Structure-based stabilization of insulin as a therapeutic protein assembly via enhanced aromatic-aromatic interactions.

Key contributions to protein structure and stability are provided by weakly polar interactions, which arise from asymmetric electronic distributions within amino acids and peptide bonds. Of particular interest are aromatic side chains whose directional π-systems commonly stabilize protein interiors and interfaces. Here, we consider aromatic-aromatic interactions within a model protein assembly: the dimer interface of insulin. Semi-classical simulations of aromatic-aromatic interactions at this interface suggested that substitution of residue TyrB26 by Trp would preserve native structure while enhancing dimerization (and hence hexamer stability). The crystal structure of a [TrpB26]insulin analog (determined as a T3Rf3 zinc hexamer at a resolution of 2.25 Å) was observed to be essentially identical to that of WT insulin. Remarkably and yet in general accordance with theoretical expectations, spectroscopic studies demonstrated a 150-fold increase in the in vitro lifetime of the variant hexamer, a critical pharmacokinetic parameter influencing design of long-acting formulations. Functional studies in diabetic rats indeed revealed prolonged action following subcutaneous injection. The potency of the TrpB26-modified analog was equal to or greater than an unmodified control. Thus, exploiting a general quantum-chemical feature of protein structure and stability, our results exemplify a mechanism-based approach to the optimization of a therapeutic protein assembly.

An ultra-stable single-chain insulin analog resists thermal inactivation and exhibits biological signaling duration equivalent to the native protein.

Thermal degradation of insulin complicates its delivery and use. Previous efforts to engineer ultra-stable analogs were confounded by prolonged cellular signaling in vivo, of unclear safety and complicating mealtime therapy. We therefore sought an ultra-stable analog whose potency and duration of action on intravenous bolus injection in diabetic rats are indistinguishable from wild-type (WT) insulin. Here, we describe the structure, function, and stability of such an analog, a 57-residue single-chain insulin (SCI) with multiple acidic substitutions. Cell-based studies revealed native-like signaling properties with negligible mitogenic activity. Its crystal structure, determined as a novel zinc-free hexamer at 2.8 Å, revealed a native insulin fold with incomplete or absent electron density in the C domain; complementary NMR studies are described in the accompanying article. The stability of the analog (ΔGU 5.0(±0.1) kcal/mol at 25 °C) was greater than that of WT insulin (3.3(±0.1) kcal/mol). On gentle agitation, the SCI retained full activity for >140 days at 45 °C and >48 h at 75 °C. These findings indicate that marked resistance to thermal inactivation in vitro is compatible with native duration of activity in vivo Further, whereas WT insulin forms large and heterogeneous aggregates above the standard 0.6 mm pharmaceutical strength, perturbing the pharmacokinetic properties of concentrated formulations, dynamic light scattering, and size-exclusion chromatography revealed only limited SCI self-assembly and aggregation in the concentration range 1-7 mm Such a combination of favorable biophysical and biological properties suggests that SCIs could provide a global therapeutic platform without a cold chain.

The Dual Regulatory Role of Amino Acids Leu480 and Gln481 of Prothrombin.

Prothrombin (FII) is activated to α-thrombin (IIa) by prothrombinase. Prothrombinase is composed of a catalytic subunit, factor Xa (fXa), and a regulatory subunit, factor Va (fVa), assembled on a membrane surface in the presence of divalent metal ions. We constructed, expressed, and purified several mutated recombinant FII (rFII) molecules within the previously determined fVa-dependent binding site for fXa (amino acid region 473-487 of FII). rFII molecules bearing overlapping deletions within this significant region first established the minimal stretch of amino acids required for the fVa-dependent recognition exosite for fXa in prothrombinase within the amino acid sequence Ser(478)-Val(479)-Leu(480)-Gln(481)-Val(482). Single, double, and triple point mutations within this stretch of rFII allowed for the identification of Leu(480) and Gln(481) as the two essential amino acids responsible for the enhanced activation of FII by prothrombinase. Unanticipated results demonstrated that although recombinant wild type α-thrombin and rIIa(S478A) were able to induce clotting and activate factor V and factor VIII with rates similar to the plasma-derived molecule, rIIa(SLQ→AAA) with mutations S478A/L480A/Q481A was deficient in clotting activity and unable to efficiently activate the pro-cofactors. This molecule was also impaired in protein C activation. Similar results were obtained with rIIa(ΔSLQ) (where rIIa(ΔSLQ) is recombinant human α-thrombin with amino acids Ser(478)/Leu(480)/Gln(481) deleted). These data provide new evidence demonstrating that amino acid sequence Leu(480)-Gln(481): 1) is crucial for proper recognition of the fVa-dependent site(s) for fXa within prothrombinase on FII, required for efficient initial cleavage of FII at Arg(320); and 2) is compulsory for appropriate tethering of fV, fVIII, and protein C required for their timely activation by IIa.

A chimeric prokaryotic pentameric ligand-gated channel reveals distinct pathways of activation.

Recent high resolution structures of several pentameric ligand-gated ion channels have provided unprecedented details of their molecular architecture. However, the conformational dynamics and structural rearrangements that underlie gating and allosteric modulation remain poorly understood. We used a combination of electrophysiology, double electron-electron resonance (DEER) spectroscopy, and x-ray crystallography to investigate activation mechanisms in a novel functional chimera with the extracellular domain (ECD) of amine-gated Erwinia chrysanthemi ligand-gated ion channel, which is activated by primary amines, and the transmembrane domain of Gloeobacter violaceus ligand-gated ion channel, which is activated by protons. We found that the chimera was independently gated by primary amines and by protons. The crystal structure of the chimera in its resting state, at pH 7.0 and in the absence of primary amines, revealed a closed-pore conformation and an ECD that is twisted with respect to the transmembrane region. Amine- and pH-induced conformational changes measured by DEER spectroscopy showed that the chimera exhibits a dual mode of gating that preserves the distinct conformational changes of the parent channels. Collectively, our findings shed light on both conserved and divergent features of gating mechanisms in this class of channels, and will facilitate the design of better allosteric modulators.

Crystal Structures of Polymorphic Prion Protein ß1 Peptides Reveal Variable Steric Zipper Conformations.

The pathogenesis of prion diseases is associated with the conformational conversion of normal, predominantly α-helical prion protein (PrP(C)) into a pathogenic form that is enriched with ß-sheets (PrP(Sc)). Several PrP(C) crystal structures have revealed ß1-mediated intermolecular sheets, suggesting that the ß1 strand may contribute to a possible initiation site for ß-sheet-mediated PrP(Sc) propagation. This ß1 strand contains the polymorphic residue 129 that influences disease susceptibility and phenotype. To investigate the effect of the residue 129 polymorphism on the conformation of amyloid-like continuous ß-sheets formed by ß1, crystal structures of ß1 peptides containing each of the polymorphic residues were determined. To probe the conformational influence of the peptide construct design, four different lengths of ß1 peptides were studied. From the 12 peptides studied, 11 yielded crystal structures ranging in resolution from 0.9 to 1.4 Å. This ensemble of ß1 crystal structures reveals conformational differences that are influenced by both the nature of the polymorphic residue and the extent of the peptide construct, indicating that comprehensive studies in which peptide constructs vary are a more rigorous approach to surveying conformational possibilities.

Bora downregulation results in radioresistance by promoting repair of double strand breaks.

Following DNA double-strand breaks cells activate several DNA-damage response protein kinases, which then trigger histone H2AX phosphorylation and the accumulation of proteins such as MDC1, p53-binding protein 1, and breast cancer gene 1 at the damage site to promote DNA double-strand breaks repair. We identified a novel biomarker, Bora (previously called C13orf34), that is associated with radiosensitivity. In the current study, we set out to investigate how Bora might be involved in response to irradiation. We found a novel function of Bora in DNA damage repair response. Bora down-regulation increased colony formation in cells exposed to irradiation. This increased resistance to irradiation in Bora-deficient cells is likely due to a faster rate of double-strand breaks repair. After irradiation, Bora-knockdown cells displayed increased G2-M cell cycle arrest and increased Chk2 phosphorylation. Furthermore, Bora specifically interacted with the tandem breast cancer gene 1 C-terminal domain of MDC1 in a phosphorylation dependent manner, and overexpression of Bora could abolish irradiation induced MDC1 foci formation. In summary, Bora may play a significant role in radiosensitivity through the regulation of MDC1 and DNA repair.

Residues required for phosphorylation of translation initiation factor eIF2α under diverse stress conditions are divergent between yeast and human.

PERK, PKR, HRI and GCN2 are the four mammalian kinases that phosphorylate the α subunit of the eukaryotic translation initiation factor 2 (eIF2α) on Ser51. This phosphorylation event is conserved among many species and attenuates protein synthesis in response to diverse stress conditions. In contrast, Saccharmyces cerevisiae expresses only the GCN2 kinase. It was demonstrated previously in S. cerevisiae that single point mutations in eIF2α's N-terminus severely impaired phosphorylation at Ser51. To assess whether similar recognition patterns are present in mammalian eIF2α, we expressed human eIF2α's with these mutations in mouse embryonic fibroblasts and assessed their phosphorylation under diverse stress conditions. Some of the mutations prevented the stress-induced phosphorylation of eIF2α by all mammalian kinases, thus defining amino acid residues in eIF2α (Gly 30, Leu 50, and Asp 83) that are required for substrate recognition. We also identified residues that were less critical or not required for recognition by the mammalian kinases (Ala 31, Met 44, Lys 79, and Tyr 81), even though they were essential for recognition of the yeast eIF2α by GCN2. We propose that mammalian eIF2α kinases evolved to maximize their interactions with the evolutionarily conserved Ser51 residue of eIF2α in response to diverse stress conditions, thus adding to the complex signaling pathways that mammalian cells have over simpler organisms.

Natriuretic peptide receptor-3 gene (NPR3): nonsynonymous polymorphism results in significant reduction in protein expression because of accelerated degradation.

BACKGROUND- The primary role of natriuretic peptide receptor-3 (NPR3) or NPR-C is in the clearance of natriuretic peptides that play an important role in modulating intravascular volume and vascular tone. Genetic variation in NPR3 has been associated with variation in blood pressure and obesity. Despite the importance of NPR3, sequence variation in the gene has not been addressed using DNA from different ethnic populations. We set out to identify and functionally characterize genetic variation in NPR3 in 3 ethnic groups. METHODS AND RESULTS- DNA samples from 96 European American, 96 African American, and 96 Han Chinese American healthy subjects were used to resequence NPR3 exons, splice junctions, and flanking regions. We identified 105 polymorphisms, 50 of which were novel, including 8 nonsynonymous single-nucleotide polymorphisms, 7 were novel. Expression constructs were created for the nonsynonymous single-nucleotide polymorphisms. HEK293 cells were transfected with constructs for wild type and variant allozymes; and recombinant proteins were measured by quantitative Western blot analysis. The most significant change in NPR3 protein was observed for the Arg146 variant allozyme, with 20% of wild-type protein, primarily because of autophagy-dependent degradation. NPR3 structural modeling confirmed that the Arg146 variant protein was not compatible with wild-type conformation and could result in protein misfolding or instability. CONCLUSIONS- Multiple novel NPR3 genetic polymorphisms were identified in 3 ethnic groups. The Arg146 allozyme displayed a significant decrease in protein quantity because of degradation mediated predominantly by autophagy. This genetic variation could have a significant effect on the metabolism of natriuretic peptides with potential clinical implications.

Impaired dimer assembly and decreased stability of naturally recurring R260C mutant A subunit for coagulation factor XIII.

Factor XIII (FXIII) consists of catalytic A subunits (FXIII-A) and carrier B subunits. Congenital FXIII deficiency is a severe bleeding disorder. We previously identified an R260C missense mutation and an exon-IV deletion in Japanese patients' F13A genes. To characterize the molecular basis of this disease, we expressed a wild-type and the mutant FXIII-A in yeast cells for detailed investigation, by taking advantage of yeast's ability for mass protein production. The mutant proteins were expressed less efficiently than the wild-type and considerably aggregated; even their non-aggregated forms became aggregated with time. Ultra-centrifugation and gel-filtration analyses revealed that the mutants were of extremely high-molecular weight, and that the wild-type formed a dimer. Notably, a part of the R260C mutant was found in monomer form. This was consistent with the prediction by molecular modelling that the mutant molecule would lose the electrostatic interaction between the two monomers, leading to their inability to form a dimer. The mutants lost enzymatic activity. The mutants were only partially converted by thrombin to the cleaved form. The wild-type was fully converted and activated. These mutants might have significantly altered conformations, resulting in their aggregation in vitro, and may ultimately lead to FXIII deficiency in vivo as well.

Human liver methionine cycle: MAT1A and GNMT gene resequencing, functional genomics, and hepatic genotype-phenotype correlation.

The "methionine cycle" plays a critical role in the regulation of concentrations of (S)-adenosylmethionine (AdoMet), the major biological methyl donor. We set out to study sequence variation in genes encoding the enzyme that synthesizes AdoMet in liver, methionine adenosyltransferase 1A (MAT1A) and the major hepatic AdoMet using enzyme, glycine N-methyltransferase (GNMT), as well as functional implications of that variation. We resequenced MAT1A and GNMT using DNA from 288 subjects of three ethnicities, followed by functional genomic and genotype-phenotype correlation studies performed with 268 hepatic biopsy samples. We identified 44 and 42 polymorphisms in MAT1A and GNMT, respectively. Quantitative Western blot analyses for the human liver samples showed large individual variation in MAT1A and GNMT protein expression. Genotype-phenotype correlation identified two genotyped single-nucleotide polymorphisms (SNPs), reference SNP (rs) 9471976 (corrected p = 3.9 × 10(-10)) and rs11752813 (corrected p = 1.8 × 10(-5)), and 42 imputed SNPs surrounding GNMT that were significantly associated with hepatic GNMT protein levels (corrected p values < 0.01). Reporter gene studies showed that variant alleles for both genotyped SNPs resulted in decreased transcriptional activity. Correlation analyses among hepatic protein levels for methionine cycle enzymes showed significant correlations between GNMT and MAT1A (p = 1.5 × 10(-3)) and between GNMT and betaine homocysteine methyltransferase (p = 1.6 × 10(-7)). Our discovery of SNPs that are highly associated with hepatic GNMT protein expression as well as the "coordinate regulation" of methionine cycle enzyme protein levels provide novel insight into the regulation of this important human liver biochemical pathway.

Molecular modeling predicts structural changes in the A subunit of factor XIII caused by two novel mutations identified in a neonate with severe congenital factor XIII deficiency.

INTRODUCTION: Coagulation factor XIII (FXIII) is a fibrin-stabilizing factor, which contributes to hemostasis, wound healing, and maintenance of pregnancy. Accordingly, patients with congenital FXIII deficiency manifest a life-long bleeding tendency, abnormal wound healing and recurrent miscarriage. In order to understand the molecular mechanisms of congenital FXIII deficiency, genetic analysis and molecular modeling were carried out in a Japanese male neonate with severe FXIII deficiency. METHODS AND RESULTS: Two novel mutations, Y204Stop (or Y204X, TAT to TAA) and S708R (AGC to AGG), were heterozygously identified by nucleotide sequencing analysis in exons V and XV of the gene for the A subunit of FXIII (FXIII-A). Y204X and S708R would lead to nonsense mediated mRNA decay and misfolding of the FXIII-A molecule, respectively. Using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, the presence of these mutations was confirmed both together in the proband and one each separately in either the maternal or paternal sides of his family. In addition, moderately decreased FXIII activity was associated with the presence of either mutation. Molecular modeling predicted that the mutant molecule of S708R would be structurally compromised by the substitution of the Ser with the larger extended bulky and positively charged Arg side-chain. CONCLUSION: It is probable that the impaired tertiary structure of the mutant S708R molecule leads to its instability, which is at least in part responsible for the FXIII deficiency of this patient. This is consistent with the fact that the mutations and the reduced FXIII activities co-segregate among the patient's family members.

Chimeric glutathione S-transferases containing inserts of kininogen peptides: potential novel protein therapeutics.

The study of synthetic peptides corresponding to discrete regions of proteins has facilitated the understanding of protein structure-activity relationships. Short peptides can also be used as powerful therapeutic agents. However, in many instances, small peptides are prone to rapid degradation or aggregation and may lack the conformation required to mimic the functional motifs of the protein. For peptides to function as pharmacologically active agents, efficient production or expression, high solubility, and retention of biological activity through purification and storage steps are required. We report here the design, expression, and functional analysis of eight engineered GST proteins (denoted GSHKTs) in which peptides ranging in size from 8 to 16 amino acids and derived from human high molecular weight kininogen (HK) domain 5 were inserted into GST (between Gly-49 and Leu-50). Peptides derived from HK are known to inhibit cell proliferation, angiogenesis, and tumor metastasis, and the biological activity of the HK peptides was dramatically (>50-fold) enhanced following insertion into GST. GSHKTs are soluble and easily purified from Escherichia coli by affinity chromatography. Functionally, these hybrid proteins cause inhibition of endothelial cell proliferation. Crystallographic analysis of GSHKT10 and GSHKT13 (harboring 10- and 13-residue HK peptides, respectively) showed that the overall GST structure was not perturbed. These results suggest that the therapeutic efficacy of short peptides can be enhanced by insertion into larger proteins that are easily expressed and purified and that GST may potentially be used as such a carrier.

Structural basis of substrate recognition in human nicotinamide N-methyltransferase.

Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide, pyridines, and other analogues using S-adenosyl-l-methionine as donor. NNMT plays a significant role in the regulation of metabolic pathways and is expressed at markedly high levels in several kinds of cancers, presenting it as a potential molecular target for cancer therapy. We have determined the crystal structure of human NNMT as a ternary complex bound to both the demethylated donor S-adenosyl-l-homocysteine and the acceptor substrate nicotinamide, to 2.7 Å resolution. These studies reveal the structural basis for nicotinamide binding and highlight several residues in the active site which may play roles in nicotinamide recognition and NNMT catalysis. The functional importance of these residues was probed by mutagenesis. Of three residues near the nicotinamide's amide group, substitution of S201 and S213 had no effect on enzyme activity while replacement of D197 dramatically decreased activity. Substitutions of Y20, whose side chain hydroxyl interacts with both the nicotinamide aromatic ring and AdoHcy carboxylate, also compromised activity. Enzyme kinetics analysis revealed k(cat)/K(m) decreases of 2-3 orders of magnitude for the D197A and Y20A mutants, confirming the functional importance of these active site residues. The mutants exhibited substantially increased K(m) for both NCA and AdoMet and modestly decreased k(cat). MD simulations revealed long-range conformational effects which provide an explanation for the large increase in K(m)(AdoMet) for the D197A mutant, which interacts directly only with nicotinamide in the ternary complex crystal structure.

Methionine adenosyltransferase 2A/2B and methylation: gene sequence variation and functional genomics.

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine, the major biological methyl donor. MAT1A and MAT2A encode two distinct MAT isoforms in mammals. MAT2A is expressed in nonhepatic tissues, whereas MAT1A is expressed in the liver. A third gene, MAT2B, encodes a MAT2A regulatory protein. We resequenced MAT2A and MAT2B exons, splice junctions, and flanking regions using 288 DNA samples from three ethnic groups and also imputed additional single nucleotide polymorphisms (SNPs) across both genes using data from the 1000 Genomes Project. For MAT2A, resequencing identified 74 polymorphisms, including two nonsynonymous (ns) SNPs. Functional genomic studies of wild type and the two MAT2A variant allozymes (Val11 and Val205) showed that the Val11 allozyme had approximately 40% decreases in levels of enzyme activity and immunoreactive protein after COS-1 cell transfection. For MAT2B, 44 polymorphisms, 2 nonsynonymous, were identified during resequencing. Neither of the two MAT2B nsSNPs displayed alterations in levels of protein. Imputation using 1000 Genomes Project data resulted in 1730 additional MAT2A and 1997 MAT2B polymorphisms within ± 200 kilobases of each gene, respectively. Coexpression of MAT2A and MAT2B in COS-1 cells resulted in significantly increased MAT enzyme activity that correlated with increased MAT2A and MAT2B immunoreactive protein, apparently as a result of decreased degradation. Finally, studies of mRNA expression in lymphoblastoid cells showed that 7 SNPs in MAT2A and 16 SNPs in MAT2B were significantly associated with mRNA expression with p < 0.01. These observations provide a foundation for future mechanistic and clinical translational pharmacogenomic studies of MAT2A/2B.

Natriuretic peptide pharmacogenetics: membrane metallo-endopeptidase (MME): common gene sequence variation, functional characterization and degradation.

Membrane metallo-endopeptidase (MME), also known as neutral endopeptidase 24.11 (EC 3.4.24.11), is involved in the metabolism of natriuretic peptides that play a key role in modulating cardiac structure and function. Common genetic variation in MME has not been addressed by resequencing the gene using DNA from different ethnic populations. We set out to identify and functionally characterize common genetic variation in MME in three ethnic groups. DNA samples from 96 European-American, 96 African-American, and 96 Han Chinese-American healthy subjects were used to resequence MME. Ninety polymorphisms, 65 novel, were identified, including 8 nonsynonymous single nucleotide polymorphisms (nsSNPs). Expression constructs for the nsSNPs were created and COS-1 cells were transfected with constructs for wild type (WT) and variant allozymes. Recombinant proteins were analyzed by quantitative Western blot analysis and by a one-step fluorometric assay. A significant reduction in enzyme activity (21% of WT) and immunoreactive protein (29% of WT) for the Val73 variant allozyme was observed. Proteasome-mediated degradation and autophagy participated in the degradation of this variant allozyme. The chaperone proteins, BiP and GRP94, were upregulated after transfection with Val73 MME, suggesting protein misfolding, compatible with conclusions based on the MME X-ray crystal structure. Multiple novel polymorphisms of MME were identified in three ethnic groups. The Val73 variant allozyme displayed a significant decrease in MME protein quantity and activity, with degradation mediated by both proteasome and autophagy pathways. This polymorphism could have a significant effect on the metabolism of natriuretic peptides.

Conformational diversity in prion protein variants influences intermolecular beta-sheet formation.

A conformational transition of normal cellular prion protein (PrP(C)) to its pathogenic form (PrP(Sc)) is believed to be a central event in the transmission of the devastating neurological diseases known as spongiform encephalopathies. The common methionine/valine polymorphism at residue 129 in the PrP influences disease susceptibility and phenotype. We report here seven crystal structures of human PrP variants: three of wild-type (WT) PrP containing V129, and four of the familial variants D178N and F198S, containing either M129 or V129. Comparison of these structures with each other and with previously published WT PrP structures containing M129 revealed that only WT PrPs were found to crystallize as domain-swapped dimers or closed monomers; the four mutant PrPs crystallized as non-swapped dimers. Three of the four mutant PrPs aligned to form intermolecular beta-sheets. Several regions of structural variability were identified, and analysis of their conformations provides an explanation for the structural features, which can influence the formation and conformation of intermolecular beta-sheets involving the M/V129 polymorphic residue.

Thiopurine S-methyltransferase pharmacogenetics: functional characterization of a novel rapidly degraded variant allozyme.

A novel human thiopurine S-methyltransferase (TPMT) variant allele, (319 T>G, 107Tyr>Asp, *27), was identified in a Thai renal transplantation recipient with reduced erythrocyte TPMT activity. The TPMT*27 variant allozyme showed a striking decrease in both immunoreactive protein level and enzyme activity after transient expression in a mammalian cell line. We set out to explore the mechanism(s) responsible for decreased expression of this novel variant of an important drug-metabolizing enzyme. We observed accelerated degradation of TPMT*27 protein in a rabbit reticulocyte lysate. TPMT*27 degradation was slowed by proteasome inhibition and involved chaperone proteins-similar to observations with regard to the degradation of the common TPMT*3A variant allozyme. TPMT*27 aggresome formation was also observed in transfected mammalian cells after proteasome inhibition. Inhibition of autophagy also decreased TPMT*27 degradation. Finally, structural analysis and molecular dynamics simulation indicated that TPMT*27 was less stable than was the wild type TPMT allozyme. In summary, TPMT*27 serves to illustrate the potential importance of protein degradation - both proteasome and autophagy-mediated degradation - for the pharmacogenetic effects of nonsynonymous SNPs.

Cytosolic 5'-nucleotidase III (NT5C3): gene sequence variation and functional genomics.

BACKGROUND: 5'-Nucleotidases play a critical role in nucleotide pool balance and in the metabolism of nucleoside analogs such as gemcitabine and cytosine arabinoside (AraC). We previously performed an expression array association study with gemcitabine and AraC cytotoxicity using 197 human lymphoblastoid cell lines. One gene that was significantly associated with gemcitabine cytotoxicity was a nucleotidase family member, NT5C3. Very little is known with regard to the pharmacogenomics of this family of enzymes. METHODS: We set out to identify common genetic variation in NT5C3 by resequencing the gene and to determine the effect of that variation on NT5C3 protein function and potential effect on response to cytidine analogs. We identified 61 NT5C3 polymorphisms, 48 of which were novel, by resequencing 240 ethnically defined DNA samples. Functional studies were performed with one nonsynonymous (G847C, Asp283His) and four synonymous cSNPs (T9C, C276T, T306C, and G759A),as well as three combined variants (T276/His283, T276/C306, T276/C9). RESULTS: The His283 and T276/His283 constructs showed decreased levels of enzyme activity and protein. Substrate kinetic analysis showed no significant differences in Km values between wild type and His283 when cytidine monophosphate, AraCMP, and GemMP were used as substrates. An association study between single nucleotide polymorphisms (SNPs) and NT5C3 expression in the 240 cell lines from which DNA was extracted to resequence NT5C3 identified four SNPs that were significantly associated with NT5C3 expression. Electrophoretic mobility shift assays showed that two of those SNPs, I4(-114) and I6(9), altered DNA-protein binding patterns. These findings suggest that genetic variation in NT5C3 might affect protein function and potentially influence drug response.

Targeting Bcl-2 based on the interaction of its BH4 domain with the inositol 1,4,5-trisphosphate receptor.

Bcl-2 is the founding member of a large family of apoptosis regulating proteins. Bcl-2 is a prime target for novel therapeutics because it is elevated in many forms of cancer and contributes to cancer progression and therapy resistance based on its ability to inhibit apoptosis. Bcl-2 interacts with proapoptotic members of the Bcl-2 family to inhibit apoptosis and small molecules that disrupt this interaction have already entered the cancer therapy arena. A separate function of Bcl-2 is to inhibit Ca2+ signals that promote apoptosis. This function is mediated through interaction of the Bcl-2 BH4 domain with the inositol 1,4,5-trisphosphate receptor (IP3R) Ca2+ channel. A novel peptide inhibitor of this interaction enhances proapoptotic Ca2+ signals. In preliminary experiments this peptide enhanced ABT-737 induced apoptosis in chronic lymphocytic leukemia cells. These findings draw attention to the BH4 domain as a potential therapeutic target. This review summarizes what is currently known about the BH4 domain of Bcl-2, its interaction with the IP3R and other proteins, and the part it plays in Bcl-2's anti-apoptotic function. In addition, we speculate on how the BH4 domain of Bcl-2 can be targeted therapeutically not only for diseases associated with apoptosis resistance, but also for diseases associated with accelerated cell death.

Crystal structure of the deglycating enzyme fructosamine oxidase (amadoriase II).

Fructosamine oxidases (FAOX) catalyze the oxidative deglycation of low molecular weight fructosamines (Amadori products). These proteins are of interest in developing an enzyme to deglycate proteins implicated in diabetic complications. We report here the crystal structures of FAOX-II from the fungi Aspergillus fumigatus, in free form and in complex with the inhibitor fructosyl-thioacetate, at 1.75 and 1.6A resolution, respectively. FAOX-II is a two domain FAD-enzyme with an overall topology that is most similar to that of monomeric sarcosine oxidase. Active site residues Tyr-60, Arg-112 and Lys-368 bind the carboxylic portion of the fructosamine, whereas Glu-280 and Arg-411 bind the fructosyl portion. From structure-guided sequence comparison, Glu-280 was identified as a signature residue for FAOX activity. Two flexible surface loops become ordered upon binding of the inhibitor in a catalytic site that is about 12A deep, providing an explanation for the very low activity of FAOX enzymes toward protein-bound fructosamines, which would have difficulty accessing the active site. Structure-based mutagenesis showed that substitution of Glu-280 and Arg-411 eliminates enzyme activity. In contrast, modification of other active site residues or of amino acids in the flexible active site loops has little effect, highlighting these regions as potential targets in designing an enzyme that will accept larger substrates.