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Biomedical subjects

Z Weng

Publications and source records attributed to Z Weng.

At least 19 recordsLinked to original sources
PubMed2000

Protein structure alignment using a genetic algorithm.

We have developed a novel, fully automatic method for aligning the three-dimensional structures of two proteins. The basic approach is to first align the proteins' secondary structure elements and then extend the alignment to include any equivalent residues found in loops or turns. The initial secondary structure element alignment is determined by a genetic algorithm. After refinement of the secondary structure element alignment, the protein backbones are superposed and a search is performed to identify any additional equivalent residues in a convergent process. Alignments are evaluated using intramolecular distance matrices. Alignments can be performed with or without sequential connectivity constraints. We have applied the method to proteins from several well-studied families: globins, immunoglobulins, serine proteases, dihydrofolate reductases, and DNA methyltransferases. Agreement with manually curated alignments is excellent. A web-based server and additional supporting information are available at http://engpub1.bu.edu/-josephs.

Algorithms↗
PubMed1999

Human transaldolase and cross-reactive viral epitopes identified by autoantibodies of multiple sclerosis patients.

Multiple sclerosis is mediated by an autoimmune process causing selective destruction of oligodendrocytes. Transaldolase, which is expressed in the brain selectively in oligodendrocytes, is a target of high affinity autoantibodies in serum and cerebrospinal fluid of multiple sclerosis patients. A three-dimensional model of human transaldolase was developed based on the crystal structure of the enzyme from Escherichia coli. To identify immunodominant epitopes, 33 peptides overlapping human transaldolase by 5 amino acids were synthesized. Ab 12484, raised against enzymatically active human transaldolase, recognized antigenic determinants corresponding to linear epitopes (residues 27-31 and 265-290) and alpha helices (residues 75-98 and 302-329). Four immunodominant peptides harboring charged amino acid residues with topographically exposed side chains were identified by sera from 13 multiple sclerosis patients with predetermined autoreactivity to transaldolase. Autoantibodies binding to the most prominent human transaldolase epitope, between residues 271 and 285, showed cross-reactivity with Epstein-Barr and herpes simplex virus type 1 capsid-derived peptides. Molecular mimicry between immunodominant autoepitopes and viral Ags may be a decisive factor in directing autoimmunity to transaldolase in multiple sclerosis patients.

Amino Acid Sequence↗
PubMed1999

Cytotoxic T cell recognition of allelic variants of HLA B35 bound to an Epstein-Barr virus epitope: influence of peptide conformation and TCR-peptide interaction.

Fine specificity analysis of HLA B35-restricted Epstein-Barr virus (EBV)-specific cytotoxic T lymphocyte (CTL) clones revealed a unique heterogeneity whereby one group of these clones cross-recognized an EBV epitope (YPLHEQHGM) on virus-infected cells expressing either HLA B*3501 or HLA B*3503, while another group cross-recognized this epitope in association with either HLA B*3502 or HLA B*3503. Peptide binding and titration studies ruled out the possibility that these differences were due to variation in the efficiency of peptide presentation by the HLA B35 alleles. Sequence analysis of the TCR genetic elements showed that these clonotypes either expressed BV12/AV3 or BV14/ADV17S1 heterodimers. Interestingly, CTL analysis with monosubstituted alanine mutants of the YPLHEQHGM epitope indicated that the BV12/AV3+ clones preferentially recognized residues towards the C terminus of the peptide, while the BV14/ADV17S1+ clones interacted with residues towards N terminus of the peptide. Molecular modelling of the MHC-peptide complexes suggests that the differences in two floor positions (114 and 116) of the HLA B35 alleles dictate different conformations of the peptide residues L3 and/or H7 and directly contribute in the discerning allele-specific immune recognition by the CTL clonotypes. These results provide evidence for a critical role for the selective interaction of the TCR with specific residues within the peptide epitope in the fine specificity of CTL recognition of allelic variants of an HLA molecule.

Alanine↗
PubMed1999

Free energy landscapes of encounter complexes in protein-protein association.

We report the computer generation of a high-density map of the thermodynamic properties of the diffusion-accessible encounter conformations of four receptor-ligand protein pairs, and use it to study the electrostatic and desolvation components of the free energy of association. Encounter complex conformations are generated by sampling the translational/rotational space of the ligand around the receptor, both at 5-A and zero surface-to-surface separations. We find that partial desolvation is always an important effect, and it becomes dominant for complexes in which one of the reactants is neutral or weakly charged. The interaction provides a slowly varying attractive force over a small but significant region of the molecular surface. In complexes with no strong charge complementarity this region surrounds the binding site, and the orientation of the ligand in the encounter conformation with the lowest desolvation free energy is similar to the one observed in the fully formed complex. Complexes with strong opposite charges exhibit two types of behavior. In the first group, represented by barnase/barstar, electrostatics exerts strong orientational steering toward the binding site, and desolvation provides some added adhesion within the local region of low electrostatic energy. In the second group, represented by the complex of kallikrein and pancreatic trypsin inhibitor, the overall stability results from the rather nonspecific electrostatic attraction, whereas the affinity toward the binding region is determined by desolvation interactions.

Animals↗
PubMed1999

Human cochlear expressed sequence tags provide insight into cochlear gene expression and identify candidate genes for deafness.

To identify candidate genes for human hearing disorders and to understand better human hearing at the molecular level, we constructed a human cochlear cDNA library. An aliquot of the unsubtracted cochlear library was contributed to the IMAGE Consortium at Lawrence Livermore National Laboratory for the generation of expressed sequence tags (ESTs) by the Merck/WashU EST project. Over 4000 ESTs were developed from the cochlear cDNA library and deposited in the GenBank EST database. Sequence clustering shows that the majority of clones are in low copy numbers, demonstrating the high complexity of the library. The sequences of 1388 cochlear ESTs (33%) match 517 known human genes. Among these are genes previously shown to cause both syndromic and non-syndromic hearing loss. A number of the cochlear ESTs show high homology to non-human genes, suggesting new gene family members or human homologs of animal genes. We also report the chromosomal map positions of 437 cochlear ESTs. These provide positional candidate genes for 18 different non-syndromic hearing disorders. A Human Cochlear EST Database web site (http://www.bwh.partners. org/pathology ) has been created to provide access to the cochlear clone data for gene discovery investigations.

Animals↗
PubMed1998

A DNA damage and stress inducible G protein-coupled receptor blocks cells in G2/M.

Cell cycle progression is monitored by highly coordinated checkpoint machinery, which is activated to induce cell cycle arrest until defects like DNA damage are corrected. We have isolated an anti-proliferative cell cycle regulator named G2A (for G2 accumulation), which is predominantly expressed in immature T and B lymphocyte progenitors and is a member of the seven membrane-spanning G protein-coupled receptor family. G2A overexpression attenuates the transformation potential of BCR-ABL and other oncogenes, and leads to accumulation of cells at G2/M independently of p53 and c-Abl. G2A can be induced in lymphocytes and to a lesser extent in nonlymphocyte cell lines or tissues by multiple stimuli including different classes of DNA-damaging agents and serves as a response to damage and cellular stimulation which functions to slow cell cycle progression.

3T3 Cells↗
PubMed1998

Computational determination of the structure of rat Fc bound to the neonatal Fc receptor.

The available crystal structure for the complex between the Fc fragment of immunoglobulin G (IgG) and the neonatal Fc receptor (FcRn) was determined at low resolution and has no electron density for a large portion of the CH2 domain of the Fc. Here, we use a well validated computational docking algorithm in conjunction with known crystallographic data to predict the orientation of CH2 when bound to FcRn, and validate the predicted structure with data from site-specific mutagenesis experiments. The predicted Fc structure indicates that the CH2 domain moves upon binding FcRn , such that the end-to-end distance of the bound Fc fragment is greater than it is in the crystal structure of isolated Fc. The calculated orientation of the bound CH2 domain is displaced by an average of 6 A from the CH2 orientation in the structure of Fc alone, and shows improved charge complementarity with FcRn. The predicted effects of 11 specific mutations in Fc and FcRn are calculated and the results are compared with experimental measurements. The predicted structure is consistent with all reported mutagenesis data, some of which are explicable only on the basis of our model. The current study predicts that FcRn-bound Fc is asymmetric due to reorientation of the CH2 domain upon FcRn binding, a rearrangement that would be likely to interfere with optimal binding of FcRn at the second binding site of the Fc homodimer.

Animals↗
PubMed1998

Genetic polymorphism in MDR-1: a tool for examining allelic expression in normal cells, unselected and drug-selected cell lines, and human tumors.

By using RNase protection analysis, residues 2677 and 2995 of MDR-1 were identified as sites of genetic polymorphism. Through use of oligonucleotide hybridization, the genomic content and expression of individual MDR-1 alleles were examined in normal tissues, unselected and drug selected cell lines, and malignant lymphomas. In normal tissues, unselected cell lines, and untreated malignant lymphoma samples, expression of MDR-1 from both alleles was similar. In contrast, in drug selected cell lines, and in relapsed malignant lymphoma samples, expression of one allele was found in a large percentage of samples. To understand how expression of one allele occurs, two multidrug resistant sublines were isolated by exposing a Burkitt lymphoma cell line to increasing concentrations of vincristine. The resistant sublines expressed only one allele and had a hybrid MDR-1 gene composed of non-MDR-1 sequences proximal to MDR-1. Previous studies showing hybrid MDR-1 genes after rearrangements provided a potential explanation for activation and expression of one MDR-1 allele. We conclude that oligonucleotide hybridization can be used as a sensitive tool to examine relative allelic expression of MDR-1, and can identify abnormal expression from a single allele. Acquired drug resistance in vitro and in patients is often associated with expression of a single MDR-1 allele, and this can be a marker of a hybrid MDR-1 gene.

Alleles↗
PubMed1998

Toward a predictive understanding of molecular recognition.

T cells circulate in blood and the lymphatic system, continually engaging cells through transient non-specific adhesion. In a normally functioning immune system, these interactions permit sufficient time for T-cell receptors (TCRs) to sample major histocompatibility complex (MHC)-peptide complexes for the presence of foreign antigen, with detection of the latter to some extent being triggered by a longer dwell time of the receptor on the complex. Precisely how this incremental stability, which may be relatively small, leads to activation is unclear, but it appears to be related to diffusion-mediated formation of ternary complex dimers. The formation of stable dimers can explain the high sensitivity of the response, but leaves a number of questions unaddressed, including the following: i) How can high sensitivity be reconciled with high specificity, and how can a short TCR dwell time be reconciled with a comparably short time for ternary complex pair formation? ii) What is the nature of the early signals on the plasma membrane that determine alternative responses e.g. proliferation at one extreme and apoptosis at the other? iii) What are the cell-surface correlates of biphasic dose response functions i.e. of responses that peak as a function of dose and then descend? This paper has two loosely coupled goals. One is to review and assess the mathematical and computational methods available for analyzing reactions with and between mobile membrane-bound receptors. These methods range from phenomenological to mechanistic, the latter being based on the details of atomic structure. The other is to apply these methods to address biological questions, such as those raised above, part of whose answer may lie in the kinetic competition between alternative reaction paths.

Animals↗
PubMed1997

Characterization of microbial cellulose from a high-producing mutagenized Acetobacter pasteurianus strain.

A wild-type Acetobacter pasteurianus was subjected to chemical mutagenesis for the induction and isolation of a cellulose overproducing strain. A mutagenized strain capable of synthesizing double amounts of cellulose compared to the wild type was obtained. Cellulose, both from the wild-type and the mutagenized strain, was extracted and purified for chemical characterization and investigation of its physico-chemical properties. The comparison of the two microbial polysaccharides shows that the putative mutation of A. pasteurianus strain had no effect on some cellulose features such as chemical structure, polymorphic form, crystallinity.

Acetobacter↗
PubMed1997

Empirical free energy calculation: comparison to calorimetric data.

An effective free energy potential, developed originally for binding free energy calculation, is compared to calorimetric data on protein unfolding, described by a linear combination of changes in polar and nonpolar surface areas. The potential consists of a molecular mechanics energy term calculated for a reference medium (vapor or nonpolar liquid), and empirical terms representing solvation and entropic effects. It is shown that, under suitable conditions, the free energy function agrees well with the calorimetric expression. An additional result of the comparison is an independent estimate of the side-chain entropy loss, which is shown to agree with a structure-based entropy scale. These findings confirm that simple functions can be used to estimate the free energy change in complex systems, and that a binding free energy evaluation model can describe the thermodynamics of protein unfolding correctly. Furthermore, it is shown that folding and binding leave the sum of solute-solute and solute-solvent van der Waals interactions nearly invariant and, due to this invariance, it may be advantageous to use a nonpolar liquid rather than vacuum as the reference medium.

Calorimetry↗
PubMed1997

Genome organization and gene expression of saguaro cactus carmovirus.

The complete sequence of the single-stranded, (+)-sense RNA genome of saguaro cactus carmovirus (SCV) has been determined. The 3879 nucleotide genome contains five open reading frames (ORFs). The 5'-proximal ORF encodes a 26 kDa protein (p26) and terminates with an amber codon which is readthrough into an in-frame p57 ORF to generate an 86 kDa fusion protein (p86). Two small, centrally located ORFs encode a 6 kDa protein (p6) and a 9 kDa protein (p9), respectively. The 3'-proximal ORF encodes a 37 kDa (p37) capsid protein (CP). Analysis of the nucleotide and predicted amino acid sequences supports the classification of SCV in the genus Carmovirus in the family Tombusviridae. All predicted SCV proteins are expressed in an in vitro translation system. SCV p26 and the readthrough fusion protein p86 are synthesized from the genomic RNA while p6, p9 and p37 CP ORFs at the 3' half of the genome are expressed from two subgenomic (sg) RNAs. The 5' termini of both sg RNAs have been mapped. The large 1614 nucleotide sg RNA contains the p6 and p9 ORFs as the first and the second ORFs respectively from its 5' end. It directs the synthesis of abundant p6 but a small amount of p9. While a synthetic transcript with the p9 ORF at the 5' end is a more efficient messenger for p9, no corresponding sg RNA has been identified in vivo. The smaller 1396 nucleotide sg RNA contains only the p37 ORF and directs the synthesis of SCV CP.

Base Sequence↗
PubMed1996

Prediction of protein complexes using empirical free energy functions.

A long sought goal in the physical chemistry of macromolecular structure, and one directly relevant to understanding the molecular basis of biological recognition, is predicting the geometry of bimolecular complexes from the geometries of their free monomers. Even when the monomers remain relatively unchanged by complex formation, prediction has been difficult because the free energies of alternative conformations of the complex have been difficult to evaluate quickly and accurately. This has forced the use of incomplete target functions, which typically do no better than to provide tens of possible complexes with no way of choosing between them. Here we present a general framework for empirical free energy evaluation and report calculations, based on a relatively complete and easily executable free energy function, that indicate that the structures of complexes can be predicted accurately from the structures of monomers, including close sequence homologues. The calculations also suggest that the binding free energies themselves may be predicted with reasonable accuracy. The method is compared to an alternative formulation that has also been applied recently to the same data set. Both approaches promise to open new opportunities in macromolecular design and specificity modification.

Crystallography, X-Ray↗
PubMed1995

Extracting hydrophobicity parameters from solute partition and protein mutation/unfolding experiments.

Hydrophobicity values for amino acids obtained from protein unfolding experiments are about twice as large as those obtained from data on the partitioning of amino acids between water and octanol. Quantitative analyses of several data sets, presented here, indicate that the difference is best explained by the most direct hypothesis, i.e. that the environment of hydrophobic groups in the interior of a protein is poorly modeled by octanol. Instead, we propose--and provide supporting evidence--that hydrocarbons are a more suitable model. First, we reanalyze data from both solute partitioning and protein unfolding experiments, taking account of the effects that were omitted previously, by introducing a volume dependence in the former and a full free energy analysis in the latter. Both changes in evaluation methodology decrease the discrepancy, but the differences remain substantial. The hydrophobicity parameter obtained from side-chain transfers between octanol and water increases from 16.7 to 22 cal/mol/Angstrom2, while that obtained from protein unfolding decreases from 34.9 to 31.2 cal/mol/Angstrom2. On the other hand, our analysis of the solubilities of pure hydrocarbons in water provides a hydrophobicity parameter of 30.8 cal/mol/Angstrom2. This apparent hydrocarbon-like environment of a protein's interior is also suggested more directly by an analysis of the contact environment of hydrophobic side chains in mutation/unfolding experiments, which have polar contact areas that are <2% of the total.

Amino Acids↗
PubMed1995

A method for accurate determination of terminal sequences of viral genomic RNA.

A combination of ligation-anchored PCR and anchored cDNA cloning techniques were used to clone the termini of the saguaro cactus virus (SCV) RNA genome. The terminal sequences of the viral genome were subsequently determined from the clones. The 5' terminus was cloned by ligation-anchored PCR, whereas the 3' terminus was obtained by a technique we term anchored cDNA cloning. In anchored cDNA cloning, an anchor oligonucleotide was prepared by phosphorylation at the 5' end, followed by addition of a dideoxynucleotide at the 3' end to block the free hydroxyl group. The 5' end of the anchor was subsequently ligated to the 3' end of SCV RNA. The anchor-ligated, chimerical viral RNA was then reverse-transcribed into cDNA using a primer complementary to the anchor. The cDNA containing the complete 3'-terminal sequence was converted into ds-cDNA, cloned, and sequenced. Two restriction sites, one within the viral sequence and one within the primer sequence, were used to facilitate cloning. The combination of these techniques proved to be an easy and accurate way to determine the terminal sequences of SCV RNA genome and should be applicable to any other RNA molecules with unknown terminal sequences.

Base Sequence↗
PubMed1995

Structure-function analysis of SH3 domains: SH3 binding specificity altered by single amino acid substitutions.

SH3 domains mediate intracellular protein-protein interactions through the recognition of proline-rich sequence motifs on cellular proteins. Structural analysis of the Src SH3 domain (Src SH3) complexed with proline-rich peptide ligands revealed three binding sites involved in this interaction: two hydrophobic interactions (between aliphatic proline dipeptides in the SH3 ligand and highly conserved aromatic residues on the surface of the SH3 domain), and one salt bridge (between Asp-99 of Src and an Arg three residues upstream of the conserved Pro-X-X-Pro motif in the ligand). We examined the importance of the arginine binding site of SH3 domains by comparing the binding properties of wild-type Src SH3 and Abl SH3 with those of a Src SH3 mutant containing a mutated arginine binding site (D99N) and Abl SH3 mutant constructs engineered to contain an arginine binding site (T98D and T98D/F91Y). We found that the D99N mutation diminished binding to most Src SH3-binding proteins in whole cell extracts; however, there was only a moderate reduction in binding to a small subset of Src SH3-binding proteins (including the Src substrate p68). p68 was shown to contain two Arg-containing Asp-99-dependent binding sites and one Asp-99-independent binding site which lacks an Arg. Moreover, substitution of Asp for Thr-98 in Abl SH3 changed the binding specificity of this domain and conferred the ability to recognize Arg-containing ligands. These results indicate that Asp-99 is important for Src SH3 binding specificity and that Asp-99-dependent binding interactions play a dominant role in Src SH3 recognition of cellular binding proteins, and they suggest the existence of two Src SH3 binding mechanisms, one requiring Asp-99 and the other independent of this residue.

3T3 Cells↗
PubMed1995

Origins of Indo-Europeans and the spread of agriculture in Europe: comparison of lexicostatistical and genetic evidence.

A series of tests was undertaken to relate lexicostatistical dissimilarities (LAN) among 48 Indo-European languages to distances representing various causal hypotheses. The comparison is limited to languages currently spoken in Europe. The putative causal distance matrices include (1) geographic (GEO) distances between the languages, (2) distances representing the origin of agriculture (OOA), (3) distances representing a model postulated by C. Renfrew (REN) concerning transformations that gave rise to the major Indo-European language families in Europe, and (4) distances representing a competing hypothesis by M. Gimbutas (GIM) concerning the origin and spread of Indo-European languages in Europe. Pairwise Mantel tests of the matrices show that OOA correlates better with LAN than does REN, supporting Renfrew's basic hypothesis of the dispersal of the Indo-European languages with the spread of agriculture but showing less effect for his postulated transformations. Partial correlation of LAN with OOA when GEO is held constant is significant at p = 0.004, whereas REN is no longer correlated with LAN when GEO is held constant. When repeated for only seven languages chosen to represent the seven major families of Indo-European languages currently spoken in Europe, the results differed appreciably, yielding a negative, albeit nonsignificant, partial correlation between OOA and LAN when GEO is held constant. This apparent contradiction led us to develop some new statistical approaches to examine, confirm, and explain the patterns. Decomposing the Mantel correlation coefficients for the 48 Indo-European languages into several additive correlation components showed that much of the positive component of the correlation coefficient was contributed by LAN, OOA correlation within language families, particularly within the Germanic family, covering up the negative contributions between language families. The differentiation of the seven major Indo-European language branches in Europe seems unrelated to the times of the origin of agriculture. This finding fails to support the fundamental assumption of Renfrew's hypothesis. There are also no significant correlations between LAN and REN or GIM. A series of Monte Carlo experiments confirmed these findings. Consideration of the accumulated evidence from genetics supports the model of demic diffusion during the origin of agriculture. However, published genetic studies and the present study lend no support to the notion that the early farmers were indeed the Indo-Europeans.

Agriculture↗