Arginine Analogues Research Articles

Profile of Michael A. Marletta

For more than three decades, Michael Marletta has traveled a serpentine road, working to elucidate a molecule whose importance to human physiology is as well recognized as his own contributions to his field. Named “Molecule of the Year” in 1992 by Science , nitric oxide, a noxious atmospheric gas, plays a pivotal role in biological functions as diverse as forming long-term memory and maintaining penile erections (1). Marletta, who was elected to the National Academy of Sciences in 2006, has shed light on the biochemistry of nitric oxide and the cellular machinery through which the molecule performs its functions. Michael A. Marletta. Marletta's work on nitric oxide not only led to a fundamental understanding of enzyme reaction mechanisms, but it has informed the development of treatments. For example, he showed that substrate lookalikes, called arginine analogs, block the activity of the enzyme nitric oxide synthase. The analogs help curb the overproduction of nitric oxide, preventing vasodilation and the precipitous drop in blood pressure experienced by patients suffering from septic shock (2). His discovery helped other researchers develop treatments for septic shock. Marletta's scientific foray began in California, where he obtained his doctorate in 1978 from the University of California, San Francisco (UCSF). That journey, with stops in Massachusetts and Michigan, has led him back to California, where he is now the Aldo DeBenedictis Distinguished Professor of Chemistry and a professor of biochemistry and molecular biology at the University of California, Berkeley. His love of science is deep-seated. Born in Rochester, NY, to parents of Italian ancestry, Marletta says the space race of the late 1950s spurred his scientific temperament. As a 6-year-old space buff, Marletta recalls watching Sputnik 1 fly overhead on a cold October night in 1957 in Rochester. “I was completely fascinated by the idea that a satellite …

ChemInform Abstract: Synthesis of Ethylene-Bridged (Nδ to Nω) Analogues of Arginine.

ChemInform Abstract: Synthesis of Ethylene-Bridged (Nδ to Nω) Analogues of Arginine.

Electrostatics of Deformable Lipid Membranes

It was recently demonstrated that significant local deformations of biological membranes take place due to the fields of charged peptides and ions, challenging the standard model of membrane electrostatics. The ability of ions to retain their immediate hydration environment, combined with the lack of sensitivity of permeability to ion type or even ion pairs, led us to question the extent to which hydration energetics and electrostatics control membrane ion permeation. Using the arginine analog methyl-guanidinium as a test case, we find that although hydrocarbon electronic polarizability causes dramatic changes in ion solvation free energy, as well as a significant change (∼0.4 V) in the membrane dipole potential, little change in membrane permeation energetics occurs. We attribute this to compensation of solvation terms from polar and polarizable nonpolar components within the membrane, and explain why the dipole potential is not fully sensed in terms of the locally deformed bilayer interface. Our descriptions provide a deeper understanding of the translocation process and allow predictions for poly-ions, ion pairs, charged lipids, and lipid flip-flop. We also report simulations of large hydrophobic-ion-like membrane defects and the ionophore valinomycin, which exhibit little membrane deformation, as well as hydrophilic defects and the ion channel gramicidin A, to provide parallels to membranes deformed by unassisted ion permeation.

Bronchial Epithelial Dysfunction In Asthma: Similarities To Metabolic Syndrome

ObjectiveObesity and metabolic syndrome are epidemiologically associated with incident and prevalent asthma. To understand its mechanisms, we determined whether bronchial epithelial dysfunction in asthma is associated with metabolic stress.MethodsWe investigated the occurrence of oxidative stress, disordered arginine and nitric oxide metabolism, mitochondrial health and epithelial injury in lungs of mice with allergic airway inflammation. We further determined whether strategies known to be effective against similar dysfunction in metabolic syndrome, i.e. L‐arginine supplementation or statin therapy, are efficacious in the asthma model.ResultsIncreased levels of asymmetric dimethyl arginine (ADMA), an endogenous arginine analog that uncouples NOS, were found in asthmatic lungs. This was positively correlated with oxo‐nitrative stress, mitochondrial dysfunction, and epithelial injury. Treatment with either L‐arginine or simvastatin was able to restore normal ADMA and nitric oxide metabolism. This was associated with reduction of oxo‐nitrative stress, improved mitochondrial health, as well as attenuation of bronchial airway injury, remodeling, and hyperresponsiveness.ConclusionAsthmatic airways show patterns of metabolic stress similar to vascular dysfunction in metabolic syndrome, and respond to treatment with L‐arginine or simvastatin.

Trypanosoma cruziamino acid transporter TcAAAP411 mediates arginine uptake in yeasts

Trypanosoma cruzi, the aetiological agent of Chagas' disease, is exposed to extremely different environment conditions during its life cycle, and transporters are key molecules for its adaptive regulation. Amino acids, and particularly arginine, are essential components in T. cruzi metabolism. In this work, a novel T. cruzi arginine permease was identified by screening different members of the AAAP family (amino acid/auxin permeases) in yeast complementation assays using a toxic arginine analogue. One gene candidate, TcAAAP411, was characterized as a very specific, high-affinity, l-arginine permease. This work is the first identification of the molecular components involved specifically in amino acid transport in T. cruzi and provides new insights for further validation of the TcAAAP family as functional permeases.

A Novel Loss-of-Function DDAH1 Promoter Polymorphism Is Associated With Increased Susceptibility to Thrombosis Stroke and Coronary Heart Disease

Asymmetrical dimethylarginine (ADMA), an endogenous arginine analogue, inhibits nitric oxide synthases and plays an important role in endothelial dysfunction. In the present study, we tested whether a novel genetic variant in dimethylarginine dimethylaminohydrolase 1 (DDAH1), an important ADMA hydrolyzing gene, was associated with stroke and coronary heart disease (CHD) susceptibility in the Chinese Han population. By resequencing, we identified a novel 4-nucleotide deletion/insertion variant in the DDAH1 promoter. The insertion allele disrupted binding of metal-regulatory transcription factor 1, which resulted in significant reduction of in vitro DDAH1 transcriptional activity and in vivo DDAH1 mRNA level, and in turn, increased plasma ADMA level and the ratio of ADMA to L-arginine. We initially genotyped the polymorphism in 1388 stroke patients and 1027 controls as well as 576 CHD patients and 557 controls and then replicated our study in additional independent case-control cohorts comprising 961 stroke patients and 822 controls and 482 CHD patients and 1072 controls. We identified that the -396 4N ins allele was significantly associated with increased risk of thrombosis stroke and CHD after adjusting for environmental factors in both samples for both diseases (thrombosis stroke discovery set: odds ratio [OR]=1.35, P=0.032; replication set: OR=1.51, P=0.006; CHD discovery set: OR=1.45, P=0.035; replication set: OR=1.47, P=0.003). Our results suggest that the DDAH1 loss-of-function polymorphism is associated with both increased risk of thrombosis stroke and CHD.

On the Roles of Anionic Lipids in Protein Localization and Permeability of Membranes

Anionic lipids, such as phosphatidylglycerol and phosphatidylserine, play important structural and functional roles in cell membranes. In particular, they appear to provide strong interactions with positively charged protein side chains to promote membrane localization of lipid binding domains and antimicrobial peptides, as well as to modulate the function of many membrane proteins. All-atom molecular dynamics simulations were used to explore the strength of these interactions and the impact they have on the ability of charged protein residues to penetrate into membranes. Using an analog of arginine and bilayers of pure phosphatidylcholine or mixtures with phosphatidylglycerol, we have computed the thermodynamics of charged side chain translocation, as well as the binding affinity of each lipid within the membrane. We found that arginine deforms the bilayer in a similar fashion, regardless of composition, and that the free energy profile for translocation is relatively unaffected by anionic lipids: the “neutralization” of the protein side chain does not reduce the large ∼20 kcal/mol barrier significantly. We decomposed these free energies to explain why anionic lipids do not play a significant role, with implications for the actions of many charged peptides and ion permeability. We also find that arginine binding to phosphatidylglycerol is more favorable by only ∼1 kcal/mol, suggesting that lipid binding domains and antimicrobial peptides likely require many charged side chains acting together to promote membrane localization.

Nitric oxide production inhibited by xenobiotic compounds in the protozoan Paramecium primaurelia

Abstract The notable increase in agricultural and industrial activities over the last decades has caused a considerable increase in anthropogenic waste and, consequently, the presence of pollutants in both water and sediments. For this reason, there is great interest in identifying alternative models and bioassays complying with the 3Rs strategy (aimed at Reducing, Refining and Replacing tests on vertebrate organisms in toxicological studies). Protozoa seem to be well suited to this strategy and it is widely accepted that assays with protozoa are relevant to the study of environmental modifications due to the presence of xenobiotic compounds. Recently, we detected the presence of nitric oxide synthase (NOS)-related NADPH-diaphorase activity and neuronal NOS-related molecules, immunologically recognized by the anti-rat brain NOS antibody, in a single-cell freshwater eukaryote, Paramecium primaurelia . In this work we have looked for the basal NO production in living cells of P. primaurelia using the specific fluorescent probe 4,5-diaminofluorescein diacetate (DAF-2 DA) and measuring the intracellular NO levels with image analysis. The NO production was sensitive to compounds modulating NOS activity such as: S-methyl-tiocitrulline, an NOS activity inhibitor, l -NAME, an analogue of arginine that inhibits NO production, arginine, an NOS substrate, or sodium nitroprusside, an NO donor. The NO production in P. primaurelia was also shown to be sensitive to μM concentrations of heavy metals (HgCl 2 and CdCl 2 ), or μM concentrations of pesticides (diazinon and AFD 25), thus representing a potential biomarker for environmental biomonitoring. The possible involvement of cellular Ca 2+ concentration, assayed by the fluorescent probe chlortetracycline hydrochloride, in NO production was examined after xenobiotic exposure.

Altered Activity and Physicochemical Properties of Short Cationic Antimicrobial Peptides by Incorporation of Arginine Analogues

The incorporation of nongenetically encoded amino acids is a well established strategy to alter the behavior of several types of promising cationic antimicrobial peptides. Generally, these elements have been improved mimics of the hydrophobic amino acids yielding peptides with increased stability and potency. In this initial study, the effect of systematic replacement of Arg in a well-defined moderately antimicrobial tripeptide library is described. It is shown that the arginine analogues need to display a strong basicity to produce active peptides. It is further revealed that the hydrophobic units needed for activity in these peptides can be effectively incorporated in the direct vicinity of the cationic charge to produce compounds with improved antibacterial properties. A well-defined facial amphiphilic structure, which remains intact upon introduction of hydrophobic elements in the cationic side chains, is seen for the majority of the tested peptides. Microcalorimetric studies revealed a peptide binding to large anionic unilamellar vesicles (LUVs) mimicking the Gram-positive bacterial membrane as well as a potentially competitive binding to human serum albumin in the low- to mid-micromolar range. No considerable alterations in binding to either albumin or the LUVs were seen for the analogue containing peptides. A neutral LUV mimicking the eukaryotic cell membrane showed no significant binding to any of the peptides. The oral absorption of this class of short lactoferricin based peptides was investigated for the first time and revealed that incorporation of weaker bases than Arg produced peptides with much improved permeability in a recently developed permeation model, the phospholipid vesicle based barrier assay. Collectively, the results presented here show that there is ample room to toggle the activity and physical properties of short cationic antimicrobial peptides by incorporation of arginine analogues.

Nitric Oxide Synthase: “Enzyme Zero” in Air Pollution–Induced Vascular Toxicity

Nitric Oxide Synthase: “Enzyme Zero” in Air Pollution–Induced Vascular Toxicity

Quality Control of a Transcriptional Regulator by SUMO-Targeted Degradation

Slx5 and Slx8 are heterodimeric RING domain-containing proteins that possess SUMO-targeted ubiquitin ligase (STUbL) activity in vitro. Slx5-Slx8 and its orthologs are proposed to target SUMO conjugates for ubiquitin-mediated proteolysis, but the only in vivo substrate identified to date is mammalian PML, and the physiological importance of SUMO-targeted ubiquitylation remains largely unknown. We previously identified mutations in SLX5 and SLX8 by selecting for suppressors of a temperature-sensitive allele of MOT1, which encodes a regulator of TATA-binding protein. Here, we demonstrate that Mot1 is SUMOylated in vivo and that disrupting the Slx5-Slx8 pathway by mutation of the target lysines in Mot1, by deletion of SLX5 or the ubiquitin E2 UBC4, or by inhibition of the proteosome suppresses mot1-301 mutant phenotypes and increases the stability of the Mot1-301 protein. The Mot1-301 mutant protein is targeted for proteolysis by SUMOylation to a much greater extent than wild-type Mot1, suggesting a quality control mechanism. In support of this idea, growth of Saccharomyces cerevisiae in the presence of the arginine analog canavanine results in increased SUMOylation and Slx5-Slx8-mediated degradation of wild-type Mot1. These results therefore demonstrate that Mot1 is an in vivo STUbL target in yeast and suggest a role for SUMO-targeted degradation in protein quality control.

Side-chain to backbone interactions dictate the conformational preferences of a cyclopentane arginine analogue.

The intrinsic conformational preferences of the nonproteinogenic amino acids constructed by incorporating the arginine side chain in the beta position of 1-aminocyclopentane-1-carboxylic acid (either in a cis or a trans orientation relative to the amino group) have been investigated by using computational methods. These compounds may be considered as constrained analogues of arginine (denoted as c(5)Arg) in which the orientation of the side chain is fixed by the cyclopentane moiety. Specifically, the N-acetyl-N'-methylamide derivatives of cis- and trans-c(5)Arg have been examined in the gas phase and in solution by using B3LYP/6-311+G(d,p) calculations and Molecular Dynamics simulations. Results indicate that the conformational space available to these compounds is highly restricted, their conformational preferences being dictated by the ability of the guanidinium group in the side chain to establish hydrogen bond interactions with the backbone. A comparison with the behavior previously described for the analogous phenylalanine derivatives is presented.

Short-Term Starvation of Immune Deficient Drosophila Improves Survival to Gram-Negative Bacterial Infections

BackgroundPrimary immunodeficiencies are inborn errors of immunity that lead to life threatening conditions. These predispositions describe human immunity in natura and highlight the important function of components of the Toll-IL-1- receptor-nuclear factor kappa B (TIR-NF-κB) pathway. Since the TIR-NF-κB circuit is a conserved component of the host defence in higher animals, genetically tractable models may contribute ideas for clinical interventions.Methodology/Principal FindingsWe used immunodeficient fruit flies (Drosophila melanogaster) to address questions pertaining to survival following bacterial infection. We describe here that flies lacking the NF-κB protein Relish, indispensable for countering Gram-negative bacteria, had a greatly improved survival to such infections when subject to dietary short-term starvation (STS) prior to immune challenge. STS induced the release of Nitric Oxide (NO), a potent molecule against pathogens in flies, mice and humans. Administering the NO Synthase-inhibitory arginine analog N-Nitro-L-Arginine-Methyl-Ester (L-NAME) but not its inactive enantiomer D-NAME increased once again sensitivity to infection to levels expected for relish mutants. Surprisingly, NO signalling required the NF-κB protein Dif, usually needed for responses against Gram-positive bacteria.Conclusions/SignificanceOur results show that NO release through STS may reflect an evolutionary conserved process. Moreover, STS could be explored to address immune phenotypes related to infection and may offer ways to boost natural immunity.

Substrate Selectivity in Adic, an E. Coli Inner Membrane Arginine-agmatine Antiporter

AdiC is a membrane antiporter that transports arginine and its decarboxylation product agmatine across E. coli inner membrane. It plays a key role in the arginine-dependent extreme acid resistance. We overexpressed AdiC in E. coli and reconstituted the purified protein into liposomes. A series of arginine analogs were tested on the transporter. The permeability sequence is as follows: arg / agm > 1,5-diaminopentane, 1,6-diamino-hexane, 1,4-diaminobutane >> argininamide, lysine, ornithine, canavinine. Kinetic analysis results are Km∼ 1mM for 1,5-diaminopentane, ∼3 mM for argininamide and lysine, ∼15 mM for canavanine.

Endogenous methyl palmitate modulates nicotinic receptor-mediated transmission in the superior cervical ganglion

Nitric oxide (NO) is identified as the endothelium-derived relaxing factor and a neurotransmitter with a superfusion bioassay cascade technique. By using a similar technique with rat superior cervical ganglion (SCG) as donor tissue and rabbit endothelium-denuded aortic ring as detector tissue, we report here that a vasodilator, which is more potent than NO, is released in the SCG upon field electrical stimulation (FES) or addition of nicotine. Release of this vasodilator was enhanced by arginine analogs, including N(omega)-nitro-l-arginine (a NO synthase inhibitor), suggesting that it is not NO. Analysis by gas chromatography/mass spectrometry identified 2 saturated fatty acids, palmitic acid methyl ester (PAME) and stearic acid methyl ester (SAME), being released from the SCG upon FES in the presence of arginine analogs. Exogenous PAME but not SAME induced significant aortic dilation (EC(50) = 0.19 nM), indicating that PAME is the potent vasodilator. Release of PAME and SAME was significantly diminished in chronically decentralized SCG but not denervated SCG, suggesting the preganglionic origin. Furthermore, release of both fatty acids was calcium- and myosin light chain kinase-dependent, suggesting that both were released from axoplasmic vesicular stores. Electrophysiological studies further demonstrated that PAME but not SAME inhibited nicotine-induced inward currents in cultured SCG and the alpha7-nicotinic acetylcholine receptor-expressing Xenopus oocytes. Endogenous PAME appears to play a role in modulation of the autonomic ganglionic transmission and to complement the vasodilator effect of NO.

AtNOS/AtNOA1 Is a Functional Arabidopsis thaliana cGTPase and Not a Nitric-oxide Synthase

AtNOS1 was previously identified as a potential nitric-oxide synthase (NOS) in Arabidopsis thaliana, despite lack of sequence similarity to animal NOSs. Although the dwarf and yellowish leaf phenotype of Atnos1 knock-out mutant plants can be rescued by treatment with exogenous NO, doubts have recently been raised as to whether AtNOS1 is a true NOS. Moreover, depending on the type of physiological responses studied, Atnos1 is not always deficient in NO induction and/or detection, as previously reported. Here, we present experimental evidence showing that AtNOS1 is unable to bind and oxidize arginine to NO. These results support the argument that AtNOS1 is not a NOS. We also show that the renamed NO-associated protein 1 (AtNOA1) is a member of the circularly permuted GTPase family (cGTPase). AtNOA1 specifically binds GTP and hydrolyzes it. Complementation experiments of Atnoa1 mutant plants with different constructs of AtNOA1 show that GTP hydrolysis is necessary but not sufficient for the physiological function of AtNOA1. Mutant AtNOA1 lacking the C-terminal domain, although retaining GTPase activity, failed to complement Atnoa1, suggesting that this domain plays a crucial role in planta. cGTPases appear to be RNA-binding proteins, and the closest homolog of AtNOA1, the Bacillus subtilis YqeH, has been shown to participate in ribosome assembly and stability. We propose a similar function for AtNOA1 and discuss it in the light of its potential role in NO accumulation and plant development.

Inducible Nitric Oxide Synthase Inhibitor SD-3651 Reduces Proteinuria in MRL/lpr Mice Deficient in the NOS2 Gene

Several studies have demonstrated the effectiveness of arginine analog nitric oxide synthase (NOS) inhibitor therapy in preventing and treating murine lupus nephritis. However, MRL/MpJ-FASlpr(MRL/lpr) mice lacking afunctional NOS2 (inducible NOS...

Strategies for the Solid-Phase Diversification of Poly-l-proline-Type II Peptide Mimic Scaffolds and Peptide Scaffolds Through Guanidinylation

A strategy for the solid-phase diversification of PPII mimic scaffolds through guanidinylation is presented. The approach involves the synthesis N-Pmc-N'-alkyl thioureas as diversification reagents. Analogues of Fmoc-Orn(Mtt)-OH can be incorporated into a growing peptide chain on Wang resin. Side chain deprotection with 1% TFA/CH2Cl2 followed by EDCI-mediated reaction of N-Pmc-N'-alkyl thioureas with the side chain amine affords arginine analogues with modified guanidine head groups. The scope, limitations, and incidental chemistry are discussed.

Partitioning of amino-acid analogues in a five-slab membrane model

The positional preferences of the twenty amino-acid residues in a phospholipid bilayer are investigated by calculating the solvation free energy of the corresponding side chain analogues using a five-slab continuum electrostatic model. The side-chain analogues of the aromatic residues tryptophan and tyrosine are found to partition in the head-group region, due to compensation between the increase of the non-polar component of the solvation free energy at the boundary with the aqueous region and the decrease in the electrostatic component. The side chain analogue of phenylalanine differs from the other aromatic molecules by being able to partition in both the head-group region and the membrane core. This finding is consistent with experimental findings of the position of phenylalanine in membrane helices. Interestingly, the charged side-chain analogues of arginine and lysine are shown to prefer the head-group region in an orientation that allows the charged moiety to interact with the aqueous layer. The orientation adopted is similar to the “snorkelling” effect seen in lysine and arginine residues in membrane helices. In contrast, the preference of the charged side-chain analogues of histidine (protonated) and aspartate (deprotonated) for the aqueous layer is shown to be due to a steep decrease in the electrostatic component of the solvation free energy at the boundary to the aqueous region. The calculations allow an understanding of the origins of side chain positioning in membranes and are thus useful in understanding membrane–protein:lipid thermodynamics.

A Proteasomal ATPase Contributes to Dislocation of Endoplasmic Reticulum-associated Degradation (ERAD) Substrates

Endoplasmic reticulum (ER)-associated degradation (ERAD) eliminates aberrant proteins from the ER by dislocating them to the cytoplasm where they are tagged by ubiquitin and degraded by the proteasome. Six distinct AAA-ATPases (Rpt1-6) at the base of the 19S regulatory particle of the 26S proteasome recognize, unfold, and translocate substrates into the 20S catalytic chamber. Here we show unique contributions of individual Rpts to ERAD by employing equivalent conservative substitutions of the invariant lysine in the ATP-binding motif of each Rpt subunit. ERAD of two substrates, luminal CPY*-HA and membrane 6myc-Hmg2, is inhibited only in rpt4R and rpt2RF mutants. Conversely, in vivo degradation of a cytosolic substrate, DeltassCPY*-GFP, as well as in vitro cleavage of Suc-LLVY-AMC are hardly affected in rpt4R mutant yet are inhibited in rpt2RF mutant. Together, we find that equivalent mutations in RPT4 and RPT2 result in different phenotypes. The Rpt4 mutation is manifested in ERAD defects, whereas the Rpt2 mutation is manifested downstream, in global proteasomal activity. Accordingly, rpt4R strain is particularly sensitive to ER stress and exhibits an activated unfolded protein response, whereas rpt2RF strain is sensitive to general stress. Further characterization of Rpt4 involvement in ERAD reveals that it participates in CPY*-HA dislocation, a function previously attributed to p97/Cdc48, another AAA-ATPase essential for ERAD of CPY*-HA but dispensable for proteasomal degradation of DeltassCPY*-GFP. Pointing to Cdc48 and Rpt4 overlapping functions, excess Cdc48 partially restores impaired ERAD in rpt4R, but not in rpt2RF. We discuss models for Cdc48 and Rpt4 cooperation in ERAD.