Conserved Amino Acid Residues Research Articles

Sequence Conservation, Domain Architectures, and Phylogenetic Distribution of the HD-GYP Type c-di-GMP Phosphodiesterases.

The HD-GYP domain, named after two of its conserved sequence motifs, was first described in 1999 as a specialized version of the widespread HD phosphohydrolase domain that had additional highly conserved amino acid residues. Domain associations of HD-GYP indicated its involvement in bacterial signal transduction and distribution patterns of this domain suggested that it could serve as a hydrolase of the bacterial second messenger c-di-GMP, in addition to or instead of the EAL domain. Subsequent studies confirmed the ability of various HD-GYP domains to hydrolyze c-di-GMP to linear pGpG and/or GMP. Certain HD-GYP-containing proteins hydrolyze another second messenger, cGAMP, and some HD-GYP domains participate in regulatory protein-protein interactions. The recently solved structures of HD-GYP domains from four distinct organisms clarified the mechanisms of c-di-GMP binding and metal-assisted hydrolysis. However, the HD-GYP domain is poorly represented in public domain databases, which causes certain confusion about its phylogenetic distribution, functions, and domain architectures. Here, we present a refined sequence model for the HD-GYP domain and describe the roles of its most conserved residues in metal and/or substrate binding. We also calculate the numbers of HD-GYPs encoded in various genomes and list the most common domain combinations involving HD-GYP, such as the RpfG (REC-HD-GYP), Bd1817 (DUF3391-HD-GYP), and PmGH (GAF-HD-GYP) protein families. We also provide the descriptions of six HD-GYP-associated domains, including four novel integral membrane sensor domains. This work is expected to stimulate studies of diverse HD-GYP-containing proteins, their N-terminal sensor domains and the signals to which they respond.

De Novo ATP1A1 Variants in an Early-Onset Complex Neurodevelopmental Syndrome.

ATP1A1 encodes the α1 subunit of the sodium-potassium ATPase, an electrogenic cation pump highly expressed in the nervous system. Pathogenic variants in other subunits of the same ATPase, encoded by ATP1A2 or ATP1A3, are associated with syndromes such as hemiplegic migraine, dystonia, or cerebellar ataxia. Worldwide, only 16 families have been reported carrying pathogenic ATP1A1 variants to date. Associated phenotypes are axonal neuropathies, spastic paraplegia, and hypomagnesemia with seizures and intellectual disability. By whole exome or genome sequencing, we identified 5 heterozygous ATP1A1 variants, c.674A>G;p.Gln225Arg, c.1003G>T;p.Gly335Cys, c.1526G>A;p.Gly509Asp, c.2152G>A;p.Gly718Ser, and c.2768T>A;p.Phe923Tyr, in 5 unrelated children with intellectual disability, spasticity, and peripheral, motor predominant neuropathy. Additional features were sensory loss, sleep disturbances, and seizures. All variants occurred de novo and are absent from control populations (MAF GnomAD = 0). Affecting conserved amino acid residues and constrained regions, all variants have high pathogenicity in silico prediction scores. In HEK cells transfected with ouabain-insensitive ATP1A1 constructs, cell viability was significantly decreased in mutants after 72h treatment with the ATPase inhibitor ouabain, demonstrating loss of ATPase function. Replicating the haploinsufficiency mechanism of disease with a gene-specific assay provides pathogenicity information and increases certainty in variant interpretation. This study further expands the genotype-phenotype spectrum of ATP1A1.

Delineation of a novel neurodevelopmental syndrome associated with PAX5 haploinsufficiency.

PAX5 is a transcription factor associated with abnormal posterior midbrain and cerebellum development in mice. PAX5 is highly loss-of-function intolerant and missense constrained, and has been identified as a candidate gene for autism spectrum disorder (ASD). We describe 16 individuals from 12 families who carry deletions involving PAX5 and surrounding genes, de novo frameshift variants that are likely to trigger nonsense-mediated mRNA decay, a rare stop-gain variant, or missense variants that affect conserved amino acid residues. Four of these individuals were published previously but without detailed clinical descriptions. All these individuals have been diagnosed with one or more neurodevelopmental phenotypes including delayed developmental milestones (DD), intellectual disability (ID), and/or ASD. Seizures were documented in four individuals. No recurrent patterns of brain magnetic resonance imaging (MRI) findings, structural birth defects, or dysmorphic features were observed. Our findings suggest that PAX5 haploinsufficiency causes a neurodevelopmental disorder whose cardinal features include DD, variable ID, and/or ASD.

Genome-wide characterization of Major Intrinsic Proteins (MIPs) family in Momordica charantia

Major intrinsic proteins (MIPs) family include the group of proteins also called channel proteins or water channels. Aquaporins facilitate the transport of water and other uncharged small molecules, forming pores in cellular boundaries which help in plant growth, development, and sometimes in plant adaptation. They usually have six tranSmembrane alpha-helical domains with conserved amino acids residues along with hydrophobic and hydrophilic regions. Though aquaporins were found both in animals and plants, also in bacteria, fungi cells, it comprises the largest and most complex group in plant species. A little is known about the aquaporins family of the plant Momordica charantia. About 25 aquaporins of its are characterized in this study. The aquaporins of M. charantia are divided into five sub-families here, they are PIPs, TIPs, XIPs, SIPs, and NIPs. There are twelve PIPs, three TIPs, eight NIPs, and one AQPs in both XIP and SIP. In the XIP subfamily, it contains SPI other than the NPA motif. Moreover, SIP and NIP subfamily also contain different motifs both in loop B and loop E other than NPA motifs. McSIPs contain NPT in loop B and McNIPs contain NPS in loop B (McNIP1; 1, McNIP1; 3 and McNIP3; 1). Again, McNIPs contain NPV in loop E (McNIP2; 2, McNIP3; 1). The conserved residues and motifs of this plant show its transportation of water and the possibility of other molecules' transportation through those channel proteins.

Molecular characterization and differential expression of an aromatic heptaketide producing type III plant polyketide synthase from Himalayan rhubarb

Rheum australe (Himalayan Rhubarb, Polygonaceae), an endangered medicinal and vegetable herb owes its age-old remedying properties to the bio-active phyto-constituents viz. anthraquinones, stilbenoids, chromones and dietary flavonoids. Polyketide pathway primarily involving the intricate Type III polyketide synthases (PKSs) contributes to the biosynthesis of these phyto-constituents. In the present study, we perform a homology-based approach to isolate an 1176 bp full-length cds sequence of the RaALS gene showing an equitable level of sequence similarity to related Type III PKSs at both nucleic acid and amino acid levels. In silico characterization revealed the presence of highly conserved amino acid residues found in nearly all Type III PKSs including the conserved active-site residues, signature motif and cyclization pocket residues with an exception of Ile256 and Gly258. Docking studies established major interactions between the starter acetyl-CoA and RaALS. Copy number analysis suggested slender evolution in Type III PKS in R. australe having a single copy of RaALS gene. qRT-PCR analyses revealed corroboration between the higher expression of RaALS in leaves followed by stem and root with that of the metabolite concentration. Expression studies further showed a direct increase of RaALS transcripts with the growing metabolite accretion in relation to altitude suggesting a probable involvement of specific Type III PKS in biosynthesis of the major phyto-constituents. Furthermore, abiotic stressors viz. methyl jasmonate, salicylic acid and UV light enhanced RaALS transcription hinting towards its role in defense mechanism in R. australe and highlighting the significance of RaALS as a prospective target for metabolic engineering.

Molecular and kinetic characterization of two acetylcholinesterases with particular focus on the roles of two amino acid substitutions (Y390N and F392W) in Bemisia tabaci

Two acetylcholinesterases (AChEs) are present in Bemisia tabaci (BtAChE1 and BtAChE2). A conserved AChE mutation conferring organophosphate (OP) resistance (F392W in BtAChE1) is saturated in field populations despite its potential fitness cost, and a highly conserved amino acid residue forming the backdoor of AChE is substituted with a unique amino acid (Y390N in BtAChE1) in B. tabaci. Thus, the roles and relationships of the two amino acid substitutions in the evolutionary adaptation of B. tabaci remain to be elucidated, and little information is available on the catalytic and molecular properties of BtAChE1 and BtAChE2. To determine which AChE is a more relevant target of OPs and carbamates, the molecular and kinetic properties of BtAChE1 and BtAChE2 were investigated. Both BtAChE1 and BtAChE2 were exclusively expressed in head and thorax but not in abdomen, bound to the membrane via GPI anchoring, and present as dimeric forms. Soluble monomeric form was detected only in BtAChE2. The catalytic activity of baculovirus-expressed BtAChE1 was 19.5-fold higher than that of BtAChE2. The inhibition assay revealed that the F392W mutation in BtAChE1 enhanced resistance to OPs. The artificial substitution of N390 (wild form) to Y (putative ancient form) led to reduced catalytic efficiency and increased inhibition by glycoalkaloids, suggesting that the Y390N substitution in BtAChE1 may have been required for Solanaceae host adaptation. BtAChE1 was proven to function as a main catalytic enzyme for ACh hydrolysis, thus being the main target of OPs and carbamates.

RAD gene family analysis in cotton provides some key genes for flowering and stress tolerance in upland cotton G. hirsutum

BackgroundRADIALIS (RAD), belongs to the MYB gene family and regulates a variety of functions including floral dorsoventral asymmetry in Antirrhinum majus and development of fruit proteins in Solanum lycopersicum. RAD genes contain an SNF2_N superfamily domain. Here, we comprehensively identified 68 RAD genes from six different species including Arabidopsis and five species of cotton.ResultsPhylogenetic analysis classified RAD genes into five groups. Gene structure, protein motifs and conserved amino acid residues indicated that GhRAD genes were highly conserved during the evolutionary process. Chromosomal location information showed that GhRAD genes were distributed unevenly on different chromosomes. Collinearity and selection pressure analysis indicated RAD gene family expansion in G. hirsutum and G. barbadense with purifying selection pressure. Further, various growth and stress related promotor cis-acting elements were observed. Tissue specific expression level indicated that most GhRAD genes were highly expressed in roots and flowers (GhRAD2, GhRAD3, GhRAD4 and GhRAD11). Next, GhRAD genes were regulated by phytohormonal stresses (JA, BL and IAA). Moreover, Ghi-miRN1496, Ghi-miR1440, Ghi-miR2111b, Ghi-miR2950a, Ghi-miR390a, Ghi-miR390b and Ghi-miR7495 were the miRNAs targeting most of GhRAD genes.ConclusionsOur study revealed that RAD genes are evolutionary conserved and might be involved in different developmental processes and hormonal stress response. Data presented in our study could be used as the basis for future studies of RAD genes in cotton.

Effect of mutations in the 2b protein of tomato aspermy virus on RNA silencing suppressor activity, virulence, and virus-induced gene silencing.

Tomato aspermy virus (TAV) and cucumber mosaic virus (CMV) belong to the genus Cucumovirus in the family Bromoviridae. The cucumovirus 2b protein is one of the first identified viral suppressors of RNA silencing (VSR). The cucumovirus 2b protein contains a conserved amino acid motif consisting of several highly conserved amino acid residues. Here, we demonstrate that the TAV 2b protein N-terminal region, Arg46, Ser40, and Ser42 as well as the CMV 2b protein are essential for VSR activity, virulence, and viral RNA accumulation. Furthermore, we developed the first TAV-induced gene silencing (VIGS) vector. In contrast to other cucumovirus vectors, such as CMV and peanut stunt virus, the TAV vector did not induce a silencing phenotype in Nicotiana benthamiana when 2b protein VSR activity was retained. These findings suggest that the cucumovirus 2b proteins share amino acid residues for VSR activity but may have different roles in VIGS induction.

Biochemical characterization and insecticidal activity of isolated peptides from the venom of the scorpion Centruroides tecomanus

The venom of scorpions is a mixture of components that constitute a source of bioactive molecules. The venom of the scorpion Centruroides tecomanus contains peptides toxic to insects, however, to date no toxin responsible for this activity has yet been isolated and fully characterized. This communication describes two new peptides Ct-IT1 and Ct-IT2 purified from this scorpion. Both peptides contain 63 amino acids with molecular weight 6857.85 for Ct-IT1 and 6987.77 Da for Ct-IT2. The soluble venom was separated using chromatographic techniques of molecular size exclusion, cationic exchange, and reverse phase chromatography, allowing the identification of at least 99 components of which in 53 the insecticidal activity was evaluated. The LD50 determined for Ct-IT1 is 3.81 μg/100 mg of cricket weight, but low amounts of peptides (0.8 μg of peptide) already cause paralysis in crickets. The relative abundance of these two peptides in the venom is 2.1% for Ct-IT1 and 1% for Ct-IT2. The molecular masses and N-terminal sequences of both insecticidal toxins were determined by mass spectrometry and Edman degradation. The primary structure of both toxins was compared with other known peptides isolated from other scorpion venoms. The analysis of the sequence alignments revealed the position of a highly conserved amino acid residue, Gly39, exclusively present in anti-insect selective depressant β-toxins (DBTXs), which in Ct-IT1 and Ct-IT2 is at position Gly40. Similarly, a three-dimensional structure of this toxins was obtained by homology modeling and compared to the structure of known insect toxins of scorpions. An important similarity of the cavity formed by the trapping apparatus region of the depressant toxin LqhIT2, isolated from the scorpion Leiurus quinquestriatus hebraeus, was found in the toxins described here. These results indicate that Ct-IT1 and Ct-IT2 toxins have a high potential to be evaluated on pests that affect economically important crops to eventually consider them as a potential biological control method.

NPTX1 mutations trigger endoplasmic reticulum stress and cause autosomal dominant cerebellar ataxia.

With more than 40 causative genes identified so far, autosomal dominant cerebellar ataxias exhibit a remarkable genetic heterogeneity. Yet, half the patients are lacking a molecular diagnosis. In a large family with nine sampled affected members, we performed exome sequencing combined with whole-genome linkage analysis. We identified a missense variant in NPTX1, NM_002522.3:c.1165G>A: p.G389R, segregating with the phenotype. Further investigations with whole-exome sequencing and an amplicon-based panel identified four additional unrelated families segregating the same variant, for whom a common founder effect could be excluded. A second missense variant, NM_002522.3:c.980A>G: p.E327G, was identified in a fifth familial case. The NPTX1-associated phenotype consists of a late-onset, slowly progressive, cerebellar ataxia, with downbeat nystagmus, cognitive impairment reminiscent of cerebellar cognitive affective syndrome, myoclonic tremor and mild cerebellar vermian atrophy on brain imaging. NPTX1 encodes the neuronal pentraxin 1, a secreted protein with various cellular and synaptic functions. Both variants affect conserved amino acid residues and are extremely rare or absent from public databases. In COS7 cells, overexpression of both neuronal pentraxin 1 variants altered endoplasmic reticulum morphology and induced ATF6-mediated endoplasmic reticulum stress, associated with cytotoxicity. In addition, the p.E327G variant abolished neuronal pentraxin 1 secretion, as well as its capacity to form a high molecular weight complex with the wild-type protein. Co-immunoprecipitation experiments coupled with mass spectrometry analysis demonstrated abnormal interactions of this variant with the cytoskeleton. In agreement with these observations, in silico modelling of the neuronal pentraxin 1 complex evidenced a destabilizing effect for the p.E327G substitution, located at the interface between monomers. On the contrary, the p.G389 residue, located at the protein surface, had no predictable effect on the complex stability. Our results establish NPTX1 as a new causative gene in autosomal dominant cerebellar ataxias. We suggest that variants in NPTX1 can lead to cerebellar ataxia due to endoplasmic reticulum stress, mediated by ATF6, and associated to a destabilization of NP1 polymers in a dominant-negative manner for one of the variants.

Effect of New Thalidomide Analogs in Acute Kidney Injury on Rats

Renal ischemia/reperfusion (I/R) injury contains multiple mechanisms involving an excessive amount of ROS that causes oxidative stress, inflammation, and rapid kidney dysfunction. This work aimed to study the ability of thalixisostere 3f on angiogenesis and antioxidant effect in I/R. There was a significant decrease in VEGF from thalix and modified thalixisostere 3f groups as compared with that of a negative control group (P < 0.001). There was a significant decrease in the mean concentration of MDA in kidney tissue obtained from thalix and thalixisostere 3f groups as compared with that of a positive control group (P < 0.001). There was a significant increase in the mean concentrations of SOD and GSH in kidney tissue of group's thalix and thalixisostere 3f groups as compared with that of a positive control group. We found that thalixisostere 3f is more effective than thalix in inhibiting the expression VEGF as pro-angiogenic factors. Molecular docking study indicated that the proper recognition of the inhibitor thalix-isostere with the conserved amino acid residues at the binding active site of VEGFR2 as one of the important enzymes to be targeted as part of antiangiogenic anticancer.

Deep Conservation of Hid-Like RHG Gene Family Homologs in Winged Insects Revealed by "Taxon Hopping" BLAST.

Together with sickle (skl), the Drosophila paralogs reaper (rpr), head involution defective (hid), and grim (RHG) control a critical switch in the induction of programmed cell death. RHG homologs have been identified in other dipteran and lepidopteran species but not beyond. Revisiting this issue with a "taxon hopping" BLAST search strategy in current genome and transcriptome resources, I detected high confidence RHG homologs in Coleoptera, Hymenoptera, Hemiptera, and Dictyoptera. Analyses of gene structure and protein sequence conservation revealed aconserved splicing pattern and highly conserved amino acid residues at both the N- and C-terminal ends that identify hid as the most ancestrally organized RHG gene family member in Drosophila. hid-like RHG homologs were also detected in mosquitoes, redefining their michelob_x (mx) genes as an expansion of derived RHG homologs. Only singleton homologs were detected in the large majority of other insect clades. Lepidopteran RHG homologs, however, stand out by producing an evolutionarily-derived splice isoform, identified in previous work, in addition to the newly detected hid-like isoform. Exceptional sequence diversification of select RHG homologs at the family- and genus-level explain their previous elusiveness in important insect genome model species like the red flour beetle Tribolium castaneum and the pea aphid Acyrthosiphon pisum. Combined, these findings expand the minimal age of the RHG gene family by about 100 million years and open new avenues for molecular cell death studies in insects.

Catalytic Fields as a Tool to Analyze Enzyme Reaction Mechanism Variants and Reaction Steps.

Catalytic fields representing the topology of the optimal molecular environment charge distribution that reduces the activation barrier have been used to examine alternative reaction variants and to determine the role of conserved catalytic residues for two consecutive reactions catalyzed by the same enzyme. Until now, most experimental and conventional top-down theoretical studies employing QM/MM or ONIOM methods have focused on the role of enzyme electric fields acting on broken bonds of reactants. In contrast, our bottom-up approach dealing with a small reactant and transition-state model allows the analysis of the opposite effects: how the catalytic field resulting from the charge redistribution during the enzyme reaction acts on conserved amino acid residues and contributes to the reduction of the activation barrier. This approach has been applied to the family of histidyl tRNA synthetases involved in the translation of the genetic code into the protein amino acid sequence. Activation energy changes related to conserved charged amino acid residues for 12 histidyl tRNA synthetases from different biological species allowed to compare on equal footing the catalytic residues involved in ATP aminoacylation and tRNA charging reactions and to analyze different reaction mechanisms proposed in the literature. A scan of the library of atomic multipoles for amino acid side-chain rotamers within the catalytic field pointed out the change in the Glu83 conformation as the critical catalytic effect, providing, at low computational cost, insight into the electrostatic preorganization of the enzyme catalytic site at a level of detail that has not yet been accessible in conventional experimental or theoretical methods. This opens the way for rational reverse biocatalyst design at a very limited computational cost without resorting to empirical methods.

Identification of damaging SNPs and their effects on Alzheimer’s disease-associated PSEN1 protein: Computational analysis

Alzheimer’s Disease (AD) is a progressive neurodegenerative disease and pathologically characterized by the presence of neurofibrillary tangles (tau aggregation) and amyloid plaques (amyloid-beta (A𝛽) aggregation). PSEN1 protein with 9 transmembrane helices acts as aspartyl protease and is one of the catalytic components of γ secretase complex, that cleaves amyloid precursor protein (APP). Furthermore, PSEN1 protein plays a significant role in the process of APP and in the generation of amyloid beta (Aβ). In the present study, it was aimed to estimate the probable deleterious effects of missense SNPs in PSEN1 gene that is associated with AD on protein stability and structure by using bioinformatics tools. SIFT, PolyPhen-2, PROVEAN, PhD-SNP, and PANTHER PSEP software were used to estimate the deleterious SNPs, whereas I-Mutant 3.0 and MUpro web tools were used to determine the effects of amino acid substitution on protein stability. Additionally, the effects of wild type and mutant amino acids on protein three-dimensional structure via modeling were predicted by Project HOPE webserver. The phylogenetic conservation of amino acid residues of PSEN1 protein was analyzed by ConSurf. In total, 386 missense SNPs were found in the human PSEN1 gene from the National Center for Biotechnology Information Single Nucleotide Polymorphism (NCBI dbSNP) database and 65 SNPs of which were determined to be deleterious or damaging. In the present study, 8 significant missense SNPs- rs63749891 (R278T), rs63750301 (P264L), rs63750353 (N135D), rs63750524 (R278S), rs63750772 (E273A), rs63751229 (P267S), rs121917807 (G266S), and rs201617677 (R157S)- were determined as high-risk pathogenic. Some differences between wild-type amino acids and mutant amino acids such as hydrophobicity, charge, size, and folding properties were determined according to the modeling findings. Our study demonstrates that high-risk pathogenic missense SNPs have the potential to alter the catalytic activity of the γ secretase complex and subsequently the amount of Aβ40 and Aβ42. Therefore, these missense SNPs may contribute to AD pathogenesis studies.

Identification and characterization of histones in Physarum polycephalum evidence a phylogenetic vicinity of Mycetozoans to the animal kingdom.

Physarum polycephalum belongs to Mycetozoans, a phylogenetic clade apart from the animal, plant and fungus kingdoms. Histones are nuclear proteins involved in genome organization and regulation and are among the most evolutionary conserved proteins within eukaryotes. Therefore, this raises the question of their conservation in Physarum and the position of this organism within the eukaryotic phylogenic tree based on histone sequences. We carried out a comprehensive study of histones in Physarum polycephalum using genomic, transcriptomic and molecular data. Our results allowed to identify the different isoforms of the core histones H2A, H2B, H3 and H4 which exhibit strong conservation of amino acid residues previously identified as subject to post-translational modifications. Furthermore, we also identified the linker histone H1, the most divergent histone, and characterized a large number of its PTMs by mass spectrometry. We also performed an in-depth investigation of histone genes and transcript structures. Histone proteins are highly conserved in Physarum and their characterization will contribute to a better understanding of the polyphyletic Mycetozoan group. Our data reinforce that P. polycephalum is evolutionary closer to animals than plants and located at the crown of the eukaryotic tree. Our study provides new insights in the evolutionary history of Physarum and eukaryote lineages.

Molecular Basis of IgE-Mediated Shrimp Allergy and Heat Desensitization.

Crustacean allergy, especially to shrimp, is the most predominant cause of seafood allergy. However, due to the high flexibility of immunoglobulin E (IgE), its three-dimensional structure remains unsolved, and the molecular mechanism of shrimp allergen recognition is unknown. Here a chimeric IgE was built in silico, and its variable region in the light chain was replaced with sequences derived from shrimp tropomyosin (TM)-allergic patients. A variety of allergenic peptides from the Chinese shrimp TM were built, treated with heating, and subjected to IgE binding in silico. Amino acid analysis shows that the amino acid residue conservation in shrimp TM contributes to eliciting an IgE-mediated immune response. In the shrimp-allergic IgE, Glu98 in the light chain and other critical residues that recognize allergens from shrimp are implicated in the molecular basis of IgE-mediated shrimp allergy. Heat treatment could alter the conformations of TM allergenic peptides, impact their intramolecular hydrogen bonding, and subsequently decrease the binding between these peptides and IgE. We found Glu98 as the characteristic amino acid residue in the light chain of IgE to recognize general shrimp-allergic sequences, and heat-induced conformational change generally desensitizes shrimp allergens.

Genetic analysis in Chinese patients with familial or young-onset amyotrophic lateral sclerosis.

The aim of our study was to investigate the genetic characteristics in patients with familial or young-onset amyotrophic lateral sclerosis (ALS) in a Chinese center. Patients with familial or young-onset (age of onset < 45years old) ALS were reviewed. The clinical data was collected. Whole-exome sequencing was performed to identify the disease-associated variants. Single-nucleotide variants and small insertions/deletions were further predicted with silico tools and compared to the Single Nucleotide Polymorphism Database, Exome Aggregation Consortium, and the 1000 Genomes Project. The evolutionary conservations were estimated, and the structures of proteins were constructed by Swiss-Model server. Immunohistochemistry was used to confirm the misfolded SOD1 protein. Three familial ALS and 5 young-onset ALS were enrolled. Genetic analysis identified related variants of SOD1 (4/6, 66.7%), FUS (1/6, 16.7%), and NEK1 (1/6, 16.7%) in 6 patients. Three of them were familial probands (3/3, 100%), and the others were sporadic young-onset patients (3/5, 60%). NEK1 c.290G > A mutation (NM_012224.2 exon4) in a patient with familial ALS and SOD1 c.362A > G mutation (NM_000454 exon5) in a young-onset ALS patient were novel. The novel mutations were predicted to be deleterious, affected evolutionarily highly conserved amino acid residue and the formation of hydrogen bonds between the mutated site and its surrounding amino acid residues. Misfolded SOD1 protein was identified in patient with SOD1 c.362A > G mutation. Two novel mutations were detected in our patients. Patients with familial or young-onset ALS often carried related gene mutations, and genetic sequencing should be thus routinely performed.

Methylation of PhoP by CheR Regulates Salmonella Virulence.

ABSTRACTThe two-component system PhoP/PhoQ is essential for Salmonella enterica serovar Typhimurium virulence. Here, we report that PhoP is methylated extensively. Two consecutive glutamate (E) and aspartate (D)/E residues, i.e., E8/D9 and E107/E108, and arginine (R) 112 can be methylated. Individual mutation of these above-mentioned residues caused impaired phosphorylation and dimerization or DNA-binding ability of PhoP to a different extent and led to attenuated bacterial virulence. With the help of specific antibodies recognizing methylated E8 and monomethylated R112, we found that the methylation levels of E8 or R112 decreased dramatically when bacteria encountered low magnesium, acidic pH, or phagocytosis by macrophages, under which PhoP can be activated. Furthermore, CheR, a bacterial chemotaxis methyltransferase, was identified to methylate R112. Overexpression of cheR decreased PhoP activity but increased PhoP stability. Together, the current study reveals that methylation plays an important role in regulating PhoP activities in response to environmental cues and, consequently, modulates Salmonella virulence.

Identification and Analysis of GhEXO Gene Family Indicated That GhEXO7_At Promotes Plant Growth and Development Through Brassinosteroid Signaling in Cotton (Gossypium hirsutum L.)

Brassinosteroids (BRs), an efficient plant endogenous hormone, significantly promotes plant nutrient growth adapting to biological and abiotic adversities. BRs mainly promote plant cell elongation by regulating gene expression patterns. EXORDIUM (EXO) genes have been characterized as the indicators of BR response genes. Cotton, an ancient crop, is of great economic value and its fibers can be made into all kinds of fabrics. However, EXO gene family genes have not been full identified in cotton. 175 EXO genes were identified in nine plant species, of which 39 GhEXO genes in Gossypium hirsutum in our study. A phylogenetic analysis grouped all of the proteins encoded by the EXO genes into five major clades. Sequence identification of conserved amino acid residues among monocotyledonous and dicotyledonous species showed a high level of conservation across the N and C terminal regions. Only 25% the GhEXO genes contain introns besides conserved gene structure and protein motifs distribution. The 39 GhEXO genes were unevenly distributed on the 18 At and Dt sub-genome chromosomes. Most of the GhEXO genes were derived from gene duplication events, while only three genes showed evidence of tandem duplication. Homologous locus relationships showed that 15 GhEXO genes are located on collinear blocks and that all orthologous/paralogous gene pairs had Ka > Ks values, indicating purifying selection pressure. The GhEXO genes showed ubiquitous expression in all eight tested cotton tissues and following exposure to three phytohormones, IAA, GA, and BL. Furthermore, GhEXO7_At was mainly expressed in response to BL treatment, and was predominantly expressed in the fibers. GhEXO7_At was found to be a plasma membrane protein, and its ectopic expression in Arabidopsis mediated BR-regulated plant growth and development with altered expression of DWF4, CPD, KCS1, and EXP5. Additionally, the functions of GhEXO7_At were confirmed by virus-induced gene silencing (VIGS) in cotton. This study will provide important genetic resources for future cotton breeding programs.

Bioinformatics and 3D homology modelling of AsAlaDH from Amycolatopsis sulphurea

Alanine dehydrogenase (AlaDH) (E.C.1.4.1.1) is an enzyme that catalyzes the interconversion of pyruvate and alanine. This enzyme has the key catalytic role for the sporulation of microorganism and synthesis of the many amino acids, proteins, and peptidoglycan layers in the microorganisms. Amycolatopsis sulphurea one of the strains of Amycolatopsis genus within the family Pseudonocardiaceae has capable to produce different antibiotics such as Ristocetin, Vancomycin, and Epoxyquinomicin as well as to biodegrade the bioplastic (poly-lactic acid (PLA) films). The 3D homology model of Alanine dehydrogenase from Amycolatopsis sulphurea was carried out through I-TASSER. The interaction of L-alanine and active site amino acids of the enzyme was determined by docking in silico via AutoDock Vina program. Protein secondary structures were predicted with EMBOSS tool garnier. Structural and functional analysis and determination of Physico-chemical properties of AsAlaDH were performed by using different bioinformatics tools. The secondary structure and multiple alignment analysis of alanine dehydrogenase displayed that there are conserved amino acid residues of AlaDH's from different microorganisms.