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1.  Role of the C-Terminus of the Photoreceptor ABCA4 Transporter in Protein Folding, Function and Retinal Degenerative Diseases* 
The Journal of biological chemistry  2008;284(6):3640-3649.
ABCA4 is an ATP binding cassette (ABC) transporter that is expressed in rod and cone photoreceptor cells and implicated in the removal of retinal derivatives from outer segments following photoexcitation. Mutations in the ABCA4 gene are responsible for a number of related retinal degenerative diseases including Stargardt macular degeneration, cone-rod dystrophy, retinitis pigmentosa, and age-related macular degeneration. In order to determine the role of the C-terminus of ABCA4 in protein structure and function and understand mechanisms by which C-terminal mutations cause retinal degenerative diseases, we have expressed and purified a series of deletion and substitution mutants of ABCA4 and ABCA1 in HEK 293T cells for analysis of their cellular localization and biochemical properties. Removal of the C-terminal 30 amino acids of ABCA4 including a conserved VFVNFA motif resulted in the loss in N-retinylidene-phosphatidylethanolamine substrate binding, ATP photoaffinity labeling, and retinal stimulated ATPase activity. This mutant was also retained in the endoplasmic reticulum (ER) of cells. Replacement of the VFVNFA motif with alanine residues also resulted in loss in function and cellular mislocalization. In contrast C-terminal deletion mutants that retain the VFVNFA motif were functionally active and localized to intracellular vesicles similar to wild-type ABCA4. Our studies indicated that the VFVNFA motif is required for the proper folding of ABCA4 into a functionally active protein. This motif also contributes to the efficient folding of ABCA1 into an active protein. Our results provide a molecular based rationale for the disease phenotype displayed by individuals with mutations in the C-terminus of ABCA4.
doi:10.1074/jbc.M806580200
PMCID: PMC4090197  PMID: 19056738 CAMSID: cams4543
2.  Glial Cell Line-derived Neurotrophic Factor Regulation of Genes Essential for Self-renewal of Mouse Spermatogonial Stem Cells Is Dependent on Src Family Kinase Signaling*S 
The Journal of biological chemistry  2007;282(35):25842-25851.
Self-renewal and differentiation by spermatogonial stem cells (SSCs) is the foundation for continual spermatogenesis. SSC self-renewal is dependent on glial cell line-derived neurotrophic factor (GDNF); however, intracellular mechanisms stimulated by GDNF in SSCs are unknown. To investigate these mechanisms we utilized a culture system that maintains a mouse undifferentiated germ cell population enriched for self-renewing SSCs. In these cultures mRNA for the transcription factors Bcl6b, Erm, and Lhx1 are up-regulated by GDNF and decreased in its absence. The expression of all three molecules was further identified in undifferentiated spermatogonia in vivo. Using small interfering RNA to reduce expression and transplantation to quantify stem cell numbers, Bcl6b, Erm, and Lhx1 were shown to be important for SSC maintenance in vitro. Next, GDNF was shown to activate both Akt and Src family kinase (SFK) signaling in SSCs, and culture of SSCs with inhibitors to Akt or SFKs followed by transplantation analysis showed significant impairment of SSC maintenance in vitro. Apoptosis analysis revealed a significant increase in the percentage of apoptotic cells when Akt, but not SFK, signaling was impaired, indicating that multiple signaling pathways are responsible for SSC self-renewal and survival. Biochemical and gene expression experiments revealed that GDNF up-regulated expression of Bcl6b, Erm, and Lhx1 transcripts is dependent on SFK signaling. Overall, these data demonstrate that GDNF up-regulation of Bcl6b, Erm, and Lhx1 expression through SFK signaling is a key component of the intracellular mechanism for SSC self-renewal.
doi:10.1074/jbc.M703474200
PMCID: PMC4083858  PMID: 17597063
3.  Phosphatidylethanolamine and Monoglucosyldiacylglycerol Are Interchangeable in Supporting Topogenesis and Function of the Polytopic Membrane Protein Lactose Permease* 
The Journal of biological chemistry  2006;281(28):19172-19178.
To determine the specific role lipids play in membrane protein topogenesis in vivo, the orientation with respect to the membrane bilayer of Escherichia coli lactose permease (LacY) transmembrane (TM) domains and their flanking extramembrane domains was compared after assembly in native membranes and membranes with genetically modified lipid content using the substituted cysteine accessibility method for determining TM domain mapping. LacY assembled in the absence of the major membrane lipid phosphatidylethanolamine (PE) does not carry out uphill transport of substrate and displays an inverted orientation for the N-terminal six-TM domain helical bundle (Bogdanov, M., Heacock, P. N., and Dowhan, W. (2002) EMBO J. 21, 2107–2116). Strikingly, the replacement of PE in vivo by the foreign lipid monoglucosyldiacylglycerol (MGlcDAG), synthesized by the Acholeplasma laidlawii MGlcDAG synthase, restored uphill transport and supported the wild type TM topology of the N-terminal helical bundle of LacY. An interchangeable role in defining membrane protein TM domain orientation and supporting function is played by the two most abundant lipids, PE and MGlcDAG, in Gram-negative and Gram-positive bacteria, respectively. Therefore, these structurally diverse lipids endow the membrane with similar properties necessary for the proper organization of protein domains in LacY that are highly sensitive to lipids as topological determinants.
doi:10.1074/jbc.M602565200
PMCID: PMC4082682  PMID: 16698795
4.  Modulation of Androgen Receptor Activation Function 2 by Testosterone and Dihydrotestosterone* 
The Journal of biological chemistry  2007;282(35):25801-25816.
The androgen receptor (AR) is transcriptionally activated by high affinity binding of testosterone (T) or its 5α-reduced metabolite, dihydrotestosterone (DHT), a more potent androgen required for male reproductive tract development. The molecular basis for the weaker activity of T was investigated by determining T-bound ligand binding domain crystal structures of wild-type AR and a prostate cancer somatic mutant complexed with the AR FXXLF or coactivator LXXLL peptide. Nearly identical interactions of T and DHT in the AR ligand binding pocket correlate with similar rates of dissociation from an AR fragment containing the ligand binding domain. However, T induces weaker AR FXXLF and coactivator LXXLL motif interactions at activation function 2 (AF2). Less effective FXXLF motif binding to AF2 accounts for faster T dissociation from full-length AR. T can nevertheless acquire DHT-like activity through an AR helix-10 H874Y prostate cancer mutation. The Tyr-874 mutant side chain mediates a new hydrogen bonding scheme from exterior helix-10 to backbone protein core helix-4 residue Tyr-739 to rescue T-induced AR activity by improving AF2 binding of FXXLF and LXXLL motifs. Greater AR AF2 activity by improved core helix interactions is supported by the effects of melanoma antigen gene protein-11, an AR coregulator that binds the AR FXXLF motif and targets AF2 for activation. We conclude that T is a weaker androgen than DHT because of less favorable T-dependent AR FXXLF and coactivator LXXLL motif interactions at AF2.
doi:10.1074/jbc.M703268200
PMCID: PMC4075031  PMID: 17591767
5.  ANALYSIS OF GLYCAN POLYMERS PRODUCED BY PEPTIDOGLYCAN GLYCOSYLTRANSFERASES 
The Journal of biological chemistry  2007;282(44):31964-31971.
Bacterial cells are surrounded by a crosslinked polymer called peptidoglycan (PG), the integrity of which is necessary for cell survival. The carbohydrate chains that form the backbone of PG are made by peptidoglycan glycosyltransferases (PGTs), highly conserved membrane-bound enzymes that are thought to be excellent targets for the development of new antibacterials. Although structural information on these enzymes recently became available, their mechanism is not well understood due to a dearth of methods to monitor PGT activity. Here we describe a direct, sensitive and quantitative SDS-PAGE method to analyze PGT reactions. We apply this method to characterize the substrate specificity and product length profile for two different PGT domains: PBP1A from Aquifex aeolicus and PBP1A from Escherichia coli. We show that both disaccharide and tetrasaccharide diphospholipids (Lipid II and Lipid IV) serve as substrates for these PGTs, but the product distributions differ significantly depending on which substrate is used as the starting material. Reactions using the disaccharide substrate are more processive and yield much longer glycan products than reactions using the tetrasaccharide substrate. We also show that the SDS-PAGE method can be applied to provide information on the roles of invariant residues in catalysis. A comprehensive mutational analysis shows that the biggest contributor to turnover of 14 mutated residues is an invariant glutamate located in the center of the active site cleft. The assay and results described provide new information about the process by which PGTs assemble bacterial cell walls.
doi:10.1074/jbc.M705440200
PMCID: PMC4048933  PMID: 17704540
6.  c-Myc Transformation Domain Recruits the Human STAGA Complex and Requires TRRAP and GCN5 Acetylase Activity for Transcription Activation* 
The Journal of biological chemistry  2003;278(22):20405-20412.
Deregulation of the c-Myc oncoprotein (Myc) is implicated in many types of cancer. Myc is a sequence-specific transcription factor that regulates transcription of genes involved in the control of cell proliferation and apoptosis via mechanisms that are still poorly understood. Cell transformation by Myc involves its association with the transformation-transactivation domain-associated protein (TRRAP) and the human histone acetyltransferase (HAT) GCN5. TRRAP and GCN5 are components of a variety of shared and distinct multiprotein HAT complexes with diverse functions. Myc induces TRRAP recruitment and histone hyperacetylation at specific Myc-activated genes in vivo. However, the identity of the HAT complexes recruited by Myc and the roles of TRRAP and GCN5 in Myc function are still unclear. Here we show that Myc co-recruits TRRAP and GCN5 via direct physical interactions of its N-terminal activation/transformation domain with the human STAGA (SPT3-TAF-GCN5 acetylase) coactivator complex. We demonstrate that GCN5 and TRRAP cooperate to enhance transcription activation by the N-terminal activation domain of Myc in vivo and that this synergy requires both the SPT3/GCN5 interaction domain of TRRAP and the HAT activity of GCN5. Thus, TRRAP might function as an adaptor within the STAGA complex, which helps recruit GCN5 HAT activity to Myc during transcription activation.
doi:10.1074/jbc.M211795200
PMCID: PMC4031917  PMID: 12660246
7.  CONVERGENCE OF P53 AND TGFβ SIGNALING ON ACTIVATING EXPRESSION OF THE TUMOR SUPPRESSOR GENE MASPIN IN MAMMARY EPITHELIAL CELLS 
The Journal of biological chemistry  2007;282(8):5661-5669.
Using two-dimensional difference gel electrophoresis, we identified the tumor suppressor gene maspin as a TGFβ target gene in human mammary epithelial cells. TGFβ upregulates maspin expression both at the RNA and protein levels. This upregulation required Smad2/3 function and intact p53 binding elements in the maspin promoter. DNA affinity immunoblot and chromatin immunoprecipitation (ChIP) revealed the presence of both Smads and p53 at the maspin promoter in TGFβ-treated cells, suggesting that both transcription factors cooperate to induce maspin transcription. TGFβ did not activate maspin-luciferase reporter in p53-mutant MDA-MB-231 breast cancer cells, which exhibit methylation of the endogenous maspin promoter. Expression of ectopic p53, however, restored ligand-induced association of Smad2/3 with a transfected maspin promoter. Stable transfection of maspin inhibited basal and TGFβ-stimulated MDA-MB-231 cell motility. Finally, knockdown of endogenous maspin in p53 wild-type MCF10A/HER2 cells enhanced basal and TGFβ-stimulated motility. Taken together, these data support cooperation between the p53 and TGFβ tumor suppressor pathways in the induction of maspin expression, thus leading to inhibition of cell migration.
doi:10.1074/jbc.M608499200
PMCID: PMC4015524  PMID: 17204482
8.  Yeast Mps1p Phosphorylates the Spindle Pole Component Spc110p in the N-terminal Domain* 
The Journal of biological chemistry  2001;276(21):17958-17967.
The yeast spindle pole body (SPB) component Spc110p (Nuf1p) undergoes specific serine/threonine phosphorylation as the mitotic spindle apparatus forms, and this phosphorylation persists until cells enter anaphase. We demonstrate that the dual-specificity kinase Mps1p is essential for the mitosis-specific phosphorylation of Spc110p in vivo and that Mps1p phosphorylates Spc110p in vitro. Phosphopeptides generated by proteolytic cleavage were identified and sequenced by mass spectrometry. Ser60, Thr64, and Thr68 are the major sites in Spc110p phosphorylated by Mps1p in vitro, and alanine substitution at these sites abolishes the mitosis-specific isoform in vivo. This is the first time that phosphorylation sites of an SPB component have been determined, and these are the first sites of Mps1p phosphorylation identified. Alanine substitution for any one of these phosphorylated residues, in conjunction with an alanine substitution at residue Ser36, is lethal in combination with alleles of SPC97, which encodes a component of the Tub4p complex. Consistent with a specific dysfunction for the alanine substitution mutations, simultaneous mutation of all four serine/threonine residues to aspartate does not confer any defect. Sites of Mps1p phosphorylation and Ser36 are located within the N-terminal globular domain of Spc110p, which resides at the inner plaque of the SPB and binds the Tub4p complex.
doi:10.1074/jbc.M010461200
PMCID: PMC4013285  PMID: 11278681
9.  Cell Adhesion to a Motif Shared by the Malaria Circumsporozoite Protein and Thrombospondin Is Mediated by Its Glycosaminoglycan-binding Region and Not by CSVTCG* 
The Journal of biological chemistry  1997;272(31):19205-19213.
The malaria circumsporozoite protein (CS), thrombospondin (TSP), and several other proteins including the terminal complement proteins and the neural adhesion molecules F-spondin and Unc-5, share a cell adhesive sequence. In CS this sequence is designated as region II-plus (EWSPCSVTCGNGIQVRIK) and in TSP it is found in the type I repeats. Previous studies aimed at fine mapping the amino acid residues required for cell adhesion have yielded discrepant results. Here we show in three different cell lines that the downstream basic residues are required for cell adhesion whereas the CSVTCG sequence is not. Using mutant Chinese hamster ovary cells selected for deficiencies in proteoglycan synthesis, we show that in wild type cells, heparan sulfate proteoglycans are the binding sites for this motif. This finding is supported by additional experiments with two other cell lines demonstrating that treatment with heparitinase but not chondroitinase abolishes cell adhesion to peptides representing this motif. Using Chinese hamster ovary cell mutants deficient in heparan sulfate proteoglycans but possessing chondroitin sulfate proteoglycans, we show that cell surface chondroitin sulfate proteoglycans can also mediate binding to this motif although higher concentrations of peptides are required for adhesion. Chondroitinase, but not heparitinase, treatment of these cells destroys cell surface-binding sites. Taken together, these results indicate that cell adhesion to this motif involves an interaction between the downstream positively-charged residues and the negatively charged glycosaminoglycan chains of heparan sulfate, or in some cases chondroitin sulfate, proteoglycans on the cell surface.
PMCID: PMC4011078  PMID: 9235912
10.  Probiotic Bacterium Prevents Cytokine-induced Apoptosis in Intestinal Epithelial Cells* 
The Journal of biological chemistry  2002;277(52):50959-50965.
Probiotic bacteria are microorganisms that benefit the host by preventing or ameliorating disease. However, little information is known regarding the scientific rationale for using probiotics as alternative medicine. The purpose of this paper is to investigate the mechanisms of probiotic beneficial effects on intestinal cell homeostasis. We now report that one such probiotic, Lactobacillus rhamnosus GG (LGG), prevents cytokine-induced apoptosis in two different intestinal epithelial cell models. Culture of LGG with either mouse or human colon cells activates the anti-apoptotic Akt/protein kinase B. This model probiotic also inhibits activation of the pro-apoptotic p38/mitogen-activated protein kinase by tumor necrosis factor, interleukin-1α, or γ-interferon. Furthermore, products recovered from LGG culture broth supernatant show concentration-dependent activation of Akt and inhibition of cytokine-induced apoptosis. These observations suggest a novel mechanism of communication between probiotic microorganisms and epithelia that increases survival of intestinal cells normally found in an environment of pro-apoptotic cytokines.
doi:10.1074/jbc.M207050200
PMCID: PMC4006994  PMID: 12393915
11.  Inhibition of Activator Protein 1 Activity and Neoplastic Transformation by Aspirin* 
The Journal of biological chemistry  1997;272(15):9962-9970.
Aspirin, along with its analgesic-antipyretic uses, is now also being considered for prevention of cardiovascular disease, cancer, and treatment of human immunodeficiency virus infection. Although many of aspirin's pharmacological actions are related to its ability to inhibit prostaglandin biosynthesis, some of its beneficial therapeutic effects are not completely understood. Transcription factor activator protein 1 (AP-1) is critical for the induction of neoplastic transformation and induction of multiple genes involved in inflammation and infection. We have used the JB6 mouse epidermal cell lines, a system that has been used extensively as an in vitro model for the study of tumor promotion and anti-tumor promotion, to study the anti-carcinogenesis effect of aspirin at the molecular level. Aspirin and aspirin-like salicylates inhibited the activation of AP-1 in the same dose range as seen for the inhibition of tumor promoter-induced transformation. The inhibition of AP-1 and tumor promoter-induced transformation in JB6 cells occurs through a prostaglandin independent- and an Erk1- or Erk2-independent pathway. The mechanism of AP-1 and transformation inhibition in this cell culture model may involve the elevation of H+ concentration. The inhibition effects on the activation of AP-1 activity by aspirin and aspirin-like salicylates may further explain the anti-carcinogenesis mechanism of action of these drugs.
PMCID: PMC4003901  PMID: 9092536
12.  H2O2-induced O2⋅‒ Production by a Non-phagocytic NAD(P)H Oxidase Causes Oxidant Injury* 
The Journal of biological chemistry  2001;276(31):29251-29256.
Non-phagocytic NAD(P)H oxidases have been implicated as major sources of reactive oxygen species in blood vessels. These oxidases can be activated by cytokines, thereby generating O2⋅‒, which is subsequently converted to H2O2 and other oxidant species. The oxidants, in turn, act as important second messengers in cell signaling cascades. We hypothesized that reactive oxygen species, themselves, can activate the non-phagocytic NAD(P)H oxidases in vascular cells to induce oxidant production and, consequently, cellular injury. The current report demonstrates that exogenous exposure of non-phagocytic cell types of vascular origin (smooth muscle cells and fibroblasts) to H2O2 activates these cell types to produce O2⋅‒ via an NAD(P)H oxidase. The ensuing endogenous production of O2⋅‒ contributes significantly to vascular cell injury following exposure to H2O2. These results suggest the existence of a feed-forward mechanism, whereby reactive oxygen species such as H2O2 can activate NAD(P)H oxidases in non-phagocytic cells to produce additional oxidant species, thereby amplifying the vascular injury process. Moreover, these findings implicate the non-phagocytic NAD(P)H oxidase as a novel therapeutic target for the amelioration of the biological effects of chronic oxidant stress.
doi:10.1074/jbc.M102124200
PMCID: PMC3974124  PMID: 11358965
13.  Regulation of Vascular Smooth Muscle Proliferation by Heparin 
The Journal of biological chemistry  2005;280(16):15682-15689.
Uncontrolled proliferation of vascular smooth muscle cells (VSMCs) contribute to intimal hyperplasia during atherosclerosis and restenosis. Heparin is an antiproliferative agent for VSMCs and has been shown to block VSMC proliferation both in tissue culture systems and in animals. Despite the well documented antiproliferative actions of heparin, its cellular targets largely remain unknown. In an effort to characterize the mechanism of the antiproliferative property of heparin, we have analyzed the effect of heparin on cell cycle in VSMC. Our results indicate that the heparin-induced block in G1 to S phase transition is imposed by p27kip1-mediated inhibition of cyclin-dependent kinase 2 activity. Further analysis of p27kip1 mRNA levels showed that the increase in p27kip1 protein levels in heparin-treated VSMC occurs at posttranscriptional levels. We present evidence that heparin causes stabilization of p27kip1 protein during G1 phase and thereby prevents activation of cyclin-dependent kinase 2.
doi:10.1074/jbc.M411458200
PMCID: PMC3972062  PMID: 15731113
14.  Human Endoplasmic Reticulum Mannosidase I Is Subject to Regulated Proteolysis* 
The Journal of biological chemistry  2006;282(7):4841-4849.
In the early secretory pathway, opportunistic cleavage of asparagine-linked oligosaccharides by endoplasmic reticulum (ER) mannosidase I targets misfolded glycoproteins for dislocation into the cytosol and destruction by 26 S proteasomes. The low basal concentration of the glycosidase is believed to coordinate the glycan cleavage with prolonged conformation-based ER retention, ensuring that terminally misfolded glycoproteins are preferentially targeted for destruction. Herein the intracellular fate of human ER mannosidase I was monitored to determine whether a post-translational process might contribute to the regulation of its intracellular concentration. The transiently expressed recombinant human glycosidase was subject to rapid intracellular turnover in mouse hepatoma cells, as was the endogenous mouse ortholog. Incubation with either chloroquine or leupeptin, but not lactacystin, led to intracellular stabilization, implicating the involvement of lysosomal acid hydrolases. Inhibition of protein synthesis with cycloheximide led to intracellular depletion of the glycosidase and concomitant ablation of asparagine-linked glycoprotein degradation, confirming the physiologic relevance of the destabilization process. Metabolic incorporation of radiolabeled phosphate, detection by anti-phosphoserine antiserum, and the stabilizing effect of general serine kinase inhibition implied that ER mannosidase I is subjected to regulated proteolysis. Stabilization in response to genetically engineered removal of the amino-terminal cytoplasmic tail, a postulated regulatory domain, and colocalization of green fluorescent protein fusion proteins with Lamp1 provided two additional lines of evidence to support the hypothesis. A model is proposed in which proteolytically driven checkpoint control of ER mannosidase I contributes to the establishment of an equitable glycoprotein quality control standard by which the efficiency of asparagine-linked glycoprotein conformational maturation is measured.
doi:10.1074/jbc.M607156200
PMCID: PMC3969733  PMID: 17166854
15.  SRC-1 and GRIP1 Coactivate Transcription with Hepatocyte Nuclear Factor 4* 
The Journal of biological chemistry  1998;273(47):30847-30850.
Hepatocyte nuclear factor-4 (HNF4), a member of the nuclear receptor superfamily, plays an important role in tissue-specific gene expression, including genes involved in hepatic glucose metabolism. In this study, we show that SRC-1 and GRIP1, which act as coactivators for various nuclear receptors, associate with HNF4 in vivo and enhance its transactivation potential. The AF-2 domain of HNF4 is required for this interaction and for the potentiation of transcriptional activity by these co-activators. p300 can also serve as a coactivator with HNF4, and it synergizes with SRC-1 to further augment the activity of HNF4. HNF4 is also a key regulator of the expression of hepatocyte nuclear factor-1 (HNF1). The overexpression of SRC-1 or GRIP1 enhances expression from a HNF1 gene promoter-reporter in HepG2 hepatoma cells, and this requires an intact HNF4-binding site in the HNF1 gene promoter. Type 1 maturity onset diabetes of young (MODY), which is characterized by abnormal glucose-mediated insulin secretion, is caused by mutations of the HNF4 gene. A mutation of the HNF4-binding site in the HNF1 gene promoter has also been associated with MODY. Thus, HNF4 is involved in the regulation of glucose homeostasis at several levels and along with the SRC-1, GRIP1, and p300 may play an important role in the pathophysiology of non-insulin-dependent diabetes mellitus.
PMCID: PMC3968904  PMID: 9812974
16.  Structural and Kinetic Evidence for an Extended Hydrogen-bonding Network in Catalysis of Methyl Group Transfer 
The Journal of biological chemistry  2006;282(9):6609-6618.
The methyltetrahydrofolate (CH3-H4folate) corrinoid-iron-sulfur protein (CFeSP) methyltransferase (MeTr) catalyzes transfer of the methyl group of CH3-H4folate to cob(I)amide. This key step in anaerobic CO and CO2 fixation is similar to the first half-reaction in the mechanisms of other cobalamin-dependent methyltransferases. Methyl transfer requires electrophilic activation of the methyl group of CH3-H4folate, which includes proton transfer to the N5 group of the pterin ring and poises the methyl group for reaction with the Co(I) nucleophile. The structure of the binary CH3-H4folate/MeTr complex (revealed here) lacks any obvious proton donor near the N5 group. Instead, an Asn residue and water molecules are found within H-bonding distance of N5. Structural and kinetic experiments described here are consistent with the involvement of an extended H-bonding network in proton transfer to N5 of the folate that includes an Asn (Asn-199 in MeTr), a conserved Asp (Asp-160), and a water molecule. This situation is reminiscent of purine nucleoside phosphorylase, which involves protonation of the purine N7 in the transition state and is accomplished by an extended H-bond network that includes water molecules, a Glu residue, and an Asn residue. In MeTr, the Asn residue swings from a distant position to within H-bonding distance of the N5 atom upon CH3-H4folate binding. An N199A variant exhibits only ~20-fold weakened affinity for CH3-H4folate but a much more marked 20,000–40,000-fold effect on catalysis, suggesting that Asn-199 plays an important role in stabilizing a transition state or high energy intermediate for methyl transfer.
doi:10.1074/jbc.M609828200
PMCID: PMC3966722  PMID: 17172470
17.  Structure and Orientation of the Mn4Ca Cluster in Plant Photosystem II Membranes Studied by Polarized Range-extended X-ray Absorption Spectroscopy*[S]♦ 
The Journal of biological chemistry  2006;282(10):7198-7208.
X-ray absorption spectroscopy has provided important insights into the structure and function of the Mn4Ca cluster in the oxygen-evolving complex of Photosystem II (PS II). The range of manganese extended x-ray absorption fine structure data collected from PSII until now has been, however, limited by the presence of iron in PS II. Using a crystal spectrometer with high energy resolution to detect solely the manganese Kα fluorescence, we are able to extend the extended x-ray absorption fine structure range beyond the onset of the iron absorption edge. This results in improvement in resolution of the manganese-backscatterer distances in PS II from 0.14 to 0.09 Å. The high resolution data obtained from oriented spinach PS II membranes in the S1 state show that there are three di-µ-oxo-bridged manganese-manganese distances of ~2.7 and ~2.8 Å in a 2:1 ratio and that these three manganese-manganese vectors are aligned at an average orientation of ~60° relative to the membrane normal. Furthermore, we are able to observe the separation of the Fourier peaks corresponding to the ~3.2 Å manganese-manganese and the ~3.4 Å manganese-calcium interactions in oriented PS II samples and determine their orientation relative to the membrane normal. The average of the manganese-calcium vectors at ~3.4 Å is aligned along the membrane normal, while the ~3.2 Å manganese-manganese vector is oriented near the membrane plane. A comparison of this structural information with the proposed Mn4Ca cluster models based on spectroscopic and diffraction data provides input for refining and selecting among these models.
doi:10.1074/jbc.M610505200
PMCID: PMC3962024  PMID: 17190828
18.  Evidence That the Heme Regulatory Motifs in Heme Oxygenase-2 Serve as a Thiol/Disulfide Redox Switch Regulating Heme Binding*s 
The Journal of biological chemistry  2007;282(29):21056-21067.
Heme oxygenase (HO) catalyzes the O2- and NADPH-dependent conversion of heme to biliverdin, CO, and iron. The two forms of HO (HO-1 and HO-2) share similar physical properties but are differentially regulated and exhibit dissimilar physiological roles and tissue distributions. Unlike HO-1, HO-2 contains heme regulatory motifs (HRMs) (McCoubrey, W. K., Jr., Huang, T. J., and Maines, M. D. (1997) J. Biol. Chem. 272, 12568–12574). Here we describe UV-visible, EPR, and differential scanning calorimetry experiments on human HO-2 variants containing single, double, and triple mutations in the HRMs. Oxidized HO-2, which contains an intramolecular disulfide bond linking Cys265 of HRM1 and Cys282 of HRM2, binds heme tightly. Reduction of the disulfide bond increases the Kd for ferric heme from 0.03 to 0.3 μm, which is much higher than the concentration of the free heme pool in cells. Although the HRMs markedly affect the Kd for heme, they do not alter the kcat for heme degradation and do not bind additional hemes. Because HO-2 plays a key role in CO generation and heme homeostasis, reduction of the disulfide bond would be expected to increase intracellular free heme and decrease CO concentrations. Thus, we propose that the HRMs in HO-2 constitute a thiol/disulfide redox switch that regulates the myriad physiological functions of HO-2, including its involvement in the hypoxic response in the carotid body, which involves interactions with a Ca2+-activated potassium channel.
doi:10.1074/jbc.M700664200
PMCID: PMC3957417  PMID: 17540772
19.  A NEW LEVEL OF ARCHITECTURAL COMPLEXITY IN THE HUMAN PYRUVATE DEHYDROGENASE COMPLEX 
The Journal of biological chemistry  2006;281(28):19772-19780.
SUMMARY
Mammalian pyruvate dehydrogenase multi-enzyme complex (PDC) is a key metabolic assembly comprising a 60- meric pentagonal dodecahedral E2 core attached to which are 30 E1 heterotetramers and 6 E3 homodimers at maximal occupancy. Stable E3 integration is mediated by an accessory E3 binding protein (E3BP) located on each of the 12 E2 icosahedral faces. Here, we present evidence for a novel subunit organisation in which dihydrolipoamide dehydrogenase (E3) and E3BP form subcomplexes with a 1:2 stoichiometry implying the existence of a network of E3 ‘cross-bridges’ linking pairs of E3BPs across the surface of the E2 core assembly. We have also determined a low resolution structure for a truncated E3BP/E3 subcomplex using small angle xray scattering showing one of the E3BP lipoyl domains docked into the E3 active site. This new level of architectural complexity in mammalian PDC contrasts with the recently published crystal structure of human E3 complexed with its cognate subunit binding domain and provides important new insights into subunit organisation, its catalytic mechanism and regulation by the intrinsic PDC kinase.
doi:10.1074/jbc.M601140200
PMCID: PMC3954457  PMID: 16679318
20.  The Binding of the Circumsporozoite Protein to Cell Surface Heparan Sulfate Proteoglycans Is Required for Plasmodium Sporozoite Attachment to Target Cells* 
The Journal of biological chemistry  2001;276(29):26784-26791.
The major surface protein of malaria sporozoites, the circumsporozoite protein, binds to heparan sulfate proteoglycans on the surface of hepatocytes. It has been proposed that this binding event is responsible for the rapid and specific localization of sporozoites to the liver after their injection into the skin by an infected anopheline mosquito. Previous in vitro studies performed under static conditions have failed to demonstrate a significant role for heparan sulfate proteogly-cans during sporozoite invasion of cells. We performed sporozoite attachment and invasion assays under more dynamic conditions and found a dramatic decrease in sporozoite attachment to cells in the presence of heparin. In contrast to its effect on attachment, heparin does not appear to have an effect on sporozoite invasion of cells. When substituted heparins were used as competitive inhibitors of sporozoite attachment, we found that sulfation of the glycosaminoglycan chains at both the N- and O-positions was important for sporozoite adhesion to cells. We conclude that the binding of the circumsporozoite protein to hepatic heparan sulfate proteoglycans is likely to function during sporozoite attachment in the liver and that this adhesion event depends on the sulfated glycosaminoglycan chains of the proteoglycans.
doi:10.1074/jbc.M104038200
PMCID: PMC3941197  PMID: 11352923
21.  STRICT PAIRING OF var PR0MOTERS AND INTRONS IS REQUIRED FOR var GENE SILENCING IN THE MALARIA PARASITE PLASMODIUM FALCIPARUM 
The Journal of biological chemistry  2006;281(15):9942-9952.
The human malaria parasite Plasmodium falciparum, maintains a persistent infection altering the proteins expressed on the surface of the infected red blood cells, thus avoiding the host’s immune response. The primary surface antigen, a protein called PfEMP1, is encoded by a multicopy gene family called var. Each individual parasite only expresses a single var gene at a time, maintaining all other members of the family in a transcriptionally silent state. Previous work using reporter genes in transiently transfected plasmid constructs implicated a conserved intron found in all var genes in the silencing process. Here we utilize episomal recombination within stably transformed parasites to generate different var promoter and intron arrangements and show that loss of the intron results in var promotor activation. Further, in multicopy plasmid concatamers, each intron could only silence a single promoter, suggesting a one to one pairing requirement for silencing. Transcriptionally active, “unpaired” promoters remained active after integration into a chromosome, however they were not recognized by the pathway that maintains mutually exclusive var gene expression. The data indicate that intron/promoter pairing is responsible for silencing each individual var gene, and that disruption of silencing of one gene does not affect the transcriptional activity of neighboring var promoters. This suggests that silencing is regulated at the level of individual genes rather than assembly of silent chromatin throughout a chromosomal region, thus providing a possible explanation of how a var gene can be maintained in a silent state while the immediately adjacent var gene is transcriptionally active.
doi:10.1074/jbc.M513067200
PMCID: PMC3941977  PMID: 16455655
22.  A Novel Role for GADD45β as a Mediator of MMP-13 Gene Expression during Chondrocyte Terminal Differentiation*S 
The Journal of biological chemistry  2005;280(46):38544-38555.
The growth arrest and DNA damage-inducible 45β (GADD45β) gene product has been implicated in the stress response, cell cycle arrest, and apoptosis. Here we demonstrated the unexpected expression of GADD45β in the embryonic growth plate and uncovered its novel role as an essential mediator of matrix metalloproteinase-13 (MMP-13) expression during terminal chondrocyte differentiation. We identified GADD45β as a prominent early response gene induced by bone morphogenetic protein-2 (BMP-2) through a Smad1/Runx2-dependent pathway. Because this pathway is involved in skeletal development, we examined mouse embryonic growth plates, and we observed expression of Gadd45β mRNA coincident with Runx2 protein in pre-hypertrophic chondrocytes, whereas GADD45β protein was localized prominently in the nucleus in late stage hypertrophic chondrocytes where Mmp-13 mRNA was expressed. In Gadd45β−/− mouse embryos, defective mineralization and decreased bone growth accompanied deficient Mmp-13 and Col10a1 gene expression in the hypertrophic zone. Transduction of small interfering RNA-GADD45β in epiphyseal chondrocytes in vitro blocked terminal differentiation and the associated expression of Mmp-13 and Col10a1 mRNA in vitro. Finally, GADD45β stimulated MMP-13 promoter activity in chondrocytes through the JNK-mediated phosphorylation of JunD, partnered with Fra2, in synergy with Runx2. These observations indicated that GADD45β plays an essential role during chondrocyte terminal differentiation.
doi:10.1074/jbc.M504202200
PMCID: PMC3937966  PMID: 16144844
23.  CYP2E1 Substrate Inhibition 
The Journal of biological chemistry  2007;283(6):3487-3496.
In this study we offer a mechanistic interpretation of the previously known but unexplained substrate inhibition observed for CYP2E1. At low substrate concentrations, p-nitrophenol (pNP) was rapidly turned over (47 min−1) with relatively low Km (24 μM); nevertheless, at concentrations of >100 μM, the rate of pNP oxidation gradually decreased as a second molecule bound to CYP2E1 through an effector site (Kss = 260 μM), which inhibited activity at the catalytic site. 4-Methylpyrazole (4MP) was a potent inhibitor for both sites through a mixed inhibition mechanism. The Ki for the catalytic site was 2.0 μM. Although we were unable to discriminate whether an EIS or ESI complex formed, the respective inhibition constants were far lower than Kss. Bicyclic indazole (IND) inhibited catalysis through a single CYP2E1 site (Ki = 0.12 μM). Similarly, 4MP and IND yielded type II binding spectra that reflected the association of either two 4MP or one IND molecule(s) to CYP2E1, respectively. Based on computational docking studies with a homology model for CYP2E1, the two sites for monocyclic molecules, pNP and 4MP, exist within a narrow channel connecting the active site to the surface of the enzyme. Because of the presence of the heme iron, one site supports catalysis, whereas the other more distal effector site binds molecules that can influence the binding orientation and egress of molecules for the catalytic site. Although IND did not bind these sites simultaneously, the presence of IND at the catalytic site blocked binding at the effector site.
doi:10.1074/jbc.M707630200
PMCID: PMC3933162  PMID: 18056994
24.  Zinc Induces Dimerization of the Class II Major Histocompatibility Complex Molecule That Leads to Cooperative Binding to a Superantigen* 
The Journal of biological chemistry  2006;282(9):5991-6000.
Dimerization of class II major histocompatibility complex (MHC) plays an important role in the MHC biological function. Mycoplasma arthritidis-derived mitogen (MAM) is a superantigen that can activate large fractions of T cells bearing specific T cell receptor Vβ elements. Here we have used structural, sedimentation, and surface plasmon resonance detection approaches to investigate the molecular interactions between MAM and the class II MHC molecule HLA-DR1 in the context of a hemagglutinin peptide-(306–318) (HA). Our results revealed that zinc ion can efficiently induce the dimerization of the HLA-DR1/HA complex. Because the crystal structure of the MAM/HLA-DR1/hemagglutinin complex in the presence of EDTA is nearly identical to the structure of the complex crystallized in the presence of zinc ion, Zn2+ is evidently not directly involved in the binding between MAM and HLA-DR1. Sedimentation and surface plasmon resonance studies further revealed that MAM binds the HLA-DR1/HA complex with high affinity in a 1:1 stoichiometry, in the absence of Zn2+. However, in the presence of Zn2+, a dimerized MAM/HLA-DR1/HA complex can arise through the Zn2+-induced DR1 dimer. In the presence of Zn2+, cooperative binding of MAM to the DR1 dimer was also observed.
doi:10.1074/jbc.M608482200
PMCID: PMC3924565  PMID: 17166841
25.  Role for A Kinase-anchoring Proteins (AKAPS) in Glutamate Receptor Trafficking and Long Term Synaptic Depression* 
The Journal of biological chemistry  2005;280(17):16962-16968.
Expression of N-methyl d-aspartate (NMDA) receptor-dependent homosynaptic long term depression at synapses in the hippocampus and neocortex requires the persistent dephosphorylation of postsynaptic protein kinase A substrates. An attractive mechanism for expression of long term depression is the loss of surface AMPA (α-amino-3-hydroxy-5-methylisoxazale-4-propio-nate) receptors at synapses. Here we show that a threshold level of NMDA receptor activation must be exceeded to trigger a stable loss of AMPA receptors from the surface of cultured hippocampal neurons. NMDA also causes displacement of protein kinase A from the synapse, and inhibiting protein kinase A (PKA) activity mimics the NMDA-induced loss of surface AMPA receptors. PKA is targeted to the synapse by an interaction with the A kinase-anchoring protein, AKAP79/150. Disruption of the PKA-AKAP interaction is sufficient to cause a long-lasting reduction in synaptic AMPA receptors in cultured neurons. In addition, we demonstrate in hippocampal slices that displacement of PKA from AKADs occludes synaptically induced long term depression. These data indicate that synaptic anchoring of PKA through association with AKAPs plays an important role in the regulation of AMPA receptor surface expression and synaptic plasticity.
doi:10.1074/jbc.M409693200
PMCID: PMC3923403  PMID: 15718245

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