Cysteine Inhibitors Research Articles

Diversely evolved xibalbin variants from remipede venom inhibit potassium channels and activate PKA-II and Erk1/2 signaling

BackgroundThe identification of novel toxins from overlooked and taxonomically exceptional species bears potential for various pharmacological applications. The remipede Xibalbanus tulumensis, an underwater cave-dwelling crustacean, is the only crustacean for which a venom system has been described. Its venom contains several xibalbin peptides that have an inhibitor cysteine knot (ICK) scaffold.ResultsOur screenings revealed that all tested xibalbin variants particularly inhibit potassium channels. Xib1 and xib13 with their eight-cysteine domain similar to spider knottins also inhibit voltage-gated sodium channels. No activity was noted on calcium channels. Expanding the functional testing, we demonstrate that xib1 and xib13 increase PKA-II and Erk1/2 sensitization signaling in nociceptive neurons, which may initiate pain sensitization. Our phylogenetic analysis suggests that xib13 either originates from the common ancestor of pancrustaceans or earlier while xib1 is more restricted to remipedes. The ten-cysteine scaffolded xib2 emerged from xib1, a result that is supported by our phylogenetic and machine learning-based analyses.ConclusionsOur functional characterization of synthesized variants of xib1, xib2, and xib13 elucidates their potential as inhibitors of potassium channels in mammalian systems. The specific interaction of xib2 with Kv1.6 channels, which are relevant to treating variants of epilepsy, shows potential for further studies. At higher concentrations, xib1 and xib13 activate the kinases PKA-II and ERK1/2 in mammalian sensory neurons, suggesting pain sensitization and potential applications related to pain research and therapy. While tested insect channels suggest that all probably act as neurotoxins, the biological function of xib1, xib2, and xib13 requires further elucidation. A novel finding on their evolutionary origin is the apparent emergence of X. tulumensis-specific xib2 from xib1. Our study is an important cornerstone for future studies to untangle the origin and function of these enigmatic proteins as important components of remipede but also other pancrustacean and arthropod venoms.

Purification and characterization of cysteine protease of Sarcocystis fusiformis from infected Egyptian water buffaloes

Sarcocystis spp. infects water buffaloes (Bubalus bubalis) causing sarcocystosis. In the present study, Sarcocystis fusiformis was recognized in Egyptian water buffaloes based on histological observation and molecular analysis of internal transcribed spacer 1 (ITS1), 18S ribosomal RNA (18S rRNA) and cytochrome c oxidase subunit I (COX-1) gene fragments. Chemotherapy and vaccines against Sarcocystis spp. could potentially target proteases because they may play a crucial role in the infection. Cysteine proteases are multifunctional enzymes involved in vital metabolic processes. However, the involvement of proteases in S. fusiform infection has not yet been characterized. Here, the purification and study on some biochemical properties of protease isolated from cysts of S. fusiform were carried out. Protease with a molecular weight of 100 kDa was purified. LC–MS/MS analyzed the protein sequence of purified protease and the data suggested that the enzyme might be related to the cysteine protease. The purified protease exhibited maximum activity at pH 6 and a temperature of 50 °C. The Michaelis–Menten constant (Km), the maximum velocity (Vmax), and the turnover number (Kcat) were determined. The complete inhibition effect of cysteine inhibitors indicated that the purified enzyme is a cysteine protease. The results suggested that S. fusiform proteolytic enzyme may be necessary for parasite survival in water buffaloes by digesting host tissues. Therefore, cysteine protease could be a suitable target for vaccinations.

Insights into DNA cleavage by MutL homologs from analysis of conserved motifs in eukaryotic Mlh1.

MutL family proteins contain an N-terminal ATPase domain (NTD), an unstructured interdomain linker, and a C-terminal domain (CTD), which mediates constitutive dimerization between subunits and often contains an endonuclease active site. Most MutL homologs direct strand-specific DNA mismatch repair by cleaving the error-containing daughter DNA strand. The strand cleavage reaction is poorly understood; however, the structure of the endonuclease active site is consistent with a two- or three-metal ion cleavage mechanism. A motif required for this endonuclease activity is present in the unstructured linker of Mlh1 and is conserved in all eukaryotic Mlh1 proteins, except those from metamonads, which also lack the almost absolutely conserved Mlh1 C-terminal phenylalanine-glutamate-arginine-cysteine (FERC) sequence. We hypothesize that the cysteine in the FERC sequence is autoinhibitory, as it sequesters the active site. We further hypothesize that the evolutionary co-occurrence of the conserved linker motif with the FERC sequence indicates a functional interaction, possibly by linker motif-mediated displacement of the inhibitory cysteine. This role is consistent with available data for interactions between the linker motif with DNA and the CTDs in the vicinity of the active site.

Peptides originally derived from Chilobrachys jingzhao tarantula venom possess beneficial effects on pancreatic beta cell health and function

Clinical approval of the glucagon-like peptide-1 (GLP-1) mimetic exenatide for the treatment of type 2 diabetes highlights the therapeutic effectiveness of venom-derived peptides. In the present study, we examined and characterised the glucose-lowering potential of synthetic Jingzhaotoxin IX and Jingzhaotoxin XI peptides, which were originally isolated from the venom of the Chinese earth tarantula Chilobrachys jingzhao. Following confirmation of lack of beta-cell toxicity of synthetic peptides, assessment of enzymatic stability and effects on in vitro beta-cell function were studied, alongside putative mechanisms. Glucose homeostatic and appetite suppressive actions of Jingzhaotoxin IX and Jingzhaotoxin XI alone, or in combination with exenatide, were then assessed in normal overnight fasted C57BL/6 mice. Synthetic Jingzhaotoxin peptides were non-toxic and exhibited a decrease in mass of 6 Da in Krebs-Ringer bicarbonate buffer suggesting inhibitor cysteine knot (ICK)-like formation, but interestingly were liable to plasma enzyme degradation. The Jingzhaotoxin peptides evoked prominent insulin secretion from BRIN BD11 beta-cells, with activity somewhat characteristic of Kv2.1 channel binding. In addition, Jingzhaotoxin peptides enhanced beta-cell proliferation and provided significant protection against cytokine-induced apoptosis. When injected co-jointly with glucose, the Jingzhaotoxin peptides slightly decreased blood-glucose levels but had no effect on appetite in overnight fasted mice. Whilst the Jingzhaotoxin peptides did not enhance exenatide-induced benefits on glucose homeostasis, they augmented exenatide-mediated suppression of appetite. Taken together, these data highlight the therapeutic potential of tarantula venom-derived peptides, such as Jingzhaotoxin IX and Jingzhaotoxin XI either alone or in combination with exenatide, for diabetes and related obesity.

Reinforcement of an alkyd resin coating incorporating a swelling clay encapsulated with L-cysteine molecules: Characterization and corrosion inhibition of Cu-36Zn alloy

Corrosion is a real industrial scourge; it is defined as the degradation of a material in corrosive environments such as seawater and acids, causing a huge economic loss, especially in the automotive, building and marine industries. To cope with this issue, a swelling clay was used as corrosion inhibiting reservoir of an amino acid (L-cysteine). For this purpose, the before-hand developed composite was characterized and incorporated into an organic coating. The clay fractions of this swelling clay were modified by the cysteine inhibitor. The modification was confirmed by X-ray diffraction (XRD), where the d002 reflection of the I-S clay fraction was increased from 4.66 Å (Na-clay) to 5.57 Å (Cys-clay). FTIR and UV–vis–NIR techniques were also used for characterization purposes. The sudied clay is a mixture of clay minerals (illite-smectite as the main component, kaolinite and chlorite) and associated minerals (quartz and calcite). The progressive release of cysteine molecules leads to a durable corrosion protection of brass in a 3 % NaCl solution, resulting in the lowest corrosion current density (10.36 μA/cm2) and reaching a maximum inhibition efficiency of about 86 %. The resulting composite was used as a charge to cover the brass surface by being mixed with an alkyd resin (5 % of the mass of the resin). The impedance modulus |Z|0.05 Hz reached a maximum value (1419 kΩ.cm2) at the end of the immersion period. The corresponding charge transfer resistance value (1.82 MΩ.cm2) was twice that of the coating containing a commercial inhibitor (0.92 MΩ.cm2), reflecting the composite efficiency and the novelty of this work. Electrochemical techniques such as open circuit potential (OCP), polarization curves and electrochemical impedance spectroscopy (Nyquist, Bode Magnitude and Bode Phase) were performed. Corrosion products and protective layers are analyzed using optical microscopy and scanning electron microscopy techniques coupled to an energy dispersive X-ray spectrometer (SEM-EDX). The development of this new smart coating will expand the range of applications for electrochemically active materials.

A novel peptide isolated from Aphonopelma chalcodes tarantula venom with benefits on pancreatic islet function and appetite control

Proof-of-concept for therapeutic application of venom-derived compounds in diabetes is exemplified by the incretin mimetic, exenatide, originally extracted from the saliva of the venomous Heloderma suspectum lizard. In this regard, we have isolated and sequenced a novel 28 amino acid peptide named Δ-theraphotoxin-Ac1 (Δ-TRTX-AC1) from venom of the Mexican Blond tarantula spider Aphonopelma chalcodes, with potential therapeutic benefits for diabetes. Following confirmation of the structure and safety profile of the synthetic peptide, assessment of enzymatic stability and effects of Δ-TRTX-AC1 on in vitro beta-cell function were studied, alongside potential mechanisms. Glucose homeostatic and satiety actions of Δ-TRTX-AC1 alone, and in combination with exenatide, were then assessed in C57BL/6 mice. Synthetic Δ-TRTX-AC1 was shown to adopt a characteristic inhibitor cysteine knot (ICK)-like structure and was non-toxic to beta-cells. Δ-TRTX-AC1 evoked glucose-dependent insulin secretion from BRIN BD11 cells with bioactivity confirmed in murine islets. Insulin secretory potency was established to be dependent on KATP and Ca2+ channel beta-cell signalling. In addition, Δ-TRTX-AC1 enhanced beta-cell proliferation and provided significant protection against cytokine-induced apoptosis. When injected co-jointly with glucose in mice at a dose of 250 nmol/kg, Δ-TRTX-AC1 decreased blood-glucose levels and evoked a significant satiating effect. Moreover, whilst Δ-TRTX-AC1 did not enhance exenatide induced benefits on glucose homeostasis, the peptide significantly augmented exenatide mediated suppression of appetite. Together these data highlight the therapeutic potential of tarantula spider venom-derived peptides, such as Δ-TRTX-Ac1, for diabetes and related obesity.

In Silico Analysis of a Drosophila Parasitoid Venom Peptide Reveals Prevalence of the Cation-Polar-Cation Clip Motif in Knottin Proteins.

As generalist parasitoid wasps, Leptopilina heterotoma are highly successful on many species of fruit flies of the genus Drosophila. The parasitoids produce specialized multi-strategy extracellular vesicle (EV)-like structures in their venom. Proteomic analysis identified several immunity-associated proteins, including the knottin peptide, LhKNOT, containing the structurally conserved inhibitor cysteine knot (ICK) fold, which is present in proteins from diverse taxa. Our structural and docking analysis of LhKNOT's 36-residue core knottin fold revealed that in addition to the knottin motif itself, it also possesses a Cation-Polar-Cation (CPC) clip. The CPC clip motif is thought to facilitate antimicrobial activity in heparin-binding proteins. Surprisingly, a majority of ICKs tested also possess the CPC clip motif, including 75 bona fide plant and arthropod knottin proteins that share high sequence and/or structural similarity with LhKNOT. Like LhKNOT and these other 75 knottin proteins, even the Drosophila Drosomycin antifungal peptide, a canonical target gene of the fly's Toll-NF-kappa B immune pathway, contains this CPC clip motif. Together, our results suggest a possible defensive function for the parasitoid LhKNOT. The prevalence of the CPC clip motif, intrinsic to the cysteine knot within the knottin proteins examined here, suggests that the resultant 3D topology is important for their biochemical functions. The CPC clip is likely a highly conserved structural motif found in many diverse proteins with reported heparin binding capacity, including amyloid proteins. Knottins are targets for therapeutic drug development, and insights into their structure-function relationships will advance novel drug design.

Vexitoxins: conotoxin-like venom peptides from predatory gastropods of the genus Vexillum.

Venoms of predatory marine cone snails are intensely studied because of the biomedical applications of the neuropeptides that they contain, termed conotoxins. Meanwhile some gastropod lineages have independently acquired secretory glands strikingly similar to the venom gland of cone snails, suggesting that they possess similar venoms. Here we focus on the most diversified of these clades, the genus Vexillum. Based on the analysis of a multi-species proteo-transcriptomic dataset, we show that Vexillum species indeed produce complex venoms dominated by highly diversified short cysteine-rich peptides, vexitoxins. Vexitoxins possess the same precursor organization, display overlapping cysteine frameworks and share several common post-translational modifications with conotoxins. Some vexitoxins show sequence similarity to conotoxins and adopt similar domain conformations, including a pharmacologically relevant inhibitory cysteine knot motif. The Vexillum envenomation gland (gL) is a notably more recent evolutionary novelty than the conoidean venom gland. Thus, we hypothesize lower divergence between vexitoxin genes, and their ancestral ‘somatic’ counterparts compared to that in conotoxins, and we find support for this hypothesis in the evolution of the vexitoxin cluster V027. We use this example to discuss how future studies on vexitoxins can inform the origin of conotoxins, and how they may help to address outstanding questions in venom evolution.

Identifying SARS-CoV-2 main protease inhibitors by a novel sandwich-like fluorescence polarization screening assay

SARS-CoV-2 main protease (Mpro) is responsible for polyprotein cleavage to release non-structural proteins (nsps) for viral genomic RNA replication, and its homologues are absent in human cells. Therefore, Mpro has been regarded as one of the ideal drug targets for the treatment of coronavirus disease 2019 (COVID-19). In this study, we first combined the fluorescence polarization (FP) technique with biotin-avidin system (BAS) to develop a novel sandwich-like FP screening assay for quick discovery of SARS-CoV-2 Mpro inhibitors from a natural product library. With this screening assay, anacardic acid (AA) and 1, 2, 3, 4, 6-O-pentagalloylglucose (PGG) were found to be the competitive inhibitor and mixed-type inhibitor targeting Mpro, respectively. Importantly, our results showed that the majority of the reported Mpro inhibitors are promiscuous cysteine inhibitors that are not specific to Mpro. In summary, this novel sandwich-like FP screening assay is simple, sensitive, and robust, which is ideal for large-scale screening. Natural products AA and PGG will be the promising lead compounds for generating more potent antiviral agents targeting Mpro, and the stringent hit validation at the early stage of drug discovery is urgently needed.

Inside the cracked kernel: establishing the molecular basis of AMG510 and MRTX849 in destabilising KRASG12C mutant switch I and II in cancer treatment

The Kirsten rat sarcoma oncoprotein (KRAS) has been punctuated by drug development failures for decades due to frequent mutations that occur mostly at codon 12 and the seemingly intractable targeting of the protein. However, with advances in covalent targeting, the oncoprotein is being expunged from the ‘undruggable’ list of proteins. This feat has seen some covalent drugs at different stages of clinical trials. The advancement of AMG510 and MRTX849 as inhibitors of cysteine mutated KRAS (KRASG12C) to phase-III clinical trials informed the biased selection of AMG510 and MRTX849 for this study. Despite this advance, the molecular and atomistic modus operandi of these drugs is yet to come to light. In this study, we employed computational tools to unravel the atomistic interactions and subsequent conformational effects of AMG510 and MRTX849 on the mutant KRASG12C. It was revealed that AMG510 and MRTX849 complexes presented similar total free binding energies, (ΔG bind), of –88.15 ± 5.96 kcal/mol and –88.71 ± 7.70 kcal/mol, respectively. Gly10, Lys16, Thr58, Gly60, Glu62, Glu63, Arg68, Asp69, Met72, His95, Tyr96, Gln99, Arg102 and Val103 interacted prominently with AMG510 and MRTX849. These residues interacted with the pharmacophoric moieties of AMG510 and MRTX849 via hydrogen bonds with decreasing bond lengths at various stages of the simulation. These interactions together with pi-pi stacking, pi-sigma and pi-alkyl interactions induced unfolding of switch I whiles compacting switch II, which could interrupt the binding of effector proteins to these interfaces. These insights present useful atomistic perspectives into the success of AMG510 and MRTX849 which could guide the design of more selective and potent KRAS inhibitors. Communicated by Ramaswamy H. Sarma

Venomics Reveals a Non-Compartmentalised Venom Gland in the Early Diverged Vermivorous Conus distans

The defensive use of cone snail venom is hypothesised to have first arisen in ancestral worm-hunting snails and later repurposed in a compartmentalised venom duct to facilitate the dietary shift to molluscivory and piscivory. Consistent with its placement in a basal lineage, we demonstrate that the C. distans venom gland lacked distinct compartmentalisation. Transcriptomics revealed C. distans expressed a wide range of structural classes, with inhibitory cysteine knot (ICK)-containing peptides dominating. To better understand the evolution of the venom gland compartmentalisation, we compared C. distans to C. planorbis, the earliest diverging species from which a defence-evoked venom has been obtained, and fish-hunting C. geographus from the Gastridium subgenus that injects distinct defensive and predatory venoms. These comparisons support the hypothesis that venom gland compartmentalisation arose in worm-hunting species and enabled repurposing of venom peptides to facilitate the dietary shift from vermivory to molluscivory and piscivory in more recently diverged cone snail lineages.

Novel Class of KLK13 Inhibitors Cysteine Knot

Novel Class of KLK13 Inhibitors Cysteine Knot

TOXIC AND IMMUNE ALLERGIC RESPONSES OF ANT VENOM TOXINS: A REVIEW

Present review article explains ant venom components and its allergic and biological effects in man and animals. Red ants or small fire ants secrete and inject venom very swiftly to defend their nest against predators, microbial pathogens, and competitors and to hunt the prey. Ant venom is a mixture of various organic compounds, including peptides, enzymes, and polypeptide toxins. It is highly toxic, allergic, invasive and venomous. It imposes sever paralytic, cytolytic, haemolytic, allergenic, pro-inflammatory, insecticidal, antimicrobial, and pain-producing pharmacologic activities after infliction. Victims show red ring-shaped allergic sign with regional swelling marked with intense pain. Ant venom also contains several hydrolases, oxidoreductases, proteases, Kunitz-like polypeptides, and inhibitor cysteine knot (ICK)-like (knottin) neurotoxins and insect defensins. Ant venom toxins/proteins generate allergic immune responses and employ eosinophils and produce Th2 cytokines, response. These compounds from ant venom could be used as a potential source of new anticonvulsants molecules. Ant venoms contain many small, linear peptides, an untapped source of bioactive peptide toxins. The remarkable insecticidal activity of ant venom could be used as a promising source of additional bio-insecticides and therapeutic agents.

Validation and invalidation of SARS-CoV-2 main protease inhibitors using the Flip-GFP and Protease-Glo luciferase assays

SARS-CoV-2 main protease (Mpro) is one of the most extensively exploited drug targets for COVID-19. Structurally disparate compounds have been reported as Mpro inhibitors, raising the question of their target specificity. To elucidate the target specificity and the cellular target engagement of the claimed Mpro inhibitors, we systematically characterize their mechanism of action using the cell-free FRET assay, the thermal shift-binding assay, the cell lysate Protease-Glo luciferase assay, and the cell-based FlipGFP assay. Collectively, our results have shown that majority of the Mpro inhibitors identified from drug repurposing including ebselen, carmofur, disulfiram, and shikonin are promiscuous cysteine inhibitors that are not specific to Mpro, while chloroquine, oxytetracycline, montelukast, candesartan, and dipyridamole do not inhibit Mpro in any of the assays tested. Overall, our study highlights the need of stringent hit validation at the early stage of drug discovery.

T cell protein tyrosine phosphatase protects intestinal barrier function by restricting epithelial tight junction remodeling.

Genome-wide association studies revealed that loss-of-function mutations in protein tyrosine phosphatase non-receptor type 2 (PTPN2) increase the risk of developing chronic immune diseases, such as inflammatory bowel disease (IBD) and celiac disease. These conditions are associated with increased intestinal permeability as an early etiological event. The aim of this study was to examine the consequences of deficient activity of the PTPN2 gene product, T cell protein tyrosine phosphatase (TCPTP), on intestinal barrier function and tight junction organization in vivo and in vitro. Here, we demonstrate that TCPTP protected against intestinal barrier dysfunction induced by the inflammatory cytokine IFN-γ by 2 mechanisms: it maintained localization of zonula occludens 1 and occludin at apical tight junctions and restricted both expression and insertion of the cation pore-forming transmembrane protein, claudin-2, at tight junctions through upregulation of the inhibitory cysteine protease, matriptase. We also confirmed that the loss-of-function PTPN2 rs1893217 SNP was associated with increased intestinal claudin-2 expression in patients with IBD. Moreover, elevated claudin-2 levels and paracellular electrolyte flux in TCPTP-deficient intestinal epithelial cells were normalized by recombinant matriptase. Our findings uncover distinct and critical roles for epithelial TCPTP in preserving intestinal barrier integrity, thereby proposing a mechanism by which PTPN2 mutations contribute to IBD.

Environmental impact and diversity of protease-producing bacteria in areas of leather tannery effluents of Sialkot, Pakistan.

Massive discharge of wastes produced by the processing of leather so far confers the most important environmental challenge facing the tanneries worldwide. Waste material from tanneries mostly consists of skin remnants and proteinaceous substances as by-products of leather processing. In these conditions, protease-producing bacteria play a vital role in degrading wastes in this sludge. Therefore, an investigation was made to study the effect of long-term tannery sludge contamination on the diversity of both protease-producing microbes and of bacterial extracellular proteases near tanneries of Sambrial and Sialkot. The high amount of carbon and nitrogen in the soil samples reflected their effect on the diversity of the microbial communities in these areas. Phylogenetic analysis based on 16S rRNA gene sequences suggest that the isolated proteolytic bacteria belonged to 9 different genera including Pseudomonas (26.19%), Proteus (19.04%), Serratia (16.66%), Klebsiella (14.28%), Providencia (9.52%), Achromobacter (7.14%), Enterobacter (2.38%), Myroides (2.38%), and Acinetobacter (2.38%). Enzyme activity showed that among all Pseudomonas and Proteus showed relatively high protease production, and inhibition studies revealed that proteases produced by all isolates were strongly inhibited by serine and/or metalloprotease inhibitors, and a smaller proportion was inhibited by inhibitors of cysteine and/or aspartic proteases. Furthermore, isolated bacteria revealed promising degradation activities against casein and/or gelatin with only a few that could hydrolyze elastin, suggesting proteases produced by these isolated bacteria belong to different classes of proteases, i.e., serine and metalloproteases. This study provided new insights on the community structure of cultivable protease-producing bacteria near tannery sludge of Sambrial and Sialkot. This study would be beneficial not only for establishing the way for effective degradation of tannery slugs but also for questing the novel properties of proteases for a future technological application.

Differences in the Inhibitory Specificity Distinguish the Efficacy of Plant Protease Inhibitors on Mouse Fibrosarcoma.

Metastasis, the primary cause of death from malignant tumors, is facilitated by multiple protease-mediated processes. Thus, effort has been invested in the development of protease inhibitors to prevent metastasis. Here, we investigated the effects of protease inhibitors including the recombinant inhibitors rBbKI (serine protease inhibitor) and rBbCI (serine and cysteine inhibitor) derived from native inhibitors identified in Bauhinia bauhinioides seeds, and EcTI (serine and metalloprotease inhibitor) isolated from the seeds of Enterolobium contortisiliquum on the mouse fibrosarcoma model (lineage L929). rBbKI inhibited 80% of cell viability of L929 cells after 48 h, while EcTI showed similar efficacy after 72 h. Both inhibitors acted in a dose and time-dependent manner. Conversely, rBbCI did not significantly affect the viability of L929 cells. Confocal microscopy revealed the binding of rBbKI and EcTI to the L929 cell surface. rBbKI inhibited approximately 63% of L929 adhesion to fibronectin, in contrast with EcTI and rBbCI, which did not significantly interfere with adhesion. None of the inhibitors interfered with the L929 cell cycle phases. The synthetic peptide RPGLPVRFESPL-NH2, based on the BbKI reactive site, inhibited 45% of the cellular viability of L929, becoming a promising protease inhibitor due to its ease of synthesis.

Biotechnological Potential of Araucaria angustifolia Pine Nuts Extract and the Cysteine Protease Inhibitor AaCI-2S.

Protease inhibitors are involved in the regulation of endogenous cysteine proteases during seed development and play a defensive role because of their ability to inhibit exogenous proteases such as those present in the digestive tracts of insects. Araucaria angustifolia seeds, which can be used in human and animal feed, were investigated for their potential for the development of agricultural biotechnology and in the field of human health. In the pine nuts extract, which blocked the activities of cysteine proteases, it was detected potent insecticidal activity against termites (Nasutitermes corniger) belonging to the most abundant termite genus in tropical regions. The cysteine inhibitor (AaCI-2S) was purified by ion-exchange, size exclusion, and reversed-phase chromatography. Its functional and structural stability was confirmed by spectroscopic and circular dichroism studies, and by detection of inhibitory activity at different temperatures and pH values. Besides having activity on cysteine proteases from C. maculatus digestive tract, AaCI-2S inhibited papain, bromelain, ficin, and cathepsin L and impaired cell proliferation in gastric and prostate cancer cell lines. These properties qualify A. angustifolia seeds as a protein source with value properties of natural insecticide and to contain a protease inhibitor with the potential to be a bioactive molecule on different cancer cells.

Isolation of Cysteine-Rich Peptides from Citrullus colocynthis.

The plant Citrullus colocynthis, a member of the squash (Cucurbitaceae) family, has a long history in traditional medicine. Based on the ancient knowledge about the healing properties of herbal preparations, plant-derived small molecules, e.g., salicylic acid, or quinine, have been integral to modern drug discovery. Additionally, many plant families, such as Cucurbitaceae, are known as a rich source for cysteine-rich peptides, which are gaining importance as valuable pharmaceuticals. In this study, we characterized the C. colocynthis peptidome using chemical modification of cysteine residues, and mass shift analysis via matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. We identified the presence of at least 23 cysteine-rich peptides in this plant, and eight novel peptides, named citcol-1 to -8, with a molecular weight between ~3650 and 4160 Da, were purified using reversed-phase high performance liquid chromatography (HPLC), and their amino acid sequences were determined by de novo assignment of b- and y-ion series of proteolytic peptide fragments. In silico analysis of citcol peptides revealed a high sequence similarity to trypsin inhibitor peptides from Cucumis sativus, Momordica cochinchinensis, Momordica macrophylla and Momordica sphaeroidea. Using genome/transcriptome mining it was possible to identify precursor sequences of this peptide family in related Cucurbitaceae species that cluster into trypsin inhibitor and antimicrobial peptides. Based on our analysis, the presence or absence of a crucial Arg/Lys residue at the putative P1 position may be used to classify these common cysteine-rich peptides by functional properties. Despite sequence homology and the common classification into the inhibitor cysteine knot family, these peptides appear to have diverse and additional bioactivities yet to be revealed.

An Integrated Proteomic and Transcriptomic Analysis Reveals the Venom Complexity of the Bullet Ant Paraponera clavata.

A critical hurdle in ant venom proteomic investigations is the lack of databases to comprehensively and specifically identify the sequence and function of venom proteins and peptides. To resolve this, we used venom gland transcriptomics to generate a sequence database that was used to assign the tandem mass spectrometry (MS) fragmentation spectra of venom peptides and proteins to specific transcripts. This was performed alongside a shotgun liquid chromatography–mass spectrometry (LC-MS/MS) analysis of the venom to confirm that these assigned transcripts were expressed as proteins. Through the combined transcriptomic and proteomic investigation of Paraponera clavata venom, we identified four times the number of proteins previously identified using 2D-PAGE alone. In addition to this, by mining the transcriptomic data, we identified several novel peptide sequences for future pharmacological investigations, some of which conform with inhibitor cysteine knot motifs. These types of peptides have the potential to be developed into pharmaceutical or bioinsecticide peptides.