Cysteine-rich Proteins Research Articles

Arabidopsis RALF4 Rapidly Halts Pollen Tube Growth by Increasing ROS and Decreasing Calcium Cytoplasmic Tip Levels

In recent years, the rapid alkalinization factor (RALF) family of cysteine-rich peptides has been reported to be crucial for several plant signaling mechanisms, including cell growth, plant immunity and fertilization. RALF4 and RALF19 (RALF4/19) pollen peptides redundantly regulate the pollen tube integrity and growth through binding to their receptors ANXUR1/2 (ANX1/2) and Buddha’s Paper Seal 1 and 2 (BUPS1/2), members of the Catharanthus roseus RLK1-like (CrRLK1L) family, and, thus, are essential for plant fertilization. However, the signaling mechanisms at the cellular level that follow these binding events remain unclear. In this study, we show that the addition of synthetic peptide RALF4 rapidly halts pollen tube growth along with the excessive deposition of plasma membrane and cell wall material at the tip. The ratiometric imaging of genetically encoded ROS and Ca2+ sensors-expressing pollen tubes shows that RALF4 treatment modulates the cytoplasmic levels of reactive oxygen species (ROS) and calcium (Ca2+) in opposite ways at the tip. Thus, we propose that pollen RALF4/19 peptides bind ANX1/2 and BUPS1/2 to regulate ROS and calcium homeostasis to ensure proper cell wall integrity and control of pollen tube growth.

A multi-domain snail metallothionein increases cadmium resistance and fitness in Caenorhabditis elegans

Metallothioneins (MTs) are a family of mostly low-molecular weight, cysteine-rich proteins capable of specific metal-ion binding that are involved in metal detoxification and homeostasis, as well as in stress response. In contrast to most other animal species which possess two-domain (bidominial) MTs, some gastropod species have evolved Cd2+-selective multidomain MTs (md-MTs) consisting of several concatenated β3 domains and a single C-terminal β1 domain. Each domain contains three-metal ion clusters and binds three metal ions. The terrestrial snail Alinda biplicata possesses, among other MT isoforms, an md-MT with nine β3 domains and a C-terminal β1 domain (termed 10md-MT), capable of binding up to 30 Cd2+ ions per protein molecule. In the present study, the Alinda biplicata 10md-MT gene and a truncated version consisting of one β3 domain and a single C-terminal β1 domain (2d-MT) were introduced into a Caenorhabditis elegans knock-out strain lacking a native MT gene (mtl-1). The two snail MT constructs consistently increased Cd2+ resistance, and partially improved morphological, life history and physiological fitness traits in the nematode model host Caenorhabditis elegans. This highlights how the engineering of transgenic Caenorhabditis elegans strains expressing snail MTs provides an enhancement of the innate metal detoxification mechanism and in doing so provides a platform for enhanced mechanistic toxicology.

Metallothionein family genes in kiwifruit: characterization and determining their roles in plant's response to different stresses.

Kiwifruit growth and development are severely affected by various biotic and abiotic stresses, especially cold stress and the bacterial disease caused by Pseudomonas syringae pv. actinidiae (Psa). Metallothioneins (MTs) are a group of cysteine-rich proteins that play crucial roles in stress response, metal detoxification, and homeostasis in plants. However, the protective role of these MTs in kiwifruit remains to be elucidated. In the present study, four AcMT genes were identified in the Hongyang kiwifruit genome, namely, two Type 2 isoforms (AcMT2 and AcMT2a) and two Type 3 isoforms (AcMT3a and AcMT3b) located separately on four different chromosomes. The hormones and stress response cis-elements within the promoter regions of these AcMTs were characterized. It was revealed that the four AcMT genes exhibited different expression patterns in different tissues: AcMT2 and AcMT2a were expressed at much higher levels in the fruit, male flower, female flower, root, and bark, while AcMT3a was expressed mainly in the fruit and AcMT3b was expressed highly in the bark. The expression patterns of these AcMT genes after exposure to Psa infection and different phytohormones, including gibberellic acid A3(GA3), ethylene (ET), and abscisic acid (ABA), were evaluated. It was revealed that in response to Psa infection, the main AcMTs in each tissue (those with expression levels higher compared to the other MTs in that tissue) were downregulated during the early stage in kiwifruits, followed by a recovery phase. In addition, most AcMTs were downregulated after exposure to ET and GA3, while type 2 AcMTs (AcMT2 and AcMT2a) were upregulated after treatment with ABA. The overexpression of AcMTs in Escherichia coli presented a higher tolerance to H2O2, heavy metals, low temperature, and high temperature. Collectively, these findings demonstrated the protective roles of AcMTs in terms of stress resistance conferred through plant hormone-related signal pathways.

Complete genomes of Asgard archaea reveal diverse integrated and mobile genetic elements.

Asgard archaea are of great interest as the progenitors of Eukaryotes, but little is known about the mobile genetic elements (MGEs) that may shape their ongoing evolution. Here, we describe MGEs that replicate in Atabeyarchaeia, a wetland Asgard archaea lineage represented by two complete genomes. We used soil depth-resolved population metagenomic data sets to track 18 MGEs for which genome structures were defined and precise chromosome integration sites could be identified for confident host linkage. Additionally, we identified a complete 20.67 kbp circular plasmid and two family-level groups of viruses linked to Atabeyarchaeia, via CRISPR spacer targeting. Closely related 40 kbp viruses possess a hypervariable genomic region encoding combinations of specific genes for small cysteine-rich proteins structurally similar to restriction-homing endonucleases. One 10.9 kbp integrative conjugative element (ICE) integrates genomically into the Atabeyarchaeum deiterrae-1 chromosome and has a 2.5 kbp circularizable element integrated within it. The 10.9 kbp ICE encodes an expressed Type IIG restriction-modification system with a sequence specificity matching an active methylation motif identified by Pacific Biosciences (PacBio) high-accuracy long-read (HiFi) metagenomic sequencing. Restriction-modification of Atabeyarchaeia differs from that of another coexisting Asgard archaea, Freyarchaeia, which has few identified MGEs but possesses diverse defense mechanisms, including DISARM and Hachiman, not found in Atabeyarchaeia. Overall, defense systems and methylation mechanisms of Asgard archaea likely modulate their interactions with MGEs, and integration/excision and copy number variation of MGEs in turn enable host genetic versatility.

Antibacterial activity of recombinant liver-expressed antimicrobial peptide-2 derived from olive flounder, Paralichthys olivaceus

Liver-expressed antimicrobial peptide-2 (LEAP-2) is a cysteine-rich peptide that plays a crucial role in the innate immune system of fish. To investigate the molecular function of LEAP-2 from olive flounder, Paralichthys olivaceus, we cloned the gene encoding LEAP-2 using PCR and expressed it in Escherichia coli. Analysis of LEAP-2 expression revealed predominant transcripts in the liver and lower levels in the intestine of olive flounder, whereas their expression levels in the liver and head kidney increased, during the initial stage of infection with the aquapathogenic bacterium Edwardsiella piscicida. Recombinant LEAP-2 (rOfLEAP-2) purified from E. coli exhibited antimicrobial activity, as demonstrated by the ultrasensitive radial diffusion assay, against both Gram-positive (Bacillus subtilis, Streptococcus parauberis, and Lactococcus garvieae) and Gram-negative (Vibrio harveyi and E. coli) bacteria, with minimum inhibitory concentrations ranging from 25 to 100 μg/mL depending on the species tested. The antibacterial activity of rOfLEAP-2 was attributed to its ability to disrupt bacterial membranes, validated by the N-phenylnaphthalen-1-amine uptake assays and scanning electron microscope analysis against E. coli, V. harveyi, B. subtilis, and L. garvieae treated with rOfLEAP-2. Furthermore, a synergistic enhancement of antibacterial activity was observed when rOfLEAP-2 was combined with ampicillin or synthetic LEAP-1 peptide, suggesting a distinct mechanism of action from those of other antimicrobial agents. These findings provide evidence for the antibacterial efficacy of LEAP-2 from olive flounder, highlighting its potential therapeutic application against pathogenic bacteria.

Systematic expression analysis of cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 protein (CAP) superfamily in Arabidopsis.

The Cysteine-rich secretory proteins (CRISPS), Antigen 5 (Ag5), and Pathogenesis-related 1 (PR-1) protein (CAP) superfamily members are found in multiple eukaryotic organisms, including yeasts, animals, and plants. Although one of the plant CAP family genes, PR-1 is known to respond to pathogen infection in plants, the functions of other CAP family genes in Arabidopsis remain largely unknown. In this study, we conducted a comprehensive analysis of the similarities, loci, and expression patterns of 22 Arabidopsis CAP genes/proteins, providing a clue to elucidate their molecular functions. According to the promoter-β-glucuronidase (GUS) analysis, members of the Arabidopsis CAP family were expressed in various young tissues or organs, such as root and shoot meristems, reproductive tissues, and particularly at the lateral root initiation site before the formation of the lateral root primordium, with distinct expression specificity. In particular, CAP51, CAP52, and CAP53 were specifically expressed in the cortical cells at the lateral root developing regions, suggesting that these genes may function in lateral root development. Thus, the expression patterns of Arabidopsis CAP family genes suggest that CAP family proteins may have certain function in the expressed organs or tissues in Arabidopsis plant.

Preparation and characterization of cysteine-rich collagen peptide and its antagonistic effect on microplastic induced damage to HK-2 cells

Cysteine (Cys) plays a significant role in the stability and antioxidant capacity of peptide. This study was to prepare Cys-rich collagen peptide and evaluate its preventive effect on microparticle (MPs)-induced HK-2 cell damage. The results showed that the Cys-rich peptides (AMP-C) were successfully prepared from collagen hydrolysate by plastein reaction in the presence of exogenous Cys. The scavenging activities of 2,2-Diphenyl-1-picrylhydrazyl and hydroxyl radical of AMP-C were significantly higher than those of original peptides without Cys. The fraction with molecular weight < 3 kDa (AMP-CA) significantly improved MPs-induced damage and apoptosis of HK-2 cells. Compared with the same fractions lacking Cys (AMP-A), AMP-CA showed better performance in reducing reactive oxygen species and increasing the total superoxide dismutase and glutathione peroxidase levels, which may be related to its down-regulation of Kelch-like ECH-associated protein 1 (Keap1) expression. In addition, AMP-CA showed strong effects on inhibiting interleukin-6, tumor necrosis factor-α, monocyte chemoattractant protein-1, kidney injury molecule-1, and nuclear factor kappa-B levels. Finally, 330 Cys-containing peptides were isolated from AMP-CA, among which Leucine-Glycine-Asparagine-Glycine-Cysteine-Proline (LGNGCP) showed the lowest binding energy with Keap1 protein. In conclusion, the Cys-rich collagen peptide with higher antioxidant activity was successfully prepared by plastein reaction, which alleviated the HK-2 cells damage induced by MPs through antioxidant and anti-inflammatory. Our findings suggested that Cys-rich collagen peptide might be used as a functional food to mitigate health hazards associated with MPs accumulation.

Overexpression of OsGASR1 promotes Al tolerance in rice

Aluminum (Al) toxicity in acid soils poses a significant threat to rice, which exhibits highly complex genetic mechanisms for both external detoxification and internal tolerance among cereal crops. Although several genes involved Al tolerance have been identified, the molecular mechanisms underlying Al tolerance in rice remain to be fully explored. Here, we functionally characterized the gibberellin-stimulated transcription gene OsGASR1, which encodes a small cysteine-rich peptide localized to the nucleus and cytoplasm and plays a significant role in Al tolerance in rice. The expression of OsGASR1 is rapidly up-regulated by Al in rice root tips but not in the shoots. Its expression is not regulated by the central regulator Aluminum Resistance Transcription Factor 1 (ART1), indicating that OsGASR1 functions as a novel gene in rice Al resistance independent of ART1. Knockout of OsGASR1 reduced root length but did not affect Al tolerance in rice, whereas overexpression of OsGASR1 enhanced Al tolerance without affecting Al distribution and accumulation and promoted the accumulation of reactive oxygen species (ROS) in the root tips. RNA-seq analysis revealed that overexpression of OsGASR1 upregulated the expression of genes associated with cell wall modification, oxidative stress, and Al tolerance. Collectively, these findings suggest that OsGASR1 is involved in Al tolerance in rice independently of ART1, and the up-regulation of this gene is necessary for rice Al tolerance.

Bioinformatic and Phenotypic Analysis of AtPCP-Ba Crucial for Silique Development in Arabidopsis.

Silique development exerts significant impacts on crop yield. CRPs (Cysteine-rich peptides) can mediate cell-cell communication during plant reproduction and development. However, the functional characterization and regulatory mechanisms of CRPs in silique development remain unclear. In this study, we identified many CRP genes downstream of the CRP gene TPD1 (TAPETUM DETERMINANT1) during silique development using a microarray assay. The novel Arabidopsis thaliana pollen-borne CRPs, the PCP-Bs (for pollen coat protein B-class) gene AtPCP-Ba, along with TPD1, are essential for silique development. The AtPCP-Ba was significantly down-regulated in tpd1 flower buds but up-regulated in OE-TPD1 flower buds and siliques. The silencing of AtPCP-Ba compromised the wider silique of OE-TPD1 plants and inhibited the morphology of OE-TPD1 siliques to the size observed in the wild type. A total of 258 CRPs were identified with the bioinformatic analysis in Arabidopsis, Brassica napus, Glycine max, Oryza sativa, Sorghum bicolor, and Zea mays. Based on the evolutionary tree classification, all CRP members can be categorized into five subgroups. Notably, 107 CRP genes were predicted to exhibit abundant expression in flowers and fruits. Most cysteine-rich peptides exhibited high expression levels in Arabidopsis and Brassica napus. These findings suggested the involvement of the CRP AtPCP-Ba in the TPD1 signaling pathway, thereby regulating silique development in Arabidopsis.

PCP-bε is a novel positive regulator of pollen germination in Arabidopsis thaliana

small cysteine-rich peptides play essential roles in different stages of the plant reproductive process. Pollen germination is a prerequisite for double fertilization and is directly related to seed formation and crop yield. However, the small cysteine-rich peptides that are involved in pollen germination remain to be identified. In this study, identification and phylogenetic analysis of PCP-Bε genes in sequenced Brassicaceae show that pollen coat protein B-class protein PCP-Bε gene is widespread in Arabidopsis and its high relatives, but lost in some Brassica species. Expression analyses display that AtPCP-Bε gene is expressed in Arabidopsis pollen. Arabidopsis PCP-Bε knockout mutants are generated by CRISPR/Cas9, Phenotypic analyses show that the absence of AtPCP-Bε obviously impairs in vitro pollen germination, but has no influence on pollen tube growth, which demonstrates that AtPCP-Bε is a novel positive regulator of pollen germination. It is speculated that AtPCP-Bε should interact with the receptor from pollen to perform its function. These findings are useful for further analysis on the molecular mechanism of pollen germination.

The Roadmap of Plant Antimicrobial Peptides Under Environmental Stress: From Farm to Bedside.

Antimicrobial peptides (AMPs) are the most favorable alternatives in overcoming multidrug resistance, alone or synergistically with conventional antibiotics. Plant-derived AMPs, as cysteine-rich peptides, widely compensate the pharmacokinetic drawbacks of peptide therapeutics. Compared to the putative genes encrypted in the genome, AMPs that are produced under stress are active forms with the ability to combat resistant microbial species. Within this study, plant-derived AMPs, namely, defensins, nodule-specific cysteine-rich peptides, snakins, lipid transfer proteins, hevein-like proteins, α-hairpinins, and aracins, expressed under biotic and abiotic stresses, are classified. We could observe that while α-hairpinins and snakins display a helix-turn-helix structure, conserved motif patterns such as β1αβ2β3 and β1β2β3 exist in plant defensins and hevein-like proteins, respectively. According to the co-expression data, several plant AMPs are expressed together to trigger synergistic effects with membrane disruption mechanisms such as toroidalpore, barrel-stave, and carpet models. The application of AMPs as an eco-friendly strategy in maintaining agricultural productivity through the development of transgenes and bio-pesticides is discussed. These AMPs can be consumed in packaging material, wound-dressing products, coating catheters, implants, and allergology. AMPs with cell-penetrating properties are verified for the clearance of intracellular pathogens. Finally, the dominant pharmacological activities of bioactive peptides derived from the gastrointestinal digestion of plant AMPs, namely, inhibitors of renin and angiotensin-converting enzymes, dipeptidyl peptidase IV and α-glucosidase inhibitors, antioxidants, anti-inflammatory, immunomodulating, and hypolipidemic peptides, are analyzed. Conclusively, as phytopathogens and human pathogens can be affected by plant-derived AMPs, they provide a bright perspective in agriculture, breeding, food, cosmetics, and pharmaceutical industries, translated as farm to bedside.

Navigating the fungal battlefield: cysteine-rich antifungal proteins and peptides from Eurotiales.

Fungi are ubiquitous in the environment and play a key role in the decomposition and recycling of nutrients. On the one hand, their special properties are a great asset for the agricultural and industrial sector, as they are used as source of nutrients, producers of enzymes, pigments, flavorings, and biocontrol agents, and in food processing, bio-remediation and plant growth promotion. On the other hand, they pose a serious challenge to our lives and the environment, as they are responsible for fungal infections in plants, animals and humans. Although host immunity opposes invading pathogens, certain factors favor the manifestation of fungal diseases. The prevalence of fungal infections is on the rise, and there is an alarming increase in the resistance of fungal pathogens to approved drugs. The limited number of antimycotics, the obstacles encountered in the development of new drugs due to the poor tolerability of antifungal agents in patients, the limited number of unique antifungal targets, and the low species specificity contribute to the gradual depletion of the antifungal pipeline and newly discovered antifungal drugs are rare. Promising candidates as next-generation therapeutics are antimicrobial proteins and peptides (AMPs) produced by numerous prokaryotic and eukaryotic organisms belonging to all kingdom classes. Importantly, filamentous fungi from the order Eurotiales have been shown to be a rich source of AMPs with specific antifungal activity. A growing number of published studies reflects the efforts made in the search for new antifungal proteins and peptides (AFPs), their efficacy, species specificity and applicability. In this review, we discuss important aspects related to fungi, their impact on our life and issues involved in treating fungal infections in plants, animals and humans. We specifically highlight the potential of AFPs from Eurotiales as promising alternative antifungal therapeutics. This article provides insight into the structural features, mode of action, and progress made toward their potential application in a clinical and agricultural setting. It also identifies the challenges that must be overcome in order to develop AFPs into therapeutics.

Redox-Active Conopeptide Li520 Has Evolved to Catalyze Oxidative Folding of Conotoxins.

The evolution of miniature conopeptide Li520 (COWC*, *: C-terminal amidation) to exhibit the disulfide isomerase activity was probed using structure, function, disulfide conformation, and the precursor gene sequence. The peptides Li520, Li504, [O2A]Li520, [W3A]Li520, and Grx506, homologues active-site motif of glutaredoxin, were chemically synthesized and assessed for their disulfide reduction potential, intrinsic folding of disulfides, and disulfide isomerization activity on α-conotoxin ImI. The reduction potential of the disulfide of peptides varies from -189 to -344 mV, which is within the range observed for the redox family of proteins that modulates the folding of protein disulfides. The oxidative folding studies confirm the significance of the tryptophan residue in engaging Li520 in disulfide-exchange reactions and the role of proline hydroxylation in extending the lifetime of Li520 in a reduced free thiol state. Studies of quenching of tryptophan fluorescence by the disulfide in situ folding reaction in conjunction with the optimized structures by density functional theory (DFT) confirm the difference in conformation of disulfides between the native and mutant peptides. Interestingly, the native peptide Li520/Li504 shares a similar disulfide conformation of (-,-)AntiRHHook with the redox family of proteins known to modulate disulfides, particularly in lieu of the tetrapeptide of glutaredoxin, deviating from its disulfide conformation compared to its naive protein. Analysis of the precursor gene sequences of M-superfamily conotoxins revealed the presence of Li520 in different cone snail species with distinct food habits and possible modes of evolution through the diversification of cysteine motifs. The results of the report suggest that the short redox conopeptide Li520 has evolved to facilitate the oxidative folding of conotoxins and may be useful to develop as reagents for the synthesis of therapeutically important cysteine-rich peptides.

AmDEF2.7, a tandem duplicated defensin gene from Ammopiptanthus mongolicus, activated by AmWRKY14, enhances the tolerance of Arabidopsis to low temperature and osmotic stress

Ammopiptanthus mongolicus is an evergreen broad-leaved shrub growing in temperate regions. Plant defensins, a type of cysteine-rich small peptides, contribute to plant defense as antimicrobial peptides. In this study, we analyzed the evolution and expression patterns of the defensin gene family in A. mongolicus, and explored the function and regulatory mechanisms of the AmDEF2.7 gene in response to abiotic stress. Seven out of ten defensin genes had undergone segmental duplication and tandem duplication, especially, AmDEF2.6, AmDEF2.7, and AmDEF2.8, which were clustered on chromosome 9. The expression of multiple defensin genes was responsive to abiotic stress, with three defensin genes, including AmDEF2.7, showing significant induction during winter. Yeast expressing AmDEF2.7 gene exhibited increased resistance to freeze-thaw cycles and osmotic stress, while transgenic Arabidopsis overexpressing the AmDEF2.7 showed improved tolerance to both freezing and drought conditions. Furthermore, AmWRKY14 bound to the AmDEF2.7 gene promoter, and activated the expression of AmDEF2.7. These results highlighted the role of defensin AmDEF2.7 in the adaptation of A. mongolicus to temperate winter climate. This study expands our knowledge of plant defensin and provides support for clarifying the molecular mechanism of the adaptation of A. mongolicus to winter climate.

Absence of mitochondrial CX9C-CX10C protein Cox12 generates oxidative and nitrosative stress in Saccharomyces cerevisiae: Implication on cellular redox homeostasis

Mitochondrial intermembrane space (IMS) harbors a series of small, evolutionarily conserved redox-active cysteine-rich proteins. These proteins are essential for the functioning of cytochrome c oxidase, but their role in maintaining cellular redox processes is unknown. Here, we find out that in the absence of two such cysteine-rich Cx9C-Cx10C proteins, cytochrome c oxidase subunit 12 (Cox12) or cytochrome c oxidase assembly factor 6 (Coa6), Saccharomyces cerevisiae cells become sensitive under the oxidative and nitrosative stress. Interestingly, knockout of COX12 generates a significant amount of endogenous reactive oxygen species (ROS) and reactive nitrogen species (RNS) as evidenced by FACS analysis. Moreover, cellular redox status, redox-active enzymes glutathione reductase, catalase, S-nitroso glutathione reductase, and protein nitration were significantly affected in Cox12 null cells. Further, we found that an overexpression of COX12 partially protects mitochondrial respiratory subunit Sdh2 under oxidative and nitrosative stress. Taken together, we provide proof of evidence that cysteine-rich proteins in the IMS dynamically control the cellular redox milieu and actively prevent reactive nitrogen and oxygen species generation.

PpyLTP36 and PpyLTP39 are involved in the transmembrane transport of cuticular wax and are associated with the occurrence of pear fruit russeting

Non-specific lipid-transfer proteins (nsLTPs) are a group of small, cysteine-rich proteins that are involved in the transport of cuticular wax and other lipid compounds. Accumulating evidence suggests that dynamic changes in cuticular waxes are strongly associated with fruit russeting, an undesirable visual quality that negatively affects consumer appeal in pears. Currently, the regulatory role of nsLTPs in cuticular wax deposition and pear fruit skin russeting remains unclear. Here, we characterized the variations of cuticular waxes in non-treated (russeted) and preharvest bagging treated (non-russeted) pear fruits throughout fruit development and confirmed that the contents of cuticular waxes were significantly negatively correlated with the occurrence of pear fruit russeting. Based on RNA-Sequencing (RNA-Seq) and quantitative real-time PCR (qRT-PCR) analyses, two nsLTP genes (PpyLTP36 and PpyLTP39) were identified, which exhibited high expression levels in non-russeted pear fruit skins and were significantly repressed during fruit skin russeting. Subcellular localization analysis demonstrated that PpyLTP36 and PpyLTP39 were localized to the plasma membrane (PM). Further, transient Virus-Induced Gene Silencing (VIGS) analyses of PpyLTP36 and PpyLTP39 in pear fruits significantly reduced cuticular wax deposition. In conclusion, PpyLTP36 and PpyLTP39 are involved in the transmembrane transport of cuticular wax and are associated with pear fruit skin russeting.

The emerging role of cysteine-rich peptides in pollen-pistil interactions.

Unlike early land plants, flowering plants have evolved a pollen tube that transports a pair of non-motile sperm cells to the female gametophyte. This process, known as siphonogamy, was first observed in gymnosperms and later became prevalent in angiosperms. However, the precise molecular mechanisms underlying the male-female interactions remain enigmatic. From the landing of the pollen grain on the stigma to gamete fusion, the male part needs to pass various tests: how does the stigma distinguish between compatible and incompatible pollen? what mechanisms guide the pollen tube towards the ovule? what factors trigger pollen tube rupture? how is polyspermy prevented? and how does the sperm cell ultimately reach the egg? Successful male-female communication is essential for surmounting these challenges, with cysteine-rich peptides (CRPs) playing a pivotal role in this dialogue. In this review, we summarize the characteristics of four distinct classes of CRPs, systematically review recent progress in the role of CRPs in four crucial stages of pollination and fertilization, consider potential applications of this knowledge in crop breeding, and conclude by suggesting avenues for future research.

Molecular Insights into the Binding and Conformational Changes of Hepcidin25 Blood Peptide with 4-Aminoantipyrine and Their Sorption Mechanism by Carboxylic-Functionalized Multiwalled Carbon Nanotubes: A Comprehensive Spectral Analysis and Molecular Dynamics Simulation Study.

In this work, the main purpose is to analyze and understand the mechanism and thermodynamic interactions of carboxylic acid-functionalized multiwalled carbon nanotubes (cf-MWCNTs) and 4-aminoantipyrine (AAP) with human hepcidine25 (Hep25) using multispectroscopic and molecular docking modeling methods, binding free energy calculations, and molecular dynamics (MD) simulations under physiological conditions. AAP belongs to a class of persistent environmental contaminants, and its residue is a potential hazard to human health, exhibiting a high binding affinity with blood peptides. Hepcidin is a 25-residue peptide hormone with four disulfide bonds that regulates the iron balance in vertebrates and contributes to host immunity as a cysteine-rich antimicrobial peptide. Due to their diverse properties and pollutant absorption capabilities, CNTs demonstrate important biological effects in biological applications, particularly in the noncovalent interactions with blood peptides. A comprehensive molecular dynamics simulation integrated with molecular docking methodologies was employed to explore the binding free energy between AAP and Hep25, identify binding sites, elucidate thermodynamic characteristics, and evaluate the binding forces governing their interaction. The investigation delved into elucidating the precise binding site of AAP within the Hep25 protein and thoroughly analyzed the impact of AAP on the microenvironment and conformational dynamics of Hep25. The circular dichroism (CD) experimental results highlight a reduction in β-sheet composition following the introduction of AAP and cf-MWCNT. In addition, outcomes from fluorescence spectroscopy demonstrate that both cf-MWCNT and AAP significantly attenuated Hep-25 fluorescence via a static quenching mechanism. According to the MD simulations, the presence of AAP induces changes in the secondary structure of Hep25 and enhances its hydrophobicity. Additionally, our findings demonstrated that alongside the alteration in protein structure and functionality induced by contaminants, cf-MWCNTs possess the capability to mitigate the contaminant-induced effects on Hep25 activity while preserving the overarching structural integrity of Hep25. Based on the distance and RDF data, we found that during the simulation the presence of the cf-MWCNT causes the AAP to move away from the Hep25, and as a result fewer and weaker interactions of the AAP with the Hep25 will be observed. Likewise, free energy calculations indicate that the binding of Hep25 to AAP and cf-MWCNT involves electrostatic, π-cationic, and π-π stacking interactions. The research findings offer invaluable insights into the intricate influence of pollutants and carbon nanotubes on protein functionality within the circulatory system and their toxicity in vivo for prospective investigations.

Phytochelatin Synthase: An In Silico Comparative Analysis in Cyanobacteria and Eukaryotic Microalgae.

Phytochelatins (PCs) are small cysteine-rich peptides involved in metal detoxification, not genetically encoded but enzymatically synthesized by phytochelatin synthases (PCSs) starting from glutathione. The constitutive PCS expression even in the absence of metal contamination, the wide phylogenetic distribution and the similarity between PCSs and the papain-type cysteine protease catalytic domain suggest a wide range of functions for PCSs. These proteins, widely studied in land plants, have not been fully analyzed in algae and cyanobacteria, although these organisms are the first to cope with heavy-metal stress in aquatic environments and can be exploited for phytoremediation. To fill this gap, we compared the features of the PCS proteins of different cyanobacterial and algal taxa by phylogenetic linkage. The analyzed sequences fall into two main, already known groups of PCS-like proteins. Contrary to previous assumptions, they are not classed as prokaryotic and eukaryotic sequences, but rather as sequences characterized by the alternative presence of asparagine and aspartic/glutamic acid residues in proximity of the catalytic cysteine. The presence of these enzymes with peculiar features suggests differences in their post-translational regulation related to cell/environmental requirements or different cell functions rather than to differences due to their belonging to different phylogenetic taxa.

Mandibular gland proteomics of the Mexican alligator lizard, Abronia graminea, and the red-lipped arboreal alligator lizard, Abronia lythrochila

A useful approach to deepen our knowledge about the origin and evolution of venom systems in Reptilia has been exploring the vast biodiversity of this clade of vertebrates in search of orally produced proteins with toxic actions, as well as their corresponding delivery systems. The occurrence of toxins in anguimorph lizards has been demonstrated experimentally or inferred from reports of the toxic effects of the oral secretions of taxa within the Varanidae and Helodermatidae families. In the present study, we have focused on two alligator lizards of the Anguidae family, the Mexican alligator lizard, Abronia graminea, and the red-lipped arboreal alligator lizard, A. lythrochila. In addition, the fine morphology of teeth of the latter species is described. The presence of a conserved set of proteins, including B-type natriuretic peptides, cysteine-rich secretory proteins, group III phospholipase A2, and kallikrein, in submandibular gland extracts was demonstrated for both Abronia species. These proteins belong to toxin families found in oral gland secretions of venomous reptile species. This finding, along with previous demonstration of toxin-producing taxa in both paleo- and neoanguimorpha clades, provides further support for the existence of a handful of conserved toxin families in oral secretions across the 100+ million years of Anguimorpha cladogenesis.