sOCP: a framework predicting smORF coding potential based on TIS and in-frame features and effectively applied in the human genome.

Small open reading frames (smORFs) have been acknowledged to play various roles on essential biological pathways and affect human beings from diabetes to tumorigenesis. Predicting smORFs in silico is quite a prerequisite for processing the omics data. Here, we proposed the smORF-coding-potential-predicting framework, sOCP, which provides functions to construct a model for predicting novel smORFs in some species. The sOCP model constructed in human was based on in-frame features and the nucleotide bias around the start codon, and the small feature subset was proved to be competent enough and avoid overfitting problems for complicated models. It showed more advanced prediction metrics than previous methods and could correlate closely with experimental evidence in a heterogeneous dataset. The model was applied to Rattus norvegicus and exhibited satisfactory performance. We then scanned smORFs with ATG and non-ATG start codons from the human genome and generated a database containing about a million novel smORFs with coding potential. Around 72 000 smORFs are located on the lncRNA regions of the genome. The smORF-encoded peptides may be involved in biological pathways rare for canonical proteins, including glucocorticoid catabolic process and the prokaryotic defense system. Our work provides a model and database for human smORF investigation and a convenient tool for further smORF prediction in other species.

Precision proteoform design for 4R tau isoform selective templated aggregation.

Prion-like spread of disease-specific tau conformers is a hallmark of all tauopathies. A 19-residue probe peptide containing a P301L mutation and spanning the R2/R3 splice junction of tau folds and stacks into seeding-competent fibrils and induces aggregation of 4R, but not 3R tau. These tau peptide fibrils propagate aggregated intracellular tau over multiple generations, have a high ß-sheet content, a colocalized lipid signal, and adopt a well-defined U-shaped fold found in 4R tauopathy brain-derived fibrils. Fully atomistic replica exchange molecular dynamics (MD) simulations were used to compute the free energy landscapes of the conformational ensemble of the peptide monomers. These identified an aggregation-prohibiting ß-hairpin structure and an aggregation-competent U-fold unique to 4R tauopathy fibrils. Guided by MD simulations, we identified that the N-terminal-flanking residues to PHF6, which slightly vary between 4R and 3R isoforms, modulate seeding. Strikingly, when a single amino acid switch at position 305 replaced the serine of 4R tau with a lysine from the corresponding position in the first repeat of 3R tau, the seeding induced by the 19-residue peptide was markedly reduced. Conversely, a 4R tau mimic with three repeats, prepared by replacing those amino acids in the first repeat with those amino acids uniquely present in the second repeat, recovered aggregation when exposed to the 19-residue peptide. These peptide fibrils function as partial prions to recruit naive 4R tau-ten times the length of the peptide-and serve as a critical template for 4R tauopathy propagation. These results hint at opportunities for tau isoform-specific therapeutic interventions.

Peptide Inhibitor Targeting the Extraterminal Domain in BRD4 Potently Suppresses Breast Cancer Both In Vitro and In Vivo.

BRD4 is associated with a variety of human diseases, including breast cancer. The crucial roles of amino-terminal bromodomains (BDs) of BRD4 in binding with acetylated histones to regulate oncogene expression make them promising drug targets. However, adverse events impede the development of the BD inhibitors. BRD4 adopts an extraterminal (ET) domain, which recruits proteins to drive oncogene expression. We discovered a peptide inhibitor PiET targeting the ET domain to disrupt BRD4/JMJD6 interaction, a protein complex critical in oncogene expression and breast cancer. The cell-permeable form of PiET, TAT-PiET, and PROTAC-modified TAT-PiET, TAT-PiET-PROTAC, potently inhibits the expression of BRD4/JMJD6 target genes and breast cancer cell growth. Combination therapy with TAT-PiET/TAT-PiET-PROTAC and JQ1, iJMJD6, or Fulvestrant exhibits synergistic effects. TAT-PiET or TAT-PiET-PROTAC treatment overcomes endocrine therapy resistance in ERα-positive breast cancer cells. Taken together, we demonstrated that targeting the ET domain is effective in suppressing breast cancer, providing a therapeutic avenue in the clinic.

Gastric digestion of the sweet-tasting plant protein thaumatin releases bitter peptides that reduce H. pylori induced pro-inflammatory IL-17A release via the TAS2R16 bitter taste receptor.

About half of the world's population is infected with the bacterium Helicobacter pylori. For colonization, the bacterium neutralizes the low gastric pH and recruits immune cells to the stomach. The immune cells secrete cytokines, i.e., the pro-inflammatory IL-17A, which directly or indirectly damage surface epithelial cells. Since (I) dietary proteins are known to be digested into bitter tasting peptides in the gastric lumen, and (II) bitter tasting compounds have been demonstrated to reduce the release of pro-inflammatory cytokines through functional involvement of bitter taste receptors (TAS2Rs), we hypothesized that the sweet-tasting plant protein thaumatin would be cleaved into anti-inflammatory bitter peptides during gastric digestion. Using immortalized human parietal cells (HGT-1 cells), we demonstrated a bitter taste receptor TAS2R16-dependent reduction of a H. pylori-evoked IL-17A release by up to 89.7 ± 21.9% (p ≤ 0.01). Functional involvement of TAS2R16 was demonstrated by the study of specific antagonists and siRNA knock-down experiments.

Glucagon-like peptide 1 agonists for type 2 diabetes, weight loss, or both?

ABSTRACT: Glucagon-like peptide 1 agonists (GLP1s) and the novel glucose-dependent insulinotropic polypeptide/glucagon-like peptide 1 agonist are effective drugs for reducing A1C and weight in patients with type 2 diabetes. However, clinicians may find it difficult to discern which drug to prescribe in specific clinical scenarios. This article discusses evidence-based clinical use of these drugs.

Maturation and Conformational Switching of a De Novo Designed Phase-Separating Polypeptide.

Cellular compartments formed by biomolecular condensation are widespread features of cell biology. These organelle-like assemblies compartmentalize macromolecules dynamically within the crowded intracellular environment. However, the intermolecular interactions that produce condensed droplets may also create arrested states and potentially pathological assemblies such as fibers, aggregates, and gels through droplet maturation. Protein liquid-liquid phase separation is a metastable process, so maturation may be an intrinsic property of phase-separating proteins, where nucleation of different phases or states arises in supersaturated condensates. Here, we describe the formation of both phase-separated droplets and proteinaceous fibers driven by a de novo designed polypeptide. We characterize the formation of supramolecular fibers in vitro and in bacterial cells. We show that client proteins can be targeted to the fibers in cells using a droplet-forming construct. Finally, we explore the interplay between phase separation and fiber formation of the de novo polypeptide, showing that the droplets mature with a post-translational switch to largely ß conformations, analogous to models of pathological phase separation.

m-nitrobenzyl alcohol supercharging reagent enhances the chromatographic separation and the charging of disulfide bond linked and His-tag peptides.

The linkages of disulfide bond (DSB) play important roles in protein stability and activity. Mass spectrometry-based (MS-based) techniques become accepted tools for DSB analysis in the recent decade. In the bottom-up approach, after enzyme digestion, the neighbouring amino acids of cysteines have great impacts on the physicochemical properties of resulting disulfide bond peptides, determining their retention behaviour on liquid chromatography (LC) and their MS ionization efficiency. In this study, the addition of supercharging reagent in LC mobile phase was used to examine the impact of supercharging reagent on the charge states of disulfide-bond peptides. The results showed that 0.1 % m-nitrobenzyl alcohol (m-NBA) in LC mobile phase increased the sensitivity and charge states of DSB peptides from our model protein, equine Interleukin-5 (eIL5), as well as the resolution of reversed-phase chromatography. Notably, also the sensitivity of C-terminal peptide with His-tag significantly improved. Our findings highlight the effectiveness of employing m-NBA as a supercharging reagent when investigating disulfide-linked peptides and the C-terminal peptide with a His-tag through nano-liquid chromatography mass spectrometry.

High Charge Density in Peptide Dendrimers is Required to Destabilize Membranes: Insights into Endosome Evasion.

Peptide dendrimers are a type of branched, symmetric, and topologically well-defined molecule that have already been used as delivery systems for nucleic acid transfection. Several of the most promising sequences showed high efficiency in many key steps of transfection, namely, binding siRNA, entering cells, and evading the endosome. However, small changes to the peptide dendrimers, such as in the hydrophobic core, the amino acid chirality, or the total available charges, led to significantly different experimental results with unclear mechanistic insights. In this work, we built a computational model of several of those peptide dendrimers (MH18, MH13, and MH47) and some of their variants to study the molecular details of the structure and function of these molecules. We performed CpHMD simulations in the aqueous phase and in interaction with a lipid bilayer to assess how conformation and protonation are affected by pH in different environments. We found that while the different peptide dendrimer sequences lead to no substantial structural differences in the aqueous phase, the total charge and, more importantly, the total charge density are key for the capacity of the dendrimer to interact and destabilize the membrane. These dendrimers become highly charged when the pH changes from 7.5 to 4.5, and the presence of a high charge density, which is decreased for MH47 that has four fewer titratable lysines, is essential to trigger membrane destabilization. These findings are in excellent agreement with the experimental data and help us to understand the high efficiency of some dendrimers and why the dendrimer MH47 is unable to complete the transfection process. This evidence provides further understanding of the mode of action of these peptide dendrimers and will be pivotal for the future design of new sequences with improved transfection capabilities.

Pepticinnamins N, O, and P, Nonribosomal Peptides from the Soil-Derived Streptomyces mirabilis P8-A2.

Cinnamoyl moiety containing nonribosomal peptides represented by pepticinnamin E are a growing family of natural products isolated from different Streptomyces species and possess diverse bioactivities. The soil bacterium Streptomyces mirabilis P8-A2 harbors a cryptic pepticinnamin biosynthetic gene cluster, producing azodyrecins as major products. Inactivation of the azodyrecin biosynthetic gene cluster by CRISPR-BEST base editing led to the activation and production of pepticinnamin E (1) and its analogues, pepticinnamins N, O, and P (2-4), the structures of which were determined by detailed NMR spectroscopy, HRMS data, and Marfey's reactions. These new compounds did not show a growth inhibitory effect against the LNCaP and C4-2B prostate cancer lines, respectively.

Assessing Weak Anion Binding to Small Peptides.

Since Hofmeister's seminal studies in the late 19th century, it has been known that salts and buffers can drastically affect the properties of peptides and proteins. These Hofmeister effects can be conceived of in terms of three distinct phenomena/mechanisms: water-salt interactions that indirectly induce the salting-out of a protein by water sequestration by the salt, and direct salt-protein interactions that can either salt-in or salt-out the protein. Unfortunately, direct salt-protein interactions responsible for Hofmeister effects are weak and difficult to quantify. As such, they are frequently construed of as being nonspecific. Nevertheless, there has been considerable effort to better specify these interactions. Here, we use pentapeptides to demonstrate the utility of the H-dimension of nuclear magnetic resonance (NMR) spectroscopy to assess anion binding using N-H signal shifts. We qualify binding using these, demonstrating the upfield shifts induced by anion association and revealing how they are much larger than the corresponding downfield shifts induced by magnetic susceptibility and other ionic strength change effects. We also qualify binding in terms of how the pattern of signal shifts changes with point mutations. In general, we find that the observed upfield shifts are small compared with those induced by anion binding to amide-based hosts, and MD simulations suggest that this is so. Thus, charge-diffuse anions associate mostly with the nonpolar regions of the peptide rather than directly interacting with the amide N-H groups. These findings reveal the utility of 1H NMR spectroscopy for qualifying affinity to peptides─even when affinity constants are very low─and serve as a benchmark for using NMR spectroscopy to study anion binding to more complex systems.

Improved assay development of pharmaceutical modalities using feedback-controlled liquid chromatography optimization.

Development of meaningful and reliable analytical assays in the (bio)pharmaceutical industry can often be challenging, involving tedious trial and error experimentation. In this work, an automated analytical workflow using an AI-based algorithm for streamlined method development and optimization is presented. Chromatographic methods are developed and optimized from start to finish by a feedback-controlled modeling approach using readily available LC instrumentation and software technologies, bypassing manual user intervention. With the use of such tools, the time requirement of the analyst is drastically minimized in the development of a method. Herein key insights on chromatography system control, automatic optimization of mobile phase conditions, and final separation landscape for challenging multicomponent mixtures are presented (e.g., small molecules drug, peptides, proteins, and vaccine products) showcased by a detailed comparison of a chiral method development process. The work presented here illustrates the power of modern chromatography instrumentation and AI-based software to accelerate the development and deployment of new separation assays across (bio)pharmaceutical modalities while yielding substantial cost-savings, method robustness, and fast analytical turnaround.

Intratumor injection of BCG Ag85A high-affinity peptides enhanced anti-tumor efficacy in PPD-positive melanoma.

As one of the scheduled immunization vaccines worldwide, virtually all individuals have been vaccinated with BCG vaccine. In order to verify the hypothesis that delivering BCG high-affinity peptides to tumor areas could activate the existing BCG memory T cells to attack tumor, we firstly predicted the HLA-A*0201 high-affinity peptides of BCG Ag85A protein (KLIANNTRV, GLPVEYLQV), and then, A375 melanoma cells and HLA-A*0201 PBMCs (from PPD-positive adults) were added to co-incubated with the predicted peptides in vitro. We found that the predicted BCG high-affinity peptides could be directly loaded onto the surface of tumor cells, enhancing the tumor-killing efficacy of PBMCs from PPD-positive volunteer. Then, we constructed PPD-positive mice model bearing B16F10 subcutaneous tumors and found that intratumor injection of BCG Ag85A high-affinity peptides (SGGANSPAL, YHPQQFVYAGAMSGLLD) enhanced the anti-tumor efficacy in PPD-positive melanoma mice. Along with the better anti-tumor efficacy, the expression of PDL1 on tumor cell surface was also increased, and stronger antitumor effects occurred when further combined with anti-PD1 antibody. For microenvironment analysis, the proportion of effector memory T cells was increased and the better treatment efficacy may be attributed to the elevated effector memory CD4 + T cells within the tumor. In conclusion, using the existing immune response of BCG vaccine by delivering high-affinity peptides of BCG to tumor area is a safe and promising therapy for cancer.

Germline homozygosity and allelic imbalance of HLA-I are common in esophagogastric adenocarcinoma and impair the repertoire of immunogenic peptides.

BACKGROUND: The individual HLA-I genotype is associated with cancer, autoimmune diseases and infections. This study elucidates the role of germline homozygosity or allelic imbalance of HLA-I loci in esophago-gastric adenocarcinoma (EGA) and determines the resulting repertoires of potentially immunogenic peptides. METHODS: HLA genotypes and sequences of either (1) 10 relevant tumor-associated antigens (TAAs) or (2) patient-specific mutation-associated neoantigens (MANAs) were used to predict good-affinity binders using an in silico approach for MHC-binding (www.iedb.org). Imbalanced or lost expression of HLA-I-A/B/C alleles was analyzed by transcriptome sequencing. FluoroSpot assays and TCR sequencing were used to determine peptide-specific T-cell responses. RESULTS: We show that germline homozygosity of HLA-I genes is significantly enriched in EGA patients (n=80) compared with an HLA-matched reference cohort (n=7605). Whereas the overall mutational burden is similar, the repertoire of potentially immunogenic peptides derived from TAAs and MANAs was lower in homozygous patients. Promiscuity of peptides binding to different HLA-I molecules was low for most TAAs and MANAs and in silico modeling of the homozygous to a heterozygous HLA genotype revealed normalized peptide repertoires. Transcriptome sequencing showed imbalanced expression of HLA-I alleles in 75% of heterozygous patients. Out of these, 33% showed complete loss of heterozygosity, whereas 66% had altered expression of only one or two HLA-I molecules. In a FluoroSpot assay, we determined that peptide-specific T-cell responses against NY-ESO-1 are derived from multiple peptides, which often exclusively bind only one HLA-I allele. CONCLUSION: The high frequency of germline homozygosity in EGA patients suggests reduced cancer immunosurveillance leading to an increased cancer risk. Therapeutic targeting of allelic imbalance of HLA-I molecules should be considered in EGA.

Applications of peptides in nanosystems for diagnosing and managing bacterial sepsis.

Sepsis represents a critical medical condition stemming from an imbalanced host immune response to infections, which is linked to a significant burden of disease. Despite substantial efforts in laboratory and clinical research, sepsis remains a prominent contributor to mortality worldwide. Nanotechnology presents innovative opportunities for the advancement of sepsis diagnosis and treatment. Due to their unique properties, including diversity, ease of synthesis, biocompatibility, high specificity, and excellent pharmacological efficacy, peptides hold great potential as part of nanotechnology approaches against sepsis. Herein, we present a comprehensive and up-to-date review of the applications of peptides in nanosystems for combating sepsis, with the potential to expedite diagnosis and enhance management outcomes. Firstly, sepsis pathophysiology, antisepsis drug targets, current modalities in management and diagnosis with their limitations, and the potential of peptides to advance the diagnosis and management of sepsis have been adequately addressed. The applications have been organized into diagnostic or managing applications, with the last one being further sub-organized into nano-delivered bioactive peptides with antimicrobial or anti-inflammatory activity, peptides as targeting moieties on the surface of nanosystems against sepsis, and peptides as nanocarriers for antisepsis agents. The studies have been grouped thematically and discussed, emphasizing the constructed nanosystem, physicochemical properties, and peptide-imparted enhancement in diagnostic and therapeutic efficacy. The strengths, limitations, and research gaps in each section have been elaborated. Finally, current challenges and potential future paths to enhance the use of peptides in nanosystems for combating sepsis have been deliberately spotlighted. This review reaffirms peptides' potential as promising biomaterials within nanotechnology strategies aimed at improving sepsis diagnosis and management.

TANGO1 inhibitors reduce collagen secretion and limit tissue scarring.

Uncontrolled secretion of ECM proteins, such as collagen, can lead to excessive scarring and fibrosis and compromise tissue function. Despite the widespread occurrence of fibrotic diseases and scarring, effective therapies are lacking. A promising approach would be to limit the amount of collagen released from hyperactive fibroblasts. We have designed membrane permeant peptide inhibitors that specifically target the primary interface between TANGO1 and cTAGE5, an interaction that is required for collagen export from endoplasmic reticulum exit sites (ERES). Application of the peptide inhibitors leads to reduced TANGO1 and cTAGE5 protein levels and a corresponding inhibition in the secretion of several ECM components, including collagens. Peptide inhibitor treatment in zebrafish results in altered tissue architecture and reduced granulation tissue formation during cutaneous wound healing. The inhibitors reduce secretion of several ECM proteins, including collagens, fibrillin and fibronectin in human dermal fibroblasts and in cells obtained from patients with a generalized fibrotic disease (scleroderma). Taken together, targeted interference of the TANGO1-cTAGE5 binding interface could enable therapeutic modulation of ERES function in ECM hypersecretion, during wound healing and fibrotic processes.

An effective antibiofilm strategy based on bacteriophages armed with silver nanoparticles.

The emerging antibiotic resistance in pathogenic bacteria is a key problem in modern medicine that has led to a search for novel therapeutic strategies. A potential approach for managing such bacteria involves the use of their natural killers, namely lytic bacteriophages. Another effective method involves the use of metal nanoparticles with antimicrobial properties. However, the use of lytic phages armed with nanoparticles as an effective antimicrobial strategy, particularly with respect to biofilms, remains unexplored. Here, we show that T7 phages armed with silver nanoparticles exhibit greater efficacy in terms of controlling bacterial biofilm, compared with phages or nanoparticles alone. We initially identified a novel silver nanoparticle-binding peptide, then constructed T7 phages that successfully displayed the peptide on the outer surface of the viral head. These recombinant, AgNP-binding phages could effectively eradicate bacterial biofilm, even when used at low concentrations. Additionally, when used at concentrations that could eradicate bacterial biofilm, T7 phages armed with silver nanoparticles were not toxic to eukaryotic cells. Our results show that the novel combination of lytic phages with phage-bound silver nanoparticles is an effective, synergistic and safe strategy for the treatment of bacterial biofilms.

MHCII-peptide presentation: an assessment of the state-of-the-art prediction methods.

Major histocompatibility complex Class II (MHCII) proteins initiate and regulate immune responses by presentation of antigenic peptides to CD4+ T-cells and self-restriction. The interactions between MHCII and peptides determine the specificity of the immune response and are crucial in immunotherapy and cancer vaccine design. With the ever-increasing amount of MHCII-peptide binding data available, many computational approaches have been developed for MHCII-peptide interaction prediction over the last decade. There is thus an urgent need to provide an up-to-date overview and assessment of these newly developed computational methods. To benchmark the prediction performance of these methods, we constructed an independent dataset containing binding and non-binding peptides to 20 human MHCII protein allotypes from the Immune Epitope Database, covering DP, DR and DQ alleles. After collecting 11 known predictors up to January 2022, we evaluated those available through a webserver or standalone packages on this independent dataset. The benchmarking results show that MixMHC2pred and NetMHCIIpan-4.1 achieve the best performance among all predictors. In general, newly developed methods perform better than older ones due to the rapid expansion of data on which they are trained and the development of deep learning algorithms. Our manuscript not only draws a full picture of the state-of-art of MHCII-peptide binding prediction, but also guides researchers in the choice among the different predictors. More importantly, it will inspire biomedical researchers in both academia and industry for the future developments in this field.

Targeted delivery of HSP90 inhibitors for efficient therapy of CD44-positive acute myeloid leukemia and solid tumor-colon cancer.

Heat shock protein 90 (HSP90) is overexpressed in numerous cancers, promotes the maturation of numerous oncoproteins and facilitates cancer cell growth. Certain HSP90 inhibitors have entered clinical trials. Although less than satisfactory clinical effects or insurmountable toxicity have compelled these trials to be terminated or postponed, these results of preclinical and clinical studies demonstrated that the prospects of targeting therapeutic strategies involving HSP90 inhibitors deserve enough attention. Nanoparticulate-based drug delivery systems have been generally supposed as one of the most promising formulations especially for targeting strategies. However, so far, no active targeting nano-formulations have succeeded in clinical translation, mainly due to complicated preparation, complex formulations leading to difficult industrialization, incomplete biocompatibility or nontoxicity. In this study, HSP90 and CD44-targeted A6 peptide functionalized biomimetic nanoparticles (A6-NP) was designed and various degrees of A6-modification on nanoparticles were fabricated to evaluate targeting ability and anticancer efficiency. With no excipients, the hydrophobic HSP90 inhibitor G2111 and A6-conjugated human serum albumin could self-assemble into nanoparticles with a uniform particle size of approximately 200 nm, easy fabrication, well biocompatibility and avoidance of hepatotoxicity. Besides, G2111 encapsulated in A6-NP was only released less than 5% in 12 h, which may avoid off-target cell toxicity before entering into cancer cells. A6 peptide modification could significantly enhance uptake within a short time. Moreover, A6-NP continues to exert the broad anticancer spectrum of Hsp90 inhibitors and displays remarkable targeting ability and anticancer efficacy both in hematological malignancies and solid tumors (with colon tumors as the model cancer) both in vitro and in vivo. Overall, A6-NP, as a simple, biomimetic and active dual-targeting (CD44 and HSP90) nanomedicine, displays high potential for clinical translation.

Marine-Derived Peptides with Anti-Hypertensive Properties: Prospects for Pharmaceuticals, Supplements, and Functional Food.

Hypertension, a major health concern linked to heart disease and premature mortality, has prompted a search for alternative treatments due to side effects of existing medications. Sustainable harvesting of low-trophic marine organisms not only enhances food security but also provides a variety of bioactive molecules, including peptides. Despite comprising only a fraction of active natural compounds, peptides are ideal for drug development due to their size, stability, and resistance to degradation. Our review evaluates the anti-hypertensive properties of peptides and proteins derived from selected marine invertebrate phyla, examining the various methodologies used and their application in pharmaceuticals, supplements, and functional food. A considerable body of research exists on the anti-hypertensive effects of certain marine invertebrates, yet many species remain unexamined. The array of assessments methods, particularly for ACE inhibition, complicates the comparison of results. The dominance of in vitro and animal in vivo studies indicates a need for more clinical research in order to transition peptides into pharmaceuticals. Our findings lay the groundwork for further exploration of these promising marine invertebrates, emphasizing the need to balance scientific discovery and marine conservation for sustainable resource use.

Protein Hydrolysis as a Way to Valorise Squid-Processing Byproducts: Obtaining and Identification of ACE, DPP-IV and PEP Inhibitory Peptides.

The industrial processing of Argentine shortfin squid to obtain rings generates a significant amount of protein-rich waste, including the skin, which is rich in collagen and attached myofibrillar proteins. This waste is generally discarded. In this study, skin was used as a source of proteins that were hydrolysed using Trypsin, Esperase® or Alcalase®, which released peptides with antioxidant potential and, in particular, antihypertensive (ACE inhibition), hypoglycemic (DPP-IV inhibition) and/or nootropic (PEP inhibition) potential. Among the three enzymes tested, Esperase® and Alcalase produced hydrolysates with potent ACE-, DPP-IV- and PEP-inhibiting properties. These hydrolysates underwent chromatography fractionation, and the composition of the most bioactive fractions was analysed using HPLC-MS-MS. The fractions with the highest bioactivity exhibited very low IC50 values (16 and 66 µg/mL for ACE inhibition, 97 µg/mL for DPP-IV inhibition and 55 µg/mL for PEP inhibition) and were mainly derived from the hydrolysate obtained using Esperase®. The presence of Leu at the C-terminal appeared to be crucial for the ACE inhibitory activity of these fractions. The DPP-IV inhibitory activity of peptides seemed to be determined by the presence of Pro or Ala in the second position from the N-terminus, and Gly and/or Pro in the last C-terminal positions. Similarly, the presence of Pro in the peptides present in the best PEP inhibitory fraction seemed to be important in the inhibitory effect. These results demonstrate that the skin of the Argentine shortfin squid is a valuable source of bioactive peptides, suitable for incorporation into human nutrition as nutraceuticals and food supplements.