Amino Acid Variations Research Articles

Insight into the function of voltage-sensing phosphatase in hindgut-derived pseudoplacenta of a viviparous teleost Xenotoca eiseni.

Nutrient absorption is essential for animal survival and development. Our previous study on zebrafish reported that nutrient absorption in lysosome-rich enterocytes (LREs) is promoted by the voltage-sensing phosphatase (VSP), which regulates phosphoinositide (PIP) homeostasis via electrical signaling in biological membranes. However, it remains unknown whether this VSP function is shared by different absorptive tissues in other species. Here, we focused on the function of VSP in a viviparous teleost Xenotoca eiseni, whose intraovarian embryos absorb nutrients from the maternal ovarian fluid through a specialized hindgut-derived pseudoplacental structure called trophotaenia. Xenotoca eiseni VSP (Xe-VSP) is expressed in trophotaenia epithelium, an absorptive tissue functionally similar to zebrafish LREs. Notably, the apical distribution of Xe-VSP in trophotaenia epithelial cells closely resembles zebrafish VSP (Dr-VSP) distribution in zebrafish LREs, suggesting a shared role for VSP in absorptive tissues between the two species. Electrophysiological analysis using a heterologous expression system revealed that Xe-VSP preserves functional voltage sensors and phosphatase activity with the leftward shifted voltage sensitivity compared with zebrafish VSP (Dr-VSP). We also identified a single amino acid variation in the S4 helix of Xe-VSP as one of the factors contributing to the leftward shifted voltage sensitivity. This study highlights the biological variation and significance of VSP in various animal species, as well as hinting at the potential role of VSP in nutrient absorption in X. eiseni trophotaenia.NEW & NOTEWORTHY We investigate the voltage-sensing phosphatase (VSP) in Xenotoca eiseni, a viviparous fish whose intraovarian embryos utilize trophotaenia for nutrient absorption. Although X. eiseni VSP (Xe-VSP) shares key features with known VSPs, its distinct voltage sensitivity arises from species-specific amino acid variation. Xe-VSP in trophotaenia epithelium suggests its involvement in nutrient absorption, similar to VSP in zebrafish enterocytes and potentially in species with similar absorptive cells. Our findings highlight the potential role of VSP across species.

Genetic variants, haplotype determination, and function of novel alleles of CYP2B6 in a Han Chinese population

Amino acid variants in protein may result in deleterious effects on enzymatic activity. In this study we investigate the DNA variants on activity of CYP2B6 gene in a Chinese Han population for potential use in precision medicine. All exons in CYP2B6 gene from 1483 Chinese Han adults (Zhejiang province) were sequenced using Sanger sequencing. The effects of nonsynonymous variants on recombinant protein catalytic activity were investigated in vitro with Sf12 system. The haplotype of novel nonsynonymous variants with other single nucleotide variants in the same allele was determined using Nanopore sequencing. Of 38 alleles listed on the Pharmacogene Variation Consortium, we detected 7 previously reported alleles and 18 novel variants, of which 11 nonsynonymous variants showed lower catalytic activity (0.00–0.60) on bupropion compared to CYP2B6*1. Further, these 11 novel star-alleles (CYP2B6*39–49) were assigned by the Pharmacogene Variation Consortium, which may be valuable for pharmacogenetic research and personalized medicine.

Quantitative trait loci identification reveals zinc finger protein CONSTANS-LIKE 4 as the key candidate gene of stigma color in watermelon (Citrullus lanatus)

Stigma color is a critical agronomic trait in watermelon that plays an important role in pollination. However, there are few reports on the regulation of stigma color in watermelon. In this study, a genetic analysis of the F2 population derived from ZXG1553 (P1, with orange stigma) and W1-17 (P2, with yellow stigma) indicated that stigma color is a quantitative trait and the orange stigma is recessive compared with the yellow stigma. Bulk segregant analysis sequencing (BSA-seq) revealed a 3.75 Mb segment on chromosome 6 that is related to stigma color. Also, a major stable effective QTL Clqsc6.1 (QTL stigma color) was detected in two years between cleaved amplified polymorphic sequencing (CAPS) markers Chr06_8338913 and Chr06_9344593 spanning a ~1.01 Mb interval that harbors 51 annotated genes. Cla97C06G117020 (annotated as zinc finger protein CONSTANS-LIKE 4) was identified as the best candidate gene for the stigma color trait through RNA-seq, quantitative real-time PCR (qRT-PCR), and gene structure alignment analysis among the natural watermelon panel. The expression level of Cla97C06G117020 in the orange stigma accession was lower than in the yellow stigma accessions with a significant difference. A nonsynonymous SNP site of the Cla97C06G117020 coding region that causes amino acid variation was related to the stigma color variation among nine watermelon accessions according to their re-sequencing data. Stigma color formation is often related to carotenoids, and we also found that the expression trend of ClCHYB (annotated as β-carotene hydroxylase) in the carotenoid metabolic pathway was consistent with Cla97C06G117020, and it was expressed in low amounts in the orange stigma accession. These data indicated that Cla97C06G117020 and ClCHYB may interact to form the stigma color. This study provides a theoretical basis for gene fine mapping and mechanisms for the regulation of stigma color in watermelon.

Abstract 631: Functional genetics of APR-246 rescuable TP53 mutants

Abstract TP53, often referred to as the guardian of the genome, encodes for the p53 tumor suppressor protein, and is one of the most frequently mutated genes in human cancers. Some cancer sub-types, such as non-small cell lung carcinoma (NSCLC) and high grade serous carcinoma (HGSC), have high TP53 mutation rates reaching up to 56% and 96%, respectively. Given its multi-faceted role in human cancers, there have been several emerging therapeutic approaches targeting mutant p53. APR-246 is a small molecule drug currently in clinical trials for the functional rescue of p53 missense mutants, the most common type of p53 mutant protein, across various cancer types. APR-246 is proposed to bind mutant p53 and promote thermodynamic stabilization of the protein allowing it to regain wild-type-like conformation and consequentially regain WT-like functionality. However, there has yet to be a functional genomic screening of p53 mutants to systematically catalog which mutants are rescuable by APR-246. To address this critical gap, we have performed functional genetic screenings using a TP53 mutagenesis library. This pooled lentiviral library contains ~8,000 amino acid variants, spanning the entirety of the TP53 gene. Following stable transduction of this library into p53-null NSCLC and HGSOC cell lines, we performed functional screenings against sublethal doses of APR-246. Next-generation sequencing of the untreated and treated cell population allows for comparison of variant representation and determination of APR-246-sensitivity of p53 mutants. Preliminary statistical analysis of our screens has identified a pool of 117 “hit” mutants which are significantly depleted following treatment, indicative of APR-246-sensitivity. Two of these mutants, C242F and G266V, are non-functional, damaging mutations which occur in patients. Identifying p53 mutants that can be rescued by APR-246 will help identify which patients may benefit most from treatment with the drug. Citation Format: Anais Saunders, Caili Tong, Anthony N. Karnezis, Gary S. Leiserowitz, Jeremy R. Chein. Functional genetics of APR-246 rescuable TP53 mutants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 631.

Abstract 4890: In silico study of enzymes involved in heparan sulfate catabolic process in cancer resistant mammals

Abstract Some mammals such as elephants, bats, whales, and blind mole rats (Spalax) are resistant to cancer. One of the cancer protective mechanisms present in such species includes a reduced degradation of heparan sulfate (HS), an important component of the extracellular matrix (ECM), present in Spalax. Such effect occurs due to a splicing variant of heparanase, called variant 36, that lacks enzymatic activity. Consequently, both HS and ECM are very stable in Spalax’s tissues. If HS is stabilized, in cancer resistant mammals, due to the decrease of heparanase or other enzymes involved in HS catabolic process is unknown. We studied total of 9 enzymes (HPSE2, GusB, IDUA, IDS, NAGLU, SGSH, GNS, Sulf-2, HGSNAT) that regulate HS catabolism and homologs in 9 mammals. Multiple sequence alignments were done by MEGA to identify substitutions present in cancer resistant species in comparison with cancer susceptible animals. Such amino acid variants were further analyzed and both their energy stability and their functional effects were predicted by using online in silico tools including MAESTROweb, Condel, PANTHER, REVEL, and CADD. Finally, homologs with deleterious predicted variants were modelled with SWISSMODEL and their 3D structures were analyzed with Pymol and ChimeraX to identify structural features likely related to its loss of function. We found two loss of function missense variants, K264Q and W626L, in Alpha-L-iduronidase (IDUA) of Spalax. In addition, most of Brandt's bat enzymes and the N-sulphoglucosamine sulphohydrolase (SGSH) of the bowhead whale were very energy destabilizing enzymes with positive and higher ΔΔG values compared to human homologs. In the other hand, although some catalytic residues in the study enzymes are conserved in cancer resistant animals, their spatial arrangement and conformation in the 3D structure are different compared to human homologs, including E182 of IDUA which is one of the catalytic residues. In general, the secondary structure is conserved across the 9 enzymes studied but some differences were observed in cancer resistant animals including one of the beta strands near the catalytic domain in human and mice IDUA that appears as a loop in Spalax. In summary, HS stabilization could raise in cancer resistant mammals through the existence of lack of enzymatic activity not only in heparanase but also in other enzymes that participate in HS catabolic process, via a set of loss of function variants and changes in secondary and tertiary structure as well as a reduction of energy stabilization. More interestingly, such mechanism could be present in other cancer resistant mammals such as bats and bowhead whales. This study demonstrates that computational approaches are useful to study biological mechanisms of cancer resistance present in some mammals. This knowledge could be translated to humans and HS catabolic process could offer a new source of therapeutic anticancer targets. Citation Format: Fabrizio Oliveri Payet, Claudia Machicado Rivero. In silico study of enzymes involved in heparan sulfate catabolic process in cancer resistant mammals [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4890.

Abstract 5292: CD28 humanized mouse model for efficacy and safety assessment of CD28-targeting therapies

Abstract Agonist antibodies targeting CD28 have proven to be effective against cancer, but also faced challenges due to severe adverse events triggered by its activation. Human (hCD28) and mouse CD28 (mCD28) have different signaling responses, with CD28 ligands and superagonists inducing pro-inflammatory cytokines upon stimulation in absence of TCR ligation in humans, but not in mice. Expression of CD28i amplifier isoform, which is thought to enhanced the production of cytokines in humans, could partially explain this difference, as it is not expressed in mice. In addition, evidence suggests that the different signaling between hCD28 and mCD28 relies on one amino acid change in the intracellular domain (ICD)[1]. Herein, we describe a CD28 humanized mouse model for assessment of CD28-targeting agents. To improve translatability, we decided to keep the expression of both canonical and CD28i human isoforms to avoid undermining the biological effects of the testing agents. Although keeping the human ICD could favor the evaluation of cytokine production and therefore the safety of the test agents, we decided to keep the mouse ICD to enable a proper interaction of CD28 with its signaling partners, allowing a physiological stimulation of CD28 in efficacy studies. The hCD28 model expresses a chimeric protein, with the whole human extracellular domain, and mouse intracellular domain. Human CD28 is expressed at physiological levels on T cells, and these cells can be activated and proliferate in response to hCD28 ligands and agonist antibodies. Tumor growth inhibition and complete tumor regressions are achieved in a MC38-TAA colorectal cancer model following prophylactic treatment with CD28-TAA bispecific antibody in combination with anti-PD-1. Extension of this study and re-challenged of the tumor-free surviving animals shows that re-challenged hCD28 mice have enhanced survival compared to naïve hCD28 mice inoculated with MC38-TAA, suggesting tumor immunological memory. Furthermore, TGN1412 administration in CD28 mice induces cytokine release, and a slightly body weight loss, but no major change in body temperature. Although the model does not fully reproduce the ability of human CD28 to produce cytokines upon activation due to the mouse ICD, the expression of the amplifier isoform CD28i might contribute to the response to TGN1412. Altogether, data suggest that hCD28 model enables assessment of efficacy and safety of CD28-targeting agents. The hCD28 model was intercrossed with CD3 humanized models to enable assessment of combination therapies and bispecific antibodies targeting both CD3 and CD28. [1] Porciello N, Grazioli P, Campese AF, et al. A non-conserved amino acid variant regulates differential signalling between human and mouse CD28. Nat Commun 2018; 9:1-16 Citation Format: Fabiane Sonego, Angela Pappalardo, Gaëlle Martin, Yacine Cherifi, Patricia Isnard-Petit, Kader Thiam. CD28 humanized mouse model for efficacy and safety assessment of CD28-targeting therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5292.

Mutational scanning pinpoints distinct binding sites of key ATGL regulators in lipolysis

ATGL is a key enzyme in intracellular lipolysis and plays an important role in metabolic and cardiovascular diseases. ATGL is tightly regulated by a known set of protein-protein interaction partners with activating or inhibiting functions in the control of lipolysis. Here, we use deep mutational protein interaction perturbation scanning and generate comprehensive profiles of single amino acid variants that affect the interactions of ATGL with its regulatory partners: CGI-58, G0S2, PLIN1, PLIN5 and CIDEC. Twenty-three ATGL amino acid variants yield a specific interaction perturbation pattern when validated in co-immunoprecipitation experiments in mammalian cells. We identify and characterize eleven highly selective ATGL switch mutations which affect the interaction of one of the five partners without affecting the others. Switch mutations thus provide distinct interaction determinants for ATGL’s key regulatory proteins at an amino acid resolution. When we test triglyceride hydrolase activity in vitro and lipolysis in cells, the activity patterns of the ATGL switch variants trace to their protein interaction profile. In the context of structural data, the integration of variant binding and activity profiles provides insights into the regulation of lipolysis and the impact of mutations in human disease.

Structural implications of BK polyomavirus sequence variations in the major viral capsid protein Vp1 and large T-antigen: a computational study.

Type and rate of amino acid variations in BKPyV may provide important insights into BKPyV diversity in human populations and an important step toward defining determinants of BKPyV-specific immunity needed to protect vulnerable patients from BKPyV diseases. Our analysis of BKPyV sequences obtained from human specimens reveals an unexpectedly high genetic variability for this double-stranded DNA virus that strongly relies on host cell DNA replication machinery with its proof reading and error correction mechanisms. BKPyV variability and immune escape should be taken into account when designing further approaches to antivirals, monoclonal antibodies, and vaccines for patients at risk of BKPyV diseases.

Integration of genome-wide association and genomic prediction for dissecting seed protein and amino acid in foxtail millet

ContextThe exploitation and utilization of genetic variation in crop germplasm benefit genomics-informed breeding programs. The integration of genome-wide association study (GWAS) and genomic prediction (GP) holds promise for illustrating genetic basis of phenotypic variation. Currently, the use of this integrative approach for dissecting grain protein and amino acid traits remains limited in crop species. ObjectiveThe research objectives were to identify significant single nucleotide polymorphisms (SNPs) associated with seed protein and amino acids and to assess the genomic predictive ability for nutritional traits in natural population of foxtail millet. MethodsIn this study, the concentrations of seed protein and 17 amino acids were assessed in a training population consisting of 238 diverse accessions of foxtail millet. The significant SNPs associated with these phenotypes were identified by GWAS with a mix-linear model. Extensive cross-validation was performed to evaluate the accuracy of genomic prediction of seed nutritional traits. Furthermore, the genomic prediction model was optimized and externally validated in 211 untested materials with the most significant SNPs obtained from GWAS. ResultsA significant genotype variance was detected for seed protein and amino acids and broad-sense heritability (h2) ranged from 0.45 to 0.87 (P < 0.05). The GWAS identified 39 significant SNPs associated with 15 amino acids (p < 1e-06), explaining an average of 12.2% phenotypic variation per locus. The number of significant SNPs ranged from 1 (Asp, Ile, Glu) to 23 for Phe amino acid, including six SNPs associated with one or multiple amino acids. In the training population, the prediction accuracies of protein and amino acids, tested using the 10-fold cross-validation model with whole genome-wide SNPs, ranged from 0.23 (Glu) to 0.60 (Lys) with an average value of 0.44%. Population structure had little effect on the prediction accuracy. In an empirical validation experiment involving 211 untested accessions, the averaged prediction accuracy increased by 12.3% across all traits when using the 1000 top SNPs obtained from GWAS, compared with the same number of randomly selected SNPs from the entire genome. ConclusionsLarge variations of seed protein and amino acids were found in a natural population of foxtail millet. GWAS identified 39 significant SNPs associated with 15 amino acids, and six SNPs were linked to more than one trait. The 10-fold cross-validation model produced the relatively higher prediction accuracies in seed quality traits of diverse accessions. By applying the significant SNPs from GWAS to the untested accessions, the use of 1000 GWAS-top SNPs resulted in more powerful predictions for complex nutritional traits than those using the same number of randomly selected SNPs across the genome. SignificanceThe research highlights the importance and effectiveness of integrative GWAS and genomic prediction in revealing the genetic architecture of seed protein and amino acid variation in foxtail millet. The results provide valuable insights into assessment and improvement of germplasm resources for advancing precision crop phenotyping and enhancing breeding programs for crop nutritional traits.

Spatio-temporal spread and evolution of Lassa virus in West Africa

BackgroundLassa fever is a hemorrhagic disease caused by Lassa virus (LASV), which has been classified by the World Health Organization as one of the top infectious diseases requiring prioritized research. Previous studies have provided insights into the classification and geographic characteristics of LASV lineages. However, the factor of the distribution and evolution characteristics and phylodynamics of the virus was still limited.MethodsTo enhance comprehensive understanding of LASV, we employed phylogenetic analysis, reassortment and recombination detection, and variation evaluation utilizing publicly available viral genome sequences.ResultsThe results showed the estimated the root of time of the most recent common ancestor (TMRCA) for large (L) segment was approximately 634 (95% HPD: [385879]), whereas the TMRCA for small (S) segment was around 1224 (95% HPD: [10301401]). LASV primarily spread from east to west in West Africa through two routes, and in route 2, the virus independently spread to surrounding countries through Liberia, resulting in a wider spread of LASV. From 1969 to 2018, the effective population size experienced two significant increased, indicating the enhanced genetic diversity of LASV. We also found the evolution rate of L segment was faster than S segment, further results showed zinc-binding protein had the fastest evolution rate. Reassortment events were detected in multiple lineages including sub-lineage IIg, while recombination events were observed within lineage V. Significant amino acid changes in the glycoprotein precursor of LASV were identified, demonstrating sequence diversity among lineages in LASV.ConclusionThis study comprehensively elucidated the transmission and evolution of LASV in West Africa, providing detailed insights into reassortment events, recombination events, and amino acid variations.

Effect of bi-enzyme hydrolysis on the properties and composition of hydrolysates of Manchurian walnut dreg protein

Walnut dreg (WD) active peptides are an important source of dietary antioxidants; however, the products of conventional hydrolysis have limited industrial output owing to poor flavour and low bioactivity. To this end, in this study, we aimed to employ bvLAP, an aminopeptidase previously identified in our research, as well as commercially available Alcalase for bi-enzyme digestion. The flavour, antioxidant activity, and structures of products resulting from various digestion methods were compared. The results showed that the bi-enzyme digestion products had enhanced antioxidant activity, increased β-sheet content, and reduced bitterness intensity from 9.65 to 6.93. Moreover, bi-enzyme hydrolysates showed a more diverse amino acid composition containing 1640 peptides with distinct sequences. These results demonstrate that bi-enzyme hydrolysis could be a potential process for converting WD into functional food ingredients. Additionally, our results provide new concepts that can be applied in waste processing and high-value utilisation of WD.

Hepatitis B virus preCore/Core region variability in pregnant women in the Republic of Guinea

Introduction. The vertical route of hepatitis B virus (HBV) transmission is a significant problem in African countries, which is characterized by late diagnosis of the disease and high mortality. The high prevalence of hepatocellular carcinoma (HCC) in Africa may be due to variability in the HBV preCore/Core region, mutations in which contribute to disease progression. Molecular genetic characterization of strains circulating among pregnant women may reflect the overall mutational profile of the pathogen in the population.&#x0D; The objective of this study was to analyze the variability of the HBV preCore/Core region circulating among pregnant women in the Republic of Guinea.&#x0D; Materials and methods. The study material included 480 plasma samples obtained from HBV-positive pregnant women from the Republic of Guinea. For all samples, the nucleotide sequences of the preCore/Core region of the HBV genome were sequenced and analyzed.&#x0D; Results. Amino acid variability in the preCore region was determined in 211 (43.96%), and in the Core region in 473 (98.54%) patients. 12 polymorphic sites of the preCore region were identified in which amino acid substitutions occurred, including 8, 2 and 5 positions identified for genotypes E, A and D, respectively. In the Core region, 67 substitution positions were identified, including 46 in samples of genotype E, 23 in HBV genotype A and 26 in genotype D. It was shown that the distribution of substitutions in the preCore and Core regions in HBV genotypes E, A and D differs significantly with a predominance in mutations among HBV genotype E — p 0.0001. Individual characteristic mutations have been identified for each genotype. The most common clinically significant mutations in the preCore/Core region in the study group were identified, including pc-H5D (27,08%), pc-W28* (35,21%), c-E64D (33,54%), c-L116I/V/G (91,46 %), c-T146N (73,13%). The double mutation A1762T/G1764A in the basal core promoter was shown in 74 samples of HBV genotype E, which accounted for 15.42% of the total group and 16.59% of patients with HBV genotype E.&#x0D; Conclusion. The frequency of clinically significant preCore/Core mutations among pregnant women in the Republic of Guinea was determined. The data obtained reflect their prevalence in the general population and can be used to predict the progression of chronic HBV among the region's population.

Metabolomic and chemometrics depict to understand the nutraceutical potential of different flaxseed varieties

Flaxseeds phytochemical and nutraceutical composition is influenced by climatic conditions that possibly impact flaxseed fingerprinting authentication. Therefore, this research aimed to discriminate flaxseeds phytochemical and nutraceutical value among eight different varieties (originating from China and Pakistan). Metabolomic fingerprint and chemometrics revealed discrimination among these varieties due to variations in fatty acids, amino acids, cyclic peptides, tocopherols, carotenoids, phenolics, and volatile compounds composition in flaxseeds. A discrimination model with p-value < 0.05 and VIP score >1 was propounded those different biomarkers such as γ-tocopherol, leutein, C18:3, Thr, Glu, 1-hexanol, 2-decenal, 1-octen-3-ol, and cyclic peptides content contribute to the discrimination of different flaxseeds varieties. Compared to RO and CH flaxseed varieties, HU, MO1, and MO2 primarily have higher total fatty acids, amino acids, tocopherols, carotenoids, and cyclic peptides. The present study significantly contributes to the quality fingerprinting of flaxseeds for food and pharmaceutical applications.

A chromosome-level genome assembly of Solanum chilense, a tomato wild relative associated with resistance to salinity and drought.

Solanum chilense is a wild relative of tomato reported to exhibit resistance to biotic and abiotic stresses. There is potential to improve tomato cultivars via breeding with wild relatives, a process greatly accelerated by suitable genomic and genetic resources. In this study we generated a high-quality, chromosome-level, de novo assembly for the S. chilense accession LA1972 using a hybrid assembly strategy with ~180 Gbp of Illumina short reads and ~50 Gbp long PacBio reads. Further scaffolding was performed using Bionano optical maps and 10x Chromium reads. The resulting sequences were arranged into 12 pseudomolecules using Hi-C sequencing. This resulted in a 901 Mbp assembly, with a completeness of 95%, as determined by Benchmarking with Universal Single-Copy Orthologs (BUSCO). Sequencing of RNA from multiple tissues resulting in ~219 Gbp of reads was used to annotate the genome assembly with an RNA-Seq guided gene prediction, and for a de novo transcriptome assembly. This chromosome-level, high-quality reference genome for S. chilense accession LA1972 will support future breeding efforts for more sustainable tomato production. Gene sequences related to drought and salt resistance were compared between S. chilense and S. lycopersicum to identify amino acid variations with high potential for functional impact. These variants were subsequently analysed in 84 resequenced tomato lines across 12 different related species to explore the variant distributions. We identified a set of 7 putative impactful amino acid variants some of which may also impact on fruit development for example the ethylene-responsive transcription factor WIN1 and ethylene-insensitive protein 2. These variants could be tested for their ability to confer functional phenotypes to cultivars that have lost these variants.

Variations of the NodB Architecture Are Attuned to Functional Specificities into and beyond the Carbohydrate Esterase Family 4.

Enzymes of the carbohydrate esterase family 4 (CE4) deacetylate a broad range of substrates, including linear, branched and mesh-like polysaccharides. Although they are enzymes of variable amino acid sequence length, they all comprise the conserved catalytic domain NodB. NodB carries the metal binding and active site residues and is characterized by a set of conserved sequence motifs, which are linked to the deacetylation activity. Besides a non-structured, flexible peptide of variable length that precedes NodB, several members of the CE4 family contain additional domains whose function or contribution to substrate specificity are not efficiently characterized. Evidence suggests that CE4 family members comprising solely the NodB domain have developed features linked to a variety of substrate specificities. To understand the NodB-based substrate diversity within the CE4 family, we perform a comparative analysis of all NodB domains structurally characterized so far. We show that amino acid sequence variations, topology diversities and excursions away from the framework structure give rise to different NodB domain classes associated with different substrate specificities and particular functions within and beyond the CE4 family. Our work reveals a link between specific NodB domain characteristics and substrate recognition. Thus, the details of the fold are clarified, and the structural basis of its variations is deciphered and associated with function. The conclusions of this work are also used to make predictions and propose specific functions for biochemically/enzymatically uncharacterized NodB-containing proteins, which have generally been considered as putative CE4 deacetylases. We show that some of them probably belong to different enzymatic families.

Substrate Promiscuity of the Triceptide Maturase XncB Leads to Incorporation of Various Amino Acids and Detection of Oxygenated Products.

Triceptides are cyclophane-containing ribosomally synthesized and post-translationally modified peptides. The characteristic cross-links are formed between an aromatic ring to Cβ on three-residue Ω1X2X3 motifs (Ω1 = aromatic). Here, we explored the promiscuity of the XYE family triceptide maturase, XncB from Xenorhabdus nematophila DSM 3370. Single amino acid variants were coexpressed with XncB in vivo in Escherichia coli, and we show that a variety of amino acids can be incorporated into the Phe-Gly-Asn cyclophane. Aromatic amino acids at the X3 position were accepted by the enzyme but yielded hydroxylated, rather than the typical cyclophane, products. These studies show that oxygen can be inserted but diverges in the final product formed relative to daropeptide maturases. Finally, truncations of the leader peptide showed that it is necessary for complete modification by XncB.

Unraveling the genetic variations underlying virulence disparities among SARS-CoV-2 strains across global regions: insights from Pakistan

Over the course of the COVID-19 pandemic, several SARS-CoV-2 variants have emerged that may exhibit different etiological effects such as enhanced transmissibility and infectivity. However, genetic variations that reduce virulence and deteriorate viral fitness have not yet been thoroughly investigated. The present study sought to evaluate the effects of viral genetic makeup on COVID-19 epidemiology in Pakistan, where the infectivity and mortality rate was comparatively lower than other countries during the first pandemic wave. For this purpose, we focused on the comparative analyses of 7096 amino-acid long polyprotein pp1ab. Comparative sequence analysis of 203 SARS-CoV-2 genomes, sampled from Pakistan during the first wave of the pandemic revealed 179 amino acid substitutions in pp1ab. Within this set, 38 substitutions were identified within the Nsp3 region of the pp1ab polyprotein. Structural and biophysical analysis of proteins revealed that amino acid variations within Nsp3’s macrodomains induced conformational changes and modified protein-ligand interactions, consequently diminishing the virulence and fitness of SARS-CoV-2. Additionally, the epistatic effects resulting from evolutionary substitutions in SARS-CoV-2 proteins may have unnoticed implications for reducing disease burden. In light of these findings, further characterization of such deleterious SARS-CoV-2 mutations will not only aid in identifying potential therapeutic targets but will also provide a roadmap for maintaining vigilance against the genetic variability of diverse SARS-CoV-2 strains circulating globally. Furthermore, these insights empower us to more effectively manage and respond to potential viral-based pandemic outbreaks of a similar nature in the future.

Omics data analysis reveals the system-level constraint on cellular amino acid composition

Proteins play a pivotal role in coordinating the functions of organisms, essentially governing their traits, as the dynamic arrangement of diverse amino acids leads to a multitude of folded configurations within peptide chains. Despite dynamic changes in amino acid composition of an individual protein (referred to as AAP) and great variance in protein expression levels under different conditions, our study, utilizing transcriptomics data from four model organisms uncovers surprising stability in the overall amino acid composition of the total cellular proteins (referred to as AACell). Although this value may vary between different species, we observed no significant differences among distinct strains of the same species. This indicates that organisms enforce system-level constraints to maintain a consistent AACell, even amid fluctuations in AAP and protein expression. Further exploration of this phenomenon promises insights into the intricate mechanisms orchestrating cellular protein expression and adaptation to varying environmental challenges.

Synthesis, biological and computational evaluation of novel cyanomethyl vinyl ether derivatives.

This work explores the biological evaluation of novel cyanomethyl vinyl ether derivatives as antiproliferative agents. Tubulin, crucial to microtubule structure and function, is a target for cancer therapies. In vitro cytotoxicity assessments revealed significant activity in SKOV3 ovarian carcinoma cells and A549 lung carcinoma cells. Structure-Activity Relationship (SAR) analysis indicated that the E isomer and specific substitutions influenced the biological activity. Computational assays predicted favorable ADME properties, highlighting potential as anticancerous agents. Molecular docking studies demonstrated that compound 12E, with the E geometry of the double bond and fused polyaromatic rings such as phenanthrene, has robust interaction with tubulin, suggesting enhanced stability due to diverse amino acid interactions. Comparative spatial distributions with colchicine further indicated potential mechanistic similarities.

The first complete genome sequence and genetic evolution analysis of bovine norovirus in Xinjiang, China

Abstract Introduction Viruses are among the main pathogens causing diarrhoea in calves. The current study found that bovine norovirus (BNoV) is one of the principal viruses causing diarrhoea in calves in Xinjiang, China. Material and Methods A total of 974 calf faecal samples from six regions in Xinjiang were tested for BNoV using reverse-transcriptase PCR. The genomic characteristics of BNoV and the genetic evolution of the VP1 gene, protein three-dimensional structure characteristics and amino acid variation were analysed using bioinformatics methods. Results Epidemiological survey results showed that the infection rate of BNoV was 19.82%, and all samples tested positive in five regions. The results of the genetic evolution analysis showed that BNoV strains from Tacheng of northern Xinjiang and Kashgar of southern Xinjiang both belonged to the GIII.2 genotype of BNoV but were not on the same cluster of evolutionary branches. Additionally, the amino acid variation of the VP1 protein was not observed to significantly affect its spatial structure. Conclusion This study is the first to report the genetic characteristics of the BNoV complete genome sequence in Xinjiang and provides a scientific basis for BNoV vaccine development and pathogenesis research.