Synthesis, screening, and sequencing of cysteine-rich one-bead one-compound peptide libraries.

Cysteine-rich peptides are valued as tags for biarsenical fluorophores and as environmentally important reagents for binding toxic heavy metals. Due to the inherent difficulties created by cysteine, the power of one-bead one-compound (OBOC) libraries has never been applied to the discovery of short cysteine-rich peptides. We have developed the first method for the synthesis, screening, and sequencing of cysteine-rich OBOC peptide libraries. First, we synthesized a heavily biased cysteine-rich OBOC library, incorporating 50% cysteine at each position (Ac-X8-KM-TentaGel). Then, we developed conditions for cysteine alkylation, cyanogen bromide cleavage, and direct MS/MS sequencing of that library at the single bead level. The sequencing efficiency of this library was comparable to a traditional cysteine-free library. To validate screening of cysteine-rich OBOC libraries, we reacted a library with the biarsenical FlAsH and identified beads bearing the known biarsenical-binding motif (CCXXCC). These results enable OBOC libraries to be used in high-throughput discovery of cysteine-rich peptides for protein tagging, environmental remediation of metal contaminants, or cysteine-rich pharmaceuticals

Negative modulation of the GABAAρ1 receptor function by l-cysteine

l-Cysteine is an endogenous sulfur-containing amino acid with multiple and varied roles in the central nervous system, including neuroprotection and the maintenance of the redox balance. However, it was also suggested as an excitotoxic agent implicated in the pathogenesis of neurological disorders such as Parkinson′s and Alzheimer′s disease. l-Cysteine can modulate the activity of ionic channels, including voltage-gated calcium channels and glutamatergic NMDA receptors, whereas its effects on GABAergic neurotransmission had not been studied before. In the present work, we analyzed the effects of l-cysteine on responses mediated by homomeric GABAAρ1 receptors, which are known for mediating tonic γ-aminobutyric acid (GABA) responses in retinal neurons. GABAAρ1 receptors were expressed in Xenopus laevis oocytes and GABA-evoked chloride currents recorded by two-electrode voltage-clamp in the presence or absence of l-cysteine. l-Cysteine antagonized GABAAρ1 receptor-mediated responses; inhibition was dose-dependent, reversible, voltage independent, and susceptible to GABA concentration. Concentration-response curves for GABA were shifted to the right in the presence of l-cysteine without a substantial change in the maximal response. l-Cysteine inhibition was insensitive to chemical protection of the sulfhydryl groups of the ρ1 subunits by the irreversible alkylating agent N-ethyl maleimide. Our results suggest that redox modulation is not involved during l-cysteine actions and that l-cysteine might be acting as a competitive antagonist of the GABAAρ1 receptors. (Figure presented.).Fil: Beltrán González, Andrea Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Vicentini, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Calvo, Daniel Juan. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin

Cell signaling promoting protein carbonylation does not cause sulfhydryl oxidation: implications to the mechanism of redox signaling

Reactive oxygen species (ROS) have been recognized as second messengers, however, targeting mechanisms for ROS in cell signaling have not been defined. While ROS oxidizing protein cysteine thiols has been the most popular proposed mechanism, our laboratory proposed that ligand/receptor-mediated cell signaling involves protein carbonylation. Peroxiredoxin-6 (Prx6) is one protein that is carbonylated at 10 min after the platelet-derived growth factor (PDGF) stimulation of human pulmonary artery smooth muscle cells. In the present study, the SulfoBiotics Protein Redox State Monitoring Kit Plus (Dojindo Molecular Technologies) was used to test if cysteine residues of Prx6 are oxidized in response to the PDGF stimulation. Human Prx6 has a molecular weight of 25 kDa and contains two cysteine residues. The Dojindo system adds the 15 kDa Protein-SHifter if these cysteine residues are reduced in the cells. Results showed that, in untreated cells, the Prx6 molecule predominantly exhibited the 55 kDa band, indicating that both cysteine residues are reduced in the cells. Treatment of cells with 1 mM H 2O 2 caused the disappearance of the 55 kDa band and the appearance of a 40 kDa band, suggesting that the high concentration of H 2O 2 oxidized one of the two cysteine residues in the Prx6 molecule. By contrast, PDGF stimulation had no effects on the thiol status of the Prx6 molecule. We concluded that protein carbonylation is a more sensitive target of ROS during ligand/receptor-mediated cell signaling than sulfhydryl oxidation

In vitro and in vivo anthelmintic efficacy of plant cysteine proteinases against the rodent gastrointestinal nematode, Trichuris muris

We examined the mechanism of action and compared the anthelmintic efficacy of cysteine proteinases from papaya, pineapple, fig, kiwi fruit and Egyptian milkweed in vitro using the rodent gastrointestinal nematode Heligmosomoides polygyrus. Within a 2 h incubation period, all the cysteine proteinases, with the exception of the kiwi fruit extract, caused marked damage to the cuticle of H. polygyrus adult male and female worms, reflected in the loss of surface cuticular layers. Efficacy was comparable for both sexes of worms, was dependent on the presence of cysteine and was completely inhibited by the cysteine proteinase inhibitor, E-64. LD50 values indicated that the purified proteinases were more efficacious than the proteinases in the crude latex, with purified ficin, papain, chymopapain, Egyptian milkweed latex extract and pineapple fruit extract, containing fruit bromelain, having the most potent effect. The mechanism of action of these plant enzymes (i.e. an attack on the protective cuticle of the worm) suggests that resistance would be slow to develop in the field. The efficacy and mode of action make plant cysteine proteinases potential candidates for a novel class of anthelmintics urgently required for the treatment of humans and domestic livestock

Proteome-wide analysis of cysteine oxidation reveals metabolic sensitivity to redox stress

Reactive oxygen species (ROS) are increasingly recognised as important signalling molecules through oxidation of protein cysteine residues. Comprehensive identification of redox-regulated proteins and pathways is crucial to understand ROS-mediated events. Here, we present stable isotope cysteine labelling with iodoacetamide (SICyLIA), a mass spectrometry-based workflow to assess proteome-scale cysteine oxidation. SICyLIA does not require enrichment steps and achieves unbiased proteome-wide sensitivity. Applying SICyLIA to diverse cellular models and primary tissues provides detailed insights into thiol oxidation proteomes. Our results demonstrate that acute and chronic oxidative stress causes oxidation of distinct metabolic proteins, indicating that cysteine oxidation plays a key role in the metabolic adaptation to redox stress. Analysis of mouse kidneys identifies oxidation of proteins circulating in biofluids, through which cellular redox stress can affect whole-body physiology. Obtaining accurate peptide oxidation profiles from complex organs using SICyLIA holds promise for future analysis of patient-derived samples to study human pathologies

The nature of CuA in cytochrome c oxidase

The isolation and purification of yeast cytochrome c oxidase is described. Characterization of the purified protein indicates that it is spectroscopically identical with cytochrome c oxidase isolated from beef heart. Preparations of isotopically substituted yeast cytochrome c oxidase are obtained incorporating [1,3-15N2]histidine or [beta,beta- 2H2]cysteine. Electron paramagnetic resonance and electron nuclear double resonance spectra of the isotopically substituted proteins identify unambiguously at least 1 cysteine and 1 histidine as ligands to CuA and suggest that substantial spin density is delocalized onto a cysteine sulfur in the oxidized protein to render the site Cu(I)-S

An ATP-binding cassette-type cysteine transporter in Campylobacter jejuni inferred from the structure of an extracytoplasmic solute receptor protein

Campylobacter jejuni is a Gram-negative food-borne pathogen associated with gastroenteritis in humans as well as cases of the autoimmune disease Guillain Barre syndrome. C. jejuni is asaccharolytic because it lacks an active glycolytic pathway for the use of sugars as a carbon source. This suggests an increased reliance on amino acids as nutrients and indeed the genome sequence of this organism indicates the presence of a number of amino acid uptake systems. Cj0982, also known as CjaA, is a putative extracytoplasmic solute receptor for one such uptake system as well as a major surface antigen and vaccine candidate. The crystal structure of Cj0982 reveals a two-domain protein with density in the enclosed cavity between the domains that clearly defines the presence of a bound cysteine ligand. Fluorescence titration experiments were used to demonstrate that Cj0982 binds cysteine tightly and specifically with a K-d of similar to 10(-7) M consistent with a role as a receptor for a high- affinity transporter. These data imply that Cj0982 is the binding protein component of an ABC-type cysteine transporter system and that cysteine uptake is important in the physiology of C. jejuni

Precise Molecular Structures of Cysteine, Cystine, Hydrogen-Bonded Dicysteine, Cysteine Dipeptide, Glutathione and Acetyl Cysteine Based on Additivity of Atomic Radii

Structures of molecules are usually represented by arbitrary line drawings, ball and stick or space filling models. In recent years, the author found that using the appropriate radii of atoms and ions, bond lengths in inorganic, organic and biomolecules and of hydrogen bonds are exact sums of the radii of the adjacent atoms and or ions. This additivity of atomic radii was shown to hold also for the bond lengths in the twenty essential amino acids. On this basis, the atomic structures of the very important molecules mentioned in the title, have been presented here for the first time. It is hoped that these precise structures and their dimensions will shed new light into their role in biochemistry, pharmaceutical chemistry, environmental chemistry, biomedicine and in the mechanism of protein folding