Scaling the Process Chemistry of a COVID-19 Antiviral Pharmaceutical Down for a Multistep Synthesis Experiment in the Undergraduate Teaching Laboratory.

Molnupiravir is an orally bioavailable direct acting antiviral agent that received emergency use authorization in late 2021 from the FDA for the treatment of patients with mild, moderate, or severe COVID-19. This prodrug is metabolized into a ribonucleoside that is incorporated into the viral RNA during replication. Its tautomerization between cytidine- and uridine-like forms ultimately causes multiple irreversible errors in the genetic code of the virus, which prevents successful viral replication. There are multiple process chemistry routes for molnupiravir synthesis published in the literature that attempt to maximize synthetic yield while minimizing cost and waste, which are goals similar to those of an implementable educational laboratory experiment for the teaching laboratory. We have developed a multiweek laboratory module for undergraduate students in which students conduct a multistep synthesis of molnupiravir. Specifically, our Organic Chemistry II Laboratory students performed the final two steps of molnupiravir synthesis using procedures derived directly from the published process chemistry literature. We utilized this opportunity to introduce students to reading and interpreting these primary experimental sources. Students obtained authentic characterization data via high pressure liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) spectroscopy to assess the conversion and purity of their products at each synthetic step. We report our in-lab activities and student generated data as well as suggestions for how this laboratory experiment could be tailored to meet similar learning objectives in other courses, such as medicinal chemistry or capstone laboratory courses, and as a function of available instrumentation.

Identification and Characterization of CC-AMP1-like and CC-AMP2-like Peptides in Capsicum spp.

Antimicrobial peptides (AMPs) are compounds with a variety of bioactive properties. Especially promising are their antibacterial activities, often toward drug-resistant pathogens. Across different AMP sources, AMPs expressed within plants are relatively underexplored with a limited number of plant AMP families identified. Recently, we identified the novel AMPs CC-AMP1 and CC-AMP2 in ghost pepper plants (Capsicum chinense x frutescens), exerting promising antibacterial activity and not classifying into any known plant AMP family. Herein, AMPs related to CC-AMP1 and CC-AMP2 were identified within both Capsicum annuum and Capsicum baccatum. In silico predictions throughout plants were utilized to illustrate that CC-AMP1-like and CC-AMP2-like peptides belong to two broader AMP families, with three-dimensional structural predictions indicating that CC-AMP1-like peptides comprise a novel subfamily of α-hairpinins. The antibacterial activities of several closely related CC-AMP1-like peptides were compared with a truncated version of CC-AMP1 possessing significantly more activity than the full peptide. This truncated peptide was further characterized to possess broad-spectrum antibacterial activity against clinically relevant ESKAPE pathogens. These findings illustrate the value in continued study of plant AMPs toward characterization of novel AMP families, with CC-AMP1-like peptides possessing promising bioactivity.

The Greater Celandine: Identification and Characterization of an Antimicrobial Peptide from Chelidonium majus.

Chelidonium majus, known as Greater Celandine, is a latex-bearing plant that has been leveraged for its anticancer and antimicrobial properties. Herein, C. majus aerial tissue is mined for the presence of antimicrobial peptides. A highly abundant cysteine-rich peptide with a length of 25 amino acids, deemed CM-AMP1, is characterized through multiple mass spectrometric approaches. Electron-activated dissociation is leveraged to differentiate between isoleucine and leucine residues and complement conventional collision-induced dissociation to gain full sequence coverage of the full-length peptide. CM-AMP1 shares little sequence similarity with any proteins in publicly available databases, highlighting the novelty of its cysteine landscape and core motif. The presence of three disulfide bonds in the native peptide confers proteolytic stability, and antimicrobial activity is greatly decreased upon the alkylation of the cysteine residues. Synthetic variants of CM-AMP1 are used to confirm the activity of the full-length sequence and the core motif. To assess the biological impact, E. coli was grown in a sublethal concentration of CM-AMP1 and quantitative proteomics was used to identify proteins produced by the bacteria under stress, ultimately suggesting a membrane lytic antimicrobial mechanism of action. This study integrates multiple analytical methods for molecular and biological characterization of a unique antimicrobial peptide identified from C. majus.

The BCKDH kinase inhibitor BT2 promotes BCAA disposal and mitochondrial proton leak in both insulin-sensitive and insulin-resistant C2C12 myotubes.

Elevated circulating branched-chain amino acids (BCAAs) have been correlated with the severity of insulin resistance, leading to recent investigations that stimulate BCAA metabolism for the potential benefit of metabolic diseases. BT2 (3,6-dichlorobenzo[b]thiophene-2-carboxylic acid), an inhibitor of branched-chain ketoacid dehydrogenase kinase, promotes BCAA metabolism by enhancing BCKDH complex activity. The purpose of this report was to investigate the effects of BT2 on mitochondrial and glycolytic metabolism, insulin sensitivity, and de novo lipogenesis both with and without insulin resistance. C2C12 myotubes were treated with or without low or moderate levels of BT2 with or without insulin resistance. Western blot and quantitative real-time polymerase chain reaction were used to assess protein and gene expression, respectively. Mitochondrial, nuclei, and lipid content were measured using fluorescent staining and microscopy. Cell metabolism was assessed via oxygen consumption and extracellular acidification rate. Liquid chromatography-mass spectrometry was used to quantify BCAA media content. BT2 treatment consistently promoted mitochondrial uncoupling following 24-h treatment, which occurred largely independent of changes in expressional profiles associated with mitochondrial biogenesis, mitochondrial dynamics, BCAA catabolism, insulin sensitivity, or lipogenesis. Acute metabolic studies revealed a significant and dose-dependent effect of BT2 on mitochondrial proton leak, suggesting BT2 functions as a small-molecule uncoupler. Additionally, BT2 treatment consistently and dose-dependently reduced extracellular BCAA levels without altering expression of BCAA catabolic enzymes or pBCKDHa activation. BT2 appears to act as a small-molecule mitochondrial uncoupler that promotes BCAA utilization, though the interplay between these two observations requires further investigation.

Physiological 4-phenylbutyrate promotes mitochondrial biogenesis and metabolism in C2C12 myotubes.

Type 2 diabetes is characterized by elevated circulating blood metabolites such as glucose, insulin, and branched chain amino acids (BCAA), which often coincide with reduced mitochondrial function. 4-Phenylbutyrate (PBA), an ammonia scavenger, has been shown to activate BCAA metabolism, resolve endoplasmic reticulum (ER) stress, and rescue BCAA-mediated insulin resistance. To determine the effect of PBA on the altered metabolic phenotype featured in type 2 diabetes, the present study investigated the effect of PBA on various metabolic parameters including mitochondrial metabolism and mitochondrial biogenesis. C2C12 myotubes were treated with PBA at 0.5 mM (representing physiologically attainable blood concentrations) or 10 mM (representing physiologically unattainable/proof-of-concept levels) for up to 24 h. Mitochondrial and glycolytic metabolism were assessed via oxygen consumption and extracellular acidification rate, respectively. Mitochondrial content, lipid content, and ER stress were measured by fluorescent staining. Metabolic gene expression was measured by qRT-PCR. Both doses of PBA increased expression of indicators of mitochondrial biogenesis, though only PBA at 0.5 mM increased mitochondrial function and content while 10 mM PBA reduced mitochondrial function and content. PBA at 0.5 mM also rescued reduced mitochondrial function during insulin resistance, though PBA also caused a reduced insulin stimulated pAkt expression during insulin resistance. PBA treatment also increased extracellular BCAA accumulation during insulin resistance despite unchanged pBCKDH expression. Taken together, PBA may increase mitochondrial biogenesis, content, and function in a dose-dependent fashion which may have implications for prevention or treatment of metabolic disease such as insulin resistance.

5-Aza-2'-deoxycytidine-mediated DNA hypomethylation with and without concurrent insulin resistance suppresses myotube mitochondrial capacity.

Type 2 diabetes is characterized by elevated blood glucose and reduced insulin sensitivity in target tissues. Moreover, reduced mitochondrial metabolism and expressional profile of genes governing mitochondrial metabolism (such as peroxisome proliferator-activated receptor gamma coactivator 1-alpha [PGC-1α]) are also reduced during insulin resistance. Epigenetic regulation via DNA methylation of genes including PGC-1α may contribute to diminished mitochondrial capacity, while hypomethylation of PGC-1α (such as that invoked by exercise) has been associated with increased PGC-1α expression and favorable metabolic outcomes. The purpose of the present report is to characterize the effects of DNA hypomethylation on myotube metabolism and expression of several related metabolic targets. C2C12 myotubes were treated with 5-Aza-2'-deoxycytidine (5-Aza) for either 24 or 72 h both with and without hyperinsulinemic-induced insulin resistance. Mitochondrial and glycolytic metabolism were measured via oxygen consumption and extracellular acidification rate, respectively. Metabolic gene and protein expression were assessed via quantitative real time polymerase chain reaction and western blot analysis, respectively. Though expression of PGC-1α and other related targets remained unaltered, insulin resistance and 5-Aza treatment significantly reduced mitochondrial metabolism. Similarly, peak glycolytic metabolism was diminished by 5-Aza-treated cells, while basal glycolytic metabolism was unaltered. 5-Aza also reduced the expression of branched-chain amino acid (BCAA) catabolic components, however BCAA utilization was enhanced during insulin resistance with 5-Aza treatment. Together the present work provides proof-of-concept evidence of the potential role of DNA methylation in the regulation of mitochondrial metabolism and the potential interactions with insulin resistance in a model of skeletal muscle.

Hydrogen peroxide-dependent oxidation of ERK2 within its D-recruitment site alters its substrate selection.

Extracellular signal-regulated kinases 1 and 2 (ERK1/2) are dysregulated in many pervasive diseases. Recently, we discovered that ERK1/2 is oxidized by signal-generated hydrogen peroxide in various cell types. Since the putative sites of oxidation lie within or near ERK1/2's ligand-binding surfaces, we investigated how oxidation of ERK2 regulates interactions with the model substrates Sub-D and Sub-F. These studies revealed that ERK2 undergoes sulfenylation at C159 on its D-recruitment site surface and that this modification modulates ERK2 activity differentially between substrates. Integrated biochemical, computational, and mutational analyses suggest a plausible mechanism for peroxide-dependent changes in ERK2-substrate interactions. Interestingly, oxidation decreased ERK2's affinity for some D-site ligands while increasing its affinity for others. Finally, oxidation by signal-generated peroxide enhanced ERK1/2's ability to phosphorylate ribosomal S6 kinase A1 (RSK1) in HeLa cells. Together, these studies lay the foundation for examining crosstalk between redox- and phosphorylation-dependent signaling at the level of kinase-substrate selection.

The effect of insulin resistance on extracellular BCAA accumulation and SLC25A44 expression in a myotube model of skeletal muscle insulin resistance.

Insulin resistance is often accompanied by elevated circulating branched-chain amino acids (BCAA). We investigated the effects of insulin resistance on the mitochondrial BCAA transporter, SLC25A44, using a myotube model of insulin resistance. Insulin sensitivity and SLC25A44 expression were assessed via Western blot. Liquid chromatography-mass spectrometry was used to evaluate extracellular BCAA media content. Insulin resistance reduced pAkt activation following insulin stimulation but did not alter SLC25A44 expression. Under select conditions, insulin resistance led to the accumulation of extracellular BCAA.

The Selective LAT1 Inhibitor JPH203 Enhances Mitochondrial Metabolism and Content in Insulin-Sensitive and Insulin-Resistant C2C12 Myotubes.

Population data have shown an association between higher circulating branched-chain amino acids (BCAA) and the severity of insulin resistance in people with diabetes. While several studies have assessed BCAA metabolism as a potential target for regulation, less attention has been paid to the role of L-type amino acid transporter 1 (LAT1), the primary transporter of BCAA in skeletal muscle. The aim of this study was to assess the impact of JPH203 (JPH), a LAT1 inhibitor, on myotube metabolism in both insulin-sensitive and insulin-resistant myotubes. C2C12 myotubes were treated with or without 1 µM or 2 µM JPH for 24 h with or without insulin resistance. Western blot and qRT-PCR were used to assess protein content and gene expression, respectively. Mitochondrial and glycolytic metabolism were measured via Seahorse Assay, and fluorescent staining was used to measure mitochondrial content. BCAA media content was quantified using liquid chromatography-mass spectrometry. JPH at 1 µM (but not 2 µM) increased mitochondrial metabolism and content without inducing changes in mRNA expression of transcripts associated with mitochondrial biogenesis or mitochondrial dynamics. Along with increased mitochondrial function, 1µM treatment also reduced extracellular leucine and valine. JPH at 2 µM reduced pAkt signaling and increased extracellular accumulation of isoleucine without inducing changes in BCAA metabolic genes. Collectively, JPH may increase mitochondrial function independent of the mitochondrial biogenic transcription pathway; however, high doses may reduce insulin signaling.

PPARß/δ Agonism with GW501516 Increases Myotube PGC-1α Content and Reduces BCAA Media Content Independent of Changes in BCAA Catabolic Enzyme Expression.

Background: Type 2 diabetes is characterized by reduced insulin sensitivity, elevated blood metabolites, and reduced mitochondrial metabolism with reduced expression of genes governing metabolism such as peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). PGC-1α regulates the expression of branched-chain amino acid (BCAA) metabolism, and thus, increased circulating BCAA in diabetics may be partially explained by reduced PGC-1α expression. PGC-1α functions in-part through interactions with peroxisome proliferator-activated receptor ß/δ (PPARß/δ). The present report examined the effects of the PPARß/δ agonism on cell metabolism and related gene/protein expression of cultured myotubes, with a primary emphasis on determining the effects of GW on BCAA disposal and catabolic enzyme expression. Methods: C2C12 myotubes were treated with GW501516 (GW) for up to 24 hours. Mitochondrial and glycolytic metabolism were measured via oxygen consumption and extracellular acidification rate, respectively. Metabolic gene and protein expression were assessed via quantitative real-time polymerase chain reaction (qRT-PCR) and western blot, respectively. Media BCAA content was assessed via liquid chromatography-mass spectrometry (LC/MS). Results: GW significantly increased PGC-1α protein expression, mitochondrial content, and mitochondrial function. GW also significantly reduced BCAA content within culture media following 24-hour treatment; however, expression of BCAA catabolic enzymes/transporter was unchanged. Conclusion: These data confirm the ability of GW to increase muscle PGC-1α content and decrease BCAA media content without affecting BCAA catabolic enzymes/transporter. These findings suggest heightened BCAA uptake (and possibly metabolism) may occur without substantial changes in the protein levels of related cell machinery.

Effect of AMPK activation and glucose availability on myotube LAT1 expression and BCAA utilization.

Those with insulin resistance often display increased circulating branched-chain amino acids (BCAA), which has been largely attributable to reduced BCAA catabolic capacity. Metabolic stimuli such as exercise activates AMP-activated kinase (AMPK), which promotes the metabolism of BCAA and induction/activation of BCAA catabolic enzymes. Though much attention has been paid to BCAA catabolic machinery, few studies have assessed the effect of AMPK activation on the predominant BCAA transporter, L-type amino acid transporter 1 (LAT1). This study assessed the effect of AMPK activation on LAT1 expression via common chemical AMPK activators in a cell model of skeletal muscle. C2C12 myotubes were treated with either 1 mM AICAR, 1 mM Metformin, or filter-sterilized water (control) for 24 h with either low- (5 mM) or high-glucose (25 mM) media. LAT1 and pAMPK protein content were measured via western blot. BCAA media content was measured using liquid chromatography-mass spectrometry. AICAR treatment significantly increased pAMPK and reduced LAT1 expression. Collectively, pAMPK and LAT1 displayed a significant inverse relationship independent of glucose levels. During low-glucose experiments, AICAR-treated cells had higher BCAA media content compared to other groups, and an inverse relationship between LAT1 and BCAA media content was observed, however, these effects were not consistently observed during high-glucose conditions. Further investigation with AICAR with and without concurrent LAT1 inhibition (via JPH203) also revealed reduced BCAA utilization in AICAR-treated cells regardless of LAT1 inhibition (which also independently reduced BCAA utilization). pAMPK activation via AICAR (but not Metformin) may reduce LAT1 expression and BCAA uptake in a glucose-dependent manner.

Insulin resistance promotes extracellular BCAA accumulation without altering LAT1 content, independent of prior BCAA treatment in a myotube model of skeletal muscle.

PURPOSE: Type 2 diabetes is characterized by reduced insulin sensitivity which correlates with increased circulating BCAA. These experiments investigated the effects of insulin resistance with and without excess BCAA on myotube insulin sensitivity and L-type amino acid transporter-1 (LAT1). METHODS: C2C12 myotubes were treated with or without excess BCAA for 1 or 6 days, both with and without insulin resistance. Western blot was used to assess insulin sensitivity and LAT1 content. Liquid chromatography-mass spectrometry was used to evaluate BCAA media content. RESULTS: Insulin resistance was associated with significantly increased extracellular BCAA accumulation independent of LAT1 content. Conversely, prior BCAA treatment was not associated with extracellular BCAA accumulation regardless of level of insulin sensitivity. CONCLUSION: These data suggest insulin resistance, but not BCAA treatment, promotes extracellular BCAA accumulation independent of changes in LAT1 content, implicating insulin resistance as a causal agent of extracellular BCAA accumulation.

Excess glutamine does not alter myotube metabolism or insulin sensitivity.

Glutamine is an amino acid previously linked with improved skeletal muscle metabolism and insulin signaling, however, past observations often use cell culture models with only supraphysiological concentrations. Additionally, past reports have yet to simultaneously investigate both metabolic outcomes and insulin signaling. The present report utilized cell culture experiments and measured the effects of both physiological and supraphysiological levels of glutamine on myotube metabolism and insulin signaling/resistance. It was hypothesized the addition of glutamine at any level would increase cell metabolism and related gene expression, as well as improve insulin signaling versus respective control cells. C2C12 myotubes were treated with glutamine ranging from 0.25 mM-4 mM (or media control) for 24 h to capture a range of physiological and supraphysiological concentrations. qRT-PCR was used to measure metabolic gene expression. Mitochondrial and glycolytic metabolism were measured via oxygen consumption and extracellular acidification rate, respectively. Insulin sensitivity (indicated by pAkt:Akt) and metabolism following glucose/insulin infusion were also assessed. Glutamine treatment consistently increased mitochondrial and glycolytic metabolism versus true controls (cells treated with media void of glutamine), however, supraphysiological glutamine did not enhance metabolism beyond that of cells with physiological levels of glutamine. Neither physiological nor supraphysiological levels of glutamine altered insulin signaling regardless of insulin stimulation or insulin resistance when compared with respective controls. These data demonstrate excess glutamine does not appear to alter myotube metabolism or glucose disposal when base levels of glutamine are present. Moreover, glutamine does not appear to alter insulin sensitivity regardless of level of insulin resistance or presence of insulin stimulation.

Proteomic response of Escherichia coli to a membrane lytic and iron chelating truncated Amaranthus tricolor defensin.

BACKGROUND: Plant defensins are a broadly distributed family of antimicrobial peptides which have been primarily studied for agriculturally relevant antifungal activity. Recent studies have probed defensins against Gram-negative bacteria revealing evidence for multiple mechanisms of action including membrane lysis and ribosomal inhibition. Herein, a truncated synthetic analog containing the γ-core motif of Amaranthus tricolor DEF2 (Atr-DEF2) reveals Gram-negative antibacterial activity and its mechanism of action is probed via proteomics, outer membrane permeability studies, and iron reduction/chelation assays. RESULTS: Atr-DEF2(G39-C54) demonstrated activity against two Gram-negative human bacterial pathogens, Escherichia coli and Klebsiella pneumoniae. Quantitative proteomics revealed changes in the E. coli proteome in response to treatment of sub-lethal concentrations of the truncated defensin, including bacterial outer membrane (OM) and iron acquisition/processing related proteins. Modification of OM charge is a common response of Gram-negative bacteria to membrane lytic antimicrobial peptides (AMPs) to reduce electrostatic interactions, and this mechanism of action was confirmed for Atr-DEF2(G39-C54) via an N-phenylnaphthalen-1-amine uptake assay. Additionally, in vitro assays confirmed the capacity of Atr-DEF2(G39-C54) to reduce Fe3+ and chelate Fe2+ at cell culture relevant concentrations, thus limiting the availability of essential enzymatic cofactors. CONCLUSIONS: This study highlights the utility of plant defensin γ-core motif synthetic analogs for characterization of novel defensin activity. Proteomic changes in E. coli after treatment with Atr-DEF2(G39-C54) supported the hypothesis that membrane lysis is an important component of γ-core motif mediated antibacterial activity but also emphasized that other properties, such as metal sequestration, may contribute to a multifaceted mechanism of action.

The same anthocyanins served four different ways: Insights into anthocyanin structure-function relationships from the wintergreen orchid, Tipularia discolor.

Over 500 unique anthocyanins have been described to date, which vary in color, antioxidant, light-attenuating, and antimicrobial properties. Identification of anthocyanin chemical structure may therefore serve as an important clue to their in situ function in plants. We characterized the histological and chemical structures of anthocyanins associated with diverse leaf color patterns in the terrestrial orchid, Tipularia discolor, as a step towards understanding their ultimate function. Tipularia discolor produces a single wintergreen leaf in autumn, which is drab brown in color during expansion. Upper (adaxial) surfaces of fully-expanded leaves may be green, purple-spotted, or solid purple, while lower (abaxial) surfaces are bright magenta. Our results showed that the same three cyanidin 3,7,3'-triglucosides, in similar concentrations and proportions, accounted for coloration in each of these cases, and that different colors result from differences in histological location of anthocyanins (i.e. abaxial/adaxial epidermis, mesophyll). Anthocyanins with 3,7,3' linkage positions are rare in plants, occurring only within the orchid subfamily Epidendroideae, to which Tipularia belongs. These results are important to the discussion of anthocyanin structure-function because they serve as a reminder that 1) plants may employ the same anthocyanins in different anatomical locations to achieve a broad range of colors (and potentially adaptive functions), and 2) anthocyanin chemical structure and anatomical location are influenced by phylogenetic inertia, as well as natural selection.

Profiling thimet oligopeptidase-mediated proteolysis in Arabidopsis thaliana.

Protein homeostasis (proteostasis) is crucial for proper cellular function, including the production of peptides with biological functions through controlled proteolysis. Proteostasis has roles in maintenance of cellular functions and plant interactions with the environment under physiological conditions. Plant stress continues to reduce agricultural yields causing substantial economic losses; thus, it is critical to understand how plants perceive stress signals to elicit responses for survival. As previously shown in Arabidopsis thaliana, thimet oligopeptidases (TOPs) TOP1 (also referred to as organellar oligopeptidase) and TOP2 (also referred to as cytosolic oligopeptidase) are essential components in plant response to pathogens, but further characterization of TOPs and their peptide substrates is required to understand their contributions to stress perception and defense signaling. Herein, label-free peptidomics via liquid chromatography-tandem mass spectrometry was used to differentially quantify 1111 peptides, originating from 369 proteins, between the Arabidopsis Col-0 wild type and top1top2 knock-out mutant. This revealed 350 peptides as significantly more abundant in the mutant, representing accumulation of these potential TOP substrates. Ten direct substrates were validated using in vitro enzyme assays with recombinant TOPs and synthetic candidate peptides. These TOP substrates are derived from proteins involved in photosynthesis, glycolysis, protein folding, biogenesis, and antioxidant defense, implicating TOP involvement in processes aside from defense signaling. Sequence motif analysis revealed TOP cleavage preference for non-polar residues in the positions surrounding the cleavage site. Identification of these substrates provides a framework for TOP signaling networks, through which the interplay between proteolytic pathways and defense signaling can be further characterized.

PepSAVI-MS Reveals a Proline-rich Antimicrobial Peptide in Amaranthus tricolor.

Traditional medicinal plants are a rich source of antimicrobials; however, the bioactive peptide constituents of most ethnobotanical species remain largely unexplored. Herein, PepSAVI-MS, a mass spectrometry-based peptidomics pipeline, was implemented for antimicrobial peptide (AMP) discovery in the medicinal plant Amaranthus tricolor. This investigation revealed a novel 1.7 kDa AMP with strong activity against Escherichia coli ATCC 25922, deemed Atr-AMP1. Initial efforts to determine the sequence of Atr-AMP1 utilized chemical derivatization and enzymatic digestion to provide information about specific residues and post-translational modifications. EThcD (electron-transfer/higher-energy collision dissociation) produced extensive backbone fragmentation and facilitated de novo sequencing, the results of which were consistent with orthogonal characterization experiments. Additionally, multistage HCD (higher-energy collisional dissociation) facilitated discrimination between isobaric leucine and isoleucine. These results revealed a positively charged proline-rich peptide present in a heterogeneous population of multiple peptidoforms, possessing several post-translational modifications including a disulfide bond, methionine oxidation, and proline hydroxylation. Additional bioactivity screening of a simplified fraction containing Atr-AMP1 revealed activity against Staphylococcus aureus LAC, demonstrating activity against both a Gram-negative and a Gram-positive bacterial species unlike many known short chain proline-rich antimicrobial peptides.

The absorptive effects of orobuccal non-liposomal nano-sized glutathione on blood glutathione parameters in healthy individuals: A pilot study.

BACKGROUND: Glutathione is an endogenous antioxidant found in oxidized (GSSG) and reduced (GSH) forms. Glutathione depletion is indicative of oxidative stress and occurs in various pathological conditions and following extreme exercise activity. Raising blood glutathione concentration has potential to attenuate and prevent chronic disease and also to improve recovery from exercise. There are a number of challenges to achieving this through traditional dietary supplements, and thus there is a need to develop optimized delivery methods to improve blood glutathione status. This study evaluated the effect of a novel glutathione formulation on blood glutathione parameters in healthy individuals. METHODS: 15 (8 male) healthy individuals (25±5y old, 78.0±14.6kg) participated in a single-blinded randomized placebo-controlled crossover study, with a minimum one-week washout period between treatments. Participants were overnight fasted and administered 1mL of a non-liposomal nano-size glutathione solution (NLNG) containing 200mg of glutathione or 1mL of placebo lacking glutathione. The solution was held in the mouth for 90 seconds before the remainder was swallowed. Blood was collected at baseline, 5, 10, 30, 60 and 120 minutes post-treatment. Protein-bound plasma and erythrocyte lysate concentrations of GSH and GSSG were measured at all time points using previously validated procedures. Linear mixed effects models were used to compare differences between baseline and post-treatment glutathione concentrations between NLNG and placebo for each parameter. RESULTS: There was a significant main effect for treatment type, such that increases in GSH concentration in erythrocyte lysate were greater following NLNG than placebo (p = 0.001). Similar significant main effects for treatment were also found for total (protein bound + erythrocyte lysate) GSH (p = 0.015) and GSSG (p = 0.037) concentration, as well as total blood glutathione pool (GSH+GSSG, p = 0.006). DISCUSSION: NLNG increased multiple blood glutathione parameters compared to placebo. Future research should examine whether NLNG can attenuate oxidative stress.

Disulfide cross-linking influences symbiotic activities of nodule peptide NCR247.

Interactions of rhizobia with legumes establish the chronic intracellular infection that underlies symbiosis. Within nodules of inverted repeat-lacking clade (IRLC) legumes, rhizobia differentiate into nitrogen-fixing bacteroids. This terminal differentiation is driven by host nodule-specific cysteine-rich (NCR) peptides that orchestrate the adaptation of free-living bacteria into intracellular residents. Medicago truncatula encodes a family of >700 NCR peptides that have conserved cysteine motifs. NCR247 is a cationic peptide with four cysteines that can form two intramolecular disulfide bonds in the oxidized forms. This peptide affects Sinorhizobium meliloti transcription, translation, and cell division at low concentrations and is antimicrobial at higher concentrations. By preparing the three possible disulfide-cross-linked NCR247 regioisomers, the reduced peptide, and a variant lacking cysteines, we performed a systematic study of the effects of intramolecular disulfide cross-linking and cysteines on the activities of an NCR peptide. The relative activities of the five NCR247 variants differed strikingly among the various bioassays, suggesting that the NCR peptide-based language used by plants to control the development of their bacterial partners during symbiosis is even greater than previously recognized. These patterns indicate that certain NCR bioactivities require cysteines whereas others do not. The results also suggest that NCR247 may exert some of its effects within the cell envelope whereas other activities occur in the cytoplasm. BacA, a membrane protein that is critical for symbiosis, provides protection against all bactericidal forms of NCR247. Oxidative folding protects NCR247 from degradation by the symbiotically relevant metalloprotease HrrP (host range restriction peptidase), suggesting that disulfide bond formation may additionally stabilize NCR peptides during symbiosis.

The Hexahistidine Motif of Host-Defense Protein Human Calprotectin Contributes to Zinc Withholding and Its Functional Versatility.

Human calprotectin (CP, S100A8/S100A9 oligomer, MRP-8/MRP-14 oligomer) is an abundant host-defense protein that is involved in the metal-withholding innate immune response. CP coordinates a variety of divalent first-row transition metal ions, which is implicated in its antimicrobial function, and its ability to sequester nutrient Zn(II) ions from microbial pathogens has been recognized for over two decades. CP has two distinct transition-metal-binding sites formed at the S100A8/S100A9 dimer interface, including a histidine-rich site composed of S100A8 residues His17 and His27 and S100A9 residues His91 and His95. In this study, we report that CP binds Zn(II) at this site using a hexahistidine motif, completed by His103 and His105 of the S100A9 C-terminal tail and previously identified as the high-affinity Mn(II) and Fe(II) coordination site. Zn(II) binding at this unique site shields the S100A9 C-terminal tail from proteolytic degradation by proteinase K. X-ray absorption spectroscopy and Zn(II) competition titrations support the formation of a Zn(II)-His6 motif. Microbial growth studies indicate that the hexahistidine motif is important for preventing microbial Zn(II) acquisition from CP by the probiotic Lactobacillus plantarum and the opportunistic human pathogen Candida albicans. The Zn(II)-His6 site of CP expands the known biological coordination chemistry of Zn(II) and provides new insight into how the human innate immune system starves microbes of essential metal nutrients.