Cryptic Peptides Research Articles

Polypharmacological Cell-Penetrating Peptides from Venomous Marine Animals Based on Immunomodulating, Antimicrobial, and Anticancer Properties.

Complex pathological diseases, such as cancer, infection, and Alzheimer's, need to be targeted by multipronged curative. Various omics technologies, with a high rate of data generation, demand artificial intelligence to translate these data into druggable targets. In this study, 82 marine venomous animal species were retrieved, and 3505 cryptic cell-penetrating peptides (CPPs) were identified in their toxins. A total of 279 safe peptides were further analyzed for antimicrobial, anticancer, and immunomodulatory characteristics. Protease-resistant CPPs with endosomal-escape ability in Hydrophis hardwickii, nuclear-localizing peptides in Scorpaena plumieri, and mitochondrial-targeting peptides from Synanceia horrida were suitable for compartmental drug delivery. A broad-spectrum S. horrida-derived antimicrobial peptide with a high binding-affinity to bacterial membranes was an antigen-presenting cell (APC) stimulator that primes cytokine release and naïve T-cell maturation simultaneously. While antibiofilm and wound-healing peptides were detected in Synanceia verrucosa, APC epitopes as universal adjuvants for antiviral vaccination were in Pterois volitans and Conus monile. Conus pennaceus-derived anticancer peptides showed antiangiogenic and IL-2-inducing properties with moderate BBB-permeation and were defined to be a tumor-homing peptide (THP) with the ability to inhibit programmed death ligand-1 (PDL-1). Isoforms of RGD-containing peptides with innate antiangiogenic characteristics were in Conus tessulatus for tumor targeting. Inhibitors of neuropilin-1 in C. pennaceus are proposed for imaging probes or therapeutic delivery. A Conus betulinus cryptic peptide, with BBB-permeation, mitochondrial-targeting, and antioxidant capacity, was a stimulator of anti-inflammatory cytokines and non-inducer of proinflammation proposed for Alzheimer's. Conclusively, we have considered the dynamic interaction of cells, their microenvironment, and proportional-orchestrating-host- immune pathways by multi-target-directed CPPs resembling single-molecule polypharmacology. This strategy might fill the therapeutic gap in complex resistant disorders and increase the candidates' clinical-translation chance.

T cells of colorectal cancer patients’ stimulated by neoantigenic and cryptic peptides better recognize autologous tumor cells

BackgroundPatients with cancers that exhibit extraordinarily high somatic mutation numbers are ideal candidates for immunotherapy and enable identifying tumor-specific peptides through stimulation of tumor-reactive T cells (Tc).MethodsColorectal cancers (CRC) HROC113...

Nilabh Shastri – Towards understanding classical and non-classical MHC-I antigen processing and presentation

Major histocompatibility complex (MHC-I) peptide antigen processing and presentation has experienced a revived interest in the context of immuno oncology, immune surveillance escape by pathogen mutations and technical advances that accelerate vaccine design. This sheds new light on the discoveries made by Nilabh Shastri and colleagues that includes the characterisation of cryptic MHC-I peptide antigen epitopes derived from untranslated regions and the N-terminal trimming of peptide antigen precursors by the aminopeptidase ERAAP (ERAP1/2 / ARTS1/LRAP) in the endoplasmic reticulum (ER) prior to the complete assembly of MHC-I complexes and their subsequent exposure to the cell surface. These scientific findings have important implications for developing novel therapeutic approaches in immunotherapy and modern vaccine design.

Blood biomarkers in ALS: challenges, applications and novel frontiers.

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease among adults. With diagnosis reached relatively late into the disease process, extensive motor cell loss narrows the window for therapeutic opportunities. Clinical heterogeneity in ALS and the lack of disease-specific biomarkers have so far led to large-sized clinical trials with long follow-up needed to define clinical outcomes. In advanced ALS patients, there is presently limited scope to use imaging or invasive cerebrospinal fluid (CSF) collection as a source of disease biomarkers. The development of more patient-friendly and accessible blood biomarker assays is hampered by analytical hurdles like the matrix effect of blood components. However, blood also provides the opportunity to identify disease-specific adaptive changes of the stoichiometry and conformation of target proteins and the endogenous immunological response to low-abundance brain peptides, such as neurofilaments (Nf). Among those biomarkers under investigation in ALS, the change in concentration before or after diagnosis of Nf has been shown to aid prognostication and to allow the a priori stratification of ALS patients into smaller sized and clinically more homogeneous cohorts, supporting more affordable clinical trials. Here, we discuss the technical hurdles affecting reproducible and sensitive biomarker measurement in blood. We also summarize the state of the art of non-CSF biomarkers in the study of prognosis, disease progression, and treatment response. We will then address the potential as disease-specific biomarkers of the newly discovered cryptic peptides which are formed down-stream of TDP-43 loss of function, the hallmark of ALS pathobiology.

A Novel Cryptic Clostridial Peptide That Kills Bacteria by a Cell Membrane Permeabilization Mechanism.

ABSTRACTThis work reports detailed characteristics of the antimicrobial peptide Intestinalin (P30), which is derived from the LysC enzyme of Clostridium intestinale strain URNW. The peptide shows a broader antibacterial spectrum than the parental enzyme, showing potent antimicrobial activity against clinical strains of Gram-positive staphylococci and Gram-negative pathogens and causing between 3.04 ± 0.12 log kill for Pseudomonas aeruginosa PAO1 and 7.10 ± 0.05 log kill for multidrug-resistant Acinetobacter baumannii KPD 581 at a 5 μM concentration. Moreover, Intestinalin (P30) prevents biofilm formation and destroys 24-h and 72-h biofilms formed by Acinetobacter baumannii CRAB KPD 205 (reduction levels of 4.28 and 2.62 log CFU/mL, respectively). The activity of Intestinalin is combined with both no cytotoxicity and little hemolytic effect against mammalian cells. The nuclear magnetic resonance and molecular dynamics (MD) data show a high tendency of Intestinalin to interact with the bacterial phospholipid cell membrane. Although positively charged, Intestinalin resides in the membrane and aggregates into small oligomers. Negatively charged phospholipids stabilize peptide oligomers to form water- and ion-permeable pores, disrupting the integrity of bacterial cell membranes. Experimental data showed that Intestinalin interacts with negatively charged lipoteichoic acid (logK based on isothermal titration calorimetry, 7.45 ± 0.44), causes membrane depolarization, and affects membrane integrity by forming large pores, all of which result in loss of bacterial viability.IMPORTANCE Antibiotic resistance is rising rapidly among pathogenic bacteria, becoming a global public health problem that threatens the effectiveness of therapies for many infectious diseases. In this respect, antimicrobial peptides appear to be an interesting alternative to combat bacterial pathogens. Here, we report the characteristics of an antimicrobial peptide (of 30 amino acids) derived from the clostridial LysC enzyme. The peptide showed killing activity against clinical strains of Gram-positive and Gram-negative pathogens. Experimental data and computational modeling showed that this peptide forms transmembrane pores, directly engaging the negatively charged phospholipids of the bacterial cell membrane. Consequently, dissipation of the electrochemical gradient across cell membranes affects many vital processes, such as ATP synthesis, motility, and transport of nutrients. This kind of dysfunction leads to the loss of bacterial viability. Our firm conviction is that the presented study will be a helpful resource in searching for novel antimicrobial peptides that could have the potential to replace conventional antibiotics.

Human Cryptic Host Defence Peptide GVF27 Exhibits Anti-Infective Properties against Biofilm Forming Members of the Burkholderia cepacia Complex.

Therapeutic solutions to counter Burkholderia cepacia complex (Bcc) bacteria are challenging due to their intrinsically high level of antibiotic resistance. Bcc organisms display a variety of potential virulence factors, have a distinct lipopolysaccharide naturally implicated in antimicrobial resistance. and are able to form biofilms, which may further protect them from both host defence peptides (HDPs) and antibiotics. Here, we report the promising anti-biofilm and immunomodulatory activities of human HDP GVF27 on two of the most clinically relevant Bcc members, Burkholderia multivorans and Burkholderia cenocepacia. The effects of synthetic and labelled GVF27 were tested on B. cenocepacia and B. multivorans biofilms, at three different stages of formation, by confocal laser scanning microscopy (CLSM). Assays on bacterial cultures and on human monocytes challenged with B. cenocepacia LPS were also performed. GVF27 exerts, at different stages of formation, anti-biofilm effects towards both Bcc strains, a significant propensity to function in combination with ciprofloxacin, a relevant affinity for LPSs isolated from B. cenocepacia as well as a good propensity to mitigate the release of pro-inflammatory cytokines in human cells pre-treated with the same endotoxin. Overall, all these findings contribute to the elucidation of the main features that a good therapeutic agent directed against these extremely leathery biofilm-forming bacteria should possess.

Generation of ENSEMBL-based proteogenomics databases boosts the identification of non-canonical peptides.

SummaryWe have implemented the pypgatk package and the pgdb workflow to create proteogenomics databases based on ENSEMBL resources. The tools allow the generation of protein sequences from novel protein-coding transcripts by performing a three-frame translation of pseudogenes, lncRNAs and other non-canonical transcripts, such as those produced by alternative splicing events. It also includes exonic out-of-frame translation from otherwise canonical protein-coding mRNAs. Moreover, the tool enables the generation of variant protein sequences from multiple sources of genomic variants including COSMIC, cBioportal, gnomAD and mutations detected from sequencing of patient samples. pypgatk and pgdb provide multiple functionalities for database handling including optimized target/decoy generation by the algorithm DecoyPyrat. Finally, we have reanalyzed six public datasets in PRIDE by generating cell-type specific databases for 65 cell lines using the pypgatk and pgdb workflow, revealing a wealth of non-canonical or cryptic peptides amounting to >5% of the total number of peptides identified.Availability and implementationThe software is freely available. pypgatk: https://github.com/bigbio/py-pgatk/ and pgdb: https://nf-co.re/pgdb.Supplementary information Supplementary data are available at Bioinformatics online.

EPCO-14. MULTIFACETED TRANSCRIPTOMIC AND PROTEOMIC ANALYSES IDENTIFIED PUTATIVE ALTERNATIVE SPLICING-DERIVED CELL SURFACE ANTIGENS IN GLIOMA

Abstract BACKGROUND To develop effective immunotherapy for gliomas, it is crucial to expand the repertoire of targetable antigens. Recent studies have suggested that alternative splicing (AS), or its deriving tumor-specific junctions (“neojunctions”), could generate cryptic amino acid sequences that can be a source of neoantigens. In this study, we investigated neojunctions based on multifaceted transcriptomic and proteomic analyses, seeking the potential cell surface antigens that may be targeted by CAR. METHODS For screening, we analyzed bulk RNA-sequencing data of TCGA-GBM/LGG with high tumor purity (n = 429) and GTEx normal tissues (n = 9,166). Cohorts of spatially mapped intratumoral samples and longitudinally collected tumors were used to determine clonality and stability of the candidate neojunctions. Nanopore long-read amplicon sequencing was deployed to confirm the full-length transcript sequence. Their protein-level expression was explored by analyzing the Clinical Proteomic Tumor Analysis Consortium (CPTAC)-GBM proteomics dataset. RESULTS In the screening analysis comparing TCGA and GTEx datasets, we identified 218 neojunctions with adequate expression, prevalence, and tumor-specificity. Of these, 12 were predicted to be cell-surface antigens. Eight of the 12, such as BCAN, DLL3, and PTPRZ1, were also observed in multiple cases of another validation dataset. In the analysis of tumors with spatially mapped intratumoral samples, 7 of the 12 were recurrently detected in no less than 50% of the samples in multiple cases. In addition, 5 of the 12 were found to be conserved in primary and recurrent pairs of tumors in multiple cases. Full-length transcript sequencing corroborated our predictions based on short reads, and also demonstrated more complex AS patterns. Finally, CPTAC-GBM proteomics analysis identified one cryptic peptide that substantiated the corresponding transcriptome-based prediction. CONCLUSION: We identified neojunctions with the potential to generate cell-surface antigens. These multifaceted transcriptomic and proteomic analyses provide the rationale to pursue the development of immunotherapy targeting neojunction-derived antigens.

Therapeutic Potential of Cancer Vaccine Based on MHC Class I Cryptic Peptides Derived from Non-Coding Regions

MHC class I molecules display intracellular peptides on cell surfaces to enable immune surveillance under pathological conditions. The source of MHC class I antigens responsible for cancer protection is not fully understood. Here, we explored the MHC class I peptidome in mouse colon cancer cells using a proteogenomic approach. We showed that cryptic peptides derived from unconventional short open reading frames accounted for part of the MHC class I peptidome. Moreover, cancer growth was significantly prevented in mice immunized with a cocktail of synthesized cryptic peptides. Together, our data showed that the source of cancer antigens was not limited to fragments of consensus proteins. Cryptic antigens were displayed by MHC molecules and mediated anti-cancer effects, suggesting their therapeutic potential for cancer prevention.

Promoter exchange of the cryptic nonribosomal peptide synthetase gene for oligopeptide production in Aspergillus oryzae.

Oligopeptides with functional activities are of current interest in the nutraceutical and medical sectors. The development of the biosynthetic process of oligopeptides through a nonribosomal peptide synthetase (NRPS) system has become more challenging. To develop a production platform for nonribosomal peptides (NRPs), reprogramming of transcriptional regulation of the acv gene encoded ACV synthetase (ACVS) was implemented in Aspergillus oryzae using the CRISPR-Cas9 system. Awakening silent acv expression was successfully achieved by promoter substitution. Among the three exchanged promoters, AoPgpdA, AoPtef1, and PtPtoxA, the replacement of the native promoter with AoPgpdA led to the highest ACV production in A. oryzae. However, the ACV production of the AoPGpdA strain was also dependent on the medium composition, in which urea was the best nitrogen source, and a C:N ratio of 20:1 was optimal for tripeptide production. In addition to cell growth, magnesium ions are an essential element for ACV production and might participate in ACVS activity. It was also found that ACV was the growth-associated product of the engineered strain that might be a result of constitutive transcriptional control by the AoPgpdA promoter. This study offers a potential strategy for nonribosomal ACV production using the fungal system, which is applicable for redesigning bioactive oligopeptides with industrial relevance.

Natural and cryptic peptides dominate the immunopeptidome of atypical teratoid rhabdoid tumors

BackgroundAtypical teratoid/rhabdoid tumors (AT/RT) are highly aggressive CNS tumors of infancy and early childhood. Hallmark is the surprisingly simple genome with inactivating mutations or deletions in the SMARCB1 gene as...

CD8+ T-cell Immune Surveillance against a Tumor Antigen Encoded by the Oncogenic Long Noncoding RNA PVT1.

CD8+ T cells recognize peptides displayed by HLA class I molecules on cell surfaces, monitoring pathologic conditions such as cancer. Advances in proteogenomic analysis of HLA ligandomes have demonstrated that cells present a subset of cryptic peptides derived from noncoding regions of the genome; however, the roles of cryptic HLA ligands in tumor immunity remain unknown. In the current study, we comprehensively and quantitatively investigated the HLA class I ligandome of a set of human colorectal cancer and matched normal tissues, showing that cryptic translation products accounted for approximately 5% of the HLA class I ligandome. We also found that a peptide encoded by the long noncoding RNA (lncRNA) PVT1 was predominantly enriched in multiple colorectal cancer tissues. The PVT1 gene is located downstream of the MYC gene in the genome and is aberrantly overexpressed across a variety of cancers, reflecting its oncogenic property. The PVT1 peptide was recognized by patient CD8+ tumor-infiltrating lymphocytes, as well as peripheral blood mononuclear cells, suggesting the presence of patient immune surveillance. Our findings show that peptides can be translated from lncRNAs and presented by HLA class I and that cancer patient T cells are capable of sensing aberrations in noncoding regions of the genome.

Abstract 1891: Targeted mass spectrometry provides direct evidence of a human endogenous retrovirus type E (HERV-E) antigen presented on the surface of clear cell renal cell carcinoma cells

Abstract Background: Previously, our group identified donor-derived T cells that mediated regression of metastatic clear cell renal cell carcinoma (ccRCC) in a patient who underwent an allogeneic hematopoietic stem cell transplant. A T cell clone isolated from this patient had direct cytotoxicity against ccRCC cells. Its target antigen was discovered to be encoded by a human endogenous retrovirus type E (named CT-RCC HERV-E) that had selective expression in ccRCC. Subsequent experiments showed this T cell clone recognized a cryptic 10-mer peptide called CT-RCC1 (ATFLGSLTWK), derived from a noncanonical open reading frame of the CT-RCC HERV-E and presented in the context of HLA-A11. This study aimed to establish a proteomic workflow using targeted parallel reaction monitoring (PRM) mass spectrometry (MS) for the direct identification of CT-RCC1 peptide in ccRCC HLA ligandomes and to use that platform to identify other CT-RCC HERV-E derived peptides presented in the context of more common HLA alleles such as HLA-A02. Methods: In this study, four ccRCC cell lines were used: HLA-A11 positive/CT-RCC HERV-E positive, HLA-A11 negative/CT-RCC HERV-E positive, HLA-A11 positive/CT-RCC HERV-E negative and HLA-A11 negative/CT-RCC HERV-E negative. Additionally, we used the HLA-A11 positive/CT-RCC HERV-E positive cell line to create both beta 2-microglobulin and CT-RCC HERV-E CRISPR knockout lines. HLA-peptide complexes were immunopurified from cell lysates with anti-HLA Class I antibodies cross-linked to Protein A Sepharose beads. Peptides were separated from HLA molecules using Sep-Pak tC18 reverse phase separation columns and subjected to targeted parallel reaction monitoring (PRM) mass spectrometry (MS) analysis. Synthetic CT-RCC1 peptide was used to generate optimum PRM transitions, determine the best collision energy, and obtain the full fragment ion spectrum. Data analysis was done using the Skyline package. Results: CT-RCC1 peptide was identified by targeted MS to be presented on the surface of HLA-A11 positive/CT-RCC HERV-E positive ccRCC cells but not in those same cells that had CT-RCC HERV-E or beta 2-microglobulin knocked out using CRISPR. Additionally, the CT-RCC1 peptide was not detected in cells which were naturally HLA-A11 negative, CT-RCC HERV-E negative, or both. Conclusions: Targeted MS identified the HERV-E derived HLA-A11 restricted peptide CT-RCC1 to be expressed on the surface of ccRCC cells. This study defines a reproducible immunopeptidomic workflow using targeted MS for identifying other antigens derived from this HERV-E that may be presented in the context of common HLA alleles. Acknowledgments: We acknowledge the NIH Intramural Research Program and Rancic O'Neill Renal Cell Cancer Research Fellowship Fund for supporting this project. Citation Format: Stefan Barisic, Elena Cherkasova, Yong Chen, Stephanie Pierre, Richard W. Childs. Targeted mass spectrometry provides direct evidence of a human endogenous retrovirus type E (HERV-E) antigen presented on the surface of clear cell renal cell carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1891.

The insulin-degrading enzyme as a link between insulin and neuropeptides metabolism

We have applied a recently developed HPLC-MS enzymatic assay to investigate the cryptic peptides generated by the action of the insulin-degrading enzyme (IDE) on some neuropeptides (NPs) involved in the development of tolerance and dependence to opioids. Particularly, the tested NPs are generated from the NPFF precursor (pro-NPFF (A)): NPFF (FLFQPQRF) and NPAF (AGEGLSSPFWSLAAPQRF). The results show that IDE is able to cleave NPFF and NPAF, generating specific cryptic peptides. As IDE is also responsible for the processing of many other peptides in the brain (amyloid beta protein among the others), we have also performed competitive degradation assays using mixtures of insulin and the above mentioned NPs. Data show that insulin is able to slow down the degradation of both NPs tested, whereas, surprisingly, NPAF is able to accelerate insulin degradation, hinting IDE as the possible link responsible of the mutual influence between insulin and NPs metabolism.

Discovery of a Cryptic Depsipeptide from Streptomyces ghanaensis via MALDI‐MS‐Guided High‐Throughput Elicitor Screening

AbstractMicrobial genomes harbor an abundance of biosynthetic gene clusters, but most are expressed at low levels and need to be activated for characterization of their cognate natural products. In this work, we report the combination of high‐throughput elicitor screening (HiTES) with matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) for the rapid identification of cryptic peptide natural products. Application to Streptomyces ghanaensis identified amygdalin as an elicitor of a novel non‐ribosomal peptide, which we term cinnapeptin. Complete structural elucidation revealed cinnapeptin as a cyclic depsipeptide with an unusual 2‐methyl‐cinnamoyl group. Insights into its biosynthesis were provided by whole genome sequencing and gene deletion studies, while bioactivity assays showed antimicrobial activity against Gram‐positive bacteria and fission yeast. MALDI‐HiTES is a broadly applicable tool for the discovery of cryptic peptides encoded in microbial genomes.

Discovery of a Cryptic Depsipeptide from Streptomyces ghanaensis via MALDI‐MS‐Guided High‐Throughput Elicitor Screening

Microbial genomes harbor an abundance of biosynthetic gene clusters, but most are expressed at low levels and need to be activated for characterization of their cognate natural products. In this work, we report the combination of high-throughput elicitor screening (HiTES) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the rapid identification of cryptic peptide natural products. Application to Streptomyces ghanaensis identified amygdalin as an elicitor of a novel non-ribosomal peptide, which we term cinnapeptin. Complete structural elucidation revealed cinnapeptin as a cyclic depsipeptide with an unusual 2-methyl-cinnamoyl group. Insights into its biosynthesis were provided by whole genome sequencing and gene deletion studies, while bioactivity assays showed antimicrobial activity against Gram-positive bacteria and fission yeast. MALDI-HiTES is a broadly applicable tool for the discovery of cryptic peptides encoded in microbial genomes.

Identification of the Cryptic HLA-I Immunopeptidome.

The success of cancer immunotherapy relies on the ability of cytotoxic T cells to specifically recognize and eliminate tumor cells based on peptides presented by HLA-I. Although the peptide epitopes that elicit the corresponding immune response often remain unidentified, it is generally assumed that neoantigens, due to tumor-specific mutations, are the most common targets. Here, we used a mass spectrometric approach to show an underappreciated class of epitopes that accounts for up to 15% of HLA-I peptides for certain HLA alleles in various tumors and patients. These peptides are translated from cryptic open reading frames in supposedly noncoding regions in the genome and are mostly unidentifiable with conventional computational analyses of mass spectrometry (MS) data. Our approach, Peptide-PRISM, identified thousands of such cryptic peptides in tumor immunopeptidomes. About 20% of these HLA-I peptides represented the C-terminus of the corresponding translation product, suggesting frequent proteasome-independent processing. Our data also revealed HLA-I allele-dependent presentation of cryptic peptides, with HLA-A*03 and HLA-A*11 presenting the highest percentage of cryptic peptides. Our analyses refute the reported frequent presentation of HLA peptides generated by proteasome-catalyzed peptide splicing. Thus, Peptide-PRISM represents an important step toward comprehensive identification of HLA-I immunopeptidomes and reveals cryptic peptides as an abundant class of epitopes with potential relevance for novel immunotherapeutic approaches.

Deciphering and predicting CD4+ T cell immunodominance of influenza virus hemagglutinin.

The importance of CD4+ T helper (Th) cells is well appreciated in view of their essential role in the elicitation of antibody and cytotoxic T cell responses. However, the mechanisms that determine the selection of immunodominant epitopes within complex protein antigens remain elusive. Here, we used ex vivo stimulation of memory T cells and screening of naive and memory T cell libraries, combined with T cell cloning and TCR sequencing, to dissect the human naive and memory CD4+ T cell repertoire against the influenza pandemic H1 hemagglutinin (H1-HA). We found that naive CD4+ T cells have a broad repertoire, being able to recognize naturally processed as well as cryptic peptides spanning the whole H1-HA sequence. In contrast, memory Th cells were primarily directed against just a few immunodominant peptides that were readily detected by mass spectrometry-based MHC-II peptidomics and predicted by structural accessibility analysis. Collectively, these findings reveal the presence of a broad repertoire of naive T cells specific for cryptic H1-HA peptides and demonstrate that antigen processing represents a major constraint determining immunodominance.

Moving Pieces in a Cellular Puzzle: A Cryptic Peptide from the Scorpion Toxin Ts14 Activates AKT and ERK Signaling and Decreases Cardiac Myocyte Contractility via Dephosphorylation of Phospholamban.

Cryptic peptides (cryptides) are biologically active peptides formed after proteolysis of native precursors present in animal venoms, for example. Proteolysis is an overlooked post-translational modification that increases venom complexity. The tripeptide KPP (Lys-Pro-Pro) is a peptide encrypted in the C-terminus of Ts14-a 25-mer peptide from the venom of the Tityus serrulatus scorpion that has a positive impact on the cardiovascular system, inducing vasodilation and reducing arterial blood pressure of hypertensive rats among other beneficial effects. A previous study reported that KPP and its native peptide Ts14 act via activation of the bradykinin receptor B2 (B2R). However, the cellular events underlying the activation of B2R by KPP are unknown. To study the cell signaling triggered by the Ts14 cryptide KPP, we incubated cardiac myocytes isolated from C57BL/6 mice with KPP (10-7 mol·L-1) for 0, 5, or 30 min and explored the proteome and phosphoproteome. Our results showed that KPP regulated cardiomyocyte proteins associated with, but not limited to, apoptosis, muscle contraction, protein turnover, and the respiratory chain. We also reported that KPP led to AKT phosphorylation, activating AKT and its downstream target nitric oxide synthase. We also observed that KPP led to dephosphorylation of phospholamban (PLN) at its activation sites (S16 and T17), leading to reduced contractility of treated cardiomyocytes. Some cellular targets reported here for KPP (e.g., AKT, PLN, and ERK) have already been reported to protect the cardiac tissue from hypoxia-induced injury. Hence, this study suggests potential beneficial effects of this scorpion cryptide that needs to be further investigated, for example, as a drug lead for cardiac infarction.

Host Defence Cryptides from Human Apolipoproteins: Applications in Medicinal Chemistry.

Several eukaryotic proteins with defined physiological roles may act as precursors of cryptic bioactive peptides released upon protein cleavage by the host and/or bacterial proteases. Based on this, the term "cryptome" has been used to define the unique portion of the proteome encompassing proteins with the ability to generate bioactive peptides (cryptides) and proteins (crypteins) upon proteolytic cleavage. Hence, the cryptome represents a source of peptides with potential pharmacological interest. Among eukaryotic precursor proteins, human apolipoproteins play an important role, since promising bioactive peptides have been identified and characterized from apolipoproteins E, B, and A-I sequences. Human apolipoproteins derived peptides have been shown to exhibit antibacterial, anti-biofilm, antiviral, anti-inflammatory, anti-atherogenic, antioxidant, or anticancer activities in in vitro assays and, in some cases, also in in vivo experiments on animal models. The most interesting Host Defence Peptides (HDPs) identified thus far in human apolipoproteins are described here with a focus on their biological activities applicable to biomedicine. Altogether, reported evidence clearly indicates that cryptic peptides represent promising templates for the generation of new drugs and therapeutics against infectious diseases.