Amino Acid Residues In Proteins Research Articles

Betacellulin regulates gap junction intercellular communication by inducing the phosphorylation of connexin 43 in human granulosa-lutein cells

BackgroundThe gap junction protein, connexin 43 (Cx43) is highly expressed in human granulosa-lutein (hGL) cells. The phosphorylation of certain amino acid residues in the Cx43 protein has been shown to be related to a decline in gap junction intercellular communication (GJIC), which subsequently affects oocyte meiotic resumption. As a member of the epidermal growth factor (EGF) family, betacellulin (BTC) mediates luteinizing hormone (LH)-induced oocyte maturation and cumulus cell expansion in mammalian follicles. Whether BTC can regulate Cx43 phosphorylation, which further reduces Cx43-coupled GJIC activity in hGL cells remains to be determined.MethodsImmortalized human granulosa cells (SVOG cells) and primary human granulosa-lutein cells obtained from women undergoing in vitro fertilization in an academic research center were used as the study models. The expression levels of Cx43 and phosphorylated Cx43 were examined following cell incubation with BTC at different time points. Several kinase inhibitors (sotrastaurin, AG1478, and U0126) and small interfering RNAs targeting EGF receptor (EGFR) and receptor tyrosine-protein kinase 4 (ErbB4) were used to verify the specificity of the effects and to investigate the molecular mechanisms. Real-time-quantitative PCR and western blot analysis were used to detect the specific mRNA and protein levels, respectively. GJIC between SVOG cells were evaluated using a scrape loading and dye transfer assay. Results were analyzed by one-way analysis of variance.ResultsThe results showed that BTC induced the rapid phosphorylation of Cx43 at serine368 without altering the expression of Cx43 in primary and immortalized hGL cells. Additionally, using a dual inhibition approach (kinase inhibitors and siRNA-based expression knockdown), we demonstrated that this effect was mainly mediated by the EGFR but not the ErbB4 receptor. Furthermore, using a protein kinase C (PKC) kinase assay and a scrape-loading and dye transfer assay, we revealed that PKC signaling is the downstream signaling pathway that mediates the increase in Cx43 phosphorylation and subsequent decrease in GJIC activity in response to BTC treatment in hGL cells.ConclusionsBTC promptly induced the phosphorylation of connexin 43 at Ser368, leading to decreased GJIC activity in hGL cells. The BTC-induced cellular activities were most likely driven by the EGFR-mediated PKC-dependent signaling pathway. Our findings shed light on the detailed molecular mechanisms by which BTC regulates the process of oocyte meiotic resumption.

Steady-state intracellular pH and recovery from NH3/NH4+-induced intracellular acid loads in primary cultures of rat hippocampal neurons and astrocytes

Intracellular pH (pH i ) homeostasis is of vital importance because pH i fluctuations alter the net charge on intracellular weak acids and bases (e.g., amino-acid residues of proteins, metabolic intermediates, solutes like inorganic phosphate), and can result in cellular dysfunction. The rate of pH i change (dpH i /dt) is determined by the balance between acid loading (e.g., metabolic production of H + , influx of H + , efflux of HCO 3 − via transmembrane proteins of the SLC4 family) and acid extrusion (e.g., efflux of H + via Na-H exchangers in the SLC9 family, influx of HCO 3 − by members of the SLC4 family). A powerful approach for studying pH i regulation is to impose an acute intracellular acid load using the NH 4 + /NH 3 prepulse technique, and then assess dpH i /dt during the subsequent pH i recovery. Using two such prepulses and recoveries (twin pulses), one can compare 2 experimental conditions. The goal of the present study is to develop a data-analysis approach that facilitates a meaningful presentation of all twin-pulse experiments in a data set. Using primary cultures (~2 weeks) of embryonic (E18–E20) rat hippocampal neurons and astrocytes on coverslips, we loaded cells with the ratiometric pH-sensitive fluorescent dye BCECF, subjected the cells to twin NH 4 + /NH 3 pulses, and calibrated BCECF at the end of each experiment with nigericin at pH=7.0. A standard 12-point nigericin calibration curve allowed us to compute pH i vs. time. Because we studied cells in 21% O 2 but the nominal absence of CO 2 /HCO 3 − , the pH i recoveries mainly reflect Na-H exchange. We fitted the pH i -recovery time course with a double exponential (DExp) to reduce bias. Nevertheless, in both neurons and astrocytes plots of dpH i /dt vs. pH i appeared rather linear, indicating that the pH i recoveries were “quasi” single exponential (SExp). To exploit the near-linearity, for each pH i recovery, we fitted the plot of dpH i /dt vs pH i with a line, the slope of which is the quasi-single exponential rate constant (kqSExp), and the x-intercept of which is the asymptotic pH i of the recovery. We summarize the twin pH i recoveries from individual cells as miniature plots (“thumbnails”) in which we display, for each recovery, both the dpH i /dt values (from the DExp fit of pH i vs. time) vs. pH i and the line of best fit for these dpH i /dt values vs. pH i . A single figure can replicate such twin-pulse experiments from many dozens of cells, so that a reader, at a glance, can surmise the essentials of each pH i recovery. The present work provides background data for additional work in which we studied pH i recoveries at a reduced [O 2 ], with or without CO 2 /HCO 3 − . R01 NS018400, R01 HL160857, R01 DK128315 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Functional gene-guided enrichment plus in situ microsphere cultivation enables isolation of new crucial ureolytic bacteria from the rumen of cattle

BackgroundRuminants can utilize urea as a dietary nitrogen source owing to their ability to recycle urea-N back to the rumen where numerous ureolytic bacteria hydrolyze urea into ammonia, which is used by numerous bacteria as their nitrogen source. Rumen ureolytic bacteria are the key microbes making ruminants the only type of animals independent of pre-formed amino acids for survival, thus having attracted much research interest. Sequencing-based studies have helped gain new insights into ruminal ureolytic bacterial diversity, but only a limited number of ureolytic bacteria have been isolated into pure cultures or studied, hindering the understanding of ureolytic bacteria with respect to their metabolism, physiology, and ecology, all of which are required to effectively improve urea-N utilization efficiency.ResultsWe established and used an integrated approach, which include urease gene (ureC) guided enrichment plus in situ agarose microsphere embedding and cultivation under rumen-simulating conditions, to isolate ureolytic bacteria from the rumen microbiome. We optimized the dilutions of the rumen microbiome during the enrichment, single-cell embedding, and then in situ cultivation of microsphere-embedded bacteria using dialysis bags placed in rumen fluid. Metabonomic analysis revealed that the dialysis bags had a fermentation profile very similar to the simulated rumen fermentation. In total, we isolated 404 unique strains of bacteria, of which 52 strains were selected for genomic sequencing. Genomic analyses revealed that 28 strains, which were classified into 12 species, contained urease genes. All these ureolytic bacteria represent new species ever identified in the rumen and represented the most abundant ureolytic species. Compared to all the previously isolated ruminal ureolytic species combined, the newly isolated ureolytic bacteria increased the number of genotypically and phenotypically characterized ureolytic species by 34.38% and 45.83%, respectively. These isolated strains have unique genes compared to the known ureolytic strains of the same species indicating their new metabolic functions, especially in energy and nitrogen metabolism. All the ureolytic species were ubiquitous in the rumen of six different species of ruminants and were correlated to dietary urea metabolism in the rumen and milk protein production. We discovered five different organizations of urease gene clusters among the new isolates, and they had varied approaches to hydrolyze urea. The key amino acid residues of the UreC protein that potentially plays critical regulatory roles in urease activation were also identified.ConclusionsWe established an integrated methodology for the efficient isolation of ureolytic bacteria, which expanded the biological resource of crucial ureolytic bacteria from the rumen. These isolates play a vital role in the incorporation of dietary nitrogen into bacterial biomass and hence contribute to ruminant growth and productivity. Moreover, this methodology can enable efficient isolation and cultivation of other bacteria of interest in the environment and help bridge the knowledge gap between genotypes and phenotypes of uncultured bacteria.6cxB9n2rooY1JSFFcvMWCJVideo abstract

In Silico Analysis of nsSNPs of Human KRAS Gene and Protein Modeling Using Bioinformatic Tools.

The KRAS gene belongs to the RAS family and codes for 188 amino acid residues of KRAS protein, with a molecular mass of 21.6 kD. Non-synonymous single-nucleotide polymorphisms (nsSNPs) have been identified within the coding region in which some are associated with different diseases. However, structural changes are not well defined yet. In this study, we first categorized SNPs in the KRAS coding area and then used computational methods to determine their impact on the protein structure and stability. In addition, the three-dimensional model of KRAS was taken from the Protein Data Bank for structural modeling. Furthermore, genomic data were extracted from a variety of sources, including the 1000 Genome Project, dbSNPs, and ENSEMBLE, and assessed through in silico methods. Based on various tools used in this study, 10 out of 48 missense SNPs with rsIDs were found deleterious. The substitution of alanine for proline at position 146 pushed several residues toward the center of the protein. Arginine instead of leucine has a minor effect on protein structure and stability. In addition, the substitution of proline for leucine at the 34th position disrupted the structure and led to a bigger size than the wild-type protein, hence interrupting the protein interaction. Using the well-intended computational approach and applying several bioinformatic tools, we characterized and identified most damaging nsSNPs and further explored the structural dynamics and stability of KRAS protein.

In Silico Structural and Functional Annotations of Hypothetical Protein All4871 as a 1-Acyl-sn-Glycerol-3-Phosphate Acyltransferase from Nostoc sp. PCC 7120

In this study, the in silico structural and functional characterization of a hypothetical protein All4871 from Nostoc sp. PCC 7120 was performed. The 287 amino acid residue protein sequence revealed considerable homology with 1-acyl-sn-glycerol-3-phosphate acyltransferase (AGPAT) from different organisms. Multiple sequence alignment of All4871 with the AGPAT isomers showed the presence of conserved motifs (motif I-IV) crucial for acyltransferase activity. The All4871 protein depicted the characteristic topology of membrane-bound AGPATs. Both N- and C-terminus of the All4871 protein face the inner side of the membrane. The predicted tertiary structure of All4871 has an N-terminal helix and αβ catalytic core, which are characteristic features of AGPATs. Amongst the CASTp predicted binding pockets, pocket 1 acts as a groove-spanning active site crucial for acyltransferase catalysis, lysophosphatidic acid (LPA), and acyl coA (MCL) binding site. Docking studies further supported pocket 1 as the binding site for both LPA and MCL, which act as substrates for acyltransferase catalysis. So, the overall analyses have pointed toward the possibility that the hypothetical protein All4871 from Nostoc sp. PCC7120 may act as a 1-acyl-sn-glycerol-3-phosphate acyltransferase.

HIV-1 Tat amino acid residues that influence Tat-TAR binding affinity: a scoping review

HIV-1 remains a global health concern and to date, nearly 38 million people are living with HIV. The complexity of HIV-1 pathogenesis and its subsequent prevalence is influenced by several factors including the HIV-1 subtype. HIV-1 subtype variation extends to sequence variation in the amino acids of the HIV-1 viral proteins. Of particular interest is the transactivation of transcription (Tat) protein due to its key function in viral transcription. The Tat protein predominantly functions by binding to the transactivation response (TAR) RNA element to activate HIV-1 transcriptional elongation. Subtype-specific Tat protein sequence variation influences Tat-TAR binding affinity. Despite several studies investigating Tat-TAR binding, it is not clear which regions of the Tat protein and/or individual Tat amino acid residues may contribute to TAR binding affinity. We, therefore, conducted a scoping review on studies investigating Tat-TAR binding. We aimed to synthesize the published data to determine (1) the regions of the Tat protein that may be involved in TAR binding, (2) key Tat amino acids involved in TAR binding and (3) if Tat subtype-specific variation influences TAR binding. A total of thirteen studies met our inclusion criteria and the key findings were that (1) both N-terminal and C-terminal amino acids outside the basic domain (47–59) may be important in increasing Tat-TAR binding affinity, (2) substitution of the amino acids Lysine and Arginine (47–59) resulted in a reduction in binding affinity to TAR, and (3) none of the included studies have investigated Tat subtype-specific substitutions and therefore no commentary could be made regarding which subtype may have a higher Tat-TAR binding affinity. Future studies investigating Tat-TAR binding should therefore use full-length Tat proteins and compare subtype-specific variations. Studies of such a nature may help explain why we see differential pathogenesis and prevalence when comparing HIV-1 subtypes.

Multi-model in silico characterization of 3-benzamidobenzoic acid derivatives as partial agonists of Farnesoid X receptor in the management of NAFLD

Non-alcoholic fatty liver disease (NAFLD) is a pathological condition which is strongly correlated with fat accumulation in the liver that has become a major health hazard globally. So far, limited treatment options are available for the management of NAFLD and partial agonism of Farnesoid X receptor (FXR) has proven to be one of the most promising strategies for treatment of NAFLD. In present work, a range of validated predictive cheminformatics and molecular modeling studies were performed with a series of 3-benzamidobenzoic acid derivatives in order to recognize their structural requirements for possessing higher potency towards FXR. 2D-QSAR models were able to extract the most significant structural attributes determining the higher activity towards the receptor. Ligand-based pharmacophore model was created with a novel and less-explored open access tool named QPhAR to acquire information regarding important 3D-pharmacophoric features that lead to higher agonistic potential towards the FXR. The alignment of the dataset compounds based on pharmacophore mapping led to 3D-QSAR models that pointed out the most crucial steric and electrostatic influence. Molecular dynamics (MD) simulation performed with the most potent and the least potent derivatives of the current dataset helped us to understand how to link the structural interpretations obtained from 2D-QSAR, 3D-QSAR and pharmacophore models with the involvement of specific amino acid residues in the FXR protein. The current study revealed that hydrogen bond interactions with carboxylate group of the ligands play an important role in the ligand receptor binding but higher stabilization of different helices close to the binding site of FXR (e.g., H5, H6 and H8) through aromatic scaffolds of the ligands should lead to higher activity for these ligands. The present work affords important guidelines towards designing novel FXR partial agonists for new therapeutic options in the management of NAFLD. Moreover, we relied mainly on open-access tools to develop the in-silico models in order to ensure their reproducibility as well as utilization.

Heterozygous ATP2A2 missense variant identified in a Shih Tzu with Darier disease.

Darier disease is caused by heterozygous loss of function variants in the ATP2A2 gene encoding the endoplasmic/sarcoplasmic reticulum Ca2+ pump ATP2A2. Defective intracellular calcium signaling in the epidermis results in a loss of desmosomal adhesion and the development of characteristic skin lesions. In this study, we investigated a Shih Tzu that developed erythematous papules on the ventrum and, over time, the dorsal neck and a nodule in the right ear canal with secondary ear infection. Histopathologic examination demonstrated discrete foci of acantholysis affecting suprabasal layers of the epidermis. Whole genome sequencing of the affected dog identified a heterozygous missense variant, p.N809H, affecting an evolutionarily conserved amino acid residue of the ATP2A2 protein. The highly characteristic clinical and histopathologic findings together with a plausible variant in the only known functional candidate gene establish the diagnosis of canine Darier disease in the studied dog and highlight the potential of genetic analyses as complementary diagnostic approach in veterinary medicine.

Poly(ADP-ribose)polymerases 1 and 2: Classical Functions and Interaction with HPF1 ‒ New Histone Poly(ADP-ribosyl)ation Factor

Poly(ADP-ribose) (PAR) is a negatively charged polymer, linear and branched, consisting of ADP-ribose monomers. This polymer is synthesized by poly(ADP-ribose)polymerase (PARP) enzymes which are activated on DNA damage and use nicotinamide adenine dinucleotide (NAD+) as a substrate. The most studied members of the PARP family, PARP1 and PARP2, are the most important nuclear proteins involved in many cellular processes, including the regulation of DNA repair. PARP1 and PARP2 catalyze both the synthesis and transfer of poly(ADP-ribose) to amino acid residues of target proteins, including autoPARylation. In view of the key role in the regulation of the DNA repair process, PARP1 and PARP2 are promising targets for chemotherapy. Recently, a novel histone PARylation factor (HPF1) has been discovered to modulate PARP1/2 activity by forming a transient joint active site with PARP1/2. In the presence of HPF1, histone modification occurs at serine residues. The general mechanism of interaction between HPF1 and PARP1/2 is only beginning to be elucidated. In this review, we consider the discovery and classical mechanism of this important process in higher eukaryotes, as well as the role of a new histone PARylation factor in this HPF1 process.

Investigating the biophysical interaction of serum albumins-gold nanorods using hybrid spectroscopic and computational approaches with the intent of enhancing cytotoxicity efficiency of targeted drug delivery

In this contribution, biophysical aspects of binding interaction, conformational changes, and molecular simulation studies of human and bovine serum albumins (HSA and BSA) exposed to gold nanorods (AuNRs) are thoroughly investigated and their bio-nanoconjugates were then used in in-vitro cytotoxic studies. The structural variations of AuNRs in the presence of serum proteins were confirmed by absorbance and high-resolution transmission electron microscopic (HR-TEM) pictures. According to the results of the steady-state emission quenching, AuNRs affect the intrinsic emission of HSA and BSA by a static mechanism. Synchronous emission spectrum results showed that the microenvironment near the tryptophan and tyrosine amino acid residues of serum proteins is altered, and the impact is stronger toward tryptophan than tyrosine. Furthermore, it was demonstrated by infrared, absorbance, and three-dimensional (3D) luminescence spectroscopic methods that the binding of AuNRs can arouse concerns and cause changes in the structural and/or microenvironmental characteristics of HSA and BSA. An outcome of the computational molecular simulation study supports the stability of the bio-nanoconjugate, and molecular docking interaction investigations suggest that the two-dimensional AuNRs surface associated with the cysteine residue of HSA and BSA contains sulphur atoms. Furthermore, using an in vitro model using the A-549 and MCF-7 cell lines, we calculated the toxicity and the effectiveness of cellular absorption of the AuNRs and their bio-nanoconjugates as a function of supplied dosage. These cytotoxicity findings demonstrate that protein adsorption affects AuNRs' physicochemical characteristics as well as enhances their subsequent biological function.

Biochemical Characterization and Functional Analysis of Glucose Regulated Protein 78 from the Silkworm Bombyx mori.

The glucose regulated protein (GRP78) is an important chaperone for various environmental and physiological stimulations. Despite the importance of GRP78 in cell survival and tumor progression, the information regarding GRP78 in silkworm Bombyx mori L. is poorly explored. We previously identified that GRP78 expression was significantly upregulated in the silkworm Nd mutation proteome database. Herein, we characterized the GRP78 protein from silkworm B. mori (hereafter, BmGRP78). The identified BmGRP78 protein encoded a 658 amino acid residues protein with a predicted molecular weight of approximately 73 kDa and comprised of two structural domains, a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD). BmGRP78 was ubiquitously expressed in all examined tissues and developmental stages by quantitative RT-PCR and Western blotting analysis. The purified recombinant BmGRP78 (rBmGRP78) exhibited ATPase activity and could inhibit the aggregating thermolabile model substrates. Heat-induction or Pb/Hg-exposure strongly stimulated the upregulation expression at the translation levels of BmGRP78 in BmN cells, whereas no significant change resulting from BmNPV infection was found. Additionally, heat, Pb, Hg, and BmNPV exposure resulted in the translocation of BmGRP78 into the nucleus. These results lay a foundation for the future identification of the molecular mechanisms related to GRP78 in silkworms.

Poly(ADP-Ribose) Polymerases 1 and 2: Classical Functions and Interaction with New Histone Poly(ADP-Ribosyl)ation Factor HPF1

Poly(ADP-ribose) (PAR) is a negatively charged polymer, linear or branched, that consists of ADP-ribose monomers. PAR is synthesized by poly(ADP-ribose)polymerase (PARP) enzymes, which are activated upon DNA damage and use nicotinamide adenine dinucleotide (NAD^(+)) as a substrate. The best-studied members of the PARP family, PARP1 and PARP2, are the most important nuclear proteins involved in many cell processes, including the regulation of DNA repair. PARP1 and PARP2 catalyze PAR synthesis and transfer to amino acid residues of target proteins, including autoPARylation. PARP1 and PARP2 are promising targets for chemotherapy in view of their key role in regulating DNA repair. A novel histone PARylation factor (HPF1) was recently discovered to modulate PARP1/2 activity by forming a transient joint active site with PARP1/2. Histones are modified at serine residues in the presence of HPF1. The general mechanism of the interaction between HPF1 and PARP1/2 is a subject of intense research now. The review considers the discovery and classical mechanism of PARylation in higher eukaryotes and the role of HPF1 in the process.

Influence of the pKa value on the antioxidant activity of licorice flavonoids under solvent-mediated effects.

Licorice flavonoids (LCFs) have been widely used in food care and medical treatment due to their significant antioxidant activities. However, the molecular mechanism of their antioxidant activity remains unclear. Therefore, network pharmacology, ADMET, density functional theory (DFT), molecular docking, and molecular dynamics (MD) simulation were employed to explore the molecular mechanism of the antioxidant effects of LCF. The network pharmacology and ADMET studies showed that the active molecules of kumatakenin (pKa = 6.18), licoflavonol (pKa = 6.86), and topazolin (pKa = 6.21) in LCF are key antioxidant components and have good biosafety. Molecular docking and MD simulation studies demonstrated that active molecules interacted with amino acid residues in target proteins to form stable protein-ligand complexes and exert their antioxidant effects. DFT studies showed that the antioxidant activity of LCF could be significantly modulated under the solvent-mediated effect. In addition, based on the derivation of the Henderson-Hasselbalch and van't Hoff formulas, the functional relationships between the reaction-free energy (ΔG) of LCF and the pH and pKa values were established. The results showed that active molecules with larger pKa values will be more conducive to the improvement of their antioxidant activity under solvent-mediated effects. In conclusion, this study found that increasing the pKa value of LCF would be an effective strategy to improve their antioxidant activity under the effect of solvent mediation. The pKa value of an LCF will be a direct standard to evaluate its solvent-mediated antioxidant activity. This study will provide theoretical guidance for the development of natural antioxidants.

The binding mechanism of oat phenolic acid to whey protein and its inhibition mechanism against AGEs as revealed using spectroscopy, chromatography and molecular docking.

Heat sterilization of dairy products can promote the formation of advanced glycation end products (AGEs), protein oxidation products (POPs) and α-dicarbonyl compounds, which have a significant influence on health due to the close association of these products with diabetes complications. In this study, eight oat phenolic acids were first analyzed for their inhibitory effect against AGEs formation. Due to their strong inhibitory effects and structural differences, caffeic acid (CA) and gallic acid (GA) were further selected to assess their anti-glycosylation mechanisms using spectroscopy, chromatography and molecular docking. CA/GA reduced the production of total AGEs and POPs in various bovine milk simulation models and protected whey proteins from structural modifications, oxidation, and cross-linking. Comparative analyses showed a structure-effect relationship between CA/GA and AGEs inhibition. Oat phenolic acids against AGEs and POPs might be related to the unique bonding of key amino acid residues in whey proteins, the inhibitory role of early fructosamine and the trapping of reactive α-dicarbonyl groups to form adducts. In conclusion, oat phenolic acids might present a promising dietary strategy to alleviate AGEs production and glycation of proteins in dairy products upon storage.

In-silico Structural Modeling of Human Immunodeficiency Virus Proteins.

Human immunodeficiency virus (HIV) is an infectious virus that depletes the CD4+ T lymphocytes of the immune system and causes a chronic life-treating disease-acquired immunodeficiency syndrome (AIDS). The HIV genome encodes different structural and accessory proteins involved in viral entry and life cycle. Determining the 3D structure of HIV proteins is essential for new target position finding, structure-based drug designing, and future planning for computational and laboratory experimentations. Hence, the study aims to predict the 3D structures of all the HIV structural and accessory proteins using computational homology modeling to understand better the structural basis of HIV proteins interacting with host cells and viral replication. The sequences of HIV capsid, matrix, nucleocapsid, p6, reverse transcriptase, invertase, protease, gp120, gp41, virus protein r, viral infectivity factor, virus protein unique, RNA splicing regulator, transactivator protein, negative regulating factor, and virus protein x proteins were retrieved from UniProt. The primary and secondary structures of HIV proteins were predicted by Expasy ProtParam and SOPMA web servers. For the homology modeling, the MODELLER predicted the 3D structures of HIV proteins using templates. Then, the modeled structures were validated by the Ramachandran plot, local and global quality estimation scores, QMEAN scores, and Z-scores. Most of the amino acid residues of HIV proteins were present in the most favored and generously allowed regions in the Ramachandran plots. The local and global quality scores and Z-scores of the HIV proteins confirmed the good quality of modeled structures. The 3D modeled structures of HIV proteins might help further investigate the possible treatment.

Molecular Docking of Shallot (Allium ascalonicum) Active Compounds to Lanosterol Enzym 14-Alpha Demethylase and Squalene Monooxygenase for Antifungi Potential Activity

<p>Shallots <em>(Allium ascalonicum</em>) are one of the important horticultural export commodities in Indonesia, but the productivity of shallots has decreased due to the attack of pathogenic fungi. This study was aimed to determine the potential of active shallot compounds as ligands for inhibiting the activity of the enzyme lanosterol 14-alpha demethylase (ID: 4LXJ) and squalene monooxygenase (ID: 6C6R) enzymes which play an important role in the biosynthesis of ergosterol or fungal cell membranes. The molecular docking used the Autodock Vina (PyRx) program, an analysis of molecule interaction used PyMol, and Discovery Studio 2019 to analyze the types of bonds between molecules. Thirty-nine ligands from shallot compounds are docked to the anti-fungal target protein. The results showed ascalonicoside A1, ascalonicoside A2, ascalonicoside B, quercetin, isorhamnetin, quercetin di glucoside, quercetin tri glucoside, ethyl palmitate and benzyl salicylate have the potential to be anti-fungal to the enzyme lanosterol 14-alpha demethylase and squalene monooxygenase enzyme that responds to the synthesis of fungi cell wall. These ligand compounds bind to the target protein's amino acid residues with hydrogen and hydrophobic bonds. This research showed that shallot was the potential to be replicated as an anti-fungus for many purposes.</p>

Oxidant-Induced Bioconjugation for Protein Labeling in Live Cells.

Chemical proteomics is a powerful technology that can be used in the studies of the functions of uncharacterized proteins in the human proteome. It relies on a suitable bioconjugation strategy for protein labeling. This could be either a UV-responsive photo-crosslinker or an electrophilic warhead embedded in chemical probes that can form covalent bonds with target proteins. Here, we report a new protein-labeling strategy in which a nitrile oxide, a highly reactive intermediate that reacts with proteins, can be efficiently generated by the treatment of oximes with a water-soluble and a minimally toxic oxidant, phenyliodine bis (trifluoroacetate) (PIFA). The resulting intermediate can rapidly bioconjugate with amino acid residues of target proteins, thus enabling target identification of oxime-containing bioactive molecules. Excellent chemoselectivity of cysteine residues by the nitrile oxide was observed, and over 4000 reactive and/or accessible cysteines, including KRAS G12C, have been successfully characterized by quantitative chemical proteomics. Some of these residues could not be detected by conventional cysteine reagents, thus demonstrating the complementary utility of this method.

Contingent Synergistic Interactions between Non-Coding RNAs and DNA-Modifying Enzymes in Myelodysplastic Syndromes.

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell disorders with maturation and differentiation defects exhibiting morphological dysplasia in one or more hematopoietic cell lineages. They are associated with peripheral blood cytopenias and by increased risk for progression into acute myelogenous leukemia. Among their multifactorial pathogenesis, age-related epigenetic instability and the error-rate DNA methylation maintenance have been recognized as critical factors for both the initial steps of their pathogenesis and for disease progression. Although lower-risk MDS is associated with an inflammatory bone marrow microenvironment, higher-risk disease is delineated by immunosuppression and clonal expansion. "Epigenetics" is a multidimensional level of gene regulation that determines the specific gene networks expressed in tissues under physiological conditions and guides appropriate chromatin rearrangements upon influence of environmental stimulation. Regulation of this level consists of biochemical modifications in amino acid residues of the histone proteins' N-terminal tails and their concomitant effects on chromatin structure, DNA methylation patterns in CpG dinucleotides and the tissue-specific non-coding RNAs repertoire, which are directed against various gene targets. The role of epigenetic modifications is widely recognized as pivotal both in gene expression control and differential molecular response to drug therapies in humans. Insights to the potential of synergistic cooperations of epigenetic mechanisms provide new avenues for treatment development to comfort human diseases with a known epigenetic shift, such as MDS. Hypomethylating agents (HMAs), such as epigenetic modulating drugs, have been widely used in the past years as first line treatment for elderly higher-risk MDS patients; however, just half of them respond to therapy and are benefited. Rational outcome predictors following epigenetic therapy in MDS and biomarkers associated with disease relapse are of high importance to improve our efforts in developing patient-tailored clinical approaches.

The role of C-terminal ionic residues in self-association of apolipoprotein A-I

Apolipoprotein A-I (apoA-I) is the main protein of high-density lipoprotein and is comprised of a helical bundle domain and a C-terminal (CT) domain encompassing the last ~65 amino acid residues of the 243-residue protein. The CT domain contains three putative helices (helix 8, 9, and 10) and is critical for initiating lipid binding and harbors sites that mediate self-association of the lipid-free protein. Three lysine residues reside in helix-8 (K195, 206, 208), and three in helix-10 (K226, 238, 239). To determine the role of each CT lysine residue in apoA-I self-association, single, double and triple lysine to glutamine mutants were engineered via site-directed mutagenesis. Circular dichroism and chemical denaturation analysis revealed all mutants retained their structural integrity. Chemical crosslinking and size-exclusion chromatography showed a small effect on self-association when helix-8 lysine residues were changed into glutamine. In contrast, mutation of the three helix-10 lysine residues resulted in a predominantly monomeric protein and K226 was identified as a critical residue. When helix-10 glutamate residues 223, 234, or 235 were substituted with glutamine, reduced self-association was observed similar to that of the helix-10 lysine variants, suggesting ionic interactions between these residues. Thus, helix-10 is a critical part of apoA-I mediating self-association, and disruption of ionic interactions changes apoA-I from an oligomeric state into a monomer. Since the helix-10 triple mutant solubilized phospholipid vesicles at higher rates compared to wild-type apoA-I, this indicates monomeric apoA-I is more potent in lipid binding, presumably because helix-10 is fully accessible to interact with lipids.

Potential Disruption of Systemic Hormone Transport by Tobacco Alkaloids Using Computational Approaches.

Tobacco/nicotine is one of the most toxic and addictive substances and continues to pose a significant threat to global public health. The harmful effects of smoking/nicotine affect every system in the human body. Nicotine has been associated with effects on endocrine homeostasis in humans such as the imbalance of gonadal steroid hormones, adrenal corticosteroid hormones, and thyroid hormones. The present study was conducted to characterize the structural binding interactions of nicotine and its three important metabolites, cotinine, trans-3'-hydroxycotinine, and 5'-hydroxycotinine, against circulatory hormone carrier proteins, i.e., sex-hormone-binding globulin (SHBG), corticosteroid-binding globulin (CBG), and thyroxine-binding globulin (TBG). Nicotine and its metabolites formed nonbonded contacts and/or hydrogen bonds with amino acid residues of the carrier proteins. For SHBG, Phe-67 and Met-139 were the most important amino acid residues for nicotine ligand binding showing the maximum number of interactions and maximum loss in ASA. For CBG, Trp-371 and Asn-264 were the most important amino acid residues, and for TBG, Ser-23, Leu-269, Lys-270, Asn-273, and Arg-381 were the most important amino acid residues. Most of the amino acid residues of carrier proteins interacting with nicotine ligands showed a commonality with the interacting residues for the native ligands of the proteins. Taken together, the results suggested that nicotine and its three metabolites competed with native ligands for binding to their carrier proteins. Thus, nicotine and its three metabolites may potentially interfere with the binding of testosterone, estradiol, cortisol, progesterone, thyroxine, and triiodothyronine to their carrier proteins and result in the disbalance of their transport and homeostasis in the blood circulation.