Amino Acid Mutations Research Articles

Dissecting the causal polymorphism of the Lr67res multipathogen resistance gene.

Partial resistance to multiple biotrophic fungal pathogens in wheat (Triticum aestivum L.) is conferred by a variant of the Lr67 gene, which encodes a hexose-proton symporter. Two mutations (G144R and V387L) differentiate the resistant and susceptible protein variants (Lr67res and Lr67sus). Lr67res lacks sugar transport capability and was associated with anion transporter-like properties when expressed in Xenopus laevis oocytes. Here, we extended this functional characterization to include yeast and in planta studies. The Lr67res allele, but not Lr67sus, induced sensitivity to ions in yeast (including NaCl, LiCl, and KI), which is consistent with our previous observations that Lr67res expression in oocytes induces novel ion fluxes. We demonstrate that another naturally occurring single amino acid variant in wheat, containing only the Lr67G144R mutation, confers rust resistance. Transgenic barley plants expressing the orthologous HvSTP13 gene carrying the G144R and V387L mutations were also more resistant to Puccinia hordei infection. NaCl treatment of pot-grown adult wheat plants with the Lr67res allele induced leaf tip necrosis and partial leaf rust resistance. An Lr67res-like function can be introduced into orthologous plant hexose transporters via single amino acid mutation, highlighting the strong possibility of generating disease resistance in other crops, especially with gene editing.

Evolutionary insights into sequence modifications governing chitin recognition and chitinase inactivity in YKL-40 (HC-gp39, CHI3L1)

YKL-40, also known as human cartilage glycoprotein-39 (HC-gp39) or CHI3L1, shares structural similarities with chitotriosidase (CHIT1), an active chitinase, but lacks chitinase activity. Despite being a biomarker for inflammatory disorders and cancer, the reasons for YKL-40's inert chitinase function have remained elusive. This study reveals that the loss of chitinase activity in YKL-40 has risen from multiple sequence modifications influencing its chitin affinity. Contrary to the common belief associating the lack of chitinase activity with amino acid substitutions in the catalytic motif, attempts to activate YKL-40 by creating two amino acid mutations in the catalytic motif (MT-YKL-40) proved ineffective. Subsequent exploration that included creating chimeras of MT-YKL-40 and CHIT1 catalytic domains (CatD) identified key exons responsible for YKL-40 inactivation. Introducing YKL-40 exons 3, 6, or 8 into CHIT1 CatD resulted in chitinase inactivation. Conversely, incorporating CHIT1 exons 3, 6, and 8 into MT-YKL-40 led to its activation. Our recombinant proteins exhibited properly formed disulfide bonds, affirming a defined structure in active molecules. Biochemical and evolutionary analysis indicated that the reduced chitinase activity of MT-YKL-40 correlates with specific amino acids in exon 3. M61I and T69W substitutions in CHIT1 CatD diminished chitinase activity and increased chitin binding. Conversely, substituting I61 with M and W69 with T in MT-YKL-40 triggered chitinase activity while reducing the chitin-binding activity. Thus, W69 plays a crucial role in a unique subsite within YKL-40. These findings emphasize that YKL-40, though retaining the structural framework of a mammalian chitinase, has evolved to recognize chitin while surrendering chitinase activity.

Establishment of a real-time fluorescent quantitative PCR detection method and phylogenetic analysis of BoAHV-1

BackgroundInfectious bovine rhinotracheitis (IBR), caused by Bovine alphaherpesvirus-1 (BoAHV-1), is an acute, highly contagious disease primarily characterized by respiratory tract lesions in infected cattle. Due to its severe pathological damage and extensive transmission, it results in significant economic losses in the cattle industry. Accurate detection of BoAHV-1 is of paramount importance. In this study, we developed a real-time fluorescent quantitative PCR detection method for detecting BoAHV-1 infections. Utilizing this method, we tested clinical samples and successfully identified and isolated a strain of BoAHV-1.1 from positive samples. Subsequently, we conducted a genetic evolution analysis on the isolate strain’s gC, TK, gG, gD, and gE genes.ResultsThe study developed a real-time quantitative PCR detection method using SYBR Green II, achieving a detection limit of 7.8 × 101 DNA copies/μL. Specificity and repeatability analyses demonstrated no cross-reactivity with other related pathogens, highlighting excellent repeatability. Using this method, 15 out of 86 clinical nasal swab samples from cattle were found to be positive (17.44%), which was higher than the results obtained from conventional PCR detection (13.95%, 12/86). The homology analysis and phylogenetic tree analysis of the gC, TK, gG, gD, and gE genes of the isolated strain indicate that the JL5 strain shares high homology with the BoAHV-1.1 reference strains. Amino acid sequence analysis revealed that gC, gE, and gG each had two amino acid mutations, while the TK gene had one synonymous mutation and one H to Y mutation, with no amino acid mutations observed in the gD gene. Phylogenetic tree analysis indicated that the JL5 strain belongs to the BoAHV-1.1 genotype and is closely related to American strains such as C33, C14, and C28.ConclusionsThe established real-time fluorescent quantitative PCR detection method exhibits good repeatability, specificity, and sensitivity. Furthermore, genetic evolution analysis of the isolated BoAHV-1 JL-5 strain indicates that it belongs to the BoAHV-1.1 subtype. These findings provide a foundation and data for the detection, prevention, and control Infectious Bovine Rhinotracheitis.

Genetic variations of penicillin-binding protein 1A: insights into the current status of amoxicillin-based regimens for Helicobacter pylori eradication in Malaysia.

Introduction. Resistance towards amoxicillin in Helicobacter pylori causes significant therapeutic impasse in healthcare settings worldwide. In Malaysia, the standard H. pylori treatment regimen includes a 14-day course of high-dose proton-pump inhibitor (rabeprazole, 20 mg) with amoxicillin (1000 mg) dual therapy.Hypothesis/Gap Statement. The high eradication rate with amoxicillin-based treatment could be attributed to the primary resistance rates of amoxicillin being relatively low at 0%, however, a low rate of secondary resistance has been documented in Malaysia recently.Aim. This study aims to investigate the amino acid mutations and related genetic variants in PBP1A of H. pylori, correlating with amoxicillin resistance in the Malaysian population.Methodology. The full-length pbp1A gene was amplified via PCR from 50 genomic DNA extracted from gastric biopsy samples of H. pylori-positive treatment-naïve Malaysian patients. The sequences were then compared with reference H. pylori strain ATCC 26695 for mutation and variant detection. A phylogenetic analysis of 50 sequences along with 43 additional sequences from the NCBI database was performed. These additional sequences included both amoxicillin-resistant strains (n=20) and amoxicillin-sensitive strains (n=23).Results. There was a total of 21 variants of amino acids, with three of them located in or near the PBP-motif (SKN402-404). The percentages of these three variants are as follows: K403X, 2%; S405I, 2% and E406K, 16%. Based on the genetic markers identified, the resistance rate for amoxicillin in our sample remained at 0%. The phylogenetic examination suggested that H. pylori might exhibit unique conserved pbp1A sequences within the Malaysian context.Conclusions. Overall, the molecular analysis of PBP1A supported the therapeutic superiority of amoxicillin-based regimens. Therefore, it is crucial to continue monitoring the amoxicillin resistance background of H. pylori with a larger sample size to ensure the sustained effectiveness of amoxicillin-based treatments in Malaysia.

BCM-7: Opioid-like Peptide with Potential Role in Disease Mechanisms.

Bovine milk is an essential supplement due to its rich energy- and nutrient-rich qualities. Caseins constitute the vast majority of the proteins in milk. Among these, β-casein comprises around 37% of all caseins, and it is an important type of casein with several different variants. The A1 and A2 variants of β-casein are the most researched genotypes due to the changes in their composition. It is accepted that the A2 variant is ancestral, while a point mutation in the 67th amino acid created the A1 variant. The digestion derived of both A1 and A2 milk is BCM-7. Digestion of A2 milk in the human intestine also forms BCM-9 peptide molecule. The opioid-like characteristics of BCM-7 are highlighted for their potential triggering effect on several diseases. Most research has been focused on gastrointestinal-related diseases; however other metabolic and nervous system-based diseases are also potentially triggered. By manipulating the mechanisms of these diseases, BCM-7 can induce certain situations, such as conformational changes, reduction in protein activity, and the creation of undesired activity in the biological system. Furthermore, the genotype of casein can also play a role in bone health, such as altering fracture rates, and calcium contents can change the characteristics of dietary products. The context between opioid molecules and BCM-7 points to a potential triggering mechanism for the central nervous system and other metabolic diseases discussed.

Screening Key Sites of Class 2 Integron Integrase that Impact Recombination Efficiency.

By capturing and expressing exogenous resistance gene cassettes through site-specific recombination, integrons play important roles in the horizontal transfer of antimicrobial resistant genes among bacteria. The characteristics of integron integrase make it to be a potential gene editing tool enzyme. In this study, a random mutation library using error-prone PCR was constructed, and amino acid residues mutants that impact on attI2 × attC or attC × attC recombination efficiency were screened and analyzed. Thirteen amino acid mutations were identified to be critical impacted on site-specific recombination of IntI2, including the predicted catalyzed site Y301. Nine of 13 mutated amino acid residues that have critically impacted on IntI2 activity were relative concentrated and near the predicted catalyzed site Y301 in the predicted three-dimensional structure indicated the importance of this area in maintain the activity of IntI2. No mutant with obviously increased recombination activity (more than four-fold as high as that of wild IntI2) was found in library screening, except P95S, R100K slightly increased (within two-fold) the excision activity of IntI2, and S243T slightly increased (within two-fold) both excision and integration activity of IntI2. These findings will provide clues for further specific modification of integron integrase to be a tool enzyme as well as establishing a new gene editing system and applied practically.

Abstract PO4-04-08: Design and Development of the APIS ESR1 Mutations Kit to Detect Eleven Mutations Relevant to Acquired Endocrine Therapy Resistance

Abstract Introduction: Estrogen receptor 1 (ER/ESR1) mutations have arisen as key biomarkers for endocrine therapy resistance in ER positive (ER+) breast cancer (BC) patients: detecting these mutations is key to guiding researchers to better understand acquired resistance during treatment. Ongoing clinical trials, which explore the prevalence of ESR1 mutations under various treatments, commonly use next-generation sequencing (NGS)-based assays. However, the NGS panels available are costly, can have long turnaround times and require specialist equipment and software. Research use only digital PCR assays are also available but have similar drawbacks to NGS. A targeted qPCR-based assay is more time and cost effective, and does not require specialist equipment. Here, we demonstrate the APIS ESR1 Mutations Kit, a targeted, sensitive, and robust qualitative qPCR assay able to detect eleven ESR1 mutations across ESR1 exons 5 (E380Q), 7 (S463P) and 8 (P535H, L536R, L536Q, L536H, L536P, Y537C, Y537S, Y537N and D538G). The assay is designed to assess circulating-free DNA (cfDNA) samples, reducing the need for invasive procedures in clinical settings and can make use of standard cfDNA extraction kits. Therefore, the APIS ESR1 Mutations Kit provides the flexibility for researchers in any conventional molecular biology laboratory to explore ESR1 mutations with high sensitivity and specificity. Methods: The APIS ESR1 Mutations Kit provides all components and controls required to assess samples. For kit development, mutation-specific DNA fragments were spiked into wildtype background DNA (either cfDNA extracted from pooled healthy donor plasma, human genomic DNA, or DNA fragments); the Limit of Blank (LoB) and Limit of Detection (LoD) were determined using these samples. To determine linearity, a dilution series of DNA fragments ranging from 5 to 10,000 copies per reaction was assessed. All PCR runs were performed using a QuantStudio™5 Dx (ThermoFisher) instrument. Results: The performance studies showed the APIS ESR1 Mutations Kit can detect D538G at 0.4 % mutant allele frequency (MAF) and Y537S at 0.06 % MAF. All other mutations can be detected at ≤ 1.0 % MAF. By using clamp and blocking technologies, each design is mutation specific, enabling identification of specific amino acid mutations. In addition, the assays can perform in high wild-type backgrounds, enabling lower limit of blanks with ≥95% confidence and threshold cut-offs to enhance the sensitivity of the assay. The linearity of each target is within 90-110% from 50 to 10,000 copies per reaction. Conclusions: The ESR1 Mutations Kit demonstrated high sensitivity and performance as a qualitative qPCR assay to detect eleven ESR1 mutations. Table. Mutations detected with the APIS ESR1 Mutations Kit Citation Format: Anna Gasior, Colette Whitfield, Anita Campbell, Christine Hoy, Sally Holdsworth, Jesse Mukose, Ryan Nana, Sahar Shammakhi, Benjamin Scales, Joanna Gorniak. Design and Development of the APIS ESR1 Mutations Kit to Detect Eleven Mutations Relevant to Acquired Endocrine Therapy Resistance [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-04-08.

Prevalence and diversity of imported severe acute respiratory syndrome coronavirus 2 variants in China, 2021-2022.

The causative agent of coronavirus disease 2019 (COVID-19), known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread accumulatively to 240 countries and continues to evolve. To gain a comprehensive understanding of the epidemiological characteristics of imported variants in China and their correlation with global circulating variants, genomic surveillance data from 11 139 imported COVID-19 cases submitted by Chinese provincial CDC laboratories between 2021 and 2022 were analyzed. Consensus sequences underwent rigorous quality checks, followed by amino acid mutations analysis using Nextclade. Sequences with satisfactory quality control status were classified according to the Pango nomenclature. The results showed that the dominant variants in imported cases reflected the global epidemic trend. An increase in the number of imported SARS-CoV-2 lineages monitored in China in the second half of 2022, and the circulating Omicron subvariants changed from the ancestral lineages of BA.5 and BA.2 into the lineages containing key amino acid mutations of spike protein. There was significant variation in the detection of Omicron subvariants among continents (χ2 = 321.968, p < 0.001) in the second half of 2022, with four lineages (BA.2.3.7, BA.2.2, BA.5.2.7, and XBB.1.2) identified through imported surveillance mainly prevalent respectively in Taiwan, China, Hong Kong SAR, China, Russian Federation, and Singapore. These findings revealed the alterations in circulating imported variants from 2021 to 2022 in China, reflecting the higher diversity of lineages in the second half of 2022, and revealed the predominant lineages of countries or regions that are in close contacts to China, providing new insights into the global prevalence of SARS-CoV-2.

Current Status and Perspectives of Therapeutic Antibodies Targeting the Spike Protein S2 Subunit against SARS-CoV-2.

The global coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has devastated public health and the global economy. New variants are continually emerging because of amino acid mutations within the SARS-CoV-2 spike protein. Existing neutralizing antibodies (nAbs) that target the receptor-binding domain (RBD) within the spike protein have been shown to have reduced neutralizing activity against these variants. In particular, the recently expanding omicron subvariants BQ 1.1 and XBB are resistant to nAbs approved for emergency use by the United States Food and Drug Administration. Therefore, it is essential to develop broad nAbs to combat emerging variants. In contrast to the massive accumulation of mutations within the RBD, the S2 subunit remains highly conserved among variants. Therefore, nAbs targeting the S2 region may provide effective cross-protection against novel SARS-CoV-2 variants. Here, we provide a detailed summary of nAbs targeting the S2 subunit: the fusion peptide, stem helix, and heptad repeats 1 and 2. In addition, we provide prospects to solve problems such as the weak neutralizing potency of nAbs targeting the S2 subunit.

Identification of novel resistance-associated mutations and discrimination within whole-genome sequences of fluoroquinolone-resistant Mycobacterium tuberculosis isolates.

This study aims to elucidate additional mutation loci associated with fluoroquinolone (FQ) resistance and evaluate the discriminatory capacity of mutation loci and allele mutation frequencies in identifying FQ-resistant Mycobacterium tuberculosis (MTB) isolates. A random selection of isolates was extracted from an ongoing collection. Drug resistance was determined using the resazurin microtiter assay (REMA) as the gold standard. Mutation loci and the burden of mutations in the quinolone resistance-determining region (QRDR) were elucidated through whole-genome sequencing (WGS). Novel amino acid mutations, namely, G520D and G520T, were identified in the gyrB and associated with FQ resistance. In the context of distinguishing FQ-resistant isolates, the AUC for the QRDR mutation frequency burden (0.969) surpassed that of the mutation locus (0.929), and this difference was statistically significant (P = 0.03). Furthermore, using the resistance mutation locus as a reference, setting the QRDR mutation frequency burden threshold at 1.31% resulted in a 3.60% increase in the accuracy of classifying FQ-resistant isolates (NRI = 3.60%, P < 0.001). The QRDR mutation frequency burden appears to offer superior diagnostic efficacy in discriminating FQ-resistant isolates compared to qualitative detection of mutant loci.IMPORTANCEFluoroquinolone (FQ) drugs are recommended as second-line drugs for the treatment of multidrug-resistant tuberculosis. With the massive use of FQ drugs in the clinical treatment of tuberculosis (TB), there is an increasing rate of drug resistance to FQ drugs. In this study, we identified and demonstrated novel amino acid mutations associated with FQ resistance in Mycobacterium tuberculosis (MTB), and we quantified the mutation sites and identified the quinolone resistance-determining region (QRDR) mutation frequency burden as a novel diagnostic method for FQ resistance. We hope that the results of this study will provide data support and a theoretical basis for the rapid diagnosis of FQ-resistant MTB.

Characterization of Amino Acid Mutations of Newcastle Disease Virus (NDV) In Swan Geese (Anser cygnoides) In East Java, Indonesia

Background: Newcastle disease (ND) is a highly contagious viral disease affecting the poultry industry. The NDV is classified into three strains based on their relative virulence, namely velogenic or highly virulent, mesogenic or moderately virulent, and lentogenic or lowly virulent. The clinical manifestations of the disease vary depending on many factors, such as host susceptibility and the virulence of the NDV strain. Objective: This study aims to analyze the amino acid mutations of the NDV in unvaccinated swan goose (Anser cygnoides) from various locations in Java. Methods: Samples were collected through cloacal swabs and isolated by inoculation in Specific Pathogen-Free (SPF) embryonated eggs that were nine days old. Hemagglutination and hemagglutination inhibition tests were conducted to confirm that the isolated virus was NDV. The isolated virus was processed using reverse transcription-polymerase chain reaction (RT-PCR) with primers that amplified partial sequences of the fusion (F) gene, which was analyzed to determine the pathotype. Results: The results indicated the presence of mutations in several regions. The amino acid changes occurred in 17 variable sites (7.2%) between RefSeq/JF950510 and ND/SW1/2018, 12 variable sites (5.1%) between RefSeq/JF950510 and ND/SW2/2018, 13 variable sites (5.5%) between RefSeq/JF950510 and ND/SW3/2018, and 19 variable sites (8.1%) between RefSeq/JF950510 and ND/SW4/2018. The amino acid sequences of the cleavage site of the fusion (F) protein revealed that all isolates had low virulence. Conclusion: The results indicated that mutations in the region outside the cleavage site not were incapable of altering the virulence of the virus.

Identification of the CesA7 Gene Encodes Brittleness Mutation Derived from IR64 Variety and Breeding for Ruminant Feeding

Rice straw presents challenges as livestock feed due to its low digestibility and the presence of chemical residues. One potential solution is to focus on breeding brittle varieties that possess disease-resistance traits. In this study, AZ1803, a brittle mutant line isolated from the IR64 mutant pool, was chosen for gene identification and breeding. The AZ1803 mutant was crossed to the TNG67 variety to generate a mapping population and to the CS11 variety for fine mapping and breeding. The gene was mapped on chr. 10 between RM467 and RM171 SSR markers and was narrowed down to RM271 and RM5392 with 600 kb proximately interval. The AZ1803 and IR64 sequencing results revealed a substitution mutant in the Exon 9th of the OsCesA7 gene, resulting in an amino acid mutation at the end of the transmembrane domain 5th of the CESA7, responsible for cellulose synthesis for the secondary cell wall. The cellulose content of AZ1803 was reduced by 25% compared with the IR64. A new brittle and disease-resistant variety was bred by using developed markers in marker-assisted selection. In addition, bending tests and bacterial blight inoculation were applied. The bacterial lesion length of the bred variety is 64% lower than that of AZ1803. The rice straw of the new variety can be used for livestock feeding, which increases farmer income and reduces pesticide residues and air pollution from straw burning.

Validating the Distinctiveness of the Omicron Lineage within the SARSCov-2 based on Protein Language Models

Introduction: Variants of concern were identified in severe acute respiratory syndrome coronavirus 2, namely Alpha, Beta, Gamma, Delta, and Omicron. This study explores the mutations of the Omicron lineage and its differences from other lineages through a protein language model. Methods: By inputting the severe acute respiratory syndrome coronavirus 2 wild-type sequence into the protein language model evolving pre-trained models-1v, this study obtained the score for each position mutating to other amino acids and calculated the overall trend of a new variant of concern mutation scores. objective: Analyze the differences in the number of Omicron amino acid mutations compared to the other four VOC mutations using statistical methods, and use the protein language model esm-1v to analyze the specificity of Omicron amino acid mutations. Results: It is found that when the proportion of unobserved mutations to observed mutations is 4:15, Omicron still generates a large number of newly emerging mutations. It was found that the overall score for the Omicron family is low, and the overall ranking for the Omicron family is low. Conclusion: Mutations in the Omicron lineage are different from amino acid mutations in other lineages. The findings of this paper deepen the understanding of the spatial distribution of spike protein amino acid mutations and overall trends of newly emerging mutations corresponding to different variants of concern. This also provides insights into simulating the evolution of the Omicron lineage.

Porcine reproductive and respiratory syndrome virus nsp4-mediated β2M downregulation contributes to SLA-I decrease and virus infection in vivo and in vitro

Porcine reproductive and respiratory syndrome virus (PRRSV) infection inhibits swine leukocyte antigen class I (SLA-I) expression in pigs, resulting in inefficient antigen presentation and subsequent low levels of cellular PRRSV-specific immunity as well as persistent viremia. We previously observed that the non-structural protein 4 (nsp4) of PRRSV contributed to inhibition of the β2-microglobulin (β2M) and SLA-I expression in cells. Here, we constructed a series of nsp4 mutants with different combination of amino acid mutations to attenuate the inhibitory effect of nsp4 on β2M and SLA-I expression. Almost all nsp4 mutants exogenously expressed in cells showed an attenuated effect on inhibition of β2M and SLA-I expression, but the recombinant PRRSV harboring these nsp4 mutants failed to be rescued with exception of the rPRRSV-nsp4-mut10 harboring three amino acid mutations. However, infection of rPRRSV-nsp4-mut10 not only enhanced β2M and SLA-I expression in both cells and pigs but also promoted the DCs to active the CD3+CD8+T lymphocytes more efficiently, as compared with its parental PRRSV (rPRRVS-nsp4-wt). These data suggested that the inhibition of nsp4-mediated β2M downregulation improved β2M/SLA-I expression in pigs.

Seasonal human coronavirus NL63 epidemics in children in Guilin, China, reveal the emergence of a new subgenotype of HCoV-NL63.

Seasonal human coronavirus NL63 (HCoV-NL63) is a frequently encountered virus linked to mild upper respiratory infections. However, its potential to cause more severe or widespread disease remains an area of concern. This study aimed to investigate a rare localized epidemic of HCoV-NL63-induced respiratory infections among pediatric patients in Guilin, China, and to understand the viral subtype distribution and genetic characteristics. In this study, 83 pediatric patients hospitalized with acute respiratory infections and positive for HCoV-NL63 were enrolled. Molecular analysis was conducted to identify the viral subgenotypes and to assess genetic variations in the receptor-binding domain of the spiking protein. Among the 83 HCoV-NL63-positive children, three subgenotypes were identified: C4, C3, and B. Notably, 21 cases exhibited a previously unreported subtype, C4. Analysis of the C4 subtype revealed a unique amino acid mutation (I507L) in the receptor-binding domain of the spiking protein, which was also observed in the previously reported C3 genotype. This mutation may suggest potential increases in viral transmissibility and pathogenicity. The findings of this study highlight the rapid mutation dynamics of HCoV-NL63 and its potential for increased virulence and epidemic transmission. The presence of a unique mutation in the C4 subtype, shared with the C3 genotype, raises concerns about the virus's evolving nature and its potential public health implications. This research contributes valuable insights into the understanding of HCoV-NL63's epidemiology and pathogenesis, which is crucial for effective disease prevention and control strategies. Future studies are needed to further investigate the biological significance of the observed mutation and its potential impact on the virus's transmissibility and pathogenicity.

Exploring a Hirudin variant from nonhematophagous leeches: Unraveling full-length sequence, alternative splicing, function, and potential as a novel anticoagulant polypeptide

Ethnopharmacological relevanceLeeches exhibit robust anticoagulant activity, making them useful for treating cardiovascular diseases in traditional Chinese medicine. Whitmania pigra, the primary source species of leech-derived medicinal compounds in China, has been demonstrated to possess formidable anticoagulant properties. Hirudin-like peptides, recognized as potent thrombin inhibitors, are prevalent in hematophagous leeches. Considering that W. pigra is a nonhematophagic leech, the following question arises: does a hirudin variant exist in this species? Aim of the studyIn this study we identified the hirudin-encoding gene (WP_HV1) in the W. pigra genome. The goal of this study was to assess its anticoagulant activity and analyze the related mechanisms. Materials and methodsIn this study, a hirudin-encoding gene, WP_HV1, was identified from the W. pigra genome, and its accurate coding sequence (CDS) was validated through cloning from cDNA extracted from fresh W. pigra specimens. The structure of WP_HV1 and the amino acids associated with its anticoagulant activity were determined by sequence and structural analysis and prediction of its binding energy to thrombin. E. coli was used for the expression of WP_HV1 and recombinant proteins with various structures and mutants. The anticoagulant activity of the synthesized recombinant proteins was then confirmed using thrombin time (TT). ResultsValidation of the WP_HV1 gene was accomplished, and three alternative splices were discovered. The TT of the blank sample exceeded that of the recombinant WP_HV1 sample by 1.74 times (0.05 mg/ml), indicating positive anticoagulant activity. The anticoagulant activity of WP_HV1 was found to be associated with its C-terminal tyrosine, along with the presence of 9 acidic amino acids on both the left and right sides. A significant reduction in the corresponding TT was observed for the mutated amino acids compared to those of the wild type, with decreases of 4.8, 6.6, and 3.9 s, respectively. In addition, the anticoagulant activity of WP_HV1 was enhanced and prolonged for 2.7 s when the lysine-67 residue was mutated to tryptophan. ConclusionOnly one hirudin-encoding variant was identified in W. pigra. The active amino acids associated with anticoagulation in WP_HV1 were resolved and validated, revealing a novel source for screening and developing new anticoagulant drugs.

Application of an alchemical free energy method for the prediction of thermostable DuraPETase variants

Non-equilibrium (NEQ) alchemical free energy calculations are an emerging tool for accurately predicting changes in protein folding free energy resulting from amino acid mutations. In this study, this method in combination with the Rosetta ddg monomer tool was applied to predict more thermostable variants of the polyethylene terephthalate (PET) degrading enzyme DuraPETase. The Rosetta ddg monomer tool efficiently enriched promising mutations prior to more accurate prediction by NEQ alchemical free energy calculations. The relative change in folding free energy of 96 single amino acid mutations was calculated by NEQ alchemical free energy calculation. Experimental validation of ten of the highest scoring variants identified two mutations (DuraPETaseS61M and DuraPETaseS223Y) that increased the melting temperature (Tm) of the enzyme by up to 1 °C. The calculated relative change in folding free energy showed an excellent correlation with experimentally determined Tm resulting in a Pearson’s correlation coefficient of r = − 0.84. Limitations in the prediction of strongly stabilizing mutations were, however, encountered and are discussed. Despite these challenges, this study demonstrates the practical applicability of NEQ alchemical free energy calculations in prospective enzyme engineering projects.Key points• Rosetta ddg monomer enriches stabilizing mutations in a library of DuraPETase variants• NEQ free energy calculations accurately predict changes in Tmof DuraPETase• The DuraPETase variants S223Y, S42M, and S61M have increased TmGraphical

PySupercharge: a python algorithm for enabling ABC transporter bacterial secretion of all proteins through amino acid mutation.

The process of producing proteins in bacterial systems and secreting them through ATP-binding cassette (ABC) transporters is an area that has been actively researched and used due to its high protein production capacity and efficiency. However, some proteins are unable to pass through the ABC transporter after synthesis, a phenomenon we previously determined to be caused by an excessive positive charge in certain regions of their amino acid sequence. If such an excessive charge is removed, the secretion of any protein through ABC transporters becomes possible. In this study, we introduce 'linear charge density' as the criteria for possibility of protein secretion through ABC transporters and confirm that this criterion can be applied to various non-secretable proteins, such as SARS-CoV-2 spike proteins, botulinum toxin light chain, and human growth factors. Additionally, we develop a new algorithm, PySupercharge, that enables the secretion of proteins containing regions with high linear charge density. It selectively converts positively charged amino acids into negatively charged or neutral amino acids after linear charge density analysis to enable protein secretion through ABC transporters. PySupercharge, which also minimizes functional/structural stability loss of the pre-mutation proteins through the use of sequence conservation data, is currently being operated on an accessible web server. We verified the efficacy of PySupercharge-driven protein supercharging by secreting various previously non-secretable proteins commonly used in research, and so suggest this tool for use in future research requiring effective protein production.

Hit discovery of potential CDK8 inhibitors and analysis of amino acid mutations for cancer therapy through computer-aided drug discovery

Cyclin-dependent kinase 8 (CDK8) has emerged as a promising target for inhibiting cancer cell function, intensifying efforts towards the development of CDK8 inhibitors as potential cancer therapeutics. Mutations in CDK8, a protein kinase, are also implicated as a primary factor associated with tumor formation. In this study, we identified potential inhibitors through virtual screening for CDK8 and single amino acid mutations in CDK8, namely D173A (Aspartate 173 mutate to Alanine), D189N (Aspartate 189 mutate to Asparagine), T196A (Threonine 196 mutate to Alanine) and T196D (Threonine 196 mutate to Aspartate). Four databases (CHEMBEL, ZINC, MCULE, and MolPort) containing 65,209,131 molecules have been searched to identify new inhibitors for CDK8 and its single mutations. In the first step, structure-based pharmacophore modeling in the Pharmit server was used to select the compounds to know the inhibitors. Then molecules with better predicted drug-like molecule properties were selected. The final filter used to select more effective inhibitors among the previously selected molecules was molecular docking. Finally, 13 hits for CDK8, 11 hits for D173A, 11 hits for D189N, 15 hits for T196A, and 12 hits for T196D were considered potential inhibitors. A majority of the virtual screening hits exhibited satisfactorily predict pharmacokinetic characteristics and toxicity properties.

Potential Pathogenicity and Genetic Characteristics of a Live-Attenuated Classical Swine Fever Virus Vaccine Derivative Variant

Classical swine fever (CSF), caused by CSF virus (CSFV), is a highly contagious disease affecting pigs and causing massive pig production losses with severe global economic recession. The immunization of live-attenuated vaccines is still one of the key measures to CSFV management in endemic countries. However, there are also strong controversies about the usage of live-attenuated vaccines, particularly in pregnant sows and young pigs, such as in Europe, where domestic pigs are routinely not vaccinated until severe outbreaks occur. Here, we report a CSF outbreak in a pig farm in China, which affected more than 90% of the delivery sows and led to ∼45% birth loss. Surprisingly, phylogenetic analysis showed that the CSFV isolate (named CSFV/HeNLY2022, GenBank No. OR195698) was clustered into subgenotype 1.1a, closely together with the live-attenuated vaccine strains. Further genomic analysis also revealed that the isolate CSFV/HeNLY2022 shared the highest nucleotide identity of 99.7% with the C/HVRI vaccine strain (C-strain, GenBank No. AY805221). Moreover, compared to the C/HVRI strain, a total of eight amino acid mutations, distributed in Erns (H436thY and S476thR), E1 (T502thI and P581thT), E2 (M979thK and A1061thS), NS5A (A2980thT), and NS5B (I3818thM), were characterized in the CSFV/HeNLY2022 isolate. Our results suggested that the CSF outbreak was most likely caused by the live-attenuated CSFV vaccine or its derivative. It raises concern that the unscientific application of CSFV vaccines could potentially lead to CSFV spread in pigs. It is needed to perform a more rigorous evaluation of the safety of the C-strain-derived vaccines in combination with other different live-attenuated vaccines.