Effects Of Amino Acid Substitutions Research Articles

​Fusarium Protein Toolkit: a web-based resource for structural and variant analysis of Fusarium species

Background​​The genus Fusarium poses significant threats to food security and safety worldwide because numerous species of the fungus cause destructive diseases and/or mycotoxin contamination in crops. The adverse effects of climate change are exacerbating some existing threats and causing new problems. These challenges highlight the need for innovative solutions, including the development of advanced tools to identify targets for control strategies.DescriptionIn response to these challenges, we developed the Fusarium Protein Toolkit (FPT), a web-based tool that allows users to interrogate the structural and variant landscape within the Fusarium pan-genome. The tool displays both AlphaFold and ESMFold-generated protein structure models from six Fusarium species. The structures are accessible through a user-friendly web portal and facilitate comparative analysis, functional annotation inference, and identification of related protein structures. Using a protein language model, FPT predicts the impact of over 270 million coding variants in two of the most agriculturally important species, Fusarium graminearum and F. verticillioides. To facilitate the assessment of naturally occurring genetic variation, FPT provides variant effect scores for proteins in a Fusarium pan-genome based on 22 diverse species. The scores indicate potential functional consequences of amino acid substitutions and are displayed as intuitive heatmaps using the PanEffect framework.ConclusionFPT fills a knowledge gap by providing previously unavailable tools to assess structural and missense variation in proteins produced by Fusarium. FPT has the potential to deepen our understanding of pathogenic mechanisms in Fusarium, and aid the identification of genetic targets for control strategies that reduce crop diseases and mycotoxin contamination. Such targets are vital to solving the agricultural problems incited by Fusarium, particularly evolving threats resulting from climate change. Thus, FPT has the potential to contribute to improving food security and safety worldwide.

Insights into Polyprotein Processing and RNA-Protein Interactions in Foot-and-Mouth Disease Virus Genome Replication.

Foot-and-mouth disease virus (FMDV) is a picornavirus, which infects cloven-hoofed animals to cause foot-and-mouth disease (FMD). The positive-sense RNA genome contains a single open reading frame, which is translated as a polyprotein that is cleaved by viral proteases to produce the viral structural and nonstructural proteins. Initial processing occurs at three main junctions to generate four primary precursors; Lpro and P1, P2, and P3 (also termed 1ABCD, 2BC, and 3AB1,2,3CD). The 2BC and 3AB1,2,3CD precursors undergo subsequent proteolysis to generate the proteins required for viral replication, including the enzymes 2C, 3Cpro, and 3Dpol. These precursors can be processed through both cis and trans (i.e., intra- and intermolecular proteolysis) pathways, which are thought to be important for controlling virus replication. Our previous studies suggested that a single residue in the 3B3-3C junction has an important role in controlling 3AB1,2,3CD processing. Here, we use in vitro based assays to show that a single amino acid substitution at the 3B3-3C boundary increases the rate of proteolysis to generate a novel 2C-containing precursor. Complementation assays showed that while this amino acid substitution enhanced production of some nonenzymatic nonstructural proteins, those with enzymatic functions were inhibited. Interestingly, replication could only be supported by complementation with mutations in cis acting RNA elements, providing genetic evidence for a functional interaction between replication enzymes and RNA elements. IMPORTANCE Foot-and-mouth disease virus (FMDV) is responsible for foot-and-mouth disease (FMD), an important disease of farmed animals, which is endemic in many parts of the world and can results in major economic losses. Replication of the virus occurs within membrane-associated compartments in infected cells and requires highly coordinated processing events to produce an array of nonstructural proteins. These are initially produced as a polyprotein that undergoes proteolysis likely through both cis and trans alternative pathways (i.e., intra- and intermolecular proteolysis). The role of alternative processing pathways may help coordination of viral replication by providing temporal control of protein production and here we analyze the consequences of amino acid substitutions that change these pathways in FMDV. Our data suggest that correct processing is required to produce key enzymes for replication in an environment in which they can interact with essential viral RNA elements. These data further the understanding of RNA genome replication.

Whole-Genome Comparison of Representatives of All Variants of SARS-CoV-2, Including Subvariant BA.2 and the GKA Clade.

Since its discovery at the end of 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly evolved into many variants, including the subvariant BA.2 and the GKA clade. Genomic clarification is needed for better management of the current pandemic as well as the possible reemergence of novel variants. The sequence of the reference genome Wuhan-Hu-1 and approximately 20 representatives of each variant were downloaded from GenBank and GISAID. Two representatives with no track of in-definitive nucleotides were selected. The sequences were aligned using muscle. The location of insertion/deletion (indel) in the genome was mapped following the open reading frame (ORF) of Wuhan-Hu-1. The phylogeny of the spike protein coding region was constructed using the maximum likelihood method. Amino acid substitutions in all ORFs were analyzed separately. There are two indel sites in ORF1AB, eight in spike, and one each in ORF3A, matrix (MA), nucleoprotein (NP), and the 3'-untranslated regions (3'UTR). Some indel sites and residues/substitutions are not unique, and some are variant-specific. The phylogeny shows that Omicron, Deltacron, and BA2 are clustered together and separated from other variants with 100% bootstrap support. In conclusion, whole-genome comparison of representatives of all variants revealed indel patterns that are specific to SARS-CoV-2 variants or subvariants. Polymorphic amino acid comparison across all coding regions also showed amino acid residues shared by specific groups of variants. Finally, the higher transmissibility of BA.2 might be due at least in part to the 48 nucleotide deletions in the 3'UTR, while the seem-to-be extinction of GKA clade is due to the lack of genetic advantages as a consequence of amino acid substitutions in various genes.

Substitutions at position 263 within the von Willebrand factor type A domain determine the functionality of complement C2 protein.

The complement system is one of the first defense lines protecting from invading pathogens. However, it may turn offensive to the body's own cells and tissues when deregulated by the presence of rare genetic variants that impair physiological regulation and/or provoke abnormal activity of key enzymatic components. Factor B and complement C2 are examples of paralogs engaged in the alternative and classical/lectin complement pathway, respectively. Pathogenic mutations in the von Willebrand factor A domain (vWA) of FB have been known for years. Despite substantial homology between two proteins and the demonstration that certain substitutions in FB translated to C2 result in analogous phenotype, there was a limited number of reports on pathogenic C2 variants in patients. Recently, we studied a cohort of patients suffering from rare kidney diseases and confirmed the existence of two gain-of-function and three loss-of-function mutations within the C2 gene sequences coding for the vWA domain (amino acids 254-452) or nearly located unstructured region (243-253) of C2 protein. Herein, we report the functional consequences of amino acid substitution of glutamine at position 263. The p.Q263G variant resulted in the gain-of-function phenotype, similarly to a homologous mutation p.D279G in FB. Conversely, the p.Q263P variant found in a patient with C3 glomerulopathy resulted in the loss of C2 function. Our results confirm that the N-terminal part of the vWA domain is a hot spot crucial for the complement C2 function.

Functional Analysis of KLK3/PSA Protease Activity in Primates

Male reproductive proteins have been the focus of evolutionary biologists due to their rapid evolutionary rates compared to other proteins in humans. However, most of the studies on their accelerated evolution and selective forces behind it rely on DNA sequencing data. The objective of this study is to experimentally compare the function of one of these reproductive proteins in humans and our close evolutionary relatives to analyze the functional consequences of amino acid substitutions, which can then help us understand the possible factors contributing to its selection. To accomplish this aim, one of the most abundant seminal plasma proteins, kallikrein‐related peptidase 3 (KLK3) was used. KLK3 is also known as prostate‐specific antigen (PSA). Its physiological function is to liquefy the seminal coagulum, an important step for fertilization, releasing sperm cells for active motility. The formation of seminal coagulum is considered as a sperm competition strategy. Higher sperm competition was found to be correlated with more nonsynonymous substitutions in many seminal proteins. Therefore, their adaptive evolution is commonly attributed to sexual selection through post‐copulatory sperm competition. Previous studies showed that several codons from KLK3 orthologs have been evolving under positive selection and there is variation in dN/dS ratios among branches of primates, these were also confirmed by our multiple sequence alignment data. Based on the literature, the equivalent of the human KLK3 gene is found as chimeric KLK (cKLK, fusion of KLK2 and KLK3) in gorillas and gibbons. However, it has not yet been clarified whether the cKLK is enzymatically active or not. Considering the role of KLK3 in dissolution of seminal coagulum, its chimeric form might be related to the mating systems of gorillas and gibbons, who likely experience very low sperm competition. We hypothesize that amino acid substitutions have caused changes in KLK3 enzymatic activity across species with varying levels of sperm competition. To test this hypothesis, coding DNA sequences of KLK3/cKLK from human, chimpanzee, their hypothetical ancestor, gorilla, gibbon, and macaque were cloned into a mammalian expression vector with a C‐terminal His6‐tag. All these recombinant proteins were successfully expressed in HEK‐293T cells and purified using Nickel columns. After purification, they were treated with thermolysin to cleave the activation sequence from the core domain of KLK3 and subsequently analyzed for the protease activity on a synthetic fluorescent PSA substrate. Varying levels of enzyme activity were observed between species, supporting our hypothesis such that even a few substitutions were sufficient to cause changes in enzymatic activity. Enzyme kinetics (Kcat/Km) were also calculated for each species’ KLK3 protease activity. However, our preliminary data did not reveal a consistent trend of enzymatic activity in species with similar levels of sperm competition. This suggests that other potential selective pressures such as pathogen response and coevolution with its substrate could also be responsible for KLK3 adaptation along with sexual selection.

A bioinformatics approach to investigating the structural and functional consequences of SNPs in TMPRSS2 for COVID‐19 infection

SARS‐CoV‐2 is a highly infectious virus that is responsible for the COVID‐19 global pandemic that swept the world in 2020. Disease outcomes range from asymptomatic to fatal. The virus initiates entry into host cells by the binding of its spike protein to the ACE2 receptor. Entry is finalized by the activation of spike glycoprotein by proteases including transmembrane protease, serine 2 (TMPRSS2) and FURIN which cleave the spike protein of the virus. Single nucleotide polymorphisms (SNPs) in TMPRSS2 may lead to functional changes which could underlie differences in disease severity. TMPRSS2 is also known to activate different respiratory illnesses including coronaviruses and influenza A (Shen et al., 2020). Previous studies have shown that knockout TMPRSS2 mice appeared healthy, experienced a decrease in viral spread within the respiratory system, and had a less severe immune response when infected with SARS‐CoV and MERS‐CoV (Baughn et al., 2020). Thus, we asked whether genetic variations in TMPRSS2 in humans lead to differences in infection rates or severity of disease symptoms of SARS‐CoV‐2. We examined the NCBI dbSNP database to identify SNPs in the TMPRSS2 gene. As of 10 December 2020, we found there were 11,023 intron variants, 393 missense variants, 186 synonymous variants, 3 in‐frame insertion variants, 2 in‐frame deletion variants, and 1 initiator codon variant reported. To narrow these down to 23 SNPs of interest, we first searched the ClinVar database to identify SNPs with general clinical significance, followed by searching the literature to determine SNPs specifically related to SARS‐CoV‐2 severity. One missense variant, rs12329760, results in an amino acid substitution, V160M, which has been predicted to alter TMPRSS2 function. A subset of these SNPs show differences in frequency in world populations, and we wondered if these SNPs had structural and functional consequences for the protein. A crystal structure of TMPRSS2 is not currently available. To visualize the structural consequences of amino acid substitutions, we performed homology modeling on TMPRSS2 (UniProt O15393) using the structure prediction software HHPred, RaptorX, and SwissModel based on the ~30% similarity to hepsin. The predicted structures of TMPRSS2 with various amino acid substitutions were then docked to the SARS‐CoV‐2 spike protein using I‐TASSER and Haddock 2.4 to observe differences in binding interactions and therefore determine which sequence changes are predicted to alter binding interactions, potentially contributing to the wide variation of symptoms caused by COVID‐19.Baughn, L. B., Sharma, N., Elhaik, E., Sekulic, A., Bryce, A. H., & Fonseca, R. (2020). Targeting TMPRSS2 in SARS‐CoV‐2 Infection. Mayo Clinic proceedings, 95(9), 1989–1999. https://doi.org/10.1016/j.mayocp.2020.06.018Shen, L.W.; Mao, H.J.;, Wu, Y.L.; Tanaka,Y.; Zhang,W. (2017) TMPRSS2: A potential target for treatment of influenza virus and coronavirus infections, Biochimie, 142, 1‐10. https://doi.org/10.1016/j.biochi.2017.07.016

Solubility of proteins.

Solubility is a fundamental protein property that has important connotations for therapeutics and use in diagnosis. Solubility of many proteins is low and affect heterologous overexpression of proteins, formulation of products and their stability. Two processes are related to soluble and solid phase relations. Solubility refers to the process where proteins have correctly folded structure, whereas aggregation is related to the formation of fibrils, oligomers or amorphous particles. Both processes are related to some diseases. Amyloid fibril formation is one of the characteristic features in several neurodegenerative diseases, but it is related to many other diseases, including cancers. Severe complex V deficiency and cataract are examples of diseases due to reduced protein solubility. Methods and approaches are described for prediction of protein solubility and aggregation, as well as predictions of consequences of amino acid substitutions. Finally, protein engineering solutions are discussed. Protein solubility can be increased, although such alterations are relatively rare and can lead to trade-off with some other properties. The aggregation prediction methods mainly aim to detect aggregation-prone sequence patches and then making them more soluble. The solubility predictors utilize a wide spectrum of features.

Identification of RNA Exosome Cofactors in Neuronal Cells to Probe Disease Mechanism

The RNA exosome, a 10‐subunit complex that mediates both RNA processing and degradation, plays a critical role in defining cellular expression profiles. This complex is ubiquitously expressed, essential, and critical for fundamental cellular functions, such as ribosomal RNA processing. Recent studies have linked mutations in genes encoding multiple subunits of the complex to tissue‐specific human disease. For example, missense mutations in the human EXOSC3 gene, which encodes an RNA exosome subunit, cause Pontocerebellar Hypoplasia type 1b (PCH1b), a disease characterized by atrophy of the pons and cerebellum. The missense mutations encode single amino acid changes in conserved regions of the EXOSC3 protein. How these amino acid substitutions confer tissue‐specific phenotypes is not known. One possible mechanism underlying the distinct disease phenotypes could be a decrease in the interaction of the RNA exosome complex with cofactors that confer specificity for RNA targets. However, most studies that identify and characterize RNA exosome cofactors have been carried out in budding yeast and thus, do not provide insight into whether tissue‐specific cofactors could exist. Our studies use immunoprecipitation from neuronal cell culture (N2A) and relevant mouse tissues to define RNA exosome cofactors. Biochemical experiments that employ cultured N2A cells were used to identify RNA exosome‐interacting proteins in both the nucleus and the cytoplasm. Preliminary results from this analysis reveal an association between the RNA exosome and a large complex of cytoplasmic tRNA ligase enzymes, which could link defects in tRNA maturation to disease pathology. We are extending these studies to explore the possibility of tissue‐specific cofactors by immunoprecipitating EXOSC3 and analyzing co‐purifying proteins from the mouse cerebellum (affected in disease) and cortex (unaffected). To complement these approaches to identify tissue‐specific RNA exosome cofactors, we are also exploring whether amino acid changes that are linked to disease alter cofactor interactions. These studies, which are being performed in N2A cells, have identified cofactors that show altered interactions with the EXOSC3 variants present in disease. These studies will provide insight into both the functional consequences of amino acid substitutions in the RNA exosome that cause disease and the role of cofactors in conferring RNA target specificity.

Assessing the Functional Properties of the TMCO1 Sequence Variants by Using In Silico Analyses

Transmembrane and Coiled-Coil Domains 1 (TMCO1) protein is encoded by TMCO1 gene consists of 7 exons. Previous studies have identified multiple TMCO1 variants in patients with cerebro-facio-thoracic dysplasia (CFTD) and TMCO1 locus was also shown to be associated with primary open angle glaucoma (POAG). However, there are limited number of research exist reporting associations of the TMCO1 gene sequence variants and majority of the findings affirm the pathogenicity of the nonsense and frameshift TMCO1 variants and their associations with clinical phenotypes. Thus functional properties of the single nucleotide variants causing amino acid changes in the TMCO1 are yet to be comprehensively elucidated. In this study, we evaluated the effects of amino acid substitutions on protein structure, identified their putative roles in post-translational modifications (PTM) and in regulatory mechanism for TMCO1 protein. We classified 41 missense variants as pathogenic based on combined scores of common in silico tools (SIFT, MutationTaster2, Polyphen2). Of these 41 variants, four (p.K211Q, p.K105E, p.S235F, p.K237R) were identified to be located in PTMs and regulatory protein binding sites; thus they were proposed to be putative functional variants. Moreover, rs1387528611 (p.Lys128Gln) had also strong evidence (RegulomeDB score=2b) for its possible regulatory function. The results of our in silico analyses highlight the functional importance of the missense TMCO1 variants that may contribute to the TMCO1-associated disease phenotypes and further in vivo evaluation yet to be needed to uncover their role in human diseases.

Identification of Tissue‐Specific RNA Exosome Cofactors as an Approach to Define Disease Mechanism

The RNA exosome, a 10‐subunit complex that mediates both RNA processing and degradation, plays a critical role in defining cellular expression profiles. This complex is ubiquitously expressed, essential, and critical for fundamental cellular functions, such as ribosomal RNA processing. Recent studies have linked mutations in genes encoding multiple subunits of the complex to tissue‐specific human disease. For example, mutations in the human EXOSC3 gene, coding for a subunit, cause Pontocerebellar Hypoplasia type 1b (PCH1b), a disease characterized by atrophy of the pons and cerebellum. The missense mutations encode single amino acid changes in conserved regions of the EXOSC3 protein. How these amino acid substitutions confer tissue‐specific phenotypes is not known. One possible mechanism underlying the distinct disease phenotypes could be a decrease in the interaction of the RNA exosome complex with cofactors that confer specificity for RNA targets. However, most studies that identify and characterize RNA exosome cofactors have been carried out in budding yeast and thus, do not provide insight into whether tissue‐specific cofactors could exist. Our studies use immunoprecipitation from neuronal cell culture (N2A) and mouse tissues to define RNA exosome cofactors. Biochemical experiments that employ cultured N2A cells identified RNA exosome interacting proteins that are enriched in both the nucleus and cytoplasm. Preliminary results from this analysis identify an association between the RNA exosome and a large complex of tRNA ligase enzymes, which could link defects in tRNA maturation to disease pathology. We are extending these studies to explore the possibility of tissue‐specific cofactors by immunoprecipitating EXOSC3 and analyzing co‐purifying proteins from the cerebellum (affected in disease) and the cortex (unaffected) in mouse brain. Finally, using CRISPR/Cas9 genome editing, we are testing whether the amino acid changes that occur in disease alter interactions with both known RNA exosome cofactors and interactors identified in our co‐immunoprecipitation studies. These studies will provide insight into both the functional consequences of amino acid substitutions in the RNA exosome that cause disease and the role of cofactors in conferring RNA target specificity.Support or Funding InformationR01GM130147This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Variant effect prediction tools assessed using independent, functional assay-based datasets: implications for discovery and diagnostics

BackgroundGenetic variant effect prediction algorithms are used extensively in clinical genomics and research to determine the likely consequences of amino acid substitutions on protein function. It is vital that we better understand their accuracies and limitations because published performance metrics are confounded by serious problems of circularity and error propagation. Here, we derive three independent, functionally determined human mutation datasets, UniFun, BRCA1-DMS and TP53-TA, and employ them, alongside previously described datasets, to assess the pre-eminent variant effect prediction tools.ResultsApparent accuracies of variant effect prediction tools were influenced significantly by the benchmarking dataset. Benchmarking with the assay-determined datasets UniFun and BRCA1-DMS yielded areas under the receiver operating characteristic curves in the modest ranges of 0.52 to 0.63 and 0.54 to 0.75, respectively, considerably lower than observed for other, potentially more conflicted datasets.ConclusionsThese results raise concerns about how such algorithms should be employed, particularly in a clinical setting. Contemporary variant effect prediction tools are unlikely to be as accurate at the general prediction of functional impacts on proteins as reported prior. Use of functional assay-based datasets that avoid prior dependencies promises to be valuable for the ongoing development and accurate benchmarking of such tools.

Optimization of amino acid replacement costs by mutational pressure in bacterial genomes

Mutations are considered a spontaneous and random process, which is important component of evolution because it generates genetic variation. On the other hand, mutations are deleterious leading to non-functional genes and energetically costly repairs. Therefore, one can expect that the mutational pressure is optimized to simultaneously generate genetic diversity and preserve genetic information. To check if empirical mutational pressures are optimized in these ways, we compared matrices of nucleotide mutation rates derived from bacterial genomes with their best possible alternatives that minimized or maximized costs of amino acid replacements associated with differences in their physicochemical properties (e.g. hydropathy and polarity). It should be noted that the studied empirical nucleotide substitution matrices and the costs of amino acid replacements are independent because these matrices were derived from sites free of selection on amino acid properties and the amino acid costs assumed only amino acid physicochemical properties without any information about mutation at the nucleotide level. Obtained results indicate that the empirical mutational matrices show a tendency to minimize costs of amino acid replacements. It implies that bacterial mutational pressures can evolve to decrease consequences of amino acid substitutions. However, the optimization is not full, which enables generation of some genetic variability.

Caenorhabditis elegans DAF-2 as a Model for Human Insulin Receptoropathies.

Human exome sequencing has dramatically increased the rate of identification of disease-associated polymorphisms. However, examining the functional consequences of those variants has created an analytic bottleneck. Insulin-like signaling in Caenorhabditis elegans has long provided a model to assess consequences of human insulin signaling mutations, but this has not been evaluated in the context of current genetic tools. We have exploited strains derived from the Million Mutation Project (MMP) and gene editing to explore further the evolutionary relationships and conservation between the human and C. elegans insulin receptors. Of 40 MMP alleles analyzed in the C. elegans insulin-like receptor gene DAF-2, 35 exhibited insulin-like signaling indistinguishable from wild-type animals, indicating tolerated mutations. Five MMP alleles proved to be novel dauer-enhancing mutations, including one new allele in the previously uncharacterized C-terminus of DAF-2. CRISPR-Cas9 genome editing was used to confirm the phenotypic consequence of six of these DAF-2 mutations and to replicate an allelic series of known human disease mutations in a highly conserved tyrosine kinase active site residue, demonstrating the utility of C. elegans for directly modeling human disease. Our results illustrate the challenges associated with prediction of the phenotypic consequences of amino acid substitutions, the value of assaying mutant isoform function in vivo, and how recently developed tools and resources afford the opportunity to expand our understanding even of highly conserved regulatory modules such as insulin signaling. This approach may prove generally useful for modeling phenotypic consequences of candidate human pathogenic mutations in conserved signaling and developmental pathways.

Homo- and hetero-dimerization of human UDP-glucuronosyltransferase 2B7 (UGT2B7) wild type and its allelic variants affect zidovudine glucuronidation activity

Most human UDP-glucuronosyltransferase (UGT; EC 2.4.1.17) genes contain non-synonymous single nucleotide polymorphisms (nsSNPs) which cause amino acid substitutions. Allelic variants caused by nsSNPs may exhibit absent or reduced enzyme activity. UGT2B7 is one of the most important UGTs that glucuronidates abundant endobiotics and xenobiotics, such as estriol, morphine, and anticancer drugs. Three nsSNPs, UGT2B7*71S (211G>T), UGT2B7*2 (802C>T) and UGT2B7*5 (1192G>A) are observed in the UGT2B7 gene, and they code for allozymes UGT2B7*71S (A71S), UGT2B7*2 (H268Y), and UGT2B7*5 (D398N). UGT2B7 has been observed to form oligomers that affect its enzymatic activity and in this study, we investigated protein–protein interactions among UGT2B7 allozymes wild type (WT), A71S, H268Y and D398N, by performing a systematic quantitative fluorescence resonance energy transfer (FRET) analysis in combination with co-immunoprecipitation assay. Quantitative FRET analysis revealed that UGT2B7 allozymes formed homo- and hetero-dimers and showed distinct features in donor–acceptor distances. Both codon 71 and codon 268 in the N-terminal domain were involved in the dimeric interaction. Co-immunoprecipitation experiments also proved that UGT2B7 allozymes formed stable dimers. The glucuronidation activities of homo- and hetero-dimers were further tested with zidovudine as the substrate. An increase in activity was observed when WT hetero-dimerized with A71S compared with homo-dimers, while both H268Y and D398N impaired the activity of WT and A71S by forming hetero-dimers. In addition, zidovudine glucuronidation activity is associated with FRET distance. These findings provide insights into the consequences of amino acid substitution in UGT2B7 on zidovudine glucuronidation and the association between protein–protein interaction and glucuronidation activity.

Prediction of the molecular consequences of aminoacid substitutions in the GALNS gene using in silico tools

Prediction of the molecular consequences of aminoacid substitutions in the GALNS gene using in silico tools

Predicting the Functional, Molecular, and Phenotypic Consequences of Amino Acid Substitutions using Hidden Markov Models

The rate at which nonsynonymous single nucleotide polymorphisms (nsSNPs) are being identified in the human genome is increasing dramatically owing to advances in whole-genome/whole-exome sequencing technologies. Automated methods capable of accurately and reliably distinguishing between pathogenic and functionally neutral nsSNPs are therefore assuming ever-increasing importance. Here, we describe the Functional Analysis Through Hidden Markov Models (FATHMM) software and server: a species-independent method with optional species-specific weightings for the prediction of the functional effects of protein missense variants. Using a model weighted for human mutations, we obtained performance accuracies that outperformed traditional prediction methods (i.e., SIFT, PolyPhen, and PANTHER) on two separate benchmarks. Furthermore, in one benchmark, we achieve performance accuracies that outperform current state-of-the-art prediction methods (i.e., SNPs&GO and MutPred). We demonstrate that FATHMM can be efficiently applied to high-throughput/large-scale human and nonhuman genome sequencing projects with the added benefit of phenotypic outcome associations. To illustrate this, we evaluated nsSNPs in wheat (Triticum spp.) to identify some of the important genetic variants responsible for the phenotypic differences introduced by intense selection during domestication. A Web-based implementation of FATHMM, including a high-throughput batch facility and a downloadable standalone package, is available at http://fathmm.biocompute.org.uk.

Glanzmann Mutations : New Allelic Variants and Role of the β3 Lys253 in the αIIb/β3 Interaction Highlighted by the Lys253Met Substitution.

Abstract 1312Poster Board I-336 IntroductionGlanzmann thrombasthenia (GT) is an autosomal recessive inherited bleeding disorder characterized by an impaired platelet aggregation. GT results from defects of the platelet fibrinogen receptor αIIbβ3. GT mutations provide useful tools for structure-function relationship studies of αIIbβ3. Patient and methodsGenomic DNA from 6 patients has been amplified for αIIb and β3 promoters and exons sequences. PCR products were directly sequenced. Potential RNA processing alterations have been studied in silico by using Genscan, NNSPLICE and ESEFinder online tools. When no RNA splicing anomaly was predicted, the effect of single point mutation on αIIbβ3 expression has been studied by using transiently transfected Cos cells. Finally, structural consequences of amino acid substitutions has been studied using the published model of αIIbβ3 (code PDB 2VDL) and structural modelling. Results7 new mutations have been characterized. 1 deletion / insertion, 2 single point mutations inducing stop codon and 1 resulting in splicing site disruption were identified. The 3 last identified single point mutations were not predicted to affect normal RNA processing but has been shown to prevent normal expression of mutant αIIbβ3 at the surface of Cos cells. The p.Meth118Arg and p.Gly221Asp substitutions that induce both important steric hindrance and charge modifications, are located inside the β-I domain of β3. So they should deeply alter the proper folding of the β-I domain, preventing the complex expression at the platelet surface. On the other hand, the p.Lys253Met protrudes from the β-I domain toward the αIIb β-propeller. A structural model of the Met253 β-I mutant has been done. An estimation of the direct electrostatic and desolvation free energies of interaction between the β-I domain surface and the αIIb β-propeller indicated that rather than the presence of a methionine, it is the lost of the Lys253 which is responsible for the complex expression defect. ConclusionSeven new GT mutations have been identified and the p.Lys253Met substitution helped to define a key role of the Lys253 in the αIIb β-propeller / β3 β-I domains interaction. DisclosuresNo relevant conflicts of interest to declare.

The C Terminus of Na+,K+-ATPase Controls Na+ Affinity on Both Sides of the Membrane through Arg935

The Na(+),K(+)-ATPase C terminus has a unique location between transmembrane segments, appearing to participate in a network of interactions. We have examined the functional consequences of amino acid substitutions in this region and deletions of the C terminus of varying lengths. Assays revealing separately the mutational effects on internally and externally facing Na(+) sites, as well as E(1)-E(2) conformational changes, have been applied. The results pinpoint the two terminal tyrosines, Tyr(1017) and Tyr(1018), as well as putative interaction partners, Arg(935) in the loop between transmembrane segments M8 and M9 and Lys(768) in transmembrane segment M5, as crucial to Na(+) activation of phosphorylation of E(1), a partial reaction reflecting Na(+) interaction on the cytoplasmic side of the membrane. Tyr(1017), Tyr(1018), and Arg(935) are furthermore indispensable to Na(+) interaction on the extracellular side of the membrane, as revealed by inability of high Na(+) concentrations to drive the transition from E(1)P to E(2)P backwards toward E(1)P and inhibit Na(+)-ATPase activity in mutants. Lys(768) is not important for Na(+) binding from the external side of the membrane but is involved in stabilization of the E(2) form. These data demonstrate that the C terminus controls Na(+) affinity on both sides of the membrane and suggest that Arg(935) constitutes an important link between the C terminus and the third Na(+) site, involving an arginine-pi stacking interaction between Arg(935) and the C-terminal tyrosines. Lys(768) may interact preferentially with the C terminus in E(1) and E(1)P forms and with the loop between transmembrane segments M6 and M7 in E(2) and E(2)P forms.

Structural consequences of amino acid substitutions causing Tay–Sachs disease

To determine the structural changes in the α-subunit of β-hexosaminidase due to amino acid substitutions causing Tay–Sachs disease, we built structural models of mutant α-subunits resulting from 33 missense mutations (24 infantile and 9 late-onset), and analyzed the influence of each amino acid replacement on the structure by calculating the number of atoms affected and determining the solvent-accessible surface area of the corresponding amino acid residue in the wild-type α-subunit. In the infantile Tay–Sachs group, the number of atoms influenced by a mutation was generally larger than that in the late-onset Tay–Sachs group in both the main chain and the side chain, and residues associated with the mutations found in the infantile Tay–Sachs group tended to be less solvent-accessible than those in the late-onset Tay–Sachs group. Furthermore, color imaging determined the distribution and degree of the structural changes caused by representative amino acid substitutions, and that there were also differences between the infantile and late-onset Tay–Sachs disease groups. Structural study is useful for elucidating the basis of Tay–Sachs disease.

Single nucleotide polymorphisms identification in expressed genes of Schistosoma mansoni

Single nucleotide polymorphism (SNP) markers have been shown to be useful in genetic investigations of medically important parasites and their hosts. In this paper, we describe the prediction and validation of SNPs in ESTs of Schistosoma mansoni. We used 107,417 public sequences of S. mansoni and identified 15,614 high-quality candidate SNPs in 12,184 contigs. The presence of predicted SNPs was observed in well characterized antigens and vaccine candidates such as those coding for myosin; Sm14 and Sm23; cathepsin B and triosephosphate isomerase (TPI). Additionally, SNPs were experimentally validated for the cathepsin B. A comparative model of the S. mansoni cathepsin B was built for predicting the possible consequences of amino acid substitutions on the protein structure. An analysis of the substitutions indicated that the amino acids were mostly located on the surface of the molecule, and we found no evidence for a significant conformational change of the enzyme. However, at least one of the substitutions could result in a structural modification of an epitope.