The Application of FAST-NMR for the Identification of Novel Drug Discovery Targets

The continued success of genome sequencing projects has resulted in a wealth of information, but 40-50% of identified genes correspond to hypothetical proteins or proteins of unknown function. The Functional Annotation Screening Technology by NMR (FAST-NMR) screen was developed to assign a biological function for these unannotated proteins with a structure solved by the Protein Structure Initiative. FAST-NMR is based on the premise that a biological function can be described by a similarity in binding sites and ligand interactions with proteins of known function. The resulting co-structure and functional assignment may provide a starting point for a drug discovery effort

Using Databases and Computational Techniques to Infer the Function of Novel Proteins

The Human Genome Project and similar efforts have resulted in the identification of an abundance of novel proteins. There is a need to expedite the process of assigning function to novel proteins. Nuclear magnetic resonance (NMR) spectroscopy can be used to infer a general biological function for a protein of unknown function by identifying compounds that preferentially bind the protein and comparing these results against proteins with defined structure and function. The Functional NMR screen generates hundreds of data sets and a manual analysis of these data sets is laborious and time- consuming. It is hypothesized that several sub-tasks of the Functional NMR can be automated successfully using an integrated database and data analysis system. Our database system integrates NMR data collection, processing, analysis, and data archiving into a unified user interface. An NMR spectra comparison algorithm is designed and implemented to compare NMR data in the presence and absence of a protein to ascertain if any compound-protein binding occurred

\u3csup\u3e1\u3c/sup\u3eH, \u3csup\u3e13\u3c/sup\u3eC, and \u3csup\u3e15\u3c/sup\u3eN NMR assignments for the \u3ci\u3eBacillus subtilis\u3c/i\u3e yndB START domain

The steroidogenic acute regulatory-related lipid transfer (START) domain is found in both eukaryotes and prokaryotes, with putative functions including signal transduction, transcriptional regulation, GTPase activation and thioester hydrolysis. Here we report the near complete 1H, 15N and 13C backbone and side chain NMR resonance assignments for the Bacillus subtilis START domain protein yndB

Multiple Novel Signals Mediate Thyroid Hormone Receptor Nuclear Import and Export

Thyroid hormone receptor (TR) is a member of the nuclear receptor superfamily that shuttles between the cytosol and nucleus. The fine balance between nuclear import and export of TR has emerged as a critical control point for modulating thyroid hormone-responsive gene expression; however, sequence motifs of TR that mediate shuttling are not fully defined. Here, we characterized multiple signals that direct TR shuttling. Along with the known nuclear localization signal in the hinge domain, we identified a novel nuclear localization signal in the A/B domain of thyroid hormone receptor a1 that is absent in thyroid hormone receptor B1 and inactive in the oncoprotein v-ErbA. Our prior studies showed that thyroid hormone receptor a1 exits the nucleus through two pathways, one dependent on the export factor CRM1 and the other CRM1-independent. Here, we identified three novel CRM1-independent nuclear export signal (NES) motifs in the ligand-binding domain as follows: a highly conserved NES in helix 12 (NES-H12) and two additional NES sequences spanning helix 3 and helix 6, respectively. Mutations predicted to disrupt the a-helical structure resulted in a significant decrease in NES-H12 activity. The high degree of conservation of helix 12 suggests that this region may function as a key NES in other nuclear receptors. Furthermore, our mutagenesis studies on NES-H12 suggest that altered shuttling of thyroid hormone receptor B1 may be a contributing factor in resistance to thyroid hormone syndrome. Taken together, our findings provide a detailed mechanistic understanding of the multiple signals that work together to regulate TR shuttling and transcriptional activity, and they provide important insights into nuclear receptor function in genera

Structure and function of \u3ci\u3ePseudomonas aeruginosa\u3ci\u3e protein PA1324 (21–170)

Pseudomonas aeruginosa is the prototypical biofilm-forming gram-negative opportunistic human pathogen. P. aeruginosa is causatively associated with nosocomial infections and with cystic fibrosis. Antibiotic resistance in some strains adds to the inherent difficulties that result from biofilm formation when treating P. aeruginosa infections. Transcriptional profiling studies suggest widespread changes in the proteome during quorum sensing and biofilm development. Many of the proteins found to be upregulated during these processes are poorly characterized from a functional standpoint. Here, we report the solution NMR structure of PA1324, a protein of unknown function identified in these studies, and provide a putative biological functional assignment based on the observed prealbumin-like fold and FAST-NMR ligand screening studies. PA1324 is postulated to be involved in the binding and transport of sugars or polysaccharides associated with the peptidoglycan matrix during biofilm formation

FAST-NMR - Functional Annotation Screening Technology Using NMR Spectroscopy

An abundance of protein structures emerging from structural genomics and the Protein Structure Initiative (PSI) are not amenable to ready functional assignment because of a lack of sequence and structural homology to proteins of known function. We describe a high-throughput NMR methodology (FAST-NMR) to annotate the biological function of novel proteins through the structural and sequence analysis of protein-ligand interactions. This is based on basic tenets of biochemistry where proteins with similar functions will have similar active sites and exhibit similar ligand binding interactions, despite global differences in sequence and structure. Protein-ligand interactions are determined through a tiered NMR screen using a library composed of compounds with known biological activity. A rapid co-structure is determined by combining the experimental identification of the ligand-binding site from NMR chemical shift perturbations with the proteinligand docking program AutoDock. Our CPASS (Comparison of Protein Active Site Structures) software and database is then used to compare this active site with proteins of known function. The methodology is demonstrated using unannotated protein SAV1430 from Staphylococcus aureus

FAST-NMR - Functional Annotation Screening Technology Using NMR Spectroscopy

An abundance of protein structures emerging from structural genomics and the Protein Structure Initiative (PSI) are not amenable to ready functional assignment because of a lack of sequence and structural homology to proteins of known function. We describe a high-throughput NMR methodology (FAST-NMR) to annotate the biological function of novel proteins through the structural and sequence analysis of protein-ligand interactions. This is based on basic tenets of biochemistry where proteins with similar functions will have similar active sites and exhibit similar ligand binding interactions, despite global differences in sequence and structure. Protein-ligand interactions are determined through a tiered NMR screen using a library composed of compounds with known biological activity. A rapid co-structure is determined by combining the experimental identification of the ligand-binding site from NMR chemical shift perturbations with the proteinligand docking program AutoDock. Our CPASS (Comparison of Protein Active Site Structures) software and database is then used to compare this active site with proteins of known function. The methodology is demonstrated using unannotated protein SAV1430 from Staphylococcus aureus

Semi-Quantitative Models for Identifying Potent and Selective Transthyretin Amyloidogenesis Inhibitors

Rate-limiting dissociation of the tetrameric protein transthyretin (TTR), followed by monomer misfolding and misassembly, appears to cause degenerative diseases in humans known as the transthyretin amyloidoses, based on human genetic, biochemical and pharmacologic evidence. Small molecules that bind to the generally unoccupied thyroxine binding pockets in the native TTR tetramer kinetically stabilize the tetramer, slowing subunit dissociation proportional to the extent that the molecules stabilize the native state over the dissociative transition state—thereby inhibiting amyloidogenesis. Herein, we use previously reported structure-activity relationship data to develop two semi-quantitative algorithms for identifying the structures of potent and selective transthyretin kinetic stabilizers/amyloidogenesis inhibitors. The viability of these prediction algorithms, in particular the more robust in silico docking model, is perhaps best validated by the clinical success of tafamidis, the first-in-class drug approved in Europe, Japan, South America, and elsewhere for treating transthyretin aggregation-associated familial amyloid polyneuropathy. Tafamidis is also being evaluated in a fully-enrolled placebo-controlled clinical trial for its efficacy against TTR cardiomyopathy. These prediction algorithms will be useful for identifying second generation TTR kinetic stabilizers, should these be needed to ameliorate the central nervous system or ophthalmologic pathology caused by TTR aggregation in organs not accessed by oral tafamidis administration

Cognitive conflicts in major depression : Between desired change and personal coherence

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposesThe notion of intrapsychic conflict has been present in psychopathology for more than a century within different theoretical orientations. However, internal conflicts have not received enough empirical attention, nor has their importance in depression been fully elaborated. This study is based on the notion of cognitive conflict, understood as implicative dilemma (ID), and on a new way of identifying these conflicts by means of the Repertory Grid Technique. Our aim was to explore the relevance of cognitive conflicts among depressive patientsPeer reviewedFinal Published versio

A systematic review of the relationship between rigidity/flexibility and transdiagnostic cognitive and behavioral processes that maintain psychopathology

An ever-growing number of transdiagnostic processes that maintain psychopathology across disorders have been identified. However, such processes are not consistently associated with psychological distress and symptoms. An understanding of what makes such processes pathological is required. One possibility is that individual differences in rigidity in the implementation of these processes determine the degree of psychopathology. The aim of this article is to examine the relationship between rigidity/flexibility and transdiagnostic maintenance processes. Initial searches were made for research examining relationships between 18 transdiagnostic processes and rigidity/flexibility. Relationships between rumination, perfectionism, impulsivity and compulsivity, and rigidity/flexibility were systemically reviewed; 50 studies met inclusion criteria. The majority of studies indicated that transdiagnostic cognitive and behavioral maintenance processes and rigidity were correlated, co-occurring, or predictive of each other. Findings are consistent with the hypothesis that it is inflexibility in the manner in which processes are employed that makes them pathologically problematic. However, further research is required to test and establish this