New Origins of Yeast, Plant and Bacterial-Derived Extracellular Vesicles to Expand and Advance Compound Delivery.

Extracellular vesicles (EVs) constitute a sophisticated molecular exchange mechanism highly regarded for their potential as a next-generation platform for compound delivery. However, identifying sustainable and biologically safe sources of EVs remains a challenge. This work explores the emergence of novel sources of plant and bacterial-based EVs, such as those obtained from food industry by-products, known as BP-EVs, and their potential to be used as safer and biocompatible nanocarriers, addressing some of the current challenges of the field. These novel sources exhibit remarkable oral bioavailability and biodistribution, with minimal cytotoxicity and a selective targeting capacity toward the central nervous system, liver, and skeletal tissues. Additionally, we review the ease of editing these recently uncovered nanocarrier-oriented vesicles using common EV editing methods, examining the cargo-loading processes applicable to these sources, which involve both passive and active functionalization methods. While the primary focus of these novel sources of endogenous EVs is on molecule delivery to the central nervous system and skeletal tissue based on their systemic target preference, their use, as reviewed here, extends beyond these key applications within the biotechnological and biomedical fields.

Substrate-binding glycine residues are major determinants for hydrolase and ligase activity of plant legumains.

Peptide asparaginyl ligases (PALs) are useful tools for precision modifications of proteins and live-cell surfaces by ligating peptides after Asn/Asp (Asx). They share high sequence and structural similarity to plant legumains that are generally known as asparaginyl endopeptidases (AEPs), thus making it challenging to identify PALs from AEPs. In this study, we investigate 875 plant species from algae to seed plants with available sequence data in public databases to identify new PALs. We conducted evolutionary trace analysis on 1500 plant legumains, including eight known PALs, to identify key residues that could differentiate ligases and proteases, followed by recombinant expression and functional validation of 16 novel legumains. Previously, we showed that the substrate-binding sequences flanking the catalytic site can strongly influence the enzymatic direction of a legumain and which we named as ligase-activity determinants (LADs). Here, we show that two conserved substrate-binding Gly residues of LADs are critical, but negative determinants for ligase activity. Our results suggest that specific glycine residues are molecular determinants to identify PALs and AEPs as two different legumain subfamilies, accounting for c. 1% and 88%, respectively.

Plant-derived nootropics and human cognition: A systematic review.

Substances with modulatory capabilities on certain aspects of human cognition have been revered as nootropics from the dawn of time. The plant kingdom provides most of the currently available nootropics of natural origin. Here, in this systematic review, we aim to provide state-of-the-art information regarding proven and unproven effects of plant-derived nootropics (PDNs) on human cognition in conditions of health and disease. Six independent searches, one for each neurocognitive domain (NCD), were performed in parallel using three independent scientific library databases: PubMed, Cochrane and Scopus. Only scientific studies and systematic reviews with humans published between January 2000 and November 2021 were reviewed, and 256 papers were included. Ginkgo biloba was the most relevant nootropic regarding perceptual and motor functions. Bacopa monnieri improves language, learning and memory. Withania somnifera (Ashwagandha) modulates anxiety and social-related cognitions. Caffeine enhances attention and executive functions. Together, the results from the compiled studies highlight the nootropic effects and the inconsistencies regarding PDNs that require further research.Supplemental data for this article is available online at https://doi.org/10.1080/10408398.2021.2021137.

The legumain McPAL1 from Momordica cochinchinensis is a highly stable Asx-specific splicing enzyme.

Legumains, also known as asparaginyl endopeptidases (AEPs), cleave peptide bonds after Asn/Asp (Asx) residues. In plants, certain legumains also have ligase activity that catalyzes biosynthesis of Asx-containing cyclic peptides. An example is the biosynthesis of MCoTI-I/II, a squash family-derived cyclic trypsin inhibitor, which involves splicing to remove the N-terminal prodomain and then N-to-C-terminal cyclization of the mature domain. To identify plant legumains responsible for the maturation of these cyclic peptides, we have isolated and characterized a legumain involved in splicing, McPAL1, from Momordica cochinchinensis (Cucurbitaceae) seeds. Functional studies show that recombinantly expressed McPAL1 displays a pH-dependent, trimodal enzymatic profile. At pH 4 to 6, McPAL1 selectively catalyzed Asp-ligation and Asn-cleavage, but at pH 6.5 to 8, Asn-ligation predominated. With peptide substrates containing N-terminal Asn and C-terminal Asp, such as is found in precursors of MCoTI-I/II, McPAL1 mediates proteolysis at the Asn site and then ligation at the Asp site at pH 5 to 6. Also, McPAL1 is an unusually stable legumain that is tolerant of heat and high pH. Together, our results support that McPAL1 is a splicing legumain at acidic pH that can mediate biosynthesis of MCoTI-I/II. We purport that the high thermal and pH stability of McPAL1 could have applications for protein engineering.

PGE1 and PGA1 bind to Nurr1 and activate its transcriptional function.

The orphan nuclear receptor Nurr1 is critical for the development, maintenance and protection of midbrain dopaminergic (mDA) neurons. Here we show that prostaglandin E1 (PGE1) and its dehydrated metabolite, PGA1, directly interact with the ligand-binding domain (LBD) of Nurr1 and stimulate its transcriptional function. We also report the crystallographic structure of Nurr1-LBD bound to PGA1 at 2.05 Å resolution. PGA1 couples covalently to Nurr1-LBD by forming a Michael adduct with Cys566, and induces notable conformational changes, including a 21° shift of the activation function-2 helix (H12) away from the protein core. Furthermore, PGE1/PGA1 exhibit neuroprotective effects in a Nurr1-dependent manner, prominently enhance expression of Nurr1 target genes in mDA neurons and improve motor deficits in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned mouse models of Parkinson's disease. Based on these results, we propose that PGE1/PGA1 represent native ligands of Nurr1 and can exert neuroprotective effects on mDA neurons, via activation of Nurr1's transcriptional function.

Structural determinants for peptide-bond formation by asparaginyl ligases.

Asparaginyl endopeptidases (AEPs) are cysteine proteases which break Asx (Asn/Asp)-Xaa bonds in acidic conditions. Despite sharing a conserved overall structure with AEPs, certain plant enzymes such as butelase 1 act as a peptide asparaginyl ligase (PAL) and catalyze Asx-Xaa bond formation in near-neutral conditions. PALs also serve as macrocyclases in the biosynthesis of cyclic peptides. Here, we address the question of how a PAL can function as a ligase rather than a protease. Based on sequence homology of butelase 1, we identified AEPs and PALs from the cyclic peptide-producing plants Viola yedoensis (Vy) and Viola canadensis (Vc) of the Violaceae family. Using a crystal structure of a PAL obtained at 2.4-Å resolution coupled to mutagenesis studies, we discovered ligase-activity determinants flanking the S1 site, namely LAD1 and LAD2 located around the S2 and S1' sites, respectively, which modulate ligase activity by controlling the accessibility of water or amine nucleophile to the S-ester intermediate. Recombinantly expressed VyPAL1-3, predicted to be PALs, were confirmed to be ligases by functional studies. In addition, mutagenesis studies on VyPAL1-3, VyAEP1, and VcAEP supported our prediction that LAD1 and LAD2 are important for ligase activity. In particular, mutagenesis targeting LAD2 selectively enhanced the ligase activity of VyPAL3 and converted the protease VcAEP into a ligase. The definition of structural determinants required for ligation activity of the asparaginyl ligases presented here will facilitate genomic identification of PALs and engineering of AEPs into PALs.

The unusual di-domain structure of Dunaliella salina glycerol-3-phosphate dehydrogenase enables direct conversion of dihydroxyacetone phosphate to glycerol.

Dunaliella has been extensively studied due to its intriguing adaptation to high salinity. Its di-domain glycerol-3-phosphate dehydrogenase (GPDH) isoform is likely to underlie the rapid production of the osmoprotectant glycerol. Here, we report the structure of the chimeric Dunaliella salina GPDH (DsGPDH) protein featuring a phosphoserine phosphatase-like domain fused to the canonical glycerol-3-phosphate (G3P) dehydrogenase domain. Biochemical assays confirm that DsGPDH can convert dihydroxyacetone phosphate (DHAP) directly to glycerol, whereas a separate phosphatase protein is required for this conversion process in most organisms. The structure of DsGPDH in complex with its substrate DHAP and co-factor nicotinamide adenine dinucleotide (NAD) allows the identification of the residues that form the active sites. Furthermore, the structure reveals an intriguing homotetramer form that likely contributes to the rapid biosynthesis of glycerol.

Ribosome protection by antibiotic resistance ATP-binding cassette protein.

The ribosome is one of the richest targets for antibiotics. Unfortunately, antibiotic resistance is an urgent issue in clinical practice. Several ATP-binding cassette family proteins confer resistance to ribosome-targeting antibiotics through a yet unknown mechanism. Among them, MsrE has been implicated in macrolide resistance. Here, we report the cryo-EM structure of ATP form MsrE bound to the ribosome. Unlike previously characterized ribosomal protection proteins, MsrE is shown to bind to ribosomal exit site. Our structure reveals that the domain linker forms a unique needle-like arrangement with two crossed helices connected by an extended loop projecting into the peptidyl-transferase center and the nascent peptide exit tunnel, where numerous antibiotics bind. In combination with biochemical assays, our structure provides insight into how MsrE binding leads to conformational changes, which results in the release of the drug. This mechanism appears to be universal for the ABC-F type ribosome protection proteins.

System-wide molecular dynamics of endothelial dysfunction in Gram-negative sepsis.

BACKGROUND: Inflammation affecting whole organism vascular networks plays a central role in the progression and establishment of several human diseases, including Gram-negative sepsis. Although the molecular mechanisms that control inflammation of specific vascular beds have been partially defined, knowledge lacks on the impact of these on the molecular dynamics of whole organism vascular beds. In this study, we have generated an in vivo model by coupling administration of lipopolysaccharide with stable isotope labeling in mammals to mimic vascular beds inflammation in Gram-negative sepsis and to evaluate its effects on the proteome molecular dynamics. Proteome molecular dynamics of individual vascular layers (glycocalyx (GC), endothelial cells (EC), and smooth muscle cells (SMC)) were then evaluated by coupling differential systemic decellularization in vivo with unbiased systems biology proteomics. RESULTS: Our data confirmed the presence of sepsis-induced disruption of the glycocalyx, and we show for the first time the downregulation of essential molecular maintenance processes in endothelial cells affecting this apical vascular coating. Similarly, a novel catabolic phenotype was identified in the newly synthesized EC proteomes that involved the impairment of protein synthesis, which affected multiple cellular mechanisms, including oxidative stress, the immune system, and exacerbated EC-specific protein turnover. In addition, several endogenous molecular protective mechanisms involving the synthesis of novel antithrombotic and anti-inflammatory proteins were also identified as active in EC. The molecular dynamics of smooth muscle cells in whole organism vascular beds revealed similar patterns of impairment as those identified in EC, although this was observed to a lesser extent. Furthermore, the dynamics of protein posttranslational modifications showed disease-specific phosphorylation sites in the EC proteomes. CONCLUSIONS: Together, the novel findings reported here provide a broader picture of the molecular dynamics that take place in whole organism vascular beds in Gram-negative sepsis inflammation. Similarly, the obtained data can pave the way for future therapeutic strategies aimed at intervening in specific protein synthesis mechanisms of the vascular unit during acute inflammatory processes.

Potentides: New Cysteine-Rich Peptides with Unusual Disulfide Connectivity from Potentilla anserina.

Cysteine-rich peptides (CRPs), which are disulfide-constrained peptides with 3 to 5 disulfide bonds and molecular weights of 2 to 6 kDa, are generally hyperstable and resistant to thermal, chemical, and enzymatic degradation. Herein, the discovery and characterization of a novel suite of CRPs, collectively named potentides pA1-pA16 from the root of the medicinal herb Potentilla anserina L, are described. Through a combination of proteomic and transcriptomic methods, it is shown that 35-residue potentide pA3, which is the most abundant member of potentides, exhibits high stability against heat, acidic, and proteolytic degradation. Transcriptomic analysis revealed that potentide precursor sequences contained four tandem repeats in the mature domain, which is the first report on tandem repeats being found in the Rosaceae family. Disulfide mapping showed that potentide pA3 displayed a novel disulfide connectivity of C1-C3, C2-C6, and C4-C5; a cystine motif that has not been reported in plant CRPs. Transcriptomic data mining and a neighbor-joining clustering analysis revealed 56 potentide homologues and their distribution in the families of Rosaceae and Ranunculaceae in angiosperm. Altogether, these results reveal a new plant CRP structure with an unusual cystine connectivity. Additionally, this study expands the families and structure diversity of CRPs as potentially active peptide pharmaceuticals.

Vascular Bed Molecular Profiling by Differential Systemic Decellularization In Vivo.

Objective- Vascular endothelial dysfunction is a key component of several major human diseases, but the molecular basis of this complex disorder has been difficult to determine in vivo. Previous attempts to identify key mediators of vascular endothelial dysfunction in experimental models have been limited by the lack of suitable methods for system-wide analyses of vascular bed biology. Here, we aimed to develop a novel method for investigating vascular endothelial dysfunction pathogenesis that enables system-wide analyses of molecular interactions between endothelial glycocalyx, endothelial cells, and smooth muscle cells in murine. Approach and Results- We developed a new technique using whole-body differential perfusion with increasing concentrations of detergent buffer to selectively solubilize distinct layers of vascular bed tissue in rodents. When combined with proteomics techniques, our novel approach of differential systemic decellularization in vivo enabled quantitative profiling of vascular beds throughout the body. Initial perfusion with phosphate buffer was used to obtain the endothelial glycocalyx, followed by subsequent extraction of endothelial cell components, and finally by smooth muscle cell constituents with increasing concentrations of detergent. Differential systemic decellularization in vivo has also been successfully applied to characterize molecular events in the vascular bed pathology of lipopolysaccharide-challenged mice. Conclusions- Together, these data indicate that differential systemic decellularization in vivo permits system-wide molecular characterization of vascular bed proteomes in rodent models and can be used to advance our current understanding of vascular endothelial dysfunction pathogenesis and progression in a wide range of disease settings.

Astratides: Insulin-Modulating, Insecticidal, and Antifungal Cysteine-Rich Peptides from Astragalus membranaceus.

Astragalus membranaceus root, Huang Qi in Chinese, is a popular medicinal herb traditionally used to regulate blood glucose. Herein, the identification and characterization of two families of cysteine-rich peptides (CRPs), designated α- and ß-astratides, from A. membranaceus roots are reported. Proteomic analysis showed that α-astratide aM1 and ß-astratide bM1 belong to two distinct CRP families. The six-cysteine-containing and proline-rich α-astratide aM1 displayed high sequence identity to Pea Albumin 1 Subunit b (PA1b), while the eight-cysteine-containing ß-astratide bM1 showed sequence similarity to plant defensins. An antifungal assay revealed that bM1 possessed potent antifungal activity. In contrast, aM1 showed a cytotoxic effect against insect Sf9 cells. More importantly, aM1 decreased insulin secretion in mouse pancreatic ß cells, suggesting it could interfere in glucose homeostasis, which accounts for the adaptogenic property of A. membranaceus. Phylogenetic clustering analysis suggested that the proline-rich aM1 is a putative prolyl oligopeptidase inhibitor and belongs to a novel subfamily of PA1b-like peptides, while bM1 belongs to a new subfamily of plant defensins. Together, the study reveals that astratides are multifunctional CRPs in plants, which expand the existing library of PA1b-like peptides and plant defensins and further our understanding of their roles in host-defense system and leads as peptidyl therapeutics.

Online Removal of Sodium Dodecyl Sulfate via Weak Cation Exchange in Liquid Chromatography-Mass Spectrometry Based Proteomics.

Biological research often requires the use of sodium dodecyl sulfate (SDS) to solubilize protein samples; however, this detergent is not compatible with direct mass spectrometry (MS) analysis. Here, we report an online high-throughput proteomics method that permits standard in-solution digestion of SDS-containing samples followed by direct liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis using weak cation-exchange chromatography (WCX). This approach, called the online removal of sodium dodecyl sulfate (Online reSDS), exploits the properties of WCX in a highly organic and mildly acidic medium to retain positively charged peptides by both hydrophilic interaction and electrostatic attraction while simultaneously repelling negative SDS molecules. This method was optimized to successfully analyze complex samples that contain up to 1% of SDS. Furthermore, online reSDS improves the identification of peptides with post-translational modifications (PTMs), such as deamidation and phosphorylation, without preliminary enrichment. In conclusion, we show that reSDS can facilitate research in proteomics by allowing the use of SDS in a wide range of LC-MS/MS applications with simplified sample-processing procedures.

Molecular diversity and function of jasmintides from Jasminum sambac.

BACKGROUND: Jasmintides jS1 and jS2 from Jasminum sambac were previously identified as a novel family of cysteine-rich peptides (CRPs) with an unusual disulfide connectivity. However, very little else is known about jasmintides, particularly their molecular diversity and functions. Here, we report the discovery and characterization of a novel suite of jasmintides from J. sambac using transcriptomic, peptidomic, structural and functional tools. RESULTS: Transcriptomic analysis of leaves, flowers and roots revealed 14 unique jasmintide precursors, all of which possess a three-domain architecture comprising a signal peptide, a pro-domain and a mature jasmintide domain. Peptidomic analysis, using fractionated mixtures of jasmintides and chemical derivatization of cysteine to pseudolysine, trypsin digestion and MS/MS sequencing, revealed an additional 86 jasmintides, some of which were post-translationally modified. NMR analysis showed that jasmintide jS3 has three anti-parallel ß-strands with a three-disulfide connectivity of CysI-CysV, CysII-CysIV and CysIII-CysVI, which is similar to jasmintide jS1. Jasmintide jS3 was able to withstand thermal, acidic and enzymatic degradation and, importantly, exhibited antifeedant activity against mealworm Tenebrio molitor. CONCLUSION: Together, this study expands the existing library of jasmintides and furthers our understanding of the molecular diversity and cystine framework of CRPs as scaffolds and tools for engineering peptides targeting pests.

Identification of Arenin, a Novel Kunitz-Like Polypeptide from the Skin Secretions of Dryophytes arenicolor.

Amphibian skin secretions are enriched with complex cocktails of bioactive molecules such as proteins, peptides, biogenic amines, alkaloids guanidine derivatives, steroids and other minor components spanning a wide spectrum of pharmacological actions exploited for centuries in folk medicine. This study presents evidence on the protein profile of the skin secretions of the canyon tree frog, Dryophytes arenicolor. At the same time, it presents the reverse-phase liquid chromatography isolation, mass spectrometry characterization and identification at mRNA level of a novel 58 amino acids Kunitz-like polypeptide from the skin secretions of Dryophytes arenicolor, arenin. Cell viability assays performed on HDFa, CaCo2 and MCF7 cells cultured with different concentrations of arenin showed a discrete effect at low concentrations (2, 4, 8 and 16 µg/mL) suggesting a multi-target interaction in a hormetic-like dose-response. Further work is required to investigate the mechanisms underlying the variable effect on cell viability produced by different concentrations of arenin.

Application of Nanosecond Laser Photolysis Protein Footprinting to Study EGFR Activation by EGF in Cells.

Mass spectrometry-based protein footprinting emerged as a useful technology to understand protein ligand interactions in vitro. We have previously demonstrated the application of footprinting in live E. coli cells. Here, we further optimized an ultrafast laser photolysis hydroxyl radical footprinting method and applied it to study the interaction of EGF and EGFR in live mammalian cells. This method used a nanosecond laser to photochemically generate a burst of hydroxyl radicals in situ in-cell suspension to oxidize the amino acids on the protein surface. Mass spectrometric analysis of the thus modified peptides was interpreted to probe the solvent-accessible surface areas of the protein in its native biological state with and without EGF activation. Our footprinting data agreed with the two relevant EGFR crystal structures, indicating that this in-cell laser photolysis footprinting technique is a valid approach to study the structural properties of integral membrane proteins directly in the native environment.

Brain ureido degenerative protein modifications are associated with neuroinflammation and proteinopathy in Alzheimer's disease with cerebrovascular disease.

BACKGROUND: Brain degenerative protein modifications (DPMs) are associated with the apparition and progression of dementia, and at the same time, Alzheimer's disease with cerebrovascular disease (AD + CVD) is the most prevalent form of dementia in the elder population. Thus, understanding the role(s) of brain DPMs in this dementia subtype may provide novel insight on the disease pathogenesis and may aid on the development of novel diagnostic and therapeutic tools. Two essential DPMs known to promote inflammation in several human diseases are the ureido DPMs (uDPMs) arginine citrullination and lysine carbamylation, although they have distinct enzymatic and non-enzymatic origins, respectively. Nevertheless, the implication of uDPMs in the neuropathology of dementia remains poorly understood. METHODS: In this study, we use the state-of-the-art, ultracentrifugation-electrostatic repulsion hydrophilic interaction chromatography (UC-ERLIC)-coupled mass spectrometry technology to undertake a comparative characterization of uDPMs in the soluble and particulate postmortem brain fractions of subjects diagnosed with AD + CVD and age-matched controls. RESULTS: An increase in the formation of uDPMs was observed in all the profiled AD + CVD brains. Citrulline-containing proteins were found more abundant in the soluble fraction of AD + CVD whereas homocitrulline-containing proteins were preferentially abundant in the particulate fraction of AD + CVD brains. Several dementia-specific citrulline residues were also identified in soluble proteins previously categorized as pro-immunogenic, which include the receptor P2X7, alpha-internexin, GFAP, CNP, MBP, and histones. Similarly, diverse dementia-specific homocitrulline residues were also observed in the particulate fractions of AD + CVD in proteins that have been vastly implicated in neuropathology. Intriguingly, we also found that the amino acids immediately flanking arginine residues may specifically influence the increase in protein citrullination. CONCLUSIONS: Taken together, these results indicate that uDPMs widely contribute to the pathophysiology of AD + CVD by promoting neuroinflammation and proteinopathy. Furthermore, the obtained results could help to identify disease-associated proteins that can act as potential targets for therapeutic intervention or as novel biomarkers of specific neuropathology.

Characterization of Glutamine Deamidation by Long-Length Electrostatic Repulsion-Hydrophilic Interaction Chromatography-Tandem Mass Spectrometry (LERLIC-MS/MS) in Shotgun Proteomics.

Deamidation of glutamine (Gln) residues is a spontaneous or enzymatic process with significant implications in aging and human pathology. Although some methods are available to identify the γ/α-glutamyl products of deamidation, none of these methods allows the characterization of this post-translational modification (PTM) from complex biological samples by shotgun proteomics. Here we present LERLIC-MS/MS, a chromatographic strategy that uses a long (50 cm) anion-exchange capillary column operating in the electrostatic repulsion-hydrophilic interaction mode (ERLIC) and coupled directly to tandem mass spectrometry (MS/MS) for proteome analysis in a single injection. Profiling of soluble extracts of brain tissues by LERLIC-MS/MS distinguished for the first time γ/α-glutamyl isomers of deamidation, encountering a 1.7 γ/α-glutamyl ratio for most Gln deamidation products. A detailed analysis of any deviation from that observed ratio allowed the identification of transglutaminase-mediated γ-glutamyl isomers as intermediate products of transamidation. Furthermore, LERLIC-MS/MS was able to simultaneously separate Gln and asparagine (Asn) deamidation products even for those peptides showing multiple deamidated proteoforms. The characterization of Asn deamidated residues by LERLIC-MS/MS also uncovered novel PIMT (protein L-isoaspartyl methyltransferase) substrate proteins in human brain tissues that deviated from the expected 3:1 isoAsp/Asp ratio. Taken together, our results demonstrate that LERLIC-MS/MS can be used to perform an in-depth study of protein deamidation on a global proteome scale. This new strategy should help to elucidate the biological implications of deamidation in aging and disease conditions.

Plasma proteome coverage is increased by unique peptide recovery from sodium deoxycholate precipitate.

The ionic detergent sodium deoxycholate (SDC) is compatible with in-solution tryptic digestion and LC-MS/MS-based shotgun proteomics by virtue of being easy to separate from the peptide products via precipitation in acidic buffers. However, it remains unclear whether unique human peptides co-precipitate with SDC during acid treatment of complex biological samples. In this study, we demonstrate for the first time that a large quantity of unique peptides in human blood plasma can be co-precipitated with SDC using an optimized sample preparation method prior to shotgun proteomic analysis. We show that the plasma peptides co-precipitated with SDC can be successfully recovered using a sequential re-solubilization and precipitation procedure, and that this approach is particularly efficient at the extraction of long peptides. Recovery of peptides from the SDC pellet dramatically increased overall proteome coverage (>60 %), thereby improving the identification of low-abundance proteins and enhancing the identification of protein components of membrane-bound organelles. In addition, when we analyzed the physiochemical properties of the co-precipitated peptides, we observed that SDC-based sample preparation improved the identification of mildly hydrophilic/hydrophobic proteins that would otherwise be lost upon discarding the pellet. These data demonstrate that the optimized SDC protocol is superior to sodium dodecyl sulfate (SDS)/urea treatment for identifying plasma biomarkers by shotgun proteomics.

Cysteine-Rich Peptide Family with Unusual Disulfide Connectivity from Jasminum sambac.

Cysteine-rich peptides (CRPs) are natural products with privileged peptidyl structures that represent a potentially rich source of bioactive compounds. Here, the discovery and characterization of a novel plant CRP family, jasmintides from Jasminum sambac of the Oleaceae family, are described. Two 27-amino acid jasmintides (jS1 and jS2) were identified at the gene and protein levels. Disulfide bond mapping of jS1 by mass spectrometry and its confirmation by NMR spectroscopy revealed disulfide bond connectivity of C-1-C-5, C-2-C-4, and C-3-C-6, a cystine motif that has not been reported in plant CRPs. Structural determination showed that jS1 displays a well-defined structure framed by three short antiparallel ß-sheets. Genomic analysis showed that jasmintides share a three-domain precursor arrangement with a C-terminal mature domain preceded by a long pro-domain of 46 residues and an intron cleavage site between the signal sequence and pro-domain. The compact cysteine-rich structure together with an N-terminal pyroglutamic acid residue confers jasmintides high resistance to heat and enzymatic degradation, including exopeptidase treatment. Collectively, these results reveal a new plant CRP structure with an unusual cystine connectivity, which could be useful as a scaffold for designing peptide drugs.