Correction to 'TMSNP: a web server to predict pathogenesis of missense mutations in the transmembrane region of membrane proteins'.

[This corrects the article DOI: 10.1093/nar/lqab008.].

TMSNP: a web server to predict pathogenesis of missense mutations in the transmembrane region of membrane proteins.

The massive amount of data generated from genome sequencing brings tons of newly identified mutations, whose pathogenic/non-pathogenic effects need to be evaluated. This has given rise to several mutation predictor tools that, in general, do not consider the specificities of the various protein groups. We aimed to develop a predictor tool dedicated to membrane proteins, under the premise that their specific structural features and environment would give different responses to mutations compared to globular proteins. For this purpose, we created TMSNP, a database that currently contains information from 2624 pathogenic and 196 705 non-pathogenic reported mutations located in the transmembrane region of membrane proteins. By computing various conservation parameters on these mutations in combination with annotations, we trained a machine-learning model able to classify mutations as pathogenic or not. TMSNP (freely available at http://lmc.uab.es/tmsnp/) improves considerably the prediction power of commonly used mutation predictors trained with globular proteins.

The mutational landscape of human olfactory G protein-coupled receptors.

BACKGROUND: Olfactory receptors (ORs) constitute a large family of sensory proteins that enable us to recognize a wide range of chemical volatiles in the environment. By contrast to the extensive information about human olfactory thresholds for thousands of odorants, studies of the genetic influence on olfaction are limited to a few examples. To annotate on a broad scale the impact of mutations at the structural level, here we analyzed a compendium of 119,069 natural variants in human ORs collected from the public domain. RESULTS: OR mutations were categorized depending on their genomic and protein contexts, as well as their frequency of occurrence in several human populations. Functional interpretation of the natural changes was estimated from the increasing knowledge of the structure and function of the G protein-coupled receptor (GPCR) family, to which ORs belong. Our analysis reveals an extraordinary diversity of natural variations in the olfactory gene repertoire between individuals and populations, with a significant number of changes occurring at the structurally conserved regions. A particular attention is paid to mutations in positions linked to the conserved GPCR activation mechanism that could imply phenotypic variation in the olfactory perception. An interactive web application (hORMdb, Human Olfactory Receptor Mutation Database) was developed for the management and visualization of this mutational dataset. CONCLUSION: We performed topological annotations and population analysis of natural variants of human olfactory receptors and provide an interactive application to explore human OR mutation data. We envisage that the utility of this information will increase as the amount of available pharmacological data for these receptors grow. This effort, together with ongoing research in the study of genetic changes in other sensory receptors could shape an emerging sensegenomics field of knowledge, which should be considered by food and cosmetic consumer product manufacturers for the benefit of the general population.

A Potent Isoprenylcysteine Carboxylmethyltransferase (ICMT) Inhibitor Improves Survival in Ras-Driven Acute Myeloid Leukemia.

Blockade of Ras activity by inhibiting its post-translational methylation catalyzed by isoprenylcysteine carboxylmethyltransferase (ICMT) has been suggested as a promising antitumor strategy. However, the paucity of inhibitors has precluded the clinical validation of this approach. In this work we report a potent ICMT inhibitor, compound 3 [UCM-1336, IC50 = 2 µM], which is selective against the other enzymes involved in the post-translational modifications of Ras. Compound 3 significantly impairs the membrane association of the four Ras isoforms, leading to a decrease of Ras activity and to inhibition of Ras downstream signaling pathways. In addition, it induces cell death in a variety of Ras-mutated tumor cell lines and increases survival in an in vivo model of acute myeloid leukemia. Because ICMT inhibition impairs the activity of the four Ras isoforms regardless of its activating mutation, compound 3 surmounts many of the common limitations of available Ras inhibitors described so far. In addition, these results validate ICMT as a valuable target for the treatment of Ras-driven tumors.

Inter-residue interactions in alpha-helical transmembrane proteins.

MOTIVATION: The number of available membrane protein structures has markedly increased in the last years and, in parallel, the reliability of the methods to detect transmembrane (TM) segments. In the present report, we characterized inter-residue interactions in α-helical membrane proteins using a dataset of 3462 TM helices from 430 proteins. This is by far the largest analysis published to date. RESULTS: Our analysis of residue-residue interactions in TM segments of membrane proteins shows that almost all interactions involve aliphatic residues and Phe. There is lack of polar-polar, polar-charged and charged-charged interactions except for those between Thr or Ser sidechains and the backbone carbonyl of aliphatic and Phe residues. The results are discussed in the context of the preferences of amino acids to be in the protein core or exposed to the lipid bilayer and to occupy specific positions along the TM segment. Comparison to datasets of ß-barrel membrane proteins and of α-helical globular proteins unveils the specific patterns of interactions and residue composition characteristic of α-helical membrane proteins that are the clue to understanding their structure. AVAILABILITY AND IMPLEMENTATION: Results data and datasets used are available at http://lmc.uab.cat/TMalphaDB/interactions.php. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

GPCR-SAS: A web application for statistical analyses on G protein-coupled receptors sequences.

G protein-coupled receptors (GPCRs) are one of the largest protein families in mammals. They mediate signal transduction across cell membranes and are important targets for the pharmaceutical industry. The G Protein-Coupled Receptors-Sequence Analysis and Statistics (GPCR-SAS) web application provides a set of tools to perform comparative analysis of sequence positions between receptors, based on a curated structural-informed multiple sequence alignment. The analysis tools include: (i) percentage of occurrence of an amino acid or motif and entropy at a position or range of positions, (ii) covariance of two positions, (iii) correlation between two amino acids in two positions (or two sequence motifs in two ranges of positions), and (iv) snake-plot representation for a specific receptor or for the consensus sequence of a group of selected receptors. The analysis of conservation of residues and motifs across transmembrane (TM) segments may guide the design of more selective ligands or help to rationalize activation mechanisms, among others. As an example, here we analyze the amino acids of the "transmission switch", that initiates receptor activation following ligand binding. The tool is freely accessible at http://lmc.uab.cat/gpcrsas/.

GPCRtm: An amino acid substitution matrix for the transmembrane region of class A G Protein-Coupled Receptors.

BACKGROUND: Protein sequence alignments and database search methods use standard scoring matrices calculated from amino acid substitution frequencies in general sets of proteins. These general-purpose matrices are not optimal to align accurately sequences with marked compositional biases, such as hydrophobic transmembrane regions found in membrane proteins. In this work, an amino acid substitution matrix (GPCRtm) is calculated for the membrane spanning segments of the G protein-coupled receptor (GPCR) rhodopsin family; one of the largest transmembrane protein family in humans with great importance in health and disease. RESULTS: The GPCRtm matrix reveals the amino acid compositional bias distinctive of the GPCR rhodopsin family and differs from other standard substitution matrices. These membrane receptors, as expected, are characterized by a high content of hydrophobic residues with regard to globular proteins. On the other hand, the presence of polar and charged residues is higher than in average membrane proteins, displaying high frequencies of replacement within themselves. CONCLUSIONS: Analysis of amino acid frequencies and values obtained from the GPCRtm matrix reveals patterns of residue replacements different from other standard substitution matrices. GPCRs prioritize the reactivity properties of the amino acids over their bulkiness in the transmembrane regions. A distinctive role is that charged and polar residues seem to evolve at different rates than other amino acids. This observation is related to the role of the transmembrane bundle in the binding of ligands, that in many cases involve electrostatic and hydrogen bond interactions. This new matrix can be useful in database search and for the construction of more accurate sequence alignments of GPCRs.

Non-meiotic chromosome instability in human immature oocytes.

Aneuploidy has been a major issue in human gametes and is closely related to fertility problems, as it is known to be present in cleavage stage embryos and gestational losses. Pre-meiotic chromosome abnormalities in women have been previously described. The aim of this study is to assess the whole-chromosome complement in immature oocytes to find those abnormalities caused by mitotic instability. For this purpose, a total of 157 oocytes at the germinal vesicle or metaphase I stage, and discarded from IVF cycles, were analysed by CGH. Fifty-six women, between 18 and 45 years old (mean 32.5 years), including 32 IVF patients (25-45 years of age) and 24 IVF oocyte donors (18-33 years of age), were included in the study. A total of 25/157 (15.9%) of the oocytes analysed, obtained from three IVF clinics, contained chromosome abnormalities, including both aneuploidy (24/157) and structural aberrations (9/157). Independently of the maternal age, the incidence of abnormal oocytes which originated before meiosis is 15.9%, and these imbalances were found in 33.9% of the females studied. This work sheds light on the relevance of mitotic instability responsible for the generation of the abnormalities present in human oocytes.

Does the S phase have an impact on the accuracy of comparative genomic hybridization profiles in single fibroblasts and human blastomeres?

OBJECTIVE: To investigate if there is an association between single-cell replicative stage and the segmental chromosome imbalances detected by comparative genomic hybridization (CGH). DESIGN: First, 135 fibroblasts from cell-line GM03184 (Coriell) at three cell stages (G0/G1, S, and G2/M) were amplified by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) or Sureplex and blindly analyzed by CGH. Second, 85 human blastomeres at the interphase and the metaphase stages, from 30 donated human cryopreserved embryos, were amplified by Sureplex and analyzed by CGH. SETTING: Academic center for reproductive medicine. PATIENT(S): None. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Incidence of aneuploidy and segmental imbalances detected at the different cell stages. RESULT(S): In DOP-PCR amplifications of fibroblasts, an increased incidence of segmental abnormalities was detected in the S phase. In Sureplex amplifications of fibroblasts and blastomeres, no differences were detected between the different cell stages. A significantly increased incidence of structural abnormalities was seen in the aneuploid blastomeres. CONCLUSION(S): The segmental imbalances detected after Sureplex amplification in 73.3% of the cryopreserved embryos analyzed are mainly nontransitory. They correspond to segmental imbalances present in the cells due to chromosome instability, rather than to replicative DNA segments.

The role of hydrophobic amino acids in the structure and function of the rhodopsin family of G protein-coupled receptors.

Recent advances in crystallization methods have permitted to resolve the molecular structure of several members of the rhodopsin family of G protein-coupled receptors (GPCRs). Comparison among these structures revealed a number of conserved polar and charged residues implicated in the receptor transduction pathways. These residues function as micro-switches in the process of receptor activation and has been the object of study of many research groups. However, hydrophobic forces, usually underappreciated, also play a major role in GPCR function. Conserved hydrophobic residues contribute significantly to receptor activation, G protein coupling, and oligomerization processes. This review focuses on the impact of the hydrophobic amino acids observed in the structure of class A GPCRs necessary for their function. This information represents a fundamental piece to complete a holistic view of the GPCR signal transduction machinery.

Sitamaquine overcomes ABC-mediated resistance to miltefosine and antimony in Leishmania.

Although oral miltefosine represented an important therapeutic advance in the treatment of leishmaniasis, the appearance of resistance remains a serious threat. LMDR1/LABCB4, a P-glycoprotein-like transporter included in the Leishmania ABC (ATP-binding cassette) family, was the first molecule shown to be involved in experimental miltefosine resistance. LMDR1 pumps drugs out of the parasite, thereby decreasing their intracellular accumulation. Sitamaquine, another promising oral drug for leishmaniasis, is currently in phase 2b clinical trials. The physicochemical features of this drug suggested to us that it could be considered for use as an LMDR1 inhibitor. Indeed, we report herein that nonleishmanicidal concentrations of sitamaquine reverse miltefosine resistance in a multidrug resistance Leishmania tropica line that overexpresses LMDR1. This reversal effect is due to modulation of the LMDR1-mediated efflux of miltefosine. In addition, sitamaquine is not a substrate of LMDR1, as this transporter does not affect sitamaquine accumulation or sensitivity in the parasite. Likewise, we show that ketoconazole, another oral leishmanicidal drug known to interact with ABC transporters, is also able to reverse LMDR1-mediated miltefosine resistance, although with a lower efficiency than sitamaquine. Molecular docking on a three-dimensional homology model of LMDR1 showed different preferential binding sites for each substrate-inhibitor pair, thus explaining this different behavior. Finally, we show that sitamaquine is also able to modulate the antimony resistance mediated by MRPA/LABCC3, another ABC transporter involved in experimental and clinical antimony resistance in this parasite. Taken together, these data suggest that the combination of sitamaquine with miltefosine or antimony could avoid the appearance of resistance mediated by these membrane transporters in Leishmania.

Risk, predictors, and clinical characteristics of lymphoma development in primary Sjögren's syndrome.

OBJECTIVE: To assess the risk and predictors of lymphoma development in a large cohort of patients with primary Sjögren's syndrome (pSS). METHODS: Cox-regression analyses were used to study the predictive value of clinical and laboratory findings at pSS diagnosis, and Kaplan-Meier survival curves to compare survival probability between patients who developed lymphoma and the total cohort. Expected risk for lymphoma was calculated by comparison with the background population. RESULTS: Eleven (4.5%) from 244 patients developed a non-Hodgkin lymphoma (NHL). Diffuse large B-cell and mucosa-associated lymphoid tissue lymphomas occurred at a similar frequency. Three (27.3%) patients died: 2 due to transformation from mucosa-associated lymphoid tissue to diffuse large B-cell. Purpura (HR 8.04, 95% confidence interval [CI] 2.33-27.67), parotidomegaly (HR 6.75, 95%CI 1.89-23.99), anemia (HR 3.43, 95%CI 1.04-11.35), leukopenia (HR 8.70, 95%CI 2.38-31.82), lymphocytopenia (HR 16.47, 95%CI 3.45-78.67), hypergammaglobulinemia (HR 4.06, 95%CI 1.06-15.58), low C3 (HR 36.65, 95%CI 10.65-126.18), and low C4 (HR 39.70, 95%CI 8.85-126.18) levels at pSS diagnosis were significant predictors of NHL development, but only hypocomplementemia and lymphocytopenia were independent risk factors. Hypocomplementemia was related to earlier development of NHL and higher mortality. The cumulative risk of developing lymphoma ranged from 3.4% in the first 5 years to 9.8% at 15 years. Standardized incidence ratio (95%CI) for NHL development was 15.6 (95%CI 8.7-28.2). CONCLUSIONS: Patients with pSS have a 16-fold increased risk of developing lymphoma. This risk increases with time. Hypocomplementemia and lymphocytopenia at pSS diagnosis are the strongest predictors. Survival is clearly reduced in patients with hypocomplementemia. Indolent lymphomas tend to evolve over time toward a more aggressive histologic type.

Development of non-peptide ligands of growth factor receptor-bound protein 2-SRC homology 2 domain using molecular modeling and NMR spectroscopy.

We report a novel series of non-peptide ligands that inhibit the growth factor receptor-bound protein 2 (Grb2)-Src homology 2 (SH2) domain binding, designed using a combined computational and NMR-driven approach. We have identified a new lead compound, 1n (IC(50) = 56 µM), which is cytotoxic in HER2-positive breast cancer cells and disrupts the interaction between HER2 and Grb2. Thus, 1n can be used as a scaffold for the development of efficient Grb2-SH2 domain binding inhibitors.

Age-related survival and clinical features in systemic sclerosis patients older or younger than 65 at diagnosis.

OBJECTIVE: To analyse the differences in SSc clinical features and survival in patients aged > or = 65 years compared with young SSc patients. METHODS: Of a total of 319 SSc patients, we identified 67 (21%) patients aged >65 years. Demographical data such as SSc subsets, the cutaneous complaint, internal organ involvement and the causes of morbidity and mortality were collected. Results of the elderly and young patients were compared. RESULTS: There were 61 (91%) women and 6 (9%) men aged > or = 65 years. The limited SSc (lSSc) subset was more prevalent in elderly than in young patients (74.6 vs 54%, P = 0.002). Pulmonary disease (86.6% in elderly vs 73.8% in young patients, P = 0.034) and cardiac involvement (70.1% in elderly vs 49.6% in young patients, P = 0.004) were significantly more prevalent in elderly patients. In contrast, signs of oesophageal involvement (43.3% in elderly vs 57.5% in young patients, P = 0.040) were less frequent in aged patients. In addition, pulmonary and heart disease appeared significantly earlier after the diagnosis in patients aged > or = 65 years. Mortality was significantly higher in elderly than in young patients (35.8 vs 19%, P = 0.005), but when standardized mortality ratios (SMRs) were analysed, there was no significant mortality increase in the elderly. CONCLUSION: In elderly patients, the lSSc subset is more prevalent than the diffuse. Pulmonary and cardiac involvement are more prevalent in aged patients and appears sooner after the disease diagnosis. SSc is clearly related to increased mortality, although it is not significant in the elderly group.

Benzimidazole derivatives as new serotonin 5-HT6 receptor antagonists. Molecular mechanisms of receptor inactivation.

On the basis of our previously described pharmacophore model for serotonin 5-HT(6) receptor (5-HT(6)R) antagonists, we have designed, synthesized, and pharmacologically characterized a series of benzimidazole derivatives 1-20 that represent a new family of potent antagonists at the human 5-HT(6)R. Site-directed mutagenesis and a beta(2)-adrenoceptor-based homology model of the 5-HT(6)R were used to predict the mode of binding of antagonist SB-258585 and the new synthesized ligands. Substitution of W6.48, F6.52, or N6.55 by Ala fully impedes compound 4 to block 5-HT-induced activation. Thus, we propose that D3.32 in TM 3 anchors the protonated piperazine ring, the benzimidazole ring expands parallel to EL 2 to hydrogen bond N6.55 in TM 6, and the aromatic ring is placed between TMs 3 and 5 in CH(2)-containing compounds and between TMs 3 and 6 in CO-containing compounds. This combined experimental and computational study has permitted to propose the molecular mechanisms by which the new benzimidazole derivatives act as 5-HT(6)R antagonists.

Influence of the g- conformation of Ser and Thr on the structure of transmembrane helices.

In order to study the influence of Ser and Thr on the structure of transmembrane helices we have analyzed a database of helix stretches extracted from crystal structures of membrane proteins and an ensemble of model helices generated by molecular dynamics simulations. Both complementary analyses show that Ser and Thr in the g- conformation induce and/or stabilize a structural distortion in the helix backbone. Using quantum mechanical calculations, we have attributed this effect to the electrostatic repulsion between the side chain Ogamma atom of Ser and Thr and the backbone carbonyl oxygen at position i-3. In order to minimize the repulsive force between these negatively charged oxygens, there is a modest increase of the helix bend angle as well as a local opening of the helix turn preceding Ser/Thr. This small distortion can be amplified through the helix, resulting in a significant displacement of the residues located at the other side of the helix. The crystal structures of aquaporin Z and the beta(2)-adrenergic receptor are used to illustrate these effects. Ser/Thr-induced structural distortions can be implicated in processes as diverse as ligand recognition, protein function and protein folding.

Comparative genomic hybridization analysis reveals new different subgroups in early-stage bladder tumors.

OBJECTIVES: To classify bladder tumors according to their genomic imbalances and evaluate their association with patient's outcome. METHODS: Sixty-three superficially and minimally invasive bladder tumors were analyzed by conventional comparative genomic hybridization. Subtelomeric screening in 15 of these tumors was performed by multiplex ligation-dependent probe amplification. RESULTS: Losses of 9q and 9p (32% and 25% of all cases, respectively) as well as gains of chromosomes Xq and Xp (28% and 25%, respectively) were the most frequent chromosome imbalances. Losses of 8p and gains in 1q and 8q were detected in >20% of cases. Tumors were classified into 3 groups according to their individualized pattern of gains and losses. The largest group was characterized by few chromosome imbalances, presenting 77% and 49% of the Ta and T1 tumors, respectively. Another group characterized by chromosomal gains, was composed of equal number of Ta and T1 tumors, with +1q and +17q gains being the most common imbalances. A minority group was characterized by chromosomal losses on 11q, 5q, and 6q. The multiplex ligation-dependent probe amplification study showed good correlation with comparative genomic hybridization results. With regard to the biological significance of this classification, a remarkable fact is that this minority group composed mainly of T1 tumors, showed a significant decrease in patient overall survival. CONCLUSIONS: Our data suggest that superficial carcinomas of the bladder can be subdivided into a larger number of subclasses than had previously been expected. Our results also demonstrate a decreased survival among patients whose tumors show more genomic losses than gains.

Synthesis of new serotonin 5-HT7 receptor ligands. Determinants of 5-HT7/5-HT1A receptor selectivity.

We report the synthesis of a new set of compounds of general structure I (1-20) with structural modifications in the pharmacophoric elements of the previously reported lead UCM-5600. The new derivatives have been evaluated for binding affinity at 5-HT(7) and 5-HT(1A) receptors. The influence of the different structural features in terms of 5-HT(7)/5-HT(1A) receptor affinity and selectivity was analyzed by computational simulations of the complexes between compounds I and beta(2)-based 3-D models of these receptors. Compound 18 (HYD(1) = 1,3-dihydro-2H-indol-2-one; spacer = -(CH(2))(4)-; HYD(2) + HYD(3) = 3,4-dihydroisoquinolin-2(1H)-yl) exhibits high 5-HT(7)R affinity (K(i) = 7 nM) and selectivity over the 5-HT(1A)R (31-fold), and has been characterized as a partial agonist of the human 5-HT(7)R.

Conformational toggle switches implicated in basal constitutive and agonist-induced activated states of 5-hydroxytryptamine-4 receptors.

The extended classic ternary complex model predicts that a G protein-coupled receptor (GPCR) exists in only two interconvertible states: an inactive R, and an active R(*). However, different structural active R(*) complexes may exist in addition to a silent inactive R ground state (Rg). Here we demonstrate, in a cellular context, that several R(*) states of 5-hydroxytryptamine-4 (5-HT(4)) receptors involve different side-chain conformational toggle switches. Using site-directed mutagenesis and molecular modeling approaches, we show that the basal constitutive receptor (R(*)basal) results from stabilization of an obligatory double toggle switch (Thr3.36 from inactive g- to active g+ and Trp6.48 from inactive g+ to active t). Mutation of either threonine or tryptophan to alanine resulted in a lowering of the activity of the R(*)basal similar to the Rg. The T3.36A mutation shows that the Thr3.36 toggle switch plays a minor role in the stabilization of R(*) induced by 5-HT (R(*)-5-HT) and BIMU8 (R(*)-BIMU8) and is fully required in the stabilization of R(*) induced by (S)-zacopride, cisapride, and 1-(4-amino-5-chloro-2-methoxyphenyl)-3-(1-butyl-4-piperidinyl)-1-propanone (RS 67333) (R(*)-benzamides). Thus, benzamides stabilize R(*)-benzamides by forming a specific hydrogen bond with Thr3.36 in the active g+ conformation. Conversely, R(*)-BIMU8 was probably the result of a direct conformational transition of Trp6.48 from inactive g+ to active t by hydrogen bonding of this residue to a carboxyl group of BIMU8. We were surprised that the Trp6.48 toggle switch was not necessary for receptor activation by the natural agonist 5-HT. R(*)-5-HT is probably attained through other routes of activation. Thus, different conformational arrangements occur during stabilization of R(*)basal, R(*)-5-HT, R(*)-benzamides, and R(*)-BIMU8.

Genomic imbalances in urothelial cancer: intratumor heterogeneity versus multifocality.

Comparative genomic hybridization and fluorescence in situ hybridization were used to define genetic changes associated with multifocal bladder cancer and to investigate whether the genetic relationship between synchronous urothelial tumors is similar to that observed within different parts of the same tumor. We investigated 8 synchronous urothelial tumors from 3 patients and macroscopically different parts of the same tumor from 2 other patients. The most frequent imbalances were gains of 1q, 2p, and 17q, and losses in 4q. The high number of chromosome imbalances detected in the present report confirms that a high level of chromosome instability could be characteristic of multicentric bladder tumors. Comparative genomic hybridization profiles obtained from independent tumors belonging to the same patient allowed us to elaborate cytogenetic pedigrees portraying the accumulation of chromosome alterations as a form of clonal evolution from a single precursor cell. The analysis of different macroscopic parts of the same tumor allowed us to detect chromosomal heterogeneity and to delineate intratumor clonal evolution. Some chromosome regions that appeared as a gain in one subpopulation were amplified in others indicating a genetic evolution process. Identical processes were observed in different tumors of the same patient. Expansion of chromosome gains and losses between different parts of the same tumor as well as in different tumors of the same patient was also observed. Our results not only provide further evidence of a clonal relationship between different synchronous bladder tumors but also show that the intratumor heterogeneity present in different subpopulations of the same tumor reproduces the behavior of independent synchronous tumors in a same patient.