Don Lorimer - Academia.edu (original) (raw)

Papers by Don Lorimer

Research paper thumbnail of Co-crystal Structure of REP3123 Bound to Clostridium difficile Methionyl tRNA Synthetase

Background: REP3123 is a potent inhibitor (K i = 20 pM) of Clostridium difficile methionyl tRNA s... more Background: REP3123 is a potent inhibitor (K i = 20 pM) of Clostridium difficile methionyl tRNA synthetase (CDMetRS) that is being developed as a treatment for C. difficile-associated disease (CDAD). Inhibition of CDMetRS by REP3123 is competitive with methionine and uncompetitive with ATP. Methods: A C-terminal truncation of CDMetRS was expressed in E. coli as a His-tagged protein and purified by metal affinity chromatography. The protein was crystallized by sitting drop vapor diffusion. X-ray diffraction data were collected, and structures were solved by molecular replacement. Results: The 3D structure of REP3123 bound to CDMetRS was solved at 1.72 Å resolution. In addition, structures of a ternary complex with REP3123 and AMPCPP (2.04 Å) and also of apo enzyme (2.16 Å) were solved. REP3123 consists of a thienopyrimidone linked to a tetrahydrochroman via a propanediamine linker. The co-crystal structure of REP3123 and CDMetRS shows the tetrahydrochroman moiety binding deep into th...

Research paper thumbnail of Expression of a mutant myosin light chain that cannot be phosphorylated increases paracellular permeability

The American journal of physiology

A murine leukemia retroviral vector was engineered to contain the DNA encoding either the wild-ty... more A murine leukemia retroviral vector was engineered to contain the DNA encoding either the wild-type, rat aorta 20-kDa myosin light chain (MLC20) or a mutant form of MLC20 in which Thr18 and Ser19 were mutated into alanines. These mutations result in a MLC20 that cannot be phosphorylated by myosin light chain kinase. An 11-amino acid epitope from c-myc was added to both MLC20 sequences to facilitate identification of these proteins. Madin-Darby canine kidney cells were stably transduced, and MLC20 expression was demonstrated by Western blot analysis using a myc-specific antibody. MLC20 exchange was demonstrated by purifying myosin from the transduced cells and repeating the Western blot analysis. Actin-activated adenosinetriphosphatase assays on the purified myosins demonstrated approximately 50% decrease in the rate of ATP hydrolysis by the myosin containing the mutant MLC20. Transepithelial electrical resistance was decreased and mannitol flux was increased across monolayers of cel...

Research paper thumbnail of Structures of prostaglandin F synthase from the protozoa Leishmania major and Trypanosoma cruzi with NADP

Acta crystallographica. Section F, Structural biology communications, 2015

The crystal structures of prostaglandin F synthase (PGF) from both Leishmania major and Trypanoso... more The crystal structures of prostaglandin F synthase (PGF) from both Leishmania major and Trypanosoma cruzi with and without their cofactor NADP have been determined to resolutions of 2.6 Å for T. cruzi PGF, 1.25 Å for T. cruzi PGF with NADP, 1.6 Å for L. major PGF and 1.8 Å for L. major PGF with NADP. These structures were determined by molecular replacement to a final R factor of less than 18.6% (Rfree of less than 22.9%). PGF in the infectious protozoa L. major and T. cruzi is a potential therapeutic target.

Research paper thumbnail of Structures of a histidine triad family protein from Entamoeba histolytica bound to sulfate, AMP and GMP

Acta crystallographica. Section F, Structural biology communications, 2015

Three structures of the histidine triad family protein from Entamoeba histolytica, the causative ... more Three structures of the histidine triad family protein from Entamoeba histolytica, the causative agent of amoebic dysentery, were solved at high resolution within the Seattle Structural Genomics Center for Infectious Disease (SSGCID). The structures have sulfate (PDB entry 3oj7), AMP (PDB entry 3omf) or GMP (PDB entry 3oxk) bound in the active site, with sulfate occupying the same space as the α-phosphate of the two nucleotides. The C(α) backbones of the three structures are nearly superimposable, with pairwise r.m.s.d.s ranging from 0.06 to 0.13 Å.

Research paper thumbnail of Expression of the catalytic domain of myosin light chain kinase increases paracellular permeability

The American journal of physiology, 1996

Contractile events resulting from phosphorylation of the 20-kDa myosin light chain (MLC20) have b... more Contractile events resulting from phosphorylation of the 20-kDa myosin light chain (MLC20) have been implicated in the regulation of epithelial tight junction permeability. To address this question, Madin-Darby canine kidney cells were transfected with a murine leukemia retroviral vector containing DNA encoding either the catalytic domain of myosin light chain kinase (tMK) or the beta-galactosidase gene (beta-gal). Autoradiograms of sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of myosin immunoprecipitated from 32Pi-labeled transfected cells demonstrated that MLC20 phosphorylation was increased 3.1 +/- 0.9-fold in cells expressing tMK compared with cells expressing beta-gal. Phosphopeptide mapping confirmed that myosin light chain kinase was responsible for the increased MLC20 phosphorylation. Transepithelial electrical resistance, a measurement of barrier function, of tMK cell monolayers was consistently < 10% (123 +/- 20 omega.cm2) of that of monolayers com...

Research paper thumbnail of Artificial Neural Networks Trained to Detect Viral and Phage Structural Proteins

PLoS Computational Biology, 2012

Phages play critical roles in the survival and pathogenicity of their hosts, via lysogenic conver... more Phages play critical roles in the survival and pathogenicity of their hosts, via lysogenic conversion factors, and in nutrient redistribution, via cell lysis. Analyses of phage-and viral-encoded genes in environmental samples provide insights into the physiological impact of viruses on microbial communities and human health. However, phage ORFs are extremely diverse of which over 70% of them are dissimilar to any genes with annotated functions in GenBank. Better identification of viruses would also aid in better detection and diagnosis of disease, in vaccine development, and generally in better understanding the physiological potential of any environment. In contrast to enzymes, viral structural protein function can be much more challenging to detect from sequence data because of low sequence conservation, few known conserved catalytic sites or sequence domains, and relatively limited experimental data. We have designed a method of predicting phage structural protein sequences that uses Artificial Neural Networks (ANNs). First, we trained ANNs to classify viral structural proteins using amino acid frequency; these correctly classify a large fraction of test cases with a high degree of specificity and sensitivity. Subsequently, we added estimates of protein isoelectric points as a feature to ANNs that classify specialized families of proteins, namely major capsid and tail proteins. As expected, these more specialized ANNs are more accurate than the structural ANNs. To experimentally validate the ANN predictions, several ORFs with no significant similarities to known sequences that are ANN-predicted structural proteins were examined by transmission electron microscopy. Some of these self-assembled into structures strongly resembling virion structures. Thus, our ANNs are new tools for identifying phage and potential prophage structural proteins that are difficult or impossible to detect by other bioinformatic analysis. The networks will be valuable when sequence is available but in vitro propagation of the phage may not be practical or possible.

Research paper thumbnail of Location and Characterization of the Human GRP Receptor Expressed by Gastrointestinal Epithelial Cells

Peptides, 1997

.-The exact location of normal gastrinreleasing peptide (GRP) receptor expression by epithelial c... more .-The exact location of normal gastrinreleasing peptide (GRP) receptor expression by epithelial cells lining the human gastrointestinal (GI) tract is not known; yet this receptor is found on upwards of 50% of GI cancers. Furthermore, the pharmacology reported for GRP receptors expressed by GI cancers varies considerably. Therefore, the purpose of this study was to determine the normal distribution of GRP receptor expression by cells lining the human GI tract, and then determine the normal pharmacology of the human receptor when ectopically expressed by the nonmalignant human colon epithelial cell line NCM460. We obtained endoscopic pinch biopsies of, and extracted the RNA from, epithelial cells lining the esophagus, stomach, jejunum, ileum, and proximal and descending colon. RT-PCR demonstrated that GRP-R expression is limited to cells lining the gastric antrum, indicating that this receptor is aberrantly expressed by GI cancers. To determine the normal pharmacology of this receptor when expressed by nonmalignant human tissues for the first time, we transfected NCM460 cells with the cDNA for the human GRP receptor. By studying three stable NCM460 cell lines expressing varying numbers of receptors, we demonstrate that agonist and antagonist binding affinity, binding kinetics, and Gprotein coupling are all independent of receptor number. Finally, by comparing GRP receptors expressed by GI cancers with those on NCM460-transfected cells, we show that the pharmacology of the aberrantly expressed receptors is significantly altered. Thus, these data demonstrate that GI cancers aberrantly express GRP receptors that then behave abnormally. ᭧ 1997 Elsevier Science Inc.

Research paper thumbnail of Specific sequences downstream from −6 are not essential for proper and efficient in Vitro utilization of the Escherichia coli lactose promoter

Journal of Molecular Biology, 1990

A series of deletion mutants of the wild-type Escherichia coli lactose promoter, with endpoints a... more A series of deletion mutants of the wild-type Escherichia coli lactose promoter, with endpoints at +25, + 19, ÷ 14, ÷ 1 and -6 (relative to the start of transcription at ÷ 1), was constructed and the deleted DNA replaced with non-lac DNA. These mutants were used to show that no specific DNA sequences downstream from -6 are required for efficient promoter utilization in vitro. In all cases transcription is dependent on the presence of the catabolite activator protein (CAP) and cAMP, and begins at + 1 at a level indistinguishable from that at the wild-type promoter. A set of lac DNA fragments deleted to -6 was constructed, having an A, C, G or T residue at + 1 and heterologous DNA downstream. These synthetic promoters allow systematic testing of the effect of the initiating nucleotide on the transcription process. Again, transcription occurs mainly from ÷ 1, at a level similar to the normal wild-type level. No substantial differences between these promoters are observed in the rates of formation of stable complexes, in the degree of complex formation, in the rate at which polymerase "escapes" from the complex or in abortive transcription products. Equivalent results are seen with a related set of constructs based on the CAP-insensitive lac UV5 promoter. Thus, lac promoter sequences including consensus hexamers at -l0 and -35, plus the spacer region between them, provide specificity and efficiency both in initiation of transcription by RNA polymerase and in CAP-polymerase interactions.

Research paper thumbnail of Fibroblast Contractility without an Increase in Basal Myosin Light Chain Phosphorylation in Wild Type Cells and Cells Expressing the Catalytic Domain of Myosin Light Chain Kinase

Journal of Biological Chemistry, 1995

Research paper thumbnail of Impact of the δF508 mutation in NBD1 of human CFTR on domain folding and structure

Journal of Biological Chemistry, Nov 2, 2004

Research paper thumbnail of Cloning and characterization of a novel human histone deacetylase, HDAC8

Biochemical Journal, 2000

Histone deacetylases (HDACs) are a growing family of enzymes implicated in transcriptional regula... more Histone deacetylases (HDACs) are a growing family of enzymes implicated in transcriptional regulation by affecting the acetylation state of core histones in the nucleus of cells. HDACs are known to have key roles in the regulation of cell proliferation Nature (London) 391, 597-600], and aberrant recruitment of an HDAC complex has been shown to be a key step in the mechanism of cell transformation in acute promyelocytic leukaemia Nature (London) 391, 815-818 ;, Nature (London) 391, [811][812][813][814]. Here we present the complete nucleotide sequence of a cDNA clone, termed HDAC8, that encodes a protein product with similarity to the RPD3 class (I) of HDACs. The predicted 377-residue HDAC8

Research paper thumbnail of Challenging the state-of-the-art in protein structure prediction : highlights of experimental target structures for the 10(th) Critical Assessment of Techniques for Protein Structure Prediction Experiment CASP10

Research paper thumbnail of Structures of prostaglandin F synthase from the protozoa Leishmania major and Trypanosoma cruzi with NADP

Acta crystallographica. Section F, Structural biology communications, 2015

The crystal structures of prostaglandin F synthase (PGF) from both Leishmania major and Trypanoso... more The crystal structures of prostaglandin F synthase (PGF) from both Leishmania major and Trypanosoma cruzi with and without their cofactor NADP have been determined to resolutions of 2.6 Å for T. cruzi PGF, 1.25 Å for T. cruzi PGF with NADP, 1.6 Å for L. major PGF and 1.8 Å for L. major PGF with NADP. These structures were determined by molecular replacement to a final R factor of less than 18.6% (Rfree of less than 22.9%). PGF in the infectious protozoa L. major and T. cruzi is a potential therapeutic target.

Research paper thumbnail of Structures of a histidine triad family protein from Entamoeba histolytica bound to sulfate, AMP and GMP

Acta crystallographica. Section F, Structural biology communications, 2015

Three structures of the histidine triad family protein from Entamoeba histolytica, the causative ... more Three structures of the histidine triad family protein from Entamoeba histolytica, the causative agent of amoebic dysentery, were solved at high resolution within the Seattle Structural Genomics Center for Infectious Disease (SSGCID). The structures have sulfate (PDB entry 3oj7), AMP (PDB entry 3omf) or GMP (PDB entry 3oxk) bound in the active site, with sulfate occupying the same space as the α-phosphate of the two nucleotides. The C(α) backbones of the three structures are nearly superimposable, with pairwise r.m.s.d.s ranging from 0.06 to 0.13 Å.

Research paper thumbnail of Structures of aspartate aminotransferases from Trypanosoma brucei , Leishmania major and Giardia lamblia

Acta Crystallographica Section F Structural Biology Communications, 2015

The structures of three aspartate aminotransferases (AATs) from eukaryotic pathogens were solved ... more The structures of three aspartate aminotransferases (AATs) from eukaryotic pathogens were solved within the Seattle Structural Genomics Center for Infectious Disease (SSGCID). Both the open and closed conformations of AAT were observed. Pyridoxal phosphate was bound to the active site via a Schiff base to a conserved lysine. An active-site mutant showed that Trypanosoma brucei AAT still binds pyridoxal phosphate even in the absence of the tethering lysine. The structures highlight the challenges for the structure-based design of inhibitors targeting the active site, while showing options for inhibitor design targeting the N-terminal arm.

Research paper thumbnail of In vivo progression of LAPC-9 and LNCaP prostate cancer models to androgen independence is associated with increased expression of insulin-like growth factor I (IGF-I) and IGF-I receptor (IGF-IR)

Cancer research, Jan 15, 2001

Androgen deprivation therapies for metastatic prostate cancer are useful initially, but progressi... more Androgen deprivation therapies for metastatic prostate cancer are useful initially, but progression to androgen independence usually results in relapse within 2 years. The molecular mechanisms underlying the clinically important transition from androgen dependence to androgen independence are poorly described. Several lines of investigation have suggested that insulin-like growth factors (IGFs) are involved in the biology of prostate cancer, but little is known about their relevance to progression to androgen independence. We used three in vivo models of androgen-dependent (AD) human prostate cancer to study this issue. Progression to androgen-independent (AI) growth was associated with a 60-fold increase in expression of IGF-I mRNA in LAPC-9 xenografts and a 28-fold increase in IGF-I expression in LNCAP xenografts, relative to the initial AD neoplasms. IGF type I receptor (IGF-IR) mRNA levels were approximately 2.5-fold and approximately 5-fold higher, respectively, in AI LAPC-9 an...

Research paper thumbnail of Structure of the Reston ebolavirus VP30 C-terminal domain

Acta Crystallographica Section F Structural Biology Communications, 2014

PDB reference: Reston ebolavirus VP30 C-terminal domain, 3v7o

Research paper thumbnail of Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit

Journal of Visualized Experiments, 2013

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortal... more Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States alone, an average of 41,400 deaths and 1.86 million hospitalizations are caused by influenza virus infection each year 1 . Point mutations in the polymerase basic protein 2 subunit (PB2) have been linked to the adaptation of the viral infection in humans 2

Research paper thumbnail of Tyrosine aminotransferase from Leishmania infantum: A new drug target candidate

International Journal for Parasitology: Drugs and Drug Resistance, 2014

Leishmania infantum is the etiological agent of zoonotic visceral leishmaniasis in the Mediterran... more Leishmania infantum is the etiological agent of zoonotic visceral leishmaniasis in the Mediterranean basin. The disease is fatal without treatment, which has been based on antimonial pentavalents for more than 60 years. Due to resistances, relapses and toxicity to current treatment, the development of new drugs is required. The structure of the L. infantum tyrosine aminotransferase (LiTAT) has been recently solved showing important differences with the mammalian orthologue. The characterization of LiTAT is reported herein. This enzyme is cytoplasmic and is over-expressed in the more infective stages and nitric oxide resistant parasites. Unlike the mammalian TAT, LiTAT is able to use ketomethiobutyrate as co-substrate. The pharmacophore model of LiTAT with this specific co-substrate is described herein. This may allow the identification of new inhibitors present in the databases. All the data obtained support that LiTAT is a good target candidate for the development of new anti-leishmanial drugs.

Research paper thumbnail of Structural analysis of H1N1 and H7N9 influenza A virus PA in the absence of PB1

Scientific Reports, 2014

Influenza A viruses cause the respiratory illness influenza, which can be mild to fatal depending... more Influenza A viruses cause the respiratory illness influenza, which can be mild to fatal depending on the strain and host immune response. The flu polymerase acidic (PA), polymerase basic 1 (PB1), and polymerase basic 2 (PB2) proteins comprise the RNA-dependent RNA polymerase complex responsible for viral genome replication. The first crystal structures of the C-terminal domain of PA (PA-CTD) in the absence of PB1-derived peptides show a number of structural changes relative to the previously reported PB1-peptide bound structures. The human A/WSN/1933 (H1N1) and avian A/Anhui1/2013 (H7N9) strain PA-CTD proteins exhibit the same global topology as other strains in the absence of PB1, but differ extensively in the PB1 binding pocket including a widening of the binding groove and the unfolding of a b-turn. Both PA-CTD proteins exhibited a significant increase in thermal stability in the presence of either a PB1-derived peptide or a previously reported inhibitor in differential scanning fluorimetry assays. These structural changes demonstrate plasticity in the PA-PB1 binding interface which may be exploited in the development of novel therapeutics.

Research paper thumbnail of Co-crystal Structure of REP3123 Bound to Clostridium difficile Methionyl tRNA Synthetase

Background: REP3123 is a potent inhibitor (K i = 20 pM) of Clostridium difficile methionyl tRNA s... more Background: REP3123 is a potent inhibitor (K i = 20 pM) of Clostridium difficile methionyl tRNA synthetase (CDMetRS) that is being developed as a treatment for C. difficile-associated disease (CDAD). Inhibition of CDMetRS by REP3123 is competitive with methionine and uncompetitive with ATP. Methods: A C-terminal truncation of CDMetRS was expressed in E. coli as a His-tagged protein and purified by metal affinity chromatography. The protein was crystallized by sitting drop vapor diffusion. X-ray diffraction data were collected, and structures were solved by molecular replacement. Results: The 3D structure of REP3123 bound to CDMetRS was solved at 1.72 Å resolution. In addition, structures of a ternary complex with REP3123 and AMPCPP (2.04 Å) and also of apo enzyme (2.16 Å) were solved. REP3123 consists of a thienopyrimidone linked to a tetrahydrochroman via a propanediamine linker. The co-crystal structure of REP3123 and CDMetRS shows the tetrahydrochroman moiety binding deep into th...

Research paper thumbnail of Expression of a mutant myosin light chain that cannot be phosphorylated increases paracellular permeability

The American journal of physiology

A murine leukemia retroviral vector was engineered to contain the DNA encoding either the wild-ty... more A murine leukemia retroviral vector was engineered to contain the DNA encoding either the wild-type, rat aorta 20-kDa myosin light chain (MLC20) or a mutant form of MLC20 in which Thr18 and Ser19 were mutated into alanines. These mutations result in a MLC20 that cannot be phosphorylated by myosin light chain kinase. An 11-amino acid epitope from c-myc was added to both MLC20 sequences to facilitate identification of these proteins. Madin-Darby canine kidney cells were stably transduced, and MLC20 expression was demonstrated by Western blot analysis using a myc-specific antibody. MLC20 exchange was demonstrated by purifying myosin from the transduced cells and repeating the Western blot analysis. Actin-activated adenosinetriphosphatase assays on the purified myosins demonstrated approximately 50% decrease in the rate of ATP hydrolysis by the myosin containing the mutant MLC20. Transepithelial electrical resistance was decreased and mannitol flux was increased across monolayers of cel...

Research paper thumbnail of Structures of prostaglandin F synthase from the protozoa Leishmania major and Trypanosoma cruzi with NADP

Acta crystallographica. Section F, Structural biology communications, 2015

The crystal structures of prostaglandin F synthase (PGF) from both Leishmania major and Trypanoso... more The crystal structures of prostaglandin F synthase (PGF) from both Leishmania major and Trypanosoma cruzi with and without their cofactor NADP have been determined to resolutions of 2.6 Å for T. cruzi PGF, 1.25 Å for T. cruzi PGF with NADP, 1.6 Å for L. major PGF and 1.8 Å for L. major PGF with NADP. These structures were determined by molecular replacement to a final R factor of less than 18.6% (Rfree of less than 22.9%). PGF in the infectious protozoa L. major and T. cruzi is a potential therapeutic target.

Research paper thumbnail of Structures of a histidine triad family protein from Entamoeba histolytica bound to sulfate, AMP and GMP

Acta crystallographica. Section F, Structural biology communications, 2015

Three structures of the histidine triad family protein from Entamoeba histolytica, the causative ... more Three structures of the histidine triad family protein from Entamoeba histolytica, the causative agent of amoebic dysentery, were solved at high resolution within the Seattle Structural Genomics Center for Infectious Disease (SSGCID). The structures have sulfate (PDB entry 3oj7), AMP (PDB entry 3omf) or GMP (PDB entry 3oxk) bound in the active site, with sulfate occupying the same space as the α-phosphate of the two nucleotides. The C(α) backbones of the three structures are nearly superimposable, with pairwise r.m.s.d.s ranging from 0.06 to 0.13 Å.

Research paper thumbnail of Expression of the catalytic domain of myosin light chain kinase increases paracellular permeability

The American journal of physiology, 1996

Contractile events resulting from phosphorylation of the 20-kDa myosin light chain (MLC20) have b... more Contractile events resulting from phosphorylation of the 20-kDa myosin light chain (MLC20) have been implicated in the regulation of epithelial tight junction permeability. To address this question, Madin-Darby canine kidney cells were transfected with a murine leukemia retroviral vector containing DNA encoding either the catalytic domain of myosin light chain kinase (tMK) or the beta-galactosidase gene (beta-gal). Autoradiograms of sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of myosin immunoprecipitated from 32Pi-labeled transfected cells demonstrated that MLC20 phosphorylation was increased 3.1 +/- 0.9-fold in cells expressing tMK compared with cells expressing beta-gal. Phosphopeptide mapping confirmed that myosin light chain kinase was responsible for the increased MLC20 phosphorylation. Transepithelial electrical resistance, a measurement of barrier function, of tMK cell monolayers was consistently < 10% (123 +/- 20 omega.cm2) of that of monolayers com...

Research paper thumbnail of Artificial Neural Networks Trained to Detect Viral and Phage Structural Proteins

PLoS Computational Biology, 2012

Phages play critical roles in the survival and pathogenicity of their hosts, via lysogenic conver... more Phages play critical roles in the survival and pathogenicity of their hosts, via lysogenic conversion factors, and in nutrient redistribution, via cell lysis. Analyses of phage-and viral-encoded genes in environmental samples provide insights into the physiological impact of viruses on microbial communities and human health. However, phage ORFs are extremely diverse of which over 70% of them are dissimilar to any genes with annotated functions in GenBank. Better identification of viruses would also aid in better detection and diagnosis of disease, in vaccine development, and generally in better understanding the physiological potential of any environment. In contrast to enzymes, viral structural protein function can be much more challenging to detect from sequence data because of low sequence conservation, few known conserved catalytic sites or sequence domains, and relatively limited experimental data. We have designed a method of predicting phage structural protein sequences that uses Artificial Neural Networks (ANNs). First, we trained ANNs to classify viral structural proteins using amino acid frequency; these correctly classify a large fraction of test cases with a high degree of specificity and sensitivity. Subsequently, we added estimates of protein isoelectric points as a feature to ANNs that classify specialized families of proteins, namely major capsid and tail proteins. As expected, these more specialized ANNs are more accurate than the structural ANNs. To experimentally validate the ANN predictions, several ORFs with no significant similarities to known sequences that are ANN-predicted structural proteins were examined by transmission electron microscopy. Some of these self-assembled into structures strongly resembling virion structures. Thus, our ANNs are new tools for identifying phage and potential prophage structural proteins that are difficult or impossible to detect by other bioinformatic analysis. The networks will be valuable when sequence is available but in vitro propagation of the phage may not be practical or possible.

Research paper thumbnail of Location and Characterization of the Human GRP Receptor Expressed by Gastrointestinal Epithelial Cells

Peptides, 1997

.-The exact location of normal gastrinreleasing peptide (GRP) receptor expression by epithelial c... more .-The exact location of normal gastrinreleasing peptide (GRP) receptor expression by epithelial cells lining the human gastrointestinal (GI) tract is not known; yet this receptor is found on upwards of 50% of GI cancers. Furthermore, the pharmacology reported for GRP receptors expressed by GI cancers varies considerably. Therefore, the purpose of this study was to determine the normal distribution of GRP receptor expression by cells lining the human GI tract, and then determine the normal pharmacology of the human receptor when ectopically expressed by the nonmalignant human colon epithelial cell line NCM460. We obtained endoscopic pinch biopsies of, and extracted the RNA from, epithelial cells lining the esophagus, stomach, jejunum, ileum, and proximal and descending colon. RT-PCR demonstrated that GRP-R expression is limited to cells lining the gastric antrum, indicating that this receptor is aberrantly expressed by GI cancers. To determine the normal pharmacology of this receptor when expressed by nonmalignant human tissues for the first time, we transfected NCM460 cells with the cDNA for the human GRP receptor. By studying three stable NCM460 cell lines expressing varying numbers of receptors, we demonstrate that agonist and antagonist binding affinity, binding kinetics, and Gprotein coupling are all independent of receptor number. Finally, by comparing GRP receptors expressed by GI cancers with those on NCM460-transfected cells, we show that the pharmacology of the aberrantly expressed receptors is significantly altered. Thus, these data demonstrate that GI cancers aberrantly express GRP receptors that then behave abnormally. ᭧ 1997 Elsevier Science Inc.

Research paper thumbnail of Specific sequences downstream from −6 are not essential for proper and efficient in Vitro utilization of the Escherichia coli lactose promoter

Journal of Molecular Biology, 1990

A series of deletion mutants of the wild-type Escherichia coli lactose promoter, with endpoints a... more A series of deletion mutants of the wild-type Escherichia coli lactose promoter, with endpoints at +25, + 19, ÷ 14, ÷ 1 and -6 (relative to the start of transcription at ÷ 1), was constructed and the deleted DNA replaced with non-lac DNA. These mutants were used to show that no specific DNA sequences downstream from -6 are required for efficient promoter utilization in vitro. In all cases transcription is dependent on the presence of the catabolite activator protein (CAP) and cAMP, and begins at + 1 at a level indistinguishable from that at the wild-type promoter. A set of lac DNA fragments deleted to -6 was constructed, having an A, C, G or T residue at + 1 and heterologous DNA downstream. These synthetic promoters allow systematic testing of the effect of the initiating nucleotide on the transcription process. Again, transcription occurs mainly from ÷ 1, at a level similar to the normal wild-type level. No substantial differences between these promoters are observed in the rates of formation of stable complexes, in the degree of complex formation, in the rate at which polymerase "escapes" from the complex or in abortive transcription products. Equivalent results are seen with a related set of constructs based on the CAP-insensitive lac UV5 promoter. Thus, lac promoter sequences including consensus hexamers at -l0 and -35, plus the spacer region between them, provide specificity and efficiency both in initiation of transcription by RNA polymerase and in CAP-polymerase interactions.

Research paper thumbnail of Fibroblast Contractility without an Increase in Basal Myosin Light Chain Phosphorylation in Wild Type Cells and Cells Expressing the Catalytic Domain of Myosin Light Chain Kinase

Journal of Biological Chemistry, 1995

Research paper thumbnail of Impact of the δF508 mutation in NBD1 of human CFTR on domain folding and structure

Journal of Biological Chemistry, Nov 2, 2004

Research paper thumbnail of Cloning and characterization of a novel human histone deacetylase, HDAC8

Biochemical Journal, 2000

Histone deacetylases (HDACs) are a growing family of enzymes implicated in transcriptional regula... more Histone deacetylases (HDACs) are a growing family of enzymes implicated in transcriptional regulation by affecting the acetylation state of core histones in the nucleus of cells. HDACs are known to have key roles in the regulation of cell proliferation Nature (London) 391, 597-600], and aberrant recruitment of an HDAC complex has been shown to be a key step in the mechanism of cell transformation in acute promyelocytic leukaemia Nature (London) 391, 815-818 ;, Nature (London) 391, [811][812][813][814]. Here we present the complete nucleotide sequence of a cDNA clone, termed HDAC8, that encodes a protein product with similarity to the RPD3 class (I) of HDACs. The predicted 377-residue HDAC8

Research paper thumbnail of Challenging the state-of-the-art in protein structure prediction : highlights of experimental target structures for the 10(th) Critical Assessment of Techniques for Protein Structure Prediction Experiment CASP10

Research paper thumbnail of Structures of prostaglandin F synthase from the protozoa Leishmania major and Trypanosoma cruzi with NADP

Acta crystallographica. Section F, Structural biology communications, 2015

The crystal structures of prostaglandin F synthase (PGF) from both Leishmania major and Trypanoso... more The crystal structures of prostaglandin F synthase (PGF) from both Leishmania major and Trypanosoma cruzi with and without their cofactor NADP have been determined to resolutions of 2.6 Å for T. cruzi PGF, 1.25 Å for T. cruzi PGF with NADP, 1.6 Å for L. major PGF and 1.8 Å for L. major PGF with NADP. These structures were determined by molecular replacement to a final R factor of less than 18.6% (Rfree of less than 22.9%). PGF in the infectious protozoa L. major and T. cruzi is a potential therapeutic target.

Research paper thumbnail of Structures of a histidine triad family protein from Entamoeba histolytica bound to sulfate, AMP and GMP

Acta crystallographica. Section F, Structural biology communications, 2015

Three structures of the histidine triad family protein from Entamoeba histolytica, the causative ... more Three structures of the histidine triad family protein from Entamoeba histolytica, the causative agent of amoebic dysentery, were solved at high resolution within the Seattle Structural Genomics Center for Infectious Disease (SSGCID). The structures have sulfate (PDB entry 3oj7), AMP (PDB entry 3omf) or GMP (PDB entry 3oxk) bound in the active site, with sulfate occupying the same space as the α-phosphate of the two nucleotides. The C(α) backbones of the three structures are nearly superimposable, with pairwise r.m.s.d.s ranging from 0.06 to 0.13 Å.

Research paper thumbnail of Structures of aspartate aminotransferases from Trypanosoma brucei , Leishmania major and Giardia lamblia

Acta Crystallographica Section F Structural Biology Communications, 2015

The structures of three aspartate aminotransferases (AATs) from eukaryotic pathogens were solved ... more The structures of three aspartate aminotransferases (AATs) from eukaryotic pathogens were solved within the Seattle Structural Genomics Center for Infectious Disease (SSGCID). Both the open and closed conformations of AAT were observed. Pyridoxal phosphate was bound to the active site via a Schiff base to a conserved lysine. An active-site mutant showed that Trypanosoma brucei AAT still binds pyridoxal phosphate even in the absence of the tethering lysine. The structures highlight the challenges for the structure-based design of inhibitors targeting the active site, while showing options for inhibitor design targeting the N-terminal arm.

Research paper thumbnail of In vivo progression of LAPC-9 and LNCaP prostate cancer models to androgen independence is associated with increased expression of insulin-like growth factor I (IGF-I) and IGF-I receptor (IGF-IR)

Cancer research, Jan 15, 2001

Androgen deprivation therapies for metastatic prostate cancer are useful initially, but progressi... more Androgen deprivation therapies for metastatic prostate cancer are useful initially, but progression to androgen independence usually results in relapse within 2 years. The molecular mechanisms underlying the clinically important transition from androgen dependence to androgen independence are poorly described. Several lines of investigation have suggested that insulin-like growth factors (IGFs) are involved in the biology of prostate cancer, but little is known about their relevance to progression to androgen independence. We used three in vivo models of androgen-dependent (AD) human prostate cancer to study this issue. Progression to androgen-independent (AI) growth was associated with a 60-fold increase in expression of IGF-I mRNA in LAPC-9 xenografts and a 28-fold increase in IGF-I expression in LNCAP xenografts, relative to the initial AD neoplasms. IGF type I receptor (IGF-IR) mRNA levels were approximately 2.5-fold and approximately 5-fold higher, respectively, in AI LAPC-9 an...

Research paper thumbnail of Structure of the Reston ebolavirus VP30 C-terminal domain

Acta Crystallographica Section F Structural Biology Communications, 2014

PDB reference: Reston ebolavirus VP30 C-terminal domain, 3v7o

Research paper thumbnail of Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit

Journal of Visualized Experiments, 2013

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortal... more Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States alone, an average of 41,400 deaths and 1.86 million hospitalizations are caused by influenza virus infection each year 1 . Point mutations in the polymerase basic protein 2 subunit (PB2) have been linked to the adaptation of the viral infection in humans 2

Research paper thumbnail of Tyrosine aminotransferase from Leishmania infantum: A new drug target candidate

International Journal for Parasitology: Drugs and Drug Resistance, 2014

Leishmania infantum is the etiological agent of zoonotic visceral leishmaniasis in the Mediterran... more Leishmania infantum is the etiological agent of zoonotic visceral leishmaniasis in the Mediterranean basin. The disease is fatal without treatment, which has been based on antimonial pentavalents for more than 60 years. Due to resistances, relapses and toxicity to current treatment, the development of new drugs is required. The structure of the L. infantum tyrosine aminotransferase (LiTAT) has been recently solved showing important differences with the mammalian orthologue. The characterization of LiTAT is reported herein. This enzyme is cytoplasmic and is over-expressed in the more infective stages and nitric oxide resistant parasites. Unlike the mammalian TAT, LiTAT is able to use ketomethiobutyrate as co-substrate. The pharmacophore model of LiTAT with this specific co-substrate is described herein. This may allow the identification of new inhibitors present in the databases. All the data obtained support that LiTAT is a good target candidate for the development of new anti-leishmanial drugs.

Research paper thumbnail of Structural analysis of H1N1 and H7N9 influenza A virus PA in the absence of PB1

Scientific Reports, 2014

Influenza A viruses cause the respiratory illness influenza, which can be mild to fatal depending... more Influenza A viruses cause the respiratory illness influenza, which can be mild to fatal depending on the strain and host immune response. The flu polymerase acidic (PA), polymerase basic 1 (PB1), and polymerase basic 2 (PB2) proteins comprise the RNA-dependent RNA polymerase complex responsible for viral genome replication. The first crystal structures of the C-terminal domain of PA (PA-CTD) in the absence of PB1-derived peptides show a number of structural changes relative to the previously reported PB1-peptide bound structures. The human A/WSN/1933 (H1N1) and avian A/Anhui1/2013 (H7N9) strain PA-CTD proteins exhibit the same global topology as other strains in the absence of PB1, but differ extensively in the PB1 binding pocket including a widening of the binding groove and the unfolding of a b-turn. Both PA-CTD proteins exhibited a significant increase in thermal stability in the presence of either a PB1-derived peptide or a previously reported inhibitor in differential scanning fluorimetry assays. These structural changes demonstrate plasticity in the PA-PB1 binding interface which may be exploited in the development of novel therapeutics.