumarate analogs on m-NAD(P)-ME are investigated. Two fumarate-insensitive mutants, m-NAD(P)-ME_R67A/R91A and m-NAD(P)-ME_K57S/E59N/K73E/D102S, as well as c-NADP-ME, were used as the negative controls. Among these analogs, mesaconate, trans-aconitate, monomethyl fumarate and monoethyl fumarate were allosteric activators of the enzyme, while oxaloacetate, diethyl oxalacetate, and dimethyl fumarate were found to be allosteric inhibitors of human m-NAD(P)-ME. The IC50 value for diethyl oxalacetate was approximately 2.5 mM. This paper suggests that the allosteric inhibitors may impede the conformational change from open form to closed form and therefore inhibit m-NAD(P)-ME enzyme activity.

Ornithine decarboxylase (ODC) is the rate-limiting enzyme in polyamine biosynthesis and a target for chemoprevention. Hydroxydibenzoylmethane (HDB), a derivative of dibenzoylmethane of licorice, is a promising chemopreventive agent. In this paper, we investigated whether HDB would inhibit the ODC pathway to enhance apoptosis in human promyelocytic leukemia HL-60 cells. We found ODC enzyme activity was reduced during HDB treatment. Overexpression of ODC in HL-60 parental cells could reduce HDB-induced apoptosis, which leads to loss of mitochondrial membrane potential (Δψ(m)), through lessening intracellular ROS. Furthermore, ODC overexpression protected cytochrome c release and the activation of caspase-3 following HDB treatment. The results demonstrated HDB-induced apoptosis was through a mechanism of down-regulation of ODC and occurred along a ROS-dependent mitochondria-mediated pathway.

Both ornithine decarboxylase (ODC) and its regulatory protein, antizyme inhibitor (AZI), can bind with antizyme (AZ), but the latter has a higher AZ-binding affinity. The results of this study clearly identify the critical amino acid residues governing the difference in AZ-binding affinities between human ODC and AZI. Inhibition experiments using a series of ODC mutants suggested that residues 125 and 140 may be the key residues responsible for the differential AZ-binding affinities. The ODC_N125K/M140K double mutant demonstrated a significant inhibition by AZ, and the IC(50) value of this mutant was 0.08 µM, three-fold smaller than that of ODC_WT. Furthermore, the activity of the AZ-inhibited ODC_N125K/M140K enzyme was hardly rescued by AZI. The dissociation constant (K(d)) of the [ODC_N125K/M140K]-AZ heterodimer was approximately 0.02 µM, which is smaller than that of WT_ODC by approximately 10-fold and is very close to the K(d) value of AZI_WT, suggesting that ODC_N125K/M140K has an AZ-binding affinity higher than that of ODC_WT and similar to that of AZI. The efficiency of the AZI_K125N/K140M double mutant in the rescue of AZ-inhibited ODC enzyme activity was less than that of AZI_WT. The K(d) value of [AZI_K125N/K140M]-AZ was 0.18 µM, nine-fold larger than that of AZI_WT and close to the K(d) value of ODC_WT, suggesting that AZI_K125N/K140M has an AZ-binding affinity lower than that of AZI_WT and similar to that of ODC. These data support the hypothesis that the differences in residues 125 and 140 in ODC and AZI are responsible for the differential AZ-binding affinities.

Ornithine decarboxylase (ODC) is a ubiquitous enzyme that is conserved in all species from bacteria to humans. Mammalian ODC is degraded by the proteasome in a ubiquitin-independent manner by direct binding to the antizyme (AZ). In contrast, Trypanosoma brucei ODC has a low binding affinity toward AZ. In this study, we identified key amino acid residues that govern the differential AZ binding affinity of human and Trypanosoma brucei ODC. Multiple sequence alignments of the ODC putative AZ-binding site highlights several key amino acid residues that are different between the human and Trypanosoma brucei ODC protein sequences, including residue 119, 124,125, 129, 136, 137 and 140 (the numbers is for human ODC). We generated a septuple human ODC mutant protein where these seven bases were mutated to match the Trypanosoma brucei ODC protein sequence. The septuple mutant protein was much less sensitive to AZ inhibition compared to the WT protein, suggesting that these amino acid residues play a role in human ODC-AZ binding. Additional experiments with sextuple mutants suggest that residue 137 plays a direct role in AZ binding, and residues 119 and 140 play secondary roles in AZ binding. The dissociation constants were also calculated to quantify the affinity of the ODC-AZ binding interaction. The K(d) value for the wild type ODC protein-AZ heterodimer ([ODC_WT]-AZ) is approximately 0.22 μM, while the K(d) value for the septuple mutant-AZ heterodimer ([ODC_7M]-AZ) is approximately 12.4 μM. The greater than 50-fold increase in [ODC_7M]-AZ binding affinity shows that the ODC-7M enzyme has a much lower binding affinity toward AZ. For the mutant proteins ODC_7M(-Q119H) and ODC_7M(-V137D), the K(d) was 1.4 and 1.2 μM, respectively. These affinities are 6-fold higher than the WT_ODC K(d), which suggests that residues 119 and 137 play a role in AZ binding.

Ornithine decarboxylase (ODC) is the first enzyme involved in polyamine biosynthesis, and it catalyzes the decarboxylation of ornithine to putrescine. ODC is a dimeric enzyme, whereas antizyme inhibitor (AZI), a positive regulator of ODC that is homologous to ODC, exists predominantly as a monomer and lacks decarboxylase activity. The goal of this paper was to identify the essential amino acid residues that determine the dimerization of AZI. The nonconserved amino acid residues in the putative dimer interface of AZI (Ser-277, Ser-331, Glu-332, and Asp-389) were substituted with the corresponding residues in the putative dimer interface of ODC (Arg-277, Tyr-331, Asp-332, and Tyr-389, respectively). Analytical ultracentrifugation analysis was used to determine the size distribution of these AZI mutants. The size-distribution analysis data suggest that residue 331 may play a major role in the dimerization of AZI. Mutating Ser-331 to Tyr in AZI (AZI-S331Y) caused a shift from a monomer configuration to a dimer. Furthermore, in comparison with the single mutant AZI-S331Y, the AZI-S331Y/D389Y double mutant displayed a further reduction in the monomer-dimer K(d), suggesting that residue 389 is also crucial for AZI dimerization. Analysis of the triple mutant AZI-S331Y/D389Y/S277R showed that it formed a stable dimer (K(d) value = 1.3 microm). Finally, a quadruple mutant, S331Y/D389Y/S277R/E332D, behaved as a dimer with a K(d) value of approximately 0.1 microm, which is very close to that of the human ODC enzyme. The quadruple mutant, although forming a dimer, could still be disrupted by antizyme (AZ), further forming a heterodimer, and it could rescue the AZ-inhibited ODC activity, suggesting that the AZ-binding ability of the AZI dimer was retained.

A growing amount of evidence implicates the involvement of apolipoprotein E (apoE) in the development of late-onset and sporadic forms of Alzheimer's disease (AD). It is now generally believed that the epsilon4 allele is associated with AD and the oxidative stress is more pronounced in AD. However, only limited data are available on apoE isoform-specificity and its relationship to both the oxidative susceptibility and conformational stability of apoE. In this article, we use site-directed mutagenesis to investigate the structural role of amino acid residue 112, which is the only differing residue between apoE3 and E4. We examine the structural variation manipulating the oxidative susceptibility and conformational stability of apolipoprotein E isoforms. Arg112 in apoE4 was changed to Ala and Glu. Previous research has reported that apoE4 is more susceptible to free radicals than apoE3. In protein oxidation experiments, apoE4-R112A becomes more resistant to free radicals to the same extent as apoE3. In contrast, apoE4-R112E becomes the most susceptible protein to free radicals among all the apoE proteins. We also examine the conformational stability and the quaternary structural change by fluorescence spectroscopy and analytical ultracentrifugation, respectively. ApoE3 and E4 show apparent three- and two-state unfolding patterns, respectively. ApoE4-R112A, similar to apoE3, demonstrates a biphasic denaturation with an intermediate that appears. The denaturation curve for apoE4-R112E, however, also displays a biphasic profile but with a slight shoulder at approximately 1.5M GdmCl, implying that an unstable intermediate existed in the denaturation equilibrium. The size distribution of apoE isoforms display similar patterns. ApoE4-R112E, however, has a greater tendency to dissociate from high-molecular-weight species to tetramers. These experimental data suggest that the amino acid residue 112 governs the differences in salt-bridges between these two isoforms and thus has a significant impact on the free radical susceptibility and structural variation of the apoE isoforms.

Recent data showed that epidermal growth factor receptor (EGFR) inhibitors, such as ZD1839, alone or in combination with chemotherapeutic agents for androgen-independent prostate cancer (AIPC) did not produce promising results in clinical settings. More effective regimens involving novel stronger inhibitor of EGFR and better combinations are needed. The anti-tumor activity of PD168393, an irreversible EGFR inhibitor, with or without chemotherapeutic agents for the treatment of AIPC was investigated in vitro. In results, both the androgen-independent cell lines PC-3 and DU145 expressed higher levels of EGFR than the androgen-dependent MDA PCa 2b and androgen-responsive LNCaP cells by Western blotting. DU145 was much more sensitive to PD168393 and ZD1839 than MDA PCa 2b. PD168393, but not ZD1839, significantly potentiated paclitaxel cytotoxicity against DU145 by MTT assay and median-effect analysis. The combination of PD168393 or ZD1839 with other cytotoxic agents including docetaxel and 5-fluorouracil, however, was either additive or antagonistic. Compared to paclitaxel alone, PD168393 significantly enhanced paclitaxel-induced DNA fragmentation, sub-G1 fraction accumulation, mitochondrial membrane dysfunction, cytochrome C release, caspase-3 activation and eventually apoptosis. These molecular events were accompanied by Bad up-regulation, p53 and p21Waf1/Cip1 induction, ERK1/2 inactivation and inhibition of EGFR phosphorylation in the presence of PD168393. These effects did not involve significant alteration in the Akt1/2 and STAT3 signaling pathway. In conclusion, the combination of paclitaxel and PD168393 produced a profound synergistic growth inhibition of AIPC cells. Combining PD168393 with paclitaxel may have clinical benefits and warrants further investigation.

Human mitochondrial NAD(P)+-dependent malic enzyme is inhibited by ATP. The X-ray crystal structures have revealed that two ATP molecules occupy both the active and exo site of the enzyme, suggesting that ATP might act as an allosteric inhibitor of the enzyme. However, mutagenesis studies and kinetic evidences indicated that the catalytic activity of the enzyme is inhibited by ATP through a competitive inhibition mechanism in the active site and not in the exo site. Three amino acid residues, Arg165, Asn259, and Glu314, which are hydrogen-bonded with NAD+ or ATP, are chosen to characterize their possible roles on the inhibitory effect of ATP for the enzyme. Our kinetic data clearly demonstrate that Arg165 is essential for catalysis. The R165A enzyme had very low enzyme activity, and it was only slightly inhibited by ATP and not activated by fumarate. The values of K(m,NAD) and K(i,ATP) to both NAD+ and malate were elevated. Elimination of the guanidino side chain of R165 made the enzyme defective on the binding of NAD+ and ATP, and it caused the charge imbalance in the active site. These effects possibly caused the enzyme to malfunction on its catalytic power. The N259A enzyme was less inhibited by ATP but could be fully activated by fumarate at a similar extent compared with the wild-type enzyme. For the N259A enzyme, the value of K(i,ATP) to NAD+ but not to malate was elevated, indicating that the hydrogen bonding between ATP and the amide side chain of this residue is important for the binding stability of ATP. Removal of this side chain did not cause any harmful effect on the fumarate-induced activation of the enzyme. The E314A enzyme, however, was severely inhibited by ATP and only slightly activated by fumarate. The values of K(m,malate), K(m,NAD), and K(i,ATP) to both NAD+ and malate for E314A were reduced to about 2-7-folds compared with those of the wild-type enzyme. It can be concluded that mutation of Glu314 to Ala eliminated the repulsive effects between Glu314 and malate, NAD+, or ATP, and thus the binding affinities of malate, NAD+, and ATP in the active site of the enzyme were enhanced.

A simple, continuous spectrophotometric assay for peptidylarginine deiminase (PAD) is described. Deimination of peptidylarginine results in the formation of peptidylcitrulline and ammonia. The ammonia released during peptidylarginine hydrolysis is coupled to the glutamate-dehydrogenase-catalyzed reductive amination of alpha-ketoglutarate to glutamate and reduced nicotinamide adenine dinucleotide (NADH) oxidation. The disappearance of absorbance at 340nm due to NADH oxidation is continuously measured. The specific activity obtained by this new protocol for highly purified human PAD is comparable to that obtained by a commonly used colorimetric procedure, which measures the ureido group of peptidylcitrulline by coupling with diacetyl monoxime. The present continuous spectrophotometric method is highly sensitive and accurate and is thus suitable for enzyme kinetic analysis of PAD. The Ca(2+) concentration for half-maximal activity of PAD obtained by this method is comparable to that previously obtained by the colorimetric procedure.

Mathematical models for the protein folding-unfolding process involving multiple intermediates have been derived. Computer fitting of the experimental data to this model generates various thermodynamic parameters for the folding-unfolding process. In this way, the complex folding-unfolding process of the multi-domain proteins can be analysed in a quantitative way. The application of the folding-unfolding model involving seven stages in human placental alkaline phosphatase is described.

This study investigated the functional roles of the N-terminal Ca(2+) ion-binding sites, in terms of enzyme catalysis and stability, of peptidylarginine deiminase 4 (PAD4). Amino acid residues located in the N-terminal Ca(2+)-binding site of PAD4 were mutated to disrupt the binding of Ca(2+) ions. Kinetic data suggest that Asp155, Asp157 and Asp179, which directly coordinate Ca3 and Ca4, are essential for catalysis in PAD4. For D155A, D157A and D179A, the k(cat)/K(m,BAEE) values were 0.02, 0.63 and 0.01 s(-1)mM(-1) (20.8 s(-1)mM(-1) for WT), respectively. Asn153 and Asp176 are directly coordinated with Ca3 and indirectly coordinated with Ca5 via a water molecule. However, N153A displayed low enzymatic activity with a k(cat) value of 0.3 s(-1) (13.3 s(-1) for wild-type), whereas D176A retained some catalytic power with a k(cat) of 9.7 s(-1). Asp168 is the direct ligand for Ca5, and Ca5 coordination by Glu252 is mediated by two water molecules. However, mutation of these two residues to Ala did not cause a reduction in the k(cat)/K(m,BAEE) values, which indicates that the binding of Ca5 may not be required for PAD4 enzymatic activity. The possible conformational changes of these PAD4 mutants were examined. Thermal stability analysis of the PAD4 mutants in the absence or presence of Ca(2+) indicated that the conformational stability of the enzyme is highly dependent on Ca(2+) ions. In addition, the results of urea-induced denaturation for the N153, D155, D157 and D179 series mutants further suggest that the binding of Ca(2+) ions in the N-terminal Ca(2+)-binding site stabilizes the overall conformational stability of PAD4. Therefore, our data strongly suggest that the N-terminal Ca(2+) ions play critical roles in the full activation of the PAD4 enzyme.