Antonio Pineda-Lucena

Single-stranded DNA mimicry in the p53 transactivation domain interaction with replication protein A

One of many protein–protein interactions modulated upon DNA damage is that of the single-stranded DNA-binding protein, replication protein A (RPA), with the p53 tumor suppressor. Here we report the crystal structure of RPA residues 1–120 (RPA70N) bound to the N-terminal transactivation domain of p53 (residues 37–57; p53N) and, by using NMR spectroscopy, characterize two mechanisms by which the RPA/p53 interaction can be modulated. RPA70N forms an oligonucleotide/oligosaccharide-binding fold, similar to that previously observed for the ssDNA-binding domains of RPA. In contrast, the N-terminal p53 transactivation domain is largely disordered in solution, but residues 37–57 fold into two amphipathic helices, H1 and H2, upon binding with RPA70N. The H2 helix of p53 structurally mimics the binding of ssDNA to the oligonucleotide/oligosaccharide-binding fold. NMR experiments confirmed that both ssDNA and an acidic peptide mimicking a phosphorylated form of RPA32N can independently compete the acidic p53N out of the binding site. Taken together, our data suggest a mechanism for DNA damage signaling that can explain a threshold response to DNA damage.

An NMR approach to structural proteomics

The influx of genomic sequence information has led to the concept of structural proteomics, the determination of protein structures on a genome-wide scale. Here we describe an approach to structural proteomics of small proteins using NMR spectroscopy. Over 500 small proteins from several organisms were cloned, expressed, purified, and evaluated by NMR. Although there was variability among proteomes, overall 20% of these proteins were found to be readily amenable to NMR structure determination. NMR sample preparation was centralized in one facility, and a distributive approach was used for NMR data collection and analysis. Twelve structures are reported here as part of this approach, which allowed us to infer putative functions for several conserved hypothetical proteins.

A Conserved Docking Surface on Calcineurin Mediates Interaction with Substrates and Immunosuppressants

The phosphatase calcineurin, a target of the immunosuppressants cyclosporin A and FK506, dephosphorylates NFAT transcription factors to promote immune activation and development of the vascular and nervous systems. NFAT interacts with calcineurin through distinct binding motifs: the PxIxIT and LxVP sites. Although many calcineurin substrates contain PxIxIT motifs, the generality of LxVP-mediated interactions is unclear. We define critical residues in the LxVP motif, and we demonstrate its binding to a hydrophobic pocket at the interface of the two calcineurin subunits. Mutations in this region disrupt binding of mammalian calcineurin to NFATC1 and the interaction of yeast calcineurin with substrates including Rcn1, which contains an LxVP motif. These mutations also interfere with calcineurin-immunosuppressant binding, and an LxVP-based peptide competes with immunosuppressant-immunophilin complexes for binding to calcineurin. These studies suggest that LxVP-type sites are a common feature of calcineurin substrates, and that immunosuppressant-immunophilin complexes inhibit calcineurin by interfering with this mode of substrate recognition.

Polymer conjugates as therapeutics: future trends, challenges and opportunities

Objective: Clinical proof of concept for polymer conjugates has already been achieved over the last 30 years, with a family of polymer–protein conjugates reaching the market and an exponentially growing list of polymer–drug conjugates currently in clinical trials. However, many challenges and opportunities still lie ahead, providing scope to develop this platform technology further. Methods: The delivery of new anticancer agents aimed at novel molecular targets and their combination, the development of both new polymeric materials with defined architectures and the treatment of diseases other than cancer are the most exciting and promising areas. The latest advances and future trends in the polymer conjugate field will be presented in this article, providing an insight into their potential in the clinics and offering a wide range of research approaches within the scientific community. Results/conclusion: Polymer therapeutics is a rapidly emerging field with exponentially growing opportunities to achieve medical treatments with highly enhanced therapeutic value.

Deciphering Biosynthesis of the RNA Polymerase Inhibitor Streptolydigin and Generation of Glycosylated Derivatives

The biosynthetic gene cluster for the dienoyltetramic acid streptolydigin was identified and characterized from the producer organism Streptomyces lydicus NRRL2433. Sequence analysis of an 80.8 kb DNA region revealed the presence of 38 ORFs, 29 of which are probably involved in streptolydigin biosynthesis and would code for all activities required for its biosynthesis. Six insertional inactivation mutants were generated in the sequenced region to prove its involvement in streptolydigin biosynthesis, to define the boundaries of the cluster, to functionally characterize some genes, and to generate novel derivatives. A model for streptolydigin biosynthesis is proposed that includes a probable domain skipping in the streptolydigin PKS and the participation of a free-standing adenylation domain protein. Some bioactive derivatives of streptolydigin with altered glycosylation pattern have been produced by combinatorial biosynthesis showing a certain degree of flexibility of the L-rhodinosyl transferase SlgG for the recognition of 2,3,6-trideoxyhexoses and 2,6-dideoxyhexoses, both in D- and L-configuration.

Small molecule inhibition of ERK dimerization prevents tumorigenesis by RAS-ERK pathway oncogenes

Nearly 50% of human malignancies exhibit unregulated RAS-ERK signaling; inhibiting it is a valid strategy for antineoplastic intervention. Upon activation, ERK dimerize, which is essential for ERK extranuclear, but not for nuclear, signaling. Here, we describe a small molecule inhibitor for ERK dimerization that, without affecting ERK phosphorylation, forestalls tumorigenesis driven by RAS-ERK pathway oncogenes. This compound is unaffected by resistance mechanisms that hamper classical RAS-ERK pathway inhibitors. Thus, ERK dimerization inhibitors provide the proof of principle for two understudied concepts in cancer therapy: (1) the blockade of sub-localization-specific sub-signals, rather than total signals, as a means of impeding oncogenic RAS-ERK signaling and (2) targeting regulatory protein-protein interactions, rather than catalytic activities, as an approach for producing effective antitumor agents.

Structure of minimal tetratricopeptide repeat domain protein Tah1 reveals mechanism of its interaction with Pih1 and Hsp90.

Background: Tah1 and Pih1 are Hsp90 interactors that form a ternary complex with the chaperone. Results: NMR structure of Tah1 revealed the presence of two tetratricopeptide repeat motifs followed by a C helix and an unstructured region. Conclusion: Tah1 can bind simultaneously two other proteins using different interaction modes. Significance: The study provides important insights into protein complex assembly. Tah1 and Pih1 are novel Hsp90 interactors. Tah1 acts as a cofactor of Hsp90 to stabilize Pih1. In yeast, Hsp90, Tah1, and Pih1 were found to form a complex that is required for ribosomal RNA processing through their effect on box C/D small nucleolar ribonucleoprotein assembly. Tah1 is a minimal tetratricopeptide repeat protein of 111 amino acid residues that binds to the C terminus of the Hsp90 molecular chaperone, whereas Pih1 consists of 344 residues of unknown fold. The NMR structure of Tah1 has been solved, and this structure shows the presence of two tetratricopeptide repeat motifs followed by a C helix and an unstructured region. The binding of Tah1 to Hsp90 is mediated by the EEVD C-terminal residues of Hsp90, which bind to a positively charged channel formed by Tah1. Five highly conserved residues, which form a two-carboxylate clamp that tightly interacts with the ultimate Asp-0 residue of the bound peptide, are also present in Tah1. Tah1 was found to bind to the C terminus of Pih1 through the C helix and the unstructured region. The C terminus of Pih1 destabilizes the protein in vitro and in vivo, whereas the binding of Tah1 to Pih1 allows for the formation of a stable complex. Based on our data, a model for an Hsp90-Tah1-Pih1 ternary complex is proposed.

Generation of potent and selective kinase inhibitors by combinatorial biosynthesis of glycosylated indolocarbazoles.

We report the generation of novel glycosylated indolocarbazoles by combinatorial biosynthesis, and the identification of two novel potent and selective compounds inhibitors of JAK2 and Ikkb kinases.

Contributions of computational chemistry and biophysical techniques to fragment-based drug discovery.

In the last decade, fragment-based drug discovery (FBDD) has evolved from a novel approach in the search of new hits to a valuable alternative to the high-throughput screening (HTS) campaigns of many pharmaceutical companies. The increasing relevance of FBDD in the drug discovery universe has been concomitant with an implementation of the biophysical techniques used for the detection of weak inhibitors, e.g. NMR, X-ray crystallography or surface plasmon resonance (SPR). At the same time, computational approaches have also been progressively incorporated into the FBDD process and nowadays several computational tools are available. These stretch from the filtering of huge chemical databases in order to build fragment-focused libraries comprising compounds with adequate physicochemical properties, to more evolved models based on different in silico methods such as docking, pharmacophore modelling, QSAR and virtual screening. In this paper we will review the parallel evolution and complementarities of biophysical techniques and computational methods, providing some representative examples of drug discovery success stories by using FBDD.

ABACUS, a Direct Method for Protein NMR Structure Computation via Assembly of Fragments

The ABACUS algorithm obtains the protein NMR structure from unassigned NOESY distance restraints. ABACUS works as an integrated approach that uses the complete set of available NMR experimental information in parallel and yields spin system typing, NOE spin pair identities, sequence specific resonance assignments, and protein structure, all at once. The protocol starts from unassigned molecular fragments (including single amino acid spin systems) derived from triple‐resonance 1H/13C/15N NMR experiments. Identifications of connected spin systems and NOEs precede the full sequence specific resonance assignments. The latter are obtained iteratively via Monte Carlo‐Metropolis and/or probabilistic sequence selections, molecular dynamics structure computation and BACUS filtering ( A. Grishaev and M. Llinás, J Biomol NMR 2004;28:1–10 ). ABACUS starts from scratch, without the requirement of an initial approximate structure, and improves iteratively the NOE identities in a self‐consistent fashion. The procedure was run as a blind test on data recorded on mth1743, a 70‐amino acid genomic protein from M. thermoautotrophicum. It converges to a structure in ca. 15 cycles of computation on a 3‐GHz processor PC. The calculated structures are very similar to the ones obtained via conventional methods (1.22 Å backbone RMSD). The success of ABACUS on mth1743 further validates BACUS as a NOESY identification protocol. Proteins 2005.