Angel R. Ortiz

The novel Cer-like protein Caronte mediates the establishment of embryonic left-right asymmetry.

In the chick embryo, left–right asymmetric patterns of gene expression in the lateral plate mesoderm are initiated by signals located in and around Hensens node. Here we show that Caronte (Car), a secreted protein encoded by a member of the Cerberus/Dan gene family, mediates the Sonic hedgehog (Shh)-dependent induction of left-specific genes in the lateral plate mesoderm. Car is induced by Shh and repressed by fibroblast growth factor-8 (FGF-8). Car activates the expression of Nodal by antagonizing a repressive activity of bone morphogenic proteins (BMPs). Our results define a complex network of antagonistic molecular interactions between Activin, FGF-8, Lefty-1, Nodal, BMPs and Car that cooperate to control left–right asymmetry in the chick embryo.

Gene Discovery in Bladder Cancer Progression using cDNA Microarrays

To identify gene expression changes along progression of bladder cancer, we compared the expression profiles of early-stage and advanced bladder tumors using cDNA microarrays containing 17,842 known genes and expressed sequence tags. The application of bootstrapping techniques to hierarchical clustering segregated early-stage and invasive transitional carcinomas into two main clusters. Multidimensional analysis confirmed these clusters and more importantly, it separated carcinoma in situ from papillary superficial lesions and subgroups within early-stage and invasive tumors displaying different overall survival. Additionally, it recognized early-stage tumors showing gene profiles similar to invasive disease. Different techniques including standard t-test, single-gene logistic regression, and support vector machine algorithms were applied to identify relevant genes involved in bladder cancer progression. Cytokeratin 20, neuropilin-2, p21, and p33ING1 were selected among the top ranked molecular targets differentially expressed and validated by immunohistochemistry using tissue microarrays (n = 173). Their expression patterns were significantly associated with pathological stage, tumor grade, and altered retinoblastoma (RB) expression. Moreover, p33ING1 expression levels were significantly associated with overall survival. Analysis of the annotation of the most significant genes revealed the relevance of critical genes and pathways during bladder cancer progression, including the overexpression of oncogenic genes such as DEK in superficial tumors or immune response genes such as Cd86 antigen in invasive disease. Gene profiling successfully classified bladder tumors based on their progression and clinical outcome. The present study has identified molecular biomarkers of potential clinical significance and critical molecular targets associated with bladder cancer progression.

Ab initio folding of proteins using restraints derived from evolutionary information.

We present our predictions in the ab initio structure prediction category of CASP3. Eleven targets were folded, using a method based on a Monte Carlo search driven by secondary and tertiary restraints derived from multiple sequence alignments. Our results can be qualitatively summarized as follows: The global fold can be considered “correct” for targets 65 and 74, “almost correct” for targets 64, 75, and 77, “half‐correct” for target 79, and “wrong” for targets 52, 56, 59, and 63. Target 72 has not yet been solved experimentally. On average, for small helical and alpha/beta proteins (on the order of 110 residues or smaller), the method predicted low resolution structures with a reasonably good prediction of the global topology. Most encouraging is that in some situations, such as with target 75 and, particularly, target 77, the method can predict a substantial portion of a rare or even a novel fold. However, the current method still fails on some beta proteins, proteins over the 110‐residue threshold, and sequences in which only a poor multiple sequence alignment can be built. On the other hand, for small proteins, the method gives results of quality at least similar to that of threading, with the advantage of not being restricted to known folds in the protein database. Overall, these results indicate that some progress has been made on the ab initio protein folding problem. Detailed information about our results can be obtained by connecting to http://www.bioinformatics.danforthcenter.org/CASP3. Proteins Suppl 1999;3:177–185.

Derivation of protein-specific pair potentials based on weak sequence fragment similarity.

A method is presented for the derivation of knowledge‐based pair potentials that corrects for the various compositions of different proteins. The resulting statistical pair potential is more specific than that derived from previous approaches as assessed by gapless threading results. Additionally, a methodology is presented that interpolates between statistical potentials when no homologous examples to the protein of interest are in the structural database used to derive the potential, to a Go‐likepotential (in which native interactions are favorable and all nonnative interactions are not) when homologous proteins are present. For cases in which no protein exceeds 30% sequence identity, pairs of weakly homologous interacting fragments are employed to enhance the specificity of the potential. In gapless threading, the mean z score increases from −10.4 for the best statistical pair potential to −12.8 when the local sequence similarity, fragment‐based pair potentials are used. Examination of the ab initio structure prediction of four representative globular proteins consistently reveals a qualitative improvement in the yield of structures in the 4 to 6 Å rmsd from native range when the fragment‐based pair potential is used relative to that when the quasichemical pair potential is employed. This suggests that such protein‐specific potentials provide a significant advantage relative to generic quasichemical potentials. Proteins 2000;38:3–16. ©2000 Wiley‐Liss, Inc.

Cooperativity Between T Cell Receptor Complexes Revealed by Conformational Mutants of CD3ɛ

A mutant CD3ɛ subunit that cannot change conformation in response to ligand binding exerts dominant-negative inhibition of T cell responses. Must Change Shape The T cell antigen receptor (TCR) complex consists of the TCR αβ subunits noncovalently associated with the δ, ɛ, γ, and ζ subunits of CD3. Engagement of the TCR by peptide bound to the major histocompatibility complex (MHC) on the surface of an antigen-presenting cell triggers the phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) in the CD3 subunits, and these motifs recruit the proteins that mediate TCR signaling. With a molecular dynamics model, Martínez-Martín et al. investigated how extracellular binding of antigen to the TCR is transduced to the inside of the cell. They identified two residues in CD3ɛ that were critical for the transmission of ligand-induced conformational changes to the intracellular portions of this subunit. CD3ɛ subunits with mutations in either of these residues blocked transmission of the conformational change, and one of these variants functioned as a dominant-negative inhibitor of TCR signaling and T cell activation even when present at very low abundance. The authors propose that the initial interaction of an antigen with a TCR may influence the conformation of oligomeric TCR complexes so that TCRs act cooperatively to transmit signals from peptide-MHC. The CD3ɛ subunit of the T cell receptor (TCR) complex undergoes a conformational change upon ligand binding that is thought to be important for the activation of T cells. To study this process, we built a molecular dynamics model of the transmission of the conformational change within the ectodomains of CD3. The model showed that the CD3 dimers underwent a stiffening effect that was funneled to the base of the CD3ɛ subunit. Mutation of two relevant amino acid residues blocked transmission of the conformational change and the differentiation and activation of T cells. Furthermore, this inhibition occurred even in the presence of excess endogenous CD3ɛ subunits. These results emphasize the importance of the conformational change in CD3ɛ for the activation of T cells and suggest the existence of unforeseen cooperativity between TCR complexes.

1H and 15N NMR assignment and solution structure of the SH3 domain of spectrin: comparison of unrefined and refined structure sets with the crystal structure.

The assignment of the 1H and 15Nnuclear magnetic resonance spectra of the Src-homology region 3 domain ofchicken brain α-spectrin has been obtained. A set of solutionstructures has been determined from distance and dihedral angle restraints,which provide a reasonable representation of the protein structure insolution, as evaluated by a principal component analysis of the globalpairwise root-mean-square deviation (rmsd) in a large set of structuresconsisting of the refined and unrefined solution structures and the crystalstructure. The solution structure is well defined, with a lower degree ofconvergence between the structures in the loop regions than in the secondarystructure elements. The average pairwise rmsd between the 15 refinedsolution structures is 0.71 ± 0.13 Å for the backbone atoms and1.43 ± 0.14 Å for all heavy atoms. The solution structure isbasically the same as the crystal structure. The average rmsd between the 15refined solution structures and the crystal structure is 0.76 Å forthe backbone atoms and 1.45 ± 0.09 Å for all heavy atoms. Thereare, however, small differences probably caused by intermolecular contactsin the crystal structure.

Crystal structure of Trbp111: a structure-specific tRNA-binding protein

Trbp111 is a 111 amino acid Aquifex aeolicus structure‐specific tRNA‐binding protein that has homologous counterparts distributed throughout evolution. A dimer is the functional unit for binding a single tRNA. Here we report the 3D structures of the A.aeolicus protein and its Escherichia coli homolog at resolutions of 2.50 and 1.87 Å, respectively. The structure shows a symmetrical dimer of two core domains and a central dimerization domain where the N‐ and C‐terminal regions of Trbp111 form an extensive dimer interface. The core of the monomer is a classical oligonucleotide/oligosaccharide‐binding (OB) fold with a five‐stranded β‐barrel and a small capping helix. This structure is similar to that seen in the anticodon‐binding domain of three class II tRNA synthetases and several other proteins. Mutational analysis identified sites important for interactions with tRNA. These residues line the inner surfaces of two clefts formed between the β‐barrel of each monomer and the dimer interface. The results are consistent with a proposed model for asymmetrical docking of the convex side of tRNA to the dimer.

Role of a nonnative interaction in the folding of the protein G B1 domain as inferred from the conformational analysis of the α-helix fragment

BACKGROUND The role of local interactions in protein folding and stability can be investigated by the conformational analysis of protein fragments. The hydrophobic staple and Schellman motifs have been described at the N and C terminus, respectively, of protein alpha-helices. These motifs are characterized by an interaction between two hydrophobic residues, one outside the helix and one within the helix, and their importance for helix stability has been analyzed in model peptides. In the alpha-helix of the protein G B1 domain, only the Schellman motif is formed--the hydrophobic staple motif is absent despite the favourable sequence pattern. We have experimentally analyzed the solution conformation of the 19-41 fragment of protein G. This peptide comprises the helical residues and contains both the hydrophobic staple and Schellman motif sequences. RESULTS In the isolated peptide in water, the hydrophobic staple motif is formed and stabilizes the helical structure as compared with a shorter peptide lacking it, but the Schellman motif is not formed. In 30% aqueous TFE, the helix is more stable than in pure water and both motifs are formed. CONCLUSIONS The results suggest that the importance of each motif for the folding and stability of protein G is different. The nonnative hydrophobic staple interaction can help to nucleate the helix at the beginning of folding but has later to be disrupted. The Schellman motif, while not providing enough energy for substantial helix stabilization in the unfolded state, could be important for determining the local fold of the sequence in the context of the rest of the protein.

Computational Approaches to Model Ligand Selectivity in Drug Design

To be effective, a designed drug must discriminate successfully the macromolecular target from alternative structures present in the organism. The last few years have witnessed the emergence of different computational tools aimed to the understanding and modeling of this process at molecular level. Although still rudimentary, these methods are shaping a coherent approach to help in the design of molecules with high affinity and specificity, both in lead discovery and in lead optimization. It is the purpose of this review to illustrate the array of computational tools available to consider selectivity in the design process, to summarize the most relevant applications, and to sketch the challenges ahead.

An aminoacyl tRNA synthetase whose sequence fits into neither of the two known classes.

Aminoacyl transfer RNA synthetases catalyse the first step of protein synthesis and establish the rules of the genetic code through the aminoacylation of tRNAs. There is a distinct synthetase for each of the 20 amino acids and throughout evolution these enzymes have been divided into two classes of ten enzymes each. These classes are defined by the distinct architectures of their active sites, which are associated with specific and universal sequence motifs. Because the synthesis of aminoacyl-tRNAs containing each of the twenty amino acids is a universally conserved, essential reaction, the absence of a recognizable gene for cysteinyl tRNA synthetase in the genomes of Archae such as Methanococcus jannaschii and Methanobacterium thermoautotrophicum has been difficult to interpret. Here we describe a different cysteinyl-tRNA synthetase from M. jannaschii and Deinococcus radiodurans and its characterization in vitro and in vivo. This protein lacks the characteristic sequence motifs seen in the more than 700 known members of the two canonical classes of tRNA synthetase and may be of ancient origin. The existence of this protein contrasts with proposals that aminoacylation with cysteine in M. jannaschii is an auxiliary function of a canonical prolyl-tRNA synthetase.