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A well-structured in silico workflow is here reported for disclosing structure-based pharmacophore models against bromodomain-containing protein 9 (BRD9), accelerating virtual screening campaigns and facilitating the identification of novel binders. Specifically, starting from 23 known ligands co-crystallized with BRD9, three-dimensional pharmacophore models, namely placed in a reference protein structure, were developed. Specifically, we here introduce a fragment-related pharmacophore model, useful for the identification of new promising small chemical probes targeting the protein region responsible of the acetyllysine recognition, and two further pharmacophore models useful for the selection of compounds featuring drug-like properties. A pharmacophore-driven virtual screening campaign was then performed to facilitate the selection of new selective BRD9 ligands, starting from a large library of commercially available molecules. The identification of a promising BRD9 binder (7) prompted us to re-iterate this computational workflow on a second focused in-house built library of synthesizable compounds and, eventually, three further novel BRD9 binders were disclosed (8-10). Moreover, all these compounds were tested among a panel comprising other nine bromodomains, showing a high selectivity for BRD9. Preclinical bioscreens for potential anticancer activity highlighted compound 7 as that showing the most promising biological effects, proving the reliability of this in silico pipeline and confirming the applicability of the here introduced structure-based three-dimensional pharmacophore models as straightforward tools for the selection of new BRD9 ligands.

Unraveling the role of post-translational modification (PTM) patterns is one of the most urgent and unresolved issues facing the scientific community. Attempts to crack the phosphorylation bio-barcode led to significant findings, which suggest that many proteins cannot be regarded as a single entity but exist as several forms which differ in their phosphorylation patterns and their functions. While protein regions that do not contain PTMs can be rather simply mimicked using peptide libraries, heavily phosphorylated regions are much harder to study using the same tools. The differences between the syntheses of simple mono-, di- and tri-phosphopeptides and the synthesis of multiphosphopeptides are dramatic. While simple phosphopeptides can be synthesized using almost standard SPPS strategies, the synthesis of multiphosphopeptides is to date a major synthetic challenge. Synthesis of multiphosphopeptides requires the insertion of several phosphate groups simultaneously or sequentially into various positions on the peptide in the presence of many other potential modification sites. These groups are bulky, unstable and cannot be easily introduced when in close proximity. Moreover, since the same protein region can possess many alternative multiphosphorylation patterns, libraries comprising a large number of peptides with different degrees and positions of phosphorylation are essential. Many strategies have been developed to provide routes to enable the preparation of multiphosphopeptides. These methods are essentially different from the methods used for the preparation of simple phosphopeptides. In this review, we specifically emphasize the challenges and importance of synthesizing multiphosphopeptides and their libraries. The historical perspective and state of the art strategies are described. We demonstrate here how the different synthetic approaches attempt to address the special problems associated with the synthesis of multiphosphopeptides. The advantages and disadvantages of each strategy are discussed in order to provide a roadmap for the synthesis of such libraries. An overview of the existing strategies and some comments regarding future directions are provided. Applications of multiphosphopeptide libraries as tools to study the effect of phosphorylation patterns on the biological function of proteins are also described.

Human tyrosine hydroxylase 1 (hTH1) activity is regulated by phosphorylation of its regulatory domain (RD-hTH1) and by an interaction with the 14-3-3 protein. The RD-hTH1 is composed of a structured region (66-169) preceded by an intrinsically disordered protein region (IDP, hTH1_65) containing two phosphorylation sites (S19 and S40) which are highly relevant for its increase in activity. The NMR signals of the IDP region in the non-phosphorylated, singly phosphorylated (pS40) and doubly phosphorylated states (pS19_pS40) were assigned by non-uniformly sampled spectra with increased dimensionality (5D). The structural changes induced by phosphorylation were analyzed by means of secondary structure propensities. The phosphorylation kinetics of the S40 and S19 by kinases PKA and PRAK respectively were monitored by non-uniformly sampled time-resolved NMR spectroscopy followed by their quantitative analysis.

Twelve samples of cheeses (three types of Saint-Nectaire PDO cheeses and Savaron cheeses) differing by manufacturing and ripening conditions, from 12 different producers, were characterised by attenuated total reflectance mid-infrared (MIR) and front-face fluorescence spectroscopies, dynamic testing rheology and physico-chemical analysis. Fluorescence spectra (tryptophan residues, vitamin A and riboflavin) and MIR (3,000–2,800 (fat region), 1,700–1,500 (protein region) and 1,500–900 cm−1 (fingerprint region)) spectra were recorded on cheese samples. The potential of the data tables was investigated for discriminating the four different groups of cheeses. The results of factorial discriminant analysis (FDA) performed on the fluorescence and mid-infrared spectra showed a good discrimination of the four cheese groups. Considering cross-validation results, the best classifications (100%) were achieved from mid-infrared and fluorescence spectra, while only 91.7 and 72.2% of correct classification were obtained by applying FDA to rheology and physico-chemical data, respectively. Spectroscopic techniques could provide useful fingerprints and allow the identification of investigated cheeses according to manufacturing conditions. Simple and rapid spectroscopic methods offer a promising approach to the authentication of cheeses.

Intrinsically disordered proteins (IDPs) constitute a recently recognized realm of atypical biologically active proteins that lack stable structure under physiological conditions, but are commonly involved in such crucial cellular processes as regulation, recognition, signaling and control. IDPs are very common among proteins associated with various diseases. Recently, we performed a systematic bioinformatics analysis of the human diseasome, a network that linked the human disease phenome (which includes all the human genetic diseases) with the human disease genome (which contains all the disease-related genes) (Goh, K. I., Cusick, M. E., Valle, D., Childs, B., Vidal, M., and Barabasi, A. L. (2007). The human disease network. Proc. Natl. Acad. Sci. U.S.A. 104, 8685-90). The analysis of this diseasome revealed that IDPs are abundant in proteins linked to human genetic diseases, and that different genetic disease classes varied dramatically in the IDP content (Midic U., Oldfield C.J., Dunker A.K., Obradovic Z., Uversky V.N. (2009) Protein disorder in the human diseasome: Unfoldomics of human genetic diseases. BMC Genomics. In press). Furthermore, many of the genetic disease- related proteins were shown to contain at least one molecular recognition feature, which is a relatively short loosely struc- tured protein region within a mostly disordered segment with the feature gaining structure upon binding to a partner. Fi- nally, alternative splicing was shown to be abundant among the diseasome genes. Based on these observations the human- genetic-disease-associated unfoldome was created. This minireview describes several illustrative examples of ordered and intrinsically disordered members of the human diseasome.

Intrinsically disordered proteins (IDPs) are ubiquitously involved in cellular processes and often implicated in human pathological conditions. The critical biological roles of these proteins, despite not adopting a well-defined fold, encouraged structural biologists to revisit their views on the protein structure-function paradigm. Unfortunately, investigating the characteristics and describing the structural behavior of IDPs is far from trivial, and inferring the function(s) of a disordered protein region remains a major challenge. Computational methods have proven particularly relevant for studying IDPs: on the sequence level their dependence on distinct characteristics determined by the local amino acid context makes sequence-based prediction algorithms viable and reliable tools for large scale analyses, while on the structure level the in silico integration of fundamentally different experimental data types is essential to describe the behavior of a flexible protein chain. Here, we offer an overview of the latest developments and computational techniques that aim to uncover how protein function is connected to intrinsic disorder., SCOPUS: sh.j, info:eu-repo/semantics/published

Cytoplasmic S4‐S5 loops have been shown to be involved in fast inactivation of voltagegate di on channels. We studied mutations in these loops and their potential cooperative effects in domains D3 (N1151C, A1152C, I1160C/A) and D4 (F1473C, L1482C/A) of the human skeletal muscle Na + channel α-subunit (hNav1.4) using expression in tsA201 cells and the whole cell patch-clamp technique. All cysteine mutations were accessible to intracellularly applied sulfhydryl reagents which considerably destabilized fast inactivation. For different combinations of corresponding D3/D4 double mutations, fast inactivation could be almost completely removed. Thermodynamic cycle analysis indicated an additive effect for N1151C/F1473C and a significant cooperative effect for I1160/L1482 double mutations. Application of oxidizing reagents such as Cu-phenanthroline to link two cysteines via a disulfide bridge did not reveal evidence for a direct physical interaction of cysteines in D3 and D4. In addition to the pronounced alterations of fast inactivation, mutations of I1160 shifted steady-state activation in the hyperpolarizing direction and slowed the kinetics of both activation and deactivation. Sulfhydryl reagents had charge-dependent effects on I1160C suggesting interaction with negative charges in another protein region. We conclude that fast inactivation of the Na + channel involves both S4‐S5 loops in D3 and D4 in a cooperative manner. D3/S4‐S5 also plays an important role in activation and deactivation.

In spite of the high similarity in amino acid sequence between rod visual pigment rhodopsin and gecko blue-sensitive pigment (gecko blue), not only the spectral sensitivities but also the thermal decay rates of the meta II- and III-intermediates are noticeably different from one another [Kojima et al. (1995) Biochemistry 34, 1096-1106]. In order to identify the protein region(s) that contain(s) key residue being responsible for the functional difference, we constructed six chimerical mutants derived from gecko blue and bovine rhodopsin, with the aid of protein production in a human embryonic kidney cell line (293S). While the absorption maximum of every mutant was located in between gecko blue (466 nm) and bovine rhodopsin (500 nm), a large blue-shift (18 nm) was observed when the helices I-III of rhodopsin were replaced with those of gecko blue. A time resolved spectroscopic study demonstrated that this replacement also accelerated the decay rate of the meta II-intermediate. The decay of the meta III-intermediate of the mutants became faster as the compartment of gecko blue was increased. Thus, the faster decay of the meta II-intermediate of gecko blue is largely attributed to residues within helices I-III, while the decay of the meta III-intermediate apparently depends on the overall structure of the protein.

Motivation: Protein–protein interactions are commonly mediated by the physical contact of distinct protein regions. Computational identification of interacting protein regions aids in the detailed understanding of protein networks and supports the prediction of novel protein interactions and the reconstruction of protein complexes. Results: We introduce an integrative approach for predicting protein region interactions using a probabilistic model fitted to an observed protein network. In particular, we consider globular domains, short linear motifs and coiled-coil regions as potential protein-binding regions. Possible cooperations between multiple regions within the same protein are taken into account. A finegrained confidence system allows for varying the impact of specific protein interactions and region annotations on the modeling process. We apply our prediction approach to a large training set using a maximum likelihood method, compare different scoring functions for region interactions and validate the predicted interactions against a collection of experimentally observed interactions. In addition, we analyze prediction performance with respect to the inclusion of different region types, the incorporation of confidence values for training data and the utilization of predicted protein interactions. Contact: mario.albrecht@mpi-inf.mpg.de Supplementary information: Supplementary data are available at Bioinformatics online.

Multiple-round in vitro transcription assays were performed using purified Escherichia coli RNA polymerase reconstituted with either wild-type or mutant final sigma70 proteins. These mutants, final sigma70(P504L) and final sigma70(S506F), bear single amino acid changes in conserved protein region 3. Behavior of the mutant enzymes on three test templates, bearing either the T7 A1, T5 N25, or T5 N25antiDSR promoter, were characterized. Transcription of all three promoter templates produced a pattern of specific abortive RNA species, which was qualitatively different for the mutants compared to the wild-type final sigma70 enzyme. Short abortive RNAs were produced at similar levels for mutant and wild-type enzymes. The production of longer abortive species was either reduced or increased by the mutant enzymes in a systematic manner that appears promoter-specific, and could be RNA length- or promoter distance-dependent. The process of abortive RNA transcription is thought to be tightly associated with that of promoter clearance. However, promoter clearance from these templates appears only slightly affected by the mutant enzymes. These mutants implicated region 3 of final sigma70 in the process of abortive transcription and suggest that the sequence of enzymatic events leading to the production of abortive or full-length RNA may be separable.

An evolutionary analysis of self peptides reported to be bound by HLA class I molecules showed that these peptides are largely derived from proteins that have been highly conserved in the history of mammals. These proteins also often have universal tissue expression and have a higher than average frequency of highly hydrophilic residues. The peptides themselves are generally still more highly conserved than the source proteins and have a higher frequency of highly hydrophobic residues, evidently often being derived from conserved hydrophobic cores of the source proteins. These results suggest that the mechanism by which peptides are derived for MHC presentation may preferentially select peptides from conserved protein regions. In the case of parasite-derived peptides, such a mechanism would be adaptive in that it would reduce the likelihood of escape mutants.

The wheat-grain proteome was investigated, as a basis for devising more efficient methods of cultivar identification or discrimination. Australian wheats (Halberd, Cranbrook, CD87 and Katepwa) were used as the basis of this study. These cultivars were selected on the basis of differences in the quality types represented, in terms of dough-processing attributes that can suit one cultivar better than another for specific types of industrial utilisation. Total wheat endosperm (flour) protein extracts were prepared from mature wheat for two-dimensional electrophoresis, across both acidic (pH 4-7) and basic (pH 6-11) pH ranges. Three particular regions of the proteome maps were chosen for close comparison, involving two sets of gluten proteins and a nongluten protein region (involving small heat shock proteins), based on previous protein characterisation. Differences in the nongluten protein regions (heat shock proteins and other unidentified polypeptides) are of particular interest as being possible targets for use in developing new approaches to cultivar discrimination, such as the development of simple immunoassays.

With the aid of gel filtration techniques (Sephadex G-200) the alkaline phosphatase (Bessey-Lowry-Brock) distribution patterns have been studied in the serum of control human subjects and of those with conditions known to affect total enzyme levels. Protein was scanned at 254 and 280 mμ and the 19S, 7S, and 4S peaks thus obtained were used as points of reference. The characteristic control serum revealed one sharp peak of enzyme activity in the 7S protein region. A similar but sharper peak was found in osteomalacia and Pagets' disease. In contrast, in malignant bone conditions 10.4%, in metastatic malignant liver disease 31.36% and in nonmalignant liver disease 12.7% of total enzyme moved with the 19S proteins. The patterns found in various conditions are reproducible, and that of metastatic malignant liver disease is particularly characteristic. The significance of the macromolecular-moving enzyme is discussed and is being investigated.

BamA is the main component of the beta-barrel assembly machinery (BAM) that folds and inserts outer membrane proteins in Gram-negative bacteria. Crystal structures have suggested that this process involves conformational changes in the transmembrane beta-barrel of BamA that allow for lateral opening, as well as large overall rearrangements of its periplasmic POTRA domains. Here, we identify local dynamics of the BamA POTRA 5 domain by solution and solid-state nuclear magnetic resonance. The protein region undergoing conformational exchange is highly conserved and contains residues critical for interaction with BamD and correct beta-barrel assembly in vivo. We show that mutations known to affect the latter processes influence the conformational equilibrium, suggesting that the plasticity of POTRA 5 is related to its interaction with BamD and possibly to substrate binding. Taken together, a view emerges in which local protein plasticity may be critically involved in the different stages of outer membrane protein folding and insertion.

The cytoplasmic concentration of Z protein (Mr approximately 12,000) was significantly reduced in a series of implanted Morris hepatomas with varying degrees of differentiation. Approximately half of the [14C]palmitoyl-CoA added to cytosol fractions from control or host livers was bound to the Z protein region whereas a much smaller proportion was bound to this region in the cytosol of nine Morris hepatomas studied. The possible implications of these findings are discussed in relation to the abnormal regulation of lipid metabolism in hepatomas.

Cowpea chlorotic mottle virus (CCMV) is a spherical plantvirus of the group of bromoviruses. An interesting feature of bromoviruses is the increase in hydrodynamic volume when the pH is raised from 5.0-7.5 at low ionic strength (