Biomedical subjects

W J Cook

Publications and source records attributed to W J Cook.

At least 37 records · Page 2Linked to original sources

PubMed1997

Crystal structure of a class I ubiquitin conjugating enzyme (Ubc7) from Saccharomyces cerevisiae at 2.9 angstroms resolution.

Ubiquitin-conjugating enzymes are a family of related proteins that participate in the ubiquitination of proteins. Previous studies on the crystal structures of Saccharomyces cerevisiae Ubc4 and Arabidopsis thaliana Ubc1 indicated that the smallest enzymes (class I), which consist entirely of the conserved core domain, share a common tertiary fold. Here we report the three-dimensional structure of the S. cerevisiae class I enzyme encoded by the UBC7 gene. The crystal structure has been solved using molecular replacement techniques and refined by simulated annealing to an R-factor of 0.183 at 2.93 A resolution. Bond lengths and angles in the molecule have root-mean-square deviations from ideal values of 0.016 A and 2.3 degrees, respectively. Ubc7 is an alpha/beta protein with four alpha-helices and a four-stranded antiparallel beta-sheet. With the exception of two regions where extra residues are present, the tertiary folding of Ubc7 is similar to those of the other two enzymes. The ubiquitin-accepting cysteine is located in a cleft between two loops. One of these loops is nonconserved, as this region of the Ubc7 molecule differs from the other two enzymes by having 13 extra residues. There is also a second single amino acid insertion that alters the orientation of the turn between the first two beta-strands. Analysis of the 13 ubiquitin-conjugating enzyme sequences in S. cerevisiae indicates that there may be two other regions where extra residues could be inserted into the common tertiary fold. Both of these other regions exhibit significant deviations in the superposition of the three structures and, like the two insertion regions in Ubc7, may represent hypervariable regions within a common tertiary fold. As ubiquitin-conjugating enzymes interact with different substrates or other accessory proteins in the ubiquitination pathway, these variable surface regions may confer distinct specificity to individual enzymes.

Amino Acid Sequence↗

PubMed1997

Refined structures of three crystal forms of toxic shock syndrome toxin-1 and of a tetramutant with reduced activity.

The structure of toxic shock syndrome toxin-1 (TSST-1), the causative agent in toxic shock syndrome, has been determined in three crystal forms. The three structural models have been refined to R-factors of 0.154, 0.150, and 0.198 at resolutions of 2.05 A, 2.90 A, and 2.75 A, respectively. One crystal form of TSST-1 contains a zinc ion bound between two symmetry-related molecules. Although not required for biological activity, zinc dramatically potentiates the mitogenicity of TSST-1 at very low concentrations. In addition, the structure of the tetramutant TSST-1H [T69I, Y80W, E132K, I140T], which is nonmitogenic and does not amplify endotoxin shock, has been determined and refined in a fourth crystal form (R-factor = 0.173 to 1.9 A resolution).

Bacterial Toxins↗

PubMed1997

Gastric duplication: endoscopic appearance and clinical features.

The case of a 13-year-old female presenting with a 6 week history of significant vomiting resulting from a gastric duplication is described. At endoscopy, an antral mass which appeared to change in size and site with peristalsis was evident. Although rare, these lesions need to be considered in the differential diagnosis of patients presenting with symptoms suggestive of gastric outlet obstruction. However, in the majority of cases, the diagnosis is rarely made prior to surgery.

Adolescent↗

PubMed1996

Regulation of neighboring gene expression by the herpes simplex virus type 1 thymidine kinase gene.

The herpes simplex virus type 1 thymidine kinase (tk) gene (UL23) lies upstream of the gH (UL22) gene with its 3' end overlapping the gH promoter, and it overlaps the UL24 gene's regulatory and coding sequences at its 5' end in a head-to-head orientation. Thus, tk expression could affect gH expression by promoter occlusion and UL24 expression by transcriptional or posttranscriptional mechanisms. To investigate these possibilities, we analyzed the effects of tk promoter mutations that reduce tk expression on gH and UL24 expression. For gH, tk promoter mutations did not increase the accumulation of gH mRNA or the rate of gH transcription. Thus, tk expression does not appear to down-regulate gH expression. In contrast, we found that decreased tk expression correlated with increased accumulation of UL24 mRNA, particularly a 1.4-kb transcript, at early times postinfection during peak expression of tk, but not at late times when tk mRNA levels have fallen. Results from viral co-infection experiments indicated that down-regulation of UL24 mRNA accumulation requires tk expression in cis. Nuclear run-off experiments did not detect differences in UL24 transcription rates in the mutant viruses. Although we cannot completely exclude a transcriptional mechanism for this down-regulation, these results can be explained by an antisense RNA mechanism acting preferentially in cis.

Animals↗

PubMed1996

Temporal regulation of herpes simplex virus type 1 UL24 mRNA expression via differential polyadenylation.

Using Northern blot, primer extension, and S1 nuclease analyses of wild-type and deletion-containing herpes simplex type 1 viruses, we found that UL24 sequences are contained in six different transcripts that originate from three previously identified mRNA start sites. Thus, the six UL24 transcripts represent three different pairs of 5' coterminal mRNAs. Each transcript pair consists of a short species whose 3' end corresponds to a polyadenylation signal located just downstream of the UL24 open reading frame, and a longer species whose 3' end corresponds to a polyadenylation signal located downstream of the UL26 gene. Maximal accumulation of the short UL24 transcripts was at early times during infection, while accumulation of the longer species did not decrease at late times. Consistent with early kinetics, the short transcripts were less sensitive to drugs that inhibited viral DNA replication than the longer transcripts which exhibited leaky-late kinetics. Quantitative S1 nuclease analysis indicated that 3' ends corresponding to the UL24 polyadenylation site were significantly more abundant at early times than at late times. Thus, differential polyadenylation determines the kinetics of accumulation of different UL24 transcripts.

Animals↗

PubMed1996

Crystal structures of nucleoside 2-deoxyribosyltransferase in native and ligand-bound forms reveal architecture of the active site.

BACKGROUND: Nucleoside 2-deoxyribosyltransferase plays an important role in the salvage pathway of nucleotide metabolism in certain organisms, catalyzing the cleavage of beta-2'-deoxyribonucleosides and the subsequent transfer of the deoxyribosyl moiety to an acceptor purine or pyrimidine base. The kinetics describe a ping-pong-bi-bi pathway involving the formation of a covalent enzyme-deoxyribose intermediate. The enzyme is produced by a limited number of microorganisms and its functions have been exploited in its use as a biocatalyst to synthesize nucleoside analogs of therapeutic interest. RESULTS: We describe the crystal structure of the enzyme with and without bound ligand. The native structure was solved by the single isomorphous replacement with anomalous scattering method (SIRAS) and refined to 2.5 A resolution resulting in a crystallographic R factor of 16.6%. The enzyme comprises a single domain that belongs to the general class of doubly-wound alpha/beta proteins; it also exhibits a unique nucleoside-binding motif. X-ray analysis of enzyme-purine and enzyme-pyrimidine complexes presented here reveals that the active site lies in a cleft formed by the edge of the beta sheet and two alpha helices and contains side chains from two subunits. CONCLUSIONS: These results indicate residues that may be important in substrate binding and catalysis and thus may serve as a framework for elucidating the mechanism of enzyme activity. In particular, the proposed nucleophile, Glu98, lies in the nucleoside-binding pocket at an appropriate position for nucleophilic attack. A comparison of the enzyme interactions with both a purine and pyrimidine ligand provides some insight into the structural basis for enzyme specificity.

Binding Sites↗

PubMed1996

Purification and crystallization of a complex between human interferon gamma receptor (extracellular domain) and human interferon gamma.

X-ray diffraction quality crystals have been obtained from a complex between interferon gamma and the extracellular domain of its high-affinity cell surface receptor. The crystals were obtained from interferon gamma/interferon gamma receptor complexes purified by size exclusion chromatography. Diffraction quality crystals required analyzing these complex samples by isoelectric focusing gels to select purified complex fractions devoid of unbound interferon gamma. These studies used interferon gamma receptor engineered with an eight amino acid N-terminal deletion to eliminate heterogeneity generated due to proteolytic cleavage. In addition, the receptor was expressed in an E. coli secretion cell line which eliminated the need to refold the protein. Hexagonal crystals were grown from 1.6 M ammonium phosphate solutions and belong to a spacegroup of P6(5)22 with unit cell dimensions a = 145.9 A and c = 180.3 A. These crystals diffract to at least 2.9 A resolution when exposed to synchrotron radiation. SDS PAGE analysis of the crystals demonstrated that both interferon gamma and the receptor were present. Analysis of the x-ray diffraction data revealed that the crystals contain complexes with a stoichiometry of 2:1 receptor: ligand within the crystallographic asymmetric unit and consist of approximately 55% solvent.

Chromatography, Gel↗

PubMed1996

Structure and humanization of a rat monoclonal Fab to human interleukin-5.

The X-ray crystal structure of a rat monoclonal Fab JES1-39D10, raised against recombinant human interleukin-5, has been determined with the use of molecular replacement techniques and refined at 2.7 A resolution by simulated annealing. The overall structure is similar to a murine Fab HyHEL-10 that is specific for hen egg white lysozyme. An interesting feature of the structure is the presence of leucine residues to support the H1 complementarity-determining region (CDR) loop. To our knowledge this is the first Fab crystal structure containing this unusual H1 loop support pattern. The activity of three humanized versions of 39D10 is explained by analysis of Fv interface residues and H1 support patterns of 39D10 and the human template HIL.

Amino Acid Sequence↗

PubMed1995

A C-terminal region of the Saccharomyces cerevisiae transcription factor ADR1 plays an important role in the regulation of peroxisome proliferation by fatty acids.

The Saccharomyces cerevisiae transcriptional activator ADR1, which controls ADH2 gene expression, was shown to be involved in the regulation of peroxisome proliferation. To study the mode of action of ADR1, we compared strains carrying the adr1-1 mutation, high or low copy numbers of the ADR1 gene, the constitutive allele ADR1-5c, and 3'-deletions of ADR1. High ADR1 gene dosage increased the transcription of genes encoding peroxisomal proteins as compared to one copy of the ADR1 gene. Furthermore, overexpression of ADR1 under ethanol growth conditions induced the proliferation of peroxisomal structures. The organelles were observed to be localized in clusters, a typical feature of peroxisomes induced by oleic acid. In contrast, the ADR1-5c allele, which induces ADH2 expression to a level comparable to that of high ADR1 gene dosage was found to have only a small effect. An analysis of functional domains of the ADR1 protein revealed that the N-terminal 220 amino acids of ADR1 were sufficient for wild-type levels of transcription of the FOX2, FOX3, and PAS1 genes, but the entire ADR1 protein was required for complete induction of the CTA1 gene and for growth oleic acid medium. Our data suggest that a functional domain of the ADR1 protein localized between residues 643 and 1323 is required for the induction of peroxisomal structures and for the utilization of oleic acid.

ATPases Associated with Diverse Cellular Activitie↗

PubMed1995

The yeast UBC4 ubiquitin conjugating enzyme monoubiquitinates itself in vivo: evidence for an E2-E2 homointeraction.

Here we report that the stress-related conjugating enzyme UBC4 from Saccharomyces cerevisiae is monoubiquintinated in vivo. The UBC4-ubiquitin conjugate was detected by the coexpression in yeast of epitope-tagged ubiquitin in combination with either untagged or epitope-tagged versions of UBC4. Under these conditions the UBC4 conjugate proved to be the most abundant conjugate detected. Using chemical mapping and site-directed mutation, the site of ubiquitination was localized to a single lysine (K144) near the carboxy terminus of UBC4. A second lysine within UBC4 (K64) was also identified whose mutation resulted in the loss of ubiquitination at K144. The mutation of either K64 or K144 had no obvious effect on the known in vivo functions associated with UBC4. In another experiment, a nonfunctional UBC4 derivative with a mutation at the active site was also found to be monoubiquitinated in a manner that depended on the expression of active UBC4. This result indicated that ubiquitin was transferred in an intermolecular reaction from one UBC4 monomer to another. Cross-linking analysis demonstrated that UBC4 monomers directly and specifically interact with one another in vitro. Both the in vivo and in vitro observations reported here, in combination with previous findings, support the view that interactions between ubiquitin conjugating enzymes represent a general phenomenon.

Amino Acid Sequence↗

PubMed1995

Crystallization and preliminary X-ray investigation of recombinant human interleukin 10.

Crystals of recombinant human interleukin 10 have been grown from solutions of ammonium sulfate. The crystals are tetragonal, space group P4(1)2(1)2 or P4(3)2(1)2; the unit cell axes are a = 36.5 A and c = 221.9 A. There is the equivalent of one polypeptide chain in the asymmetric unit. The crystals are stable to X-rays and diffract to at least 2.5 A resolution.

Ammonium Sulfate↗

PubMed1995

Rapid renal deterioration secondary to oxalate in a patient with diabetic gastroenteropathy.

We report the case of a white woman with insulin-dependent diabetes for 12 years who had rapid deterioration in renal function over a 7-month period. A renal biopsy showed widespread deposition of a polarizing crystalline material consistent with calcium oxalate. Fat malabsorption due to diabetic diarrhea was first documented 5 years earlier when renal function was normal. Chronic malabsorption can lead to chronic interstitial nephritis secondary to oxalate deposition, but rarely leads to acute deterioration in renal function. This entity should be considered in individuals with steatorrhea and no other cause for their renal failure.

Adult↗

PubMed1995

Initiator elements and regulated expression of the herpes simplex virus thymidine kinase gene.

Regulated expression of thymidine kinase mRNA in herpes simplex virus mutants harboring thymidine kinase promoters that lacked functional TATA boxes was largely unaffected by additional sequence alterations around the transcriptional start site. A strong initiator element increased the regulated expression of a TATA-containing promoter by 50% but did not affect that of the TATA-less promoter. Thus, initiator elements exert only small effects in this promoter context.

Animals↗

PubMed1995

Induction of transcription by a viral regulatory protein depends on the relative strengths of functional TATA boxes.

The mechanisms by which viral regulatory proteins activate the cellular transcription apparatus without binding to specific DNA elements are not fully understood. Several lines of evidence suggest that activation by one such regulatory protein, herpes simplex virus ICP4, could be mediated, at least in part, by TFIID. To test this model, we replaced the TATA box of the ICP4-responsive viral thymidine kinase gene with functional TATA boxes that displayed different apparent affinities for TATA-box-binding protein as measured by DNase I footprinting. We measured the effects of these TATA boxes on ICP4 induction by constructing ICP4-deficient recombinant viruses containing the different TATA alleles and comparing their expression in cells lacking or expressing ICP4. Overall, ICP4 induced weak TATA boxes (those that displayed low apparent affinity for TATA-box-binding protein and low basal expression) the most (18- to 41-fold) and strong TATA boxes the least (7- to 10-fold). Therefore, ICP4 induction correlated inversely with TATA box strength. Using a reconstituted in vitro transcription assay, we determined that the relative levels of induction by ICP4 of the different TATA alleles were similar to those measured in vivo, suggesting that ICP4 was the only viral protein required for induction. These results fit a model in which ICP4 acts in part to enhance binding of TFIID to the TATA box. We compare and contrast these results with those observed with the viral regulatory proteins adenovirus E1a and simian virus 40 large T antigen and the cellular coactivator PC4.

Base Sequence↗

PubMed1994

Drug binding by calmodulin: crystal structure of a calmodulin-trifluoperazine complex.

The crystal structure of calmodulin (CaM) bound to trifluoperazine (TFP) has been determined and refined to a resolution of 2.45 A. Only one TFP is bound to CaM, but that is sufficient to cause distortion of the central alpha-helix and juxtaposition of the N- and C-terminal domains similar to that seen in CaM-polypeptide complexes. The drug makes extensive contacts with residues in the C-terminal domain of CaM but only a few contacts with one residue in the N-terminal domain. The structure suggests that substrate binding to the C-terminal domain is sufficient to cause the conformational changes in calmodulin that lead to activation of its targets.

Animals↗

PubMed1994

Mutations in the zinc-finger region of the yeast regulatory protein ADR1 affect both DNA binding and transcriptional activation.

The expression of the yeast ADH2 gene is controlled by the transcriptional activator ADR1, a zinc-finger protein that binds to an upstream activating sequence (UAS1) in the ADH2 promoter. We report here the isolation of seven mutations in the ADR1-5c allele, defining five different amino acid changes, that suppress the enhanced ADH2 expression caused by the ADR1-5c allele. Each of the mutations was shown to reduce the activation of ADH2 by a wild-type ADR1 gene, suggesting the mutations disrupt a domain important to the function of both the ADR1 and ADR1-5c proteins. All five amino acid changes occurred within the DNA-binding domain of ADR1 and were shown to severely inhibit the ability of ADR1 to bind UAS1 in vitro. These mutations were found, however, to also affect the ability of ADR1 to activate transcription independent of its ability to bind DNA. These results indicate that the DNA-binding region of ADR1 is involved in both transactivation and DNA binding.

Amino Acid Sequence↗

PubMed1994

Structure of tetraubiquitin shows how multiubiquitin chains can be formed.

Eukaryotic proteins are targeted for degradation by covalent ligation of multiubiquitin chains. In these multiubiquitin chains, successive ubiquitins are linked by an isopeptide bond involving the side chain of Lys48 and the carboxyl group of the C-terminus (Gly76). The crystal structure of a tetraubiquitin chain (Ub4) has been determined and refined at 2.4 A resolution. The molecule exhibits both translational and 2-fold rotational symmetry; each pair of (rotationally symmetric) ubiquitin molecules in Ub4 is related to the next pair by a simple translation. The 2-fold symmetry in each pair of ubiquitin molecules is quite different from the 2-fold symmetry observed in the previously determined structure of isolated diubiquitin. There are multiple hydrophilic contacts among the four ubiquitin molecules, but the hydrophobic surface formed in the middle of diubiquitin is not seen. The structure of the tetraubiquitin chain demonstrates how a multiubiquitin chain of any length can be formed.

Biopolymers↗

PubMed1994

The yeast CCR4 protein is neither regulated by nor associated with the SPT6 and SPT10 proteins and forms a functionally distinct complex from that of the SNF/SWI transcription factors.

The CCR4 protein is specifically required for the increased transcription at the ADH2 locus resulting from mutations in the SPT10 (CRE1) and SPT6 (CRE2) genes and is also required for the expression of ADH2 and other genes under non-fermentative growth conditions. The mechanism by which mutations in CCR4 suppress defects in SPT10 and SPT6 was examined. The SPT10 and SPT6 genes were shown not to control CCR4 mRNA or protein expression nor did SPT10 and SPT6 proteins co-immuneprecipitate with CCR4. CCR4 association with two other proteins, 195 and 185 kDa in size, was unaffected by either spt10 or spt6 mutations. Also, the ability of CCR4 to activate transcription when fused to the LexA DNA binding domain was not specifically enhanced by defects in either SPT10 or SPT6. These results suggest that SPT10 and SPT6, in negatively regulating transcription at ADH2, act through a factor that requires CCR4 function, but do not regulate CCR4 expression, control its activity, physically interact with it, or affect its binding to other factors. The relationship of CCR4 to the group of general transcription factors, SNF2, SNF5, SNF6 and SWI1 and SWI3, which comprise a multisubunit complex required for ADH2 and other genes' expression, was also examined. CCR4 protein expression was not controlled by these factors nor did they co-immuneprecipitate or associate with CCR4. In addition, a ccr4 mutation had little effect on an ADH2 promoter alteration in contrast to the large effects displayed by mutations in SNF2 and SNF5. These data suggest that CCR4 acts by a separate mechanism from that used by the SNF/SWI general transcription factors in affecting gene expression.

Alcohol Dehydrogenase↗