Amino Acid Residues Research Articles

Identification and Design of Novel Potential Antimicrobial Peptides Targeting Mycobacterial Protein Kinase PknB.

Antimicrobial peptides have gradually gained advantages over small molecule inhibitors for their multifunctional effects, synthesising accessibility and target specificity. The current study aims to determine an antimicrobial peptide to inhibit PknB, a serine/threonine protein kinase (STPK), by binding efficiently at the helically oriented hinge region. A library of 5626 antimicrobial peptides from publicly available repositories has been prepared and categorised based on the length. Molecular docking using ADCP helped to find the multiple conformations of the subjected peptides. For each peptide served as input the tool outputs 100 poses of the subjected peptide. To maintain an efficient binding for relatively a longer duration, only those peptides were chosen which were seen to bind constantly to the active site of the receptor protein over all the poses observed. Each peptide had different number of constituent amino acid residues; the peptides were classified based on the length into five groups. In each group the peptide length incremented upto four residues from the initial length form. Five peptides were selected for Molecular Dynamic simulation in Gromacs based on higher binding affinity. Post-dynamic analysis and the frame comparison inferred that neither the shorter nor the longer peptide but an intermediate length of 15 mer peptide bound well to the receptor. Residual substitution to the selected peptides was performed to enhance the targeted interaction. The new complexes considered were further analysed using the Elastic Network Model (ENM) for the functional site's intrinsic dynamic movement to estimate the new peptide's role. The study sheds light on prospects that besides the length of peptides, the combination of constituent residues equally plays a pivotal role in peptide-based inhibitor generation. The study envisages the challenges of fine-tuned peptide recovery and the scope of Machine Learning (ML) and Deep Learning (DL) algorithm development. As the study was primarily meant for generation of therapeutics for Tuberculosis (TB), the peptide proposed by this study demands meticulous invitro analysis prior to clinical applications.

Discovery and Characterization of Panaxatriol as a Novel Thrombin Inhibitor from Panax notoginseng Using a Combination of Computational and Experimental Approaches.

Thrombin is a crucial enzyme in the coagulation cascade, and inhibitors of thrombin have been extensively studied as potential antithrombotic agents. The objective of this study was to identify natural inhibitors of thrombin from Panax notoginseng and evaluate their biological activity in vitro and binding characteristics. A combined approach involving molecular docking, thrombin inhibition assays, surface plasmon resonance, and molecular dynamics simulation was utilized to identify natural thrombin inhibitors. The results demonstrated that panaxatriol directly inhibits thrombin, with an IC50 of 10.3 µM. Binding studies using surface plasmon resonance revealed that panaxatriol interacts with thrombin, with a KD value of 7.8 µM. Molecular dynamics analysis indicated that the thrombin-panaxatriol system reached equilibrium rapidly with minimal fluctuations, and the calculated binding free energy was - 23.8 kcal/mol. The interaction between panaxatriol and thrombin involves the amino acid residues Glu146, Glu192, Gly216, Gly219, Tyr60A, and Trp60D. This interaction provides a mechanistic basis for further optimizing panaxatriol as a thrombin inhibitor. Our study has shown that panaxatriol serves as a direct thrombin inhibitor, laying the groundwork for further research and development of novel thrombin inhibitors.

EFCAB4B (CRACR2A/Rab46) Genetic Variants Associated with COVID-19 Fatality

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in more than 692 million cases worldwide and nearly 7 million deaths (August 2023). Severe COVID-19 is characterised in part by vascular thrombosis and a cytokine storm due to increased plasma concentrations of pro-thrombotic proteins such as von Willebrand factor and cytokines secreted from endothelial and T-cells. EFCAB4B is a gene that encodes for two proteins (CRACR2A and Rab46) that play important roles in endothelial and T-cell secretion. In this study, using patient data recorded in the UK Biobank, we demonstrate the importance of variants in the EFCAB4B genetic sequence with COVID-19 fatality. Using logistic regression analysis, we determined that three single-nucleotide polymorphisms (SNPs) in the gene cause missense variations in CRACR2A and Rab46, which are associated with COVID-19 fatality (rs9788233: p = 0.004, odds ratio = 1.511; rs17836273: p = 0.012, odds ratio = 1.433; rs36030417: p = 0.013, odds ratio = 1.393). All three SNPs cause changes in amino acid residues that are highly conserved across species, indicating their importance in protein structure and function. Two SNPs, rs17836273 (A98T) and rs36030417 (H212Q), cause amino acid substitutions in important functional domains: the EF-hand and coiled-coil domain, respectively. Molecular modelling shows minimal impact by the substitution of threonine at position 98 on the structure of the EF-hand. Since Rab46 is a GTPase that regulates both endothelial cell secretion and T-cell signalling, these missense variants may play a role in the molecular mechanisms underlying the thrombotic and inflammatory characteristics observed in patients with severe COVID-19 outcomes.

The 3,3'-dimethoxy-4,4'-dihydroxy-stilbene Triazole (STT) Inhibits Liver Cancer Cell Growth by Targeting Akt/mTOR Pathway.

The present study was aimed to investigate the proliferation inhibitory ability of 3,3'-dimethoxy-4,4'-dihydroxy-stilbene triazole (STT) on SNU449 and Huh7 cells. Moreover, the mechanism associated with the suppression of liver cancer cell proliferation by STT was also studied. The results revealed that STT suppresses proliferation of SNU449 and Huh7 cells to 28 and 21%, respectively treatment with 20 µM. The clonogenic survival of SNU449 and Huh7 cells was also significantly reduced after incubation with STT compared to the control cultures. In comparison to the control, STT treatment significantly decreased the invasive potential of SNU449 cells. Treatment with STT led to a prominent suppression in p62 and increase in LC3B protein expression in SNU449 cells compared to the control cells. The STT treatment dramatically decreased p-Akt and p-mTOR protein expression in SNU449 cells. Docking study revealed that STT interacts via traditional hydrogen bonding with the glutamine, phenylalanine, leucine, serine, arginine, aspartic acid, and lysine residues of Akt protein. In summary, the current study demonstrates that STT effectively suppresses the viability of SNU449 and Huh7 liver cancer cells. Moreover, STT treatment of the liver cancer cells also significantly reduces the clonogenic survival and invasive potential of SNU449 cells. Treatment of liver cancer cells with STT increases the expression of autophagic, targets anti-autophagic protein expression and down-regulates Akt/mTOR pathway to inhibit cancer growth and proliferation. Thus, STT exhibits prominent anticancer effect and needs to be investigated further as a potential candidate for the treatment of liver cancer.

Saturation Mutagenesis and Molecular Modeling: The Impact of Methionine 182 Substitutions on the Stability of β-Lactamase TEM-1.

Serine β-lactamase TEM-1 is the first β-lactamase discovered and is still common in Gram-negative pathogens resistant to β-lactam antibiotics. It hydrolyzes penicillins and cephalosporins of early generations. Some of the emerging TEM-1 variants with one or several amino acid substitutions have even broader substrate specificity and resistance to known covalent inhibitors. Key amino acid substitutions affect catalytic properties of the enzyme, and secondary mutations accompany them. The occurrence of the secondary mutation M182T, called a "global suppressor", has almost doubled over the last decade. Therefore, we performed saturating mutagenesis at position 182 of TEM-1 to determine the influence of this single amino acid substitution on the catalytic properties, thermal stability, and ability for thermoreactivation. Steady-state parameters for penicillin, cephalothin, and ceftazidime are similar for all TEM-1 M182X variants, whereas melting temperature and ability to reactivate after incubation at a higher temperature vary significantly. The effects are multidirectional and depend on the particular amino acid at position 182. The M182E variant of β-lactamase TEM-1 demonstrates the highest residual enzymatic activity, which is 1.5 times higher than for the wild-type enzyme. The 3D structure of the side chain of residue 182 is of particular importance as observed from the comparison of the M182I and M182L variants of TEM-1. Both of these amino acid residues have hydrophobic side chains of similar size, but their residual activity differs by three-fold. Molecular dynamic simulations add a mechanistic explanation for this phenomenon. The important structural element is the V159-R65-E177 triad that exists due to both electrostatic and hydrophobic interactions. Amino acid substitutions that disturb this triad lead to a decrease in the ability of the β-lactamase to be reactivated.

Clickable tryptophan modification for late-stage diversification of native peptides.

As the least abundant residue in proteins, tryptophan widely exists in peptide drugs and bioactive natural products and contributes to drug-target interactions in multiple ways. We report here a clickable tryptophan modification for late-stage diversification of native peptides, via catalyst-free C2-sulfenylation with 8-quinoline thiosulfonate reagents in trifluoroacetic acid (TFA). A wide range of groups including trifluoromethylthio (SCF3), difluoromethylthio (SCF2H), (ethoxycarbonyl)difluoromethylthio (SCF2CO2Et), alkylthio, and arylthio were readily incorporated. The rapid reaction kinetics of Trp modification and full tolerance with other 19 proteinogenic amino acids, as well as the super dissolving capability of TFA, render this method suitable for all kinds of Trp-containing peptides without limitations from sequences, hydrophobicity, and aggregation propensity. The late-stage modification of 15 therapeutic peptides (1.0 to 7.6 kilodaltons) and the improved bioactivity and serum stability of SCF3- and SCF2H-modified melittin analogs illustrated the effectiveness of this method and its potential in pharmacokinetic property improvement.

Clickable tryptophan modification for late-stage diversification of native peptides.

As the least abundant residue in proteins, tryptophan widely exists in peptide drugs and bioactive natural products and contributes to drug-target interactions in multiple ways. We report here a clickable tryptophan modification for late-stage diversification of native peptides, via catalyst-free C2-sulfenylation with 8-quinoline thiosulfonate reagents in trifluoroacetic acid (TFA). A wide range of groups including trifluoromethylthio (SCF3), difluoromethylthio (SCF2H), (ethoxycarbonyl)difluoromethylthio (SCF2CO2Et), alkylthio, and arylthio were readily incorporated. The rapid reaction kinetics of Trp modification and full tolerance with other 19 proteinogenic amino acids, as well as the super dissolving capability of TFA, render this method suitable for all kinds of Trp-containing peptides without limitations from sequences, hydrophobicity, and aggregation propensity. The late-stage modification of 15 therapeutic peptides (1.0 to 7.6 kilodaltons) and the improved bioactivity and serum stability of SCF3- and SCF2H-modified melittin analogs illustrated the effectiveness of this method and its potential in pharmacokinetic property improvement.

Identification of natural inhibitors targeting Trehalase of Anopheles funestus in the management of malaria: A Biocomputational assessment.

Anopheles funestus is playing an increasingly important role in malaria transmission in sub-Saharan Africa. Trehalase, an enzyme required for trehalose breakdown, is important for mosquito flight and stress adaptation. Hence, its inhibition has emerged as a promising malaria management strategy. A collection of 1900 natural compounds from the ZINC database were screened against the 3D modeled structure of the A. funestus trehalase protein using in-silico tools. ADMET-AI, a web-based platform, was used to predict the absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties of the selected compounds. Here in this study, we report 5 natural compounds namely, ZINC00488388, ZINC00488525, ZINC00488566, ZINC00488304, and ZINC00488456 demonstrated strong binding affinity to the trehalase protein. These compounds interacted with critical residues of the trehalase protein and exhibited good drug-like characteristics. These compounds show promise as trehalase protein inhibitors for malaria management. Nonetheless, additional experimental studies are required to optimize these compounds as potential trehalase inhibitors.

Sirt3-Mediated Opa1 Deacetylation Protects Against Sepsis-Induced Acute Lung Injury by Inhibiting Alveolar Macrophage Pro-Inflammatory Polarization.

Aims: Mitochondrial dynamics in alveolar macrophages (AMs) are associated with sepsis-induced acute lung injury (ALI). In this study, we aimed to investigate whether changes in mitochondrial dynamics could alter the polarization of AMs in sepsis-induced ALI and to explore the regulatory mechanism of mitochondrial dynamics by focusing on sirtuin (SIRT)3-induced optic atrophy protein 1 (OPA1) deacetylation. Results: The AMs of sepsis-induced ALI showed imbalanced mitochondrial dynamics and polarization to the M1 macrophage phenotype. In sepsis, SIRT3 overexpression promotes mitochondrial dynamic equilibrium in AMs. However, 3-(1H-1, 2, 3-triazol-4-yl) pyridine (3TYP)-specific inhibition of SIRT3 increased the mitochondrial dynamic imbalance and pro-inflammatory polarization of AMs and further aggravated sepsis-induced ALI. OPA1 is directly bound to and deacetylated by SIRT3 in AMs. In AMs of sepsis-induced ALI, SIRT3 protein expression was decreased and OPA1 acetylation was increased. OPA1 acetylation at the lysine 792 amino acid residue (OPA1-K792) promotes self-cleavage and is associated with an imbalance in mitochondrial dynamics. However, decreased acetylation of OPA1-K792 reversed the pro-inflammatory polarization of AMs and protected the barrier function of alveolar epithelial cells in sepsis-induced ALI. Innovation: Our study revealed, for the first time, the regulation of mitochondrial dynamics and AM polarization by SIRT3-mediated deacetylation of OPA1 in sepsis-induced ALI, which may serve as an intervention target for precision therapy of the disease. Conclusions: Our data suggest that imbalanced mitochondrial dynamics promote pro-inflammatory polarization of AMs in sepsis-induced ALI and that deacetylation of OPA1 mediated by SIRT3 improves mitochondrial dynamic equilibrium, thereby ameliorating lung injury.

A mimetic peptide of ACE2 protects against SARS-CoV-2 infection and decreases pulmonary inflammation related to COVID-19

Since human angiotensin-converting enzyme 2 (ACE2) serves as a primary receptor for SARS-CoV-2, characterizing ACE2 regions that allow SARS-CoV-2 to enter human cells is essential for designing peptide-based antiviral blockers and elucidating the pathogenesis of the virus. We identified and synthesized a 25-mer mimetic peptide (encompassing positions 22-46 of the ACE2 alpha-helix α1) implicated in the S1 receptor-binding domain (RBD)-ACE2 interface. The mimetic (wild-type, WT) ACE2 peptide significantly inhibited SARS-CoV-2 infection of human pulmonary Calu-3 cells in vitro. In silico protein modeling predicted that residues F28, K31, F32, F40, and Y41 of the ACE2 alpha-helix α1 are critical for the original, Delta, and Omicron strains of SARS-CoV-2 to establish the Spike RBD-ACE2 interface. Substituting these residues with alanine (A) or aspartic acid (D) abrogated the antiviral protective effect of the peptides, indicating that these positions are critical for viral entry into pulmonary cells. WT ACE2 peptide, but not the A or D mutated peptides, exhibited significant interaction with the SARS-CoV-2 S1 RBD, as shown through molecular dynamics simulations. Through identifying the critical amino acid residues of the ACE2 alpha-helix α1, which is necessary for the Spike RBD-ACE2 interface and mobilized during the in vitro viral infection of cells, we demonstrated that the WT ACE2 peptide protects susceptible K18-hACE2 mice against in vivo SARS-CoV-2 infection and is effective for the treatment of COVID-19.

Phytochromobilin Binding and Specific Amino Acid Residues Near The Chromophore Contribute To Orange Light Perception By The Dualchrome Phytochrome Region.

A novel photoreceptor dualchrome 1 (DUC1), containing a fused structure of cryptochrome and phytochrome, was discovered in the marine green alga Pycnococcus provasolli. The DUC1 phytochrome region (PpDUC1-N) binds to the bilin (linear tetrapyrrole) chromophores, phytochromobilin (PΦB) or phycocyanobilin (PCB), and reversibly photoconverts between the orange-absorbing dark-adapted state and the far-red-absorbing photoproduct state. This contrasts with typical phytochromes, which photoconvert between the red-absorbing dark-adapted and far-red-absorbing photoproduct states. In this study, we examined the molecular mechanism of PpDUC1-N to sense orange light by identifying the chromophore species synthesized by P. provasolli and the amino acid residues within the PpDUC1-N responsible for sensing orange light in the dark-adapted state. We focused on the PcyA homolog of P. provasolli (PpPcyA). Coexpression with the photoreceptors followed by an enzymatic assay revealed that PpPcyA synthesized PCB. Next, we focused on the PpDUC1-N GAF domain responsible for chromophore binding and light sensing. Ten amino acid residues were selected as the mutagenesis target near the chromophore. Replacement of these residues with those conserved in typical phytochromes revealed that three mutations (F290Y/M304S/L353M) resulted in a 23-nm red-shift in the dark-adapted state. Finally, we combined these constructs to obtain the PΦB-binding F290Y/M304S/L353M mutant and a 38-nm red-shift was observed compared with the PCB-binding wild-type PpDUC1. The binding chromophore species and the key residues near the chromophore contribute to blue-shifted orange light sensing in the dark-adapted state of the PpDUC1-N.

Mono-ADP-ribosylation of peptides: an overview of synthetic and chemoenzymatic methodologies.

Adenosine diphosphate (ADP)-ribosylation is a ubiquitous post-translational modification that regulates vital biological processes like histone reorganization and DNA-damage repair through the modification of various amino acid residues. Due to advances in mass-spectrometry, the collection of long-known ADP-ribose (ADPr) acceptor sites, e.g. arginine, cysteine and glutamic acid, has been expanded with serine, tyrosine and histidine, among others. Well-defined ADPr-peptides are valuable tools for investigating the exact structures, mechanisms of action and interaction partners of the different flavors of this modification. This review provides a comprehensive overview of synthetic and chemoenzymatic methodologies that enabled the construction of peptides mono-ADP-ribosylated on various amino acids, and close mimetics thereof.

Active Site Characterization of a Campylobacter jejuni Nitrate Reductase Variant Provides Insight into the Enzyme Mechanism.

Mo K-edge X-ray absorption spectroscopy (XAS) is used to probe the structure of wild-type Campylobacter jejuni nitrate reductase NapA and the C176A variant. The results of extended X-ray absorption fine structure (EXAFS) experiments on wt NapA support an oxidized Mo(VI) hexacoordinate active site coordinated by a single terminal oxo donor, four sulfur atoms from two separate pyranopterin dithiolene ligands, and an additional S atom from a conserved cysteine amino acid residue. We found no evidence of a terminal sulfido ligand in wt NapA. EXAFS analysis shows the C176A active site to be a 6-coordinate structure, and this is supported by EPR studies on C176A and small molecule analogs of Mo(V) enzyme forms. The SCys is replaced by a hydroxide or water ligand in C176A, and we find no evidence of a coordinated sulfhydryl (SH) ligand. Kinetic studies show that this variant has completely lost its catalytic activity toward nitrate. Taken together, the results support a critical role for the conserved C176 in catalysis and an oxygen atom transfer mechanism for the catalytic reduction of nitrate to nitrite that does not employ a terminal sulfido ligand in the catalytic cycle.

Identification of Potent SFRP1 Inhibitors for Colorectal Cancer using a Comprehensive Computational Approach.

The incidence of CRC has increased worldwide over the past decade. The statistics report from WHO highlights the increased severity and fatality rate of CRC among the populations. Wnt/β-catenin is recognized as the resource for cell regeneration and cancer signaling pathways driven by frizzled receptor cofactors. Aberrant regulation of Wnt/β- catenin suppression is an important challenge in treating CRC management. The SFRP1 comprises a cysteine-rich region that is homologous to the putative Wnt-binding sites of Frizzled proteins, with the potential to impede and alter the cascade of Wnt signaling. Indirect regulation, like targeting Wnt antagonist SFRP1, is an alternative strategy to suppress the cancer signals by enhancing the apoptotic activity. Hence, this study aimed to approach the SFRP1 protein as a therapeutic target to inhibit Wnt signaling in colorectal cancer. Further, it aimed to identify the lead compounds against the SFRP1 protein, which inhibit the oncogenic expression of CRC, which might be possible using computational approaches, recognizing the importance of the SFRP1 protein role in CRC. The homology-modeled SFRP1 structure was refined, and virtual screening was performed against the anti-cancer drugs and natural drug databases to find the best hit molecules. The molecular docking, MD, and MMGBSA analysis confirmed the firm binding of SFRP1 complexes to identify the potent CRC inhibitors. The amino acid residues Arg5, Arg11, Ala13, Lys 245, Lys274, Phe147, Pro99, and Ser277 are essential for ligand binding and show similar interactions for SFRP1 complexes. The ADME/T profile for top hits is acceptable in range and obtains the drug-likeness property. The 100ns run for MD simulation confirms the stability of protein complexes. Overall, the findings of this study reveal that the lead compounds screened are capable of inhibiting SFRP1 against CRC. Targeting SFRP1 paves the way for new platforms in the field of cancer and the therapeutic sector for new approachable finds.

Active Site Characterization of a Campylobacter jejuni Nitrate Reductase Variant Provides Insight into the Enzyme Mechanism.

Mo K-edge X-ray absorption spectroscopy (XAS) is used to probe the structure of wild-type Campylobacter jejuni nitrate reductase NapA and the C176A variant. The results of extended X-ray absorption fine structure (EXAFS) experiments on wt NapA support an oxidized Mo(VI) hexacoordinate active site coordinated by a single terminal oxo donor, four sulfur atoms from two separate pyranopterin dithiolene ligands, and an additional S atom from a conserved cysteine amino acid residue. We found no evidence of a terminal sulfido ligand in wt NapA. EXAFS analysis shows the C176A active site to be a 6-coordinate structure, and this is supported by EPR studies on C176A and small molecule analogs of Mo(V) enzyme forms. The SCys is replaced by a hydroxide or water ligand in C176A, and we find no evidence of a coordinated sulfhydryl (SH) ligand. Kinetic studies show that this variant has completely lost its catalytic activity toward nitrate. Taken together, the results support a critical role for the conserved C176 in catalysis and an oxygen atom transfer mechanism for the catalytic reduction of nitrate to nitrite that does not employ a terminal sulfido ligand in the catalytic cycle.

Atomistic Prediction of Structures, Conformational Ensembles and Binding Energetics for the SARS-CoV-2 Spike JN.1, KP.2 and KP.3 Variants Using AlphaFold2 and Molecular Dynamics Simulations: Mutational Profiling and Binding Free Energy Analysis Reveal Epistatic Hotspots of the ACE2 Affinity and Immune Escape.

The most recent wave of SARS-CoV-2 Omicron variants descending from BA.2 and BA.2.86 exhibited improved viral growth and fitness due to convergent evolution of functional hotspots. These hotspots operate in tandem to optimize both receptor binding for effective infection and immune evasion efficiency, thereby maintaining overall viral fitness. The lack of molecular details on structure, dynamics and binding energetics of the latest FLiRT and FLuQE variants with the ACE2 receptor and antibodies provides a considerable challenge that is explored in this study. We combined AlphaFold2-based atomistic predictions of structures and conformational ensembles of the SARS-CoV-2 Spike complexes with the host receptor ACE2 for the most dominant Omicron variants JN.1, KP.1, KP.2 and KP.3 to examine the mechanisms underlying the role of convergent evolution hotspots in balancing ACE2 binding and antibody evasion. Using the ensemble-based mutational scanning of the spike protein residues and computations of binding affinities, we identified binding energy hotspots and characterized molecular basis underlying epistatic couplings between convergent mutational hotspots. The results suggested that the existence of epistatic interactions between convergent mutational sites at L455, F456, Q493 positions that enable to protect and restore ACE2 binding affinity while conferring beneficial immune escape. To examine immune escape mechanisms, we performed structure-based mutational profiling of the spike protein binding with several classes of antibodies that displayed impaired neutralization against BA.2.86, JN.1, KP.2 and KP.3. The results confirmed the experimental data that JN.1, KP.2 and KP.3 harboring the L455S and F456L mutations can significantly impair the neutralizing activity of class-1 monoclonal antibodies, while the epistatic effects mediated by F456L can facilitate the subsequent convergence of Q493E changes to rescue ACE2 binding. Structural and energetic analysis provided a rationale to the experimental results showing that BD55-5840 and BD55-5514 antibodies that bind to different binding epitopes can retain neutralizing efficacy against all examined variants BA.2.86, JN.1, KP.2 and KP.3. The results support the notion that evolution of Omicron variants may favor emergence of lineages with beneficial combinations of mutations involving mediators of epistatic couplings that control balance of high ACE2 affinity and immune evasion.

Toxin Homology Domain in Plant Type 2 Prolyl 4-Hydroxylases Acts as a Golgi Localization Domain.

Prolyl 4-hydroxylase (P4H) generates hydroxyproline residues in proteins. Two classes of P4H have been found in plants. Type 1 P4H has a signal anchor at the N-terminus, while type 2 P4H has both an N-terminal signal peptide and a C-terminal toxin homology domain (Tox1 domain) with six conserved cysteine residues. We analyzed the localization of tobacco type 2 P4H (NtP4H2.2) in tobacco BY-2 cells. Cell fractionation studies, immunostaining of cells, and GFP fusion study indicated that NtP4H2.2 localizes predominantly to the Golgi apparatus and is a peripheral membrane protein associated with the luminal side of organelles. Expression of the GFP-Tox1 domains of NtP4H2.2 and another tobacco type 2 P4H NtP4H2.1 in BY-2 cells and Arabidopsis epidermal cells indicated that these proteins were targeted to the Golgi. The Tox1 domains from Arabidopsis and rice type 2 P4Hs also directed GFP to the Golgi in tobacco BY-2 cells. The Tox1 domain of NtP4H2.2 increased the membrane association of GFP, and mutation of the cysteine residues in this domain abolished Golgi localization. Furthermore, the catalytic domain of NtP4H2.2 also directed GFP to the Golgi. Thus, the Tox1 domains of plant P4Hs are the Golgi localization domains, and tobacco P4H2.2 localizes to the Golgi by the action of both this domain and the catalytic domain.

Unveiling Charge-Pair Salt-Bridge Interaction Between GAGs and Collagen Protein in Cartilage: Atomic Evidence from DNP-Enhanced ssNMR at Natural Isotopic Abundance.

The interactions between glycosaminoglycans (GAGs) and proteins are essential in numerous biochemical processes that involve ion-pair interactions. However, there is no evidence of direct and specific interactions between GAGs and collagen proteins in native cartilage. The resolution of solid-state NMR (ssNMR) can offer such information but the detection of GAG interactions in cartilage is limited by the sensitivity of the experiments when 13C and 15N isotopes are at natural abundance. In this communication, this limitation is overcome by taking advantage of dynamic nuclear polarization (DNP)-enhanced magic-angle spinning (MAS) experiments to obtain two-dimensional (2D) 15N-13C and 13C-13C correlations on native samples at natural abundance. These experiments unveiled inter-residue correlations in the aliphatic regions of the collagen protein previously unobserved. Additionally, our findings provide direct evidence of charge-pair salt-bridge interactions between negatively charged GAGs and positively charged arginine (Arg) residues of collagen protein. We also identified potential hydrogen bonding interactions between hydroxyproline (Hyp) and GAGs, offering atomic insights into the biochemical interactions within the extracellular matrix of native cartilage. Our approach may provide a new avenue for the structural characterization of other native systems.

Unveiling Charge-Pair Salt-Bridge Interaction Between GAGs and Collagen Protein in Cartilage: Atomic Evidence from DNP-Enhanced ssNMR at Natural Isotopic Abundance.

The interactions between glycosaminoglycans (GAGs) and proteins are essential in numerous biochemical processes that involve ion-pair interactions. However, there is no evidence of direct and specific interactions between GAGs and collagen proteins in native cartilage. The resolution of solid-state NMR (ssNMR) can offer such information but the detection of GAG interactions in cartilage is limited by the sensitivity of the experiments when 13C and 15N isotopes are at natural abundance. In this communication, this limitation is overcome by taking advantage of dynamic nuclear polarization (DNP)-enhanced magic-angle spinning (MAS) experiments to obtain two-dimensional (2D) 15N-13C and 13C-13C correlations on native samples at natural abundance. These experiments unveiled inter-residue correlations in the aliphatic regions of the collagen protein previously unobserved. Additionally, our findings provide direct evidence of charge-pair salt-bridge interactions between negatively charged GAGs and positively charged arginine (Arg) residues of collagen protein. We also identified potential hydrogen bonding interactions between hydroxyproline (Hyp) and GAGs, offering atomic insights into the biochemical interactions within the extracellular matrix of native cartilage. Our approach may provide a new avenue for the structural characterization of other native systems.

In Silico Investigation against Inhibitors of Alpha-Amylase Using Structure-based Screening, Molecular Docking, and Molecular Simulations Studies.

Type-II diabetes mellitus is a chronic disorder that results from fluctuations in the glucose level leading to hyperglycemia with severe adverse effects increasing worldwide. Alpha-Amylase is the key enzyme involved in the mechanism of glucose formation therefore Alpha-Amylase inhibitors have become a therapeutic target in the development of new leads as they have the potential to suppress glucose levels. Existing drugs targeting Alpha-Amylase highlight major drawbacks in terms of poor absorption rate that causes several gastrointestinal issues. So, this research is aimed to develop novel inhibitors interacting with Alpha-Amylase's active site using structural-based screening, binding pattern analysis, and molecular dynamic simulation. Hence, to search for a potential lead, we analyzed a total of 133 valiolamine derivatives and 535 desoxynojirimycin derivatives that exhibited drug-like properties screened through Lipinski filters. Virtual screening followed by binding interaction analysis we identified ten compounds that exhibited better binding energy scores compared to the standard drugs voglibose and miglitol, used in our study. The docking analysis, ADMET and metabolic site prediction estimated the best top two compounds with good drug profiles. Further, top compounds VG9 and VG15 were promoted to simulation study using the Biovia Discovery study to access the stability at a time interval of 100 ns. MD simulation results revealed that our compound VG9 possesses better conformational stability in the complex to the active site residues of Alpha-Amylase target protein than standard drug voglibose. Thus, our investigation revealed that compound VG9 also exhibits the best pharmacokinetic as well as binding affinity results and could act as a potential lead compound targeting Alpha-Amylase for Type II diabetes.