Unraveling a Small Secreted Peptide SUBPEP3 That Positively Regulates Salt-Stress Tolerance in Pyrus betulifolia.

Small secreted peptides (SSPs) play important roles in regulating plants' growth and development in response to external stimulus, but the genes and functions of SSPs in many species are still unknown. Therefore, it is particularly significant to characterize and annotate SSP genes in plant genomes. As a widely used stock of pears, Pyrus betulifolia has strong resistance to biotic and abiotic stresses. In this study, we analyzed the SSPs genes in the genome of P. betulifolia according to their characteristics and homology. A total of 1195 SSP genes were identified, and most of them are signaling molecules. Among these, we identified a new SSP, subtilase peptide 3 (SUBPEP3), which derived from the PA region of preSUBPEP3, increasing the expression level under salt stress. Both adding synthetic peptide SUBPEP3 to the culture medium of pears and the overexpression of SUBPEP3 in tobacco can improve the salt tolerance of plants. In summary, we annotated the SSP genes in the P. betulifolia genome and identified a small secreted peptide SUBPEP3 that regulates the salt tolerance of P. betulifolia, which provides an important theoretical basis for further revealing the function of SSPs.

Peptides Targeting the IF1-ATP Synthase Complex Modulate the Permeability Transition Pore in Cancer HeLa Cells.

The mitochondrial protein IF1 is upregulated in many tumors and acts as a pro-oncogenic protein through its interaction with the ATP synthase and the inhibition of apoptosis. We have recently characterized the molecular nature of the IF1-Oligomycin Sensitivity Conferring Protein (OSCP) subunit interaction; however, it remains to be determined whether this interaction could be targeted for novel anti-cancer therapeutic intervention. We generated mitochondria-targeting peptides to displace IF1 from the OSCP interaction. The use of one selective peptide led to displacement of the inhibitor IF1 from ATP synthase, as shown by immunoprecipitation. NMR spectroscopy analysis, aimed at clarifying whether these peptides were able to directly bind to the OSCP protein, identified a second peptide which showed affinity for the N-terminal region of this subunit overlapping the IF1 binding region. In situ treatment with the membrane-permeable derivatives of these peptides in HeLa cells, that are silenced for the IF1 inhibitor protein, showed significant inhibition in mitochondrial permeability transition and no effects on mitochondrial respiration. These peptides mimic the effects of the IF1 inhibitor protein in cancer HeLa cells and confirm that the IF1-OSCP interaction inhibits apoptosis. A third peptide was identified which counteracts the anti-apoptotic role of IF1, showing that OSCP is a promising target for anti-cancer therapies.

Rpt5-Derived Analogs Stimulate Human Proteasome Activity in Cells and Degrade Proteins Forming Toxic Aggregates in Age-Related Diseases.

Aging and age-related diseases are associated with a decline in the capacity of protein turnover. Intrinsically disordered proteins, as well as proteins misfolded and oxidatively damaged, prone to aggregation, are preferentially digested by the ubiquitin-independent proteasome system (UIPS), a major component of which is the 20S proteasome. Therefore, boosting 20S activity constitutes a promising strategy to counteract a decrease in total proteasome activity during aging. One way to enhance the proteolytic removal of unwanted proteins appears to be the use of peptide-based activators of the 20S. In this study, we synthesized a series of peptides and peptidomimetics based on the C-terminus of the Rpt5 subunit of the 19S regulatory particle. Some of them efficiently stimulated human 20S proteasome activity. The attachment of the cell-penetrating peptide TAT allowed them to penetrate the cell membrane and stimulate proteasome activity in HEK293T cells, which was demonstrated using a cell-permeable substrate of the proteasome, TAS3. Furthermore, the best activator enhanced the degradation of aggregation-prone α-synuclein and Tau-441. The obtained compounds may therefore have the potential to compensate for the unbalanced proteostasis found in aging and age-related diseases.

Dishevelled2 activates WGEF via its interaction with a unique internal peptide motif of the GEF.

The Wnt-planar cell polarity (Wnt-PCP) pathway is crucial in establishing cell polarity during development and tissue homoeostasis. This pathway is found to be dysregulated in many pathological conditions, including cancer and autoimmune disorders. The central event in Wnt-PCP pathway is the activation of Weak-similarity guanine nucleotide exchange factor (WGEF) by the adapter protein Dishevelled (Dvl). The PDZ domain of Dishevelled2 (Dvl2PDZ) binds and activates WGEF by releasing it from its autoinhibitory state. However, the actual Dvl2PDZ binding site of WGEF and the consequent activation mechanism of the GEF have remained elusive. Using biochemical and molecular dynamics studies, we show that a unique "internal-PDZ binding motif" (IPM) of WGEF mediates the WGEF-Dvl2PDZ interaction to activate the GEF. The residues at P2, P0, P-2 and P-3 positions of IPM play an important role in stabilizing the WGEFpep-Dvl2PDZ interaction. Furthermore, MD simulations of modelled Dvl2PDZ-WGEFIPM peptide complexes suggest that WGEF-Dvl2PDZ interaction may differ from the reported Dvl2PDZ-IPM interactions. Additionally, the apo structure of human Dvl2PDZ shows conformational dynamics different from its IPM peptide bound state, suggesting an induced fit mechanism for the Dvl2PDZ-peptide interaction. The current study provides a model for Dvl2 induced activation of WGEF.

Two Novel Angiotensin-Converting Enzyme (ACE) Inhibitory and ACE2 Upregulating Peptides from the Hydrolysate of Pumpkin (Cucurbita moschata) Seed Meal.

Pumpkin (Cucurbita moschata) seed meal (PSM), the major byproduct of pumpkin seed oil industry, was used to prepare angiotensin-converting enzyme (ACE) inhibitory and angiotensin-converting enzyme 2 (ACE2) upregulating peptides. These peptides were isolated and purified from the PSM hydrolysate prepared using Neutrase 5.0 BG by ultrafiltration, Sephadex G-15 column chromatography, and reversed-phase high-performance liquid chromatography. Two peptides with significant ACE inhibition activity were identified as SNHANQLDFHP and PVQVLASAYR with IC50 values of 172.07 and 90.69 µM, respectively. The C-terminal tripeptides of the two peptides contained Pro, Phe, and Tyr, respectively, and PVQVLASAYR also had Val in its N-terminal tripeptide, which was a favorable structure for ACE inhibition. Molecular docking results declared that the two peptides could interact with ACE through hydrogen bonds and hydrophobic interactions. Furthermore, the two peptides performed protective function on EA.hy926 cells by decreasing the secretion of endothelin-1, increasing the release of nitric oxide, and regulating the ACE2 activity. In vitro simulated gastrointestinal digestion showed the two peptides exhibited good stability against gastrointestinal enzyme digestion. In conclusion, PSM is a promising material for preparing antihypertensive peptides.

Membrane-bound chemoreception of bitter bile acids and peptides is mediated by the same subset of bitter taste receptors.

The vertebrate sense of taste allows rapid assessment of the nutritional quality and potential presence of harmful substances prior to ingestion. Among the five basic taste qualities, salty, sour, sweet, umami, and bitter, bitterness is associated with the presence of putative toxic substances and elicits rejection behaviors in a wide range of animals including humans. However, not all bitter substances are harmful, some are thought to be health-beneficial and nutritious. Among those compound classes that elicit a bitter taste although being non-toxic and partly even essential for humans are bitter peptides and L-amino acids. Using functional heterologous expression assays, we observed that the 5 dominant human bitter taste receptors responsive to bitter peptides and amino acids are activated by bile acids, which are notorious for their extreme bitterness. We further demonstrate that the cross-reactivity of bitter taste receptors for these two different compound classes is evolutionary conserved and can be traced back to the amphibian lineage. Moreover, we show that the cross-detection by some receptors relies on "structural mimicry" between the very bitter peptide L-Trp-Trp-Trp and bile acids, whereas other receptors exhibit a phylogenetic conservation of this trait. As some bile acid-sensitive bitter taste receptor genes fulfill dual-roles in gustatory and non-gustatory systems, we suggest that the phylogenetic conservation of the rather surprising cross-detection of the two substance classes could rely on a gene-sharing-like mechanism in which the non-gustatory function accounts for the bitter taste response to amino acids and peptides.

Affinity-Driven Aryl Diazonium Labeling of Peptide Receptors on Living Cells.

Peptide-receptor interactions play critical roles in a wide variety of physiological processes. Methods to link bioactive peptides covalently to unmodified receptors on the surfaces of living cells are valuable for studying receptor signaling, dynamics, and trafficking and for identifying novel peptide-receptor interactions. Here, we utilize peptide analogues bearing deactivated aryl diazonium groups for the affinity-driven labeling of unmodified receptors. We demonstrate that aryl diazonium-bearing peptide analogues can covalently label receptors on the surface of living cells using both the neurotensin and the glucagon-like peptide 1 receptor systems. Receptor labeling occurs in the complex environment of the cell surface in a sequence-specific manner. We further demonstrate the utility of this covalent labeling approach for the visualization of peptide receptors by confocal fluorescence microscopy and for the enrichment and identification of labeled receptors by mass spectrometry-based proteomics. Aryl diazonium-based affinity-driven receptor labeling is attractive due to the high abundance of tyrosine and histidine residues susceptible to azo coupling in the peptide binding sites of receptors, the ease of incorporation of aryl diazonium groups into peptides, and the relatively small size of the aryl diazonium group. This approach should prove to be a powerful and relatively general method to study peptide-receptor interactions in cellular contexts.

Measurement of Forces Acting on Single T-Cell Receptors.

Molecular forces are increasingly recognized as an important parameter to understand cellular signaling processes. In the recent years, evidence accumulated that also T-cells exert tensile forces via their T-cell receptor during the antigen recognition process. To measure such intercellular pulling forces, one can make use of the elastic properties of spider silk peptides, which act similar to Hookean springs: increased strain corresponds to increased stress applied to the peptide. Combined with Förster resonance energy transfer (FRET) to read out the strain, such peptides represent powerful and versatile nanoscopic force sensing tools. In this paper, we provide a detailed protocol how to synthesize a molecular force sensor for application in T-cell antigen recognition and hands-on guidelines on experiments and analysis of obtained single molecule FRET data.

Enzyme engineering lets us play with new building blocks in non-ribosomal peptide synthesis.

In a recent issue of Nature Chemical Biology, Folger et al. demonstrated a high-throughput approach for engineering peptide bond forming domains from non-ribosomal peptide synthesis. A non-ribosomal peptide synthetase module from surfactin biosynthesis was reprogrammed to accept a fatty acid substrate into peptide biosynthesis, thus illustrating the potential of this approach for generating novel bioactive peptides.

Targeting the Plasmodium falciparum UCHL3 ubiquitin hydrolase using chemically constrained peptides.

The ubiquitin-proteasome system is essential to all eukaryotes and has been shown to be critical to parasite survival as well, including Plasmodium falciparum, the causative agent of the deadliest form of malarial disease. Despite the central role of the ubiquitin-proteasome pathway to parasite viability across its entire life-cycle, specific inhibitors targeting the individual enzymes mediating ubiquitin attachment and removal do not currently exist. The ability to disrupt P. falciparum growth at multiple developmental stages is particularly attractive as this could potentially prevent both disease pathology, caused by asexually dividing parasites, as well as transmission which is mediated by sexually differentiated parasites. The deubiquitinating enzyme PfUCHL3 is an essential protein, transcribed across both human and mosquito developmental stages. PfUCHL3 is considered hard to drug by conventional methods given the high level of homology of its active site to human UCHL3 as well as to other UCH domain enzymes. Here, we apply the RaPID mRNA display technology and identify constrained peptides capable of binding to PfUCHL3 with nanomolar affinities. The two lead peptides were found to selectively inhibit the deubiquitinase activity of PfUCHL3 versus HsUCHL3. NMR spectroscopy revealed that the peptides do not act by binding to the active site but instead block binding of the ubiquitin substrate. We demonstrate that this approach can be used to target essential protein-protein interactions within the Plasmodium ubiquitin pathway, enabling the application of chemically constrained peptides as a novel class of antimalarial therapeutics.

Substrate displacement of CK1 C-termini regulates kinase specificity.

CK1 kinases participate in many signaling pathways, and their regulation is of meaningful biological consequence. CK1s autophosphorylate their C-terminal noncatalytic tails, and eliminating these tails increases substrate phosphorylation in vitro, suggesting that the autophosphorylated C-termini act as inhibitory pseudosubstrates. To test this prediction, we comprehensively identified the autophosphorylation sites on Schizosaccharomyces pombe Hhp1 and human CK1ε. Phosphoablating mutations increased Hhp1 and CK1ε activity toward substrates. Peptides corresponding to the C-termini interacted with the kinase domains only when phosphorylated, and substrates competitively inhibited binding of the autophosphorylated tails to the substrate binding grooves. Tail autophosphorylation influenced the catalytic efficiency with which CK1s targeted different substrates, and truncating the tail of CK1δ broadened its linear peptide substrate motif, indicating that tails contribute to substrate specificity as well. Considering autophosphorylation of both T220 in the catalytic domain and C-terminal sites, we propose a displacement specificity model to describe how autophosphorylation modulates substrate specificity for the CK1 family.

The Development of CDC25A-Derived Phosphoseryl Peptides That Bind 14-3-3ε with High Affinities.

Overexpression of the 14-3-3ε protein is associated with suppression of apoptosis in cutaneous squamous cell carcinoma (cSCC). This antiapoptotic activity of 14-3-3ε is dependent on its binding to CDC25A; thus, inhibiting 14-3-3ε - CDC25A interaction is an attractive therapeutic approach to promote apoptosis in cSCC. In this regard, designing peptide inhibitors of 14-3-3ε - CDC25A interactions is of great interest. This work reports the rational design of peptide analogs of pS, a CDC25A-derived peptide that has been shown to inhibit 14-3-3ε-CDC25A interaction and promote apoptosis in cSCC with micromolar IC50. We designed new peptide analogs in silico by shortening the parent pS peptide from 14 to 9 amino acid residues; then, based on binding motifs of 14-3-3 proteins, we introduced modifications in the pS(174-182) peptide. We studied the binding of the peptides using conventional molecular dynamics (MD) and steered MD simulations, as well as biophysical methods. Our results showed that shortening the pS peptide from 14 to 9 amino acids reduced the affinity of the peptide. However, substituting Gln176 with either Phe or Tyr amino acids rescued the binding of the peptide. The optimized peptides obtained in this work can be candidates for inhibition of 14-3-3ε - CDC25A interactions in cSCC.

RVG29 Peptide-Modified Exosomes Loaded with Mir-133b Mediate the RhoA-ROCK Pathway to Improve Motor and Neurological Symptoms in Parkinson's Disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder worldwide. Drug delivery to the brain through the blood-brain barrier (BBB) is a significant challenge in PD treatment. Exosomes, which can efficiently traverse the BBB, which many drugs cannot penetrate, are ideal natural carriers for drug delivery. In this study, the BBB shuttle peptide was modified on the exosome surfaces. Three types of exosomes were constructed, each modified with a distinct peptide (RVG29, TAT, or Ang2) and loaded with miR-133b. The safety and brain-targeting capabilities of these peptide-modified exosomes were then evaluated. Finally, the mechanism by which RVG29-Exo-133b regulates the RhoA-ROCK signaling pathway was investigated. The findings indicate that the three peptide-modified exosomes were adequately tolerated, safe, and effectively assimilated in vivo and ex vivo, with RVG29 exhibiting superior targeting to the brain. Furthermore, RVG29-Exo-133b decreased the phosphorylation level of the Tau protein by targeting the RhoA-ROCK signaling pathway. It also enhanced the motor function in mice with PD, thereby reducing the degree of depression, improving dopaminergic neuron function, and attenuating 6-OHDA-induced nerve damage. In this study, we developed a stable drug delivery mechanism that targets the intracerebral region using exosomes. Furthermore, a novel strategy was developed to manage PD and can potentially serve as a preclinical basis for utilizing exosomes in the diagnosis and treatment of neurodegenerative conditions.

Diversification of Phage-Displayed Peptide Libraries with Noncanonical Amino Acid Mutagenesis and Chemical Modification.

Sitting on the interface between biologics and small molecules, peptides represent an emerging class of therapeutics. Numerous techniques have been developed in the past 30 years to take advantage of biological methods to generate and screen peptide libraries for the identification of therapeutic compounds, with phage display being one of the most accessible techniques. Although traditional phage display can generate billions of peptides simultaneously, it is limited to expression of canonical amino acids. Recently, several groups have successfully undergone efforts to apply genetic code expansion to introduce noncanonical amino acids (ncAAs) with novel reactivities and chemistries into phage-displayed peptide libraries. In addition to biological methods, several different chemical approaches have also been used to install noncanonical motifs into phage libraries. This review focuses on these recent advances that have taken advantage of both biological and chemical means for diversification of phage libraries with ncAAs.

Genome-scale annotation of protein binding sites via language model and geometric deep learning.

Revealing protein binding sites with other molecules, such as nucleic acids, peptides, or small ligands, sheds light on disease mechanism elucidation and novel drug design. With the explosive growth of proteins in sequence databases, how to accurately and efficiently identify these binding sites from sequences becomes essential. However, current methods mostly rely on expensive multiple sequence alignments or experimental protein structures, limiting their genome-scale applications. Besides, these methods haven't fully explored the geometry of the protein structures. Here, we propose GPSite, a multi-task network for simultaneously predicting binding residues of DNA, RNA, peptide, protein, ATP, HEM, and metal ions on proteins. GPSite was trained on informative sequence embeddings and predicted structures from protein language models, while comprehensively extracting residual and relational geometric contexts in an end-to-end manner. Experiments demonstrate that GPSite substantially surpasses state-of-the-art sequence-based and structure-based approaches on various benchmark datasets, even when the structures are not well-predicted. The low computational cost of GPSite enables rapid genome-scale binding residue annotations for over 568,000 sequences, providing opportunities to unveil unexplored associations of binding sites with molecular functions, biological processes, and genetic variants. The GPSite webserver and annotation database can be freely accessed at https://bio-web1.nscc-gz.cn/app/GPSite.

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.

Characteristics of saltiness-enhancing peptides derived from yeast proteins and elucidation of their mechanism of action by molecular docking.

This study aimed to identify saltiness-enhancing peptides from yeast protein and elucidate their mechanisms by molecular docking. Yeast protein hydrolysates with optimal saltiness-enhancing effects were prepared under conditions determined using an orthogonal test. Ten saltiness-enhancing peptide candidates were screened using an integrated virtual screening strategy. Sensory evaluation demonstrated that these peptides exhibited diverse taste characteristics (detection thresholds: 0.13-0.50 mmol/L). Peptides NKF, LGLR, WDL, NMKF, FDSL and FDGK synergistically or additively enhanced the saltiness of a 0.30% NaCl solution. Molecular docking revealed that these peptides predominantly interacted with TMC4 by hydrogen bonding, with hydrophilic amino acids from both peptides and TMC4 playing a pivotal role in their binding. Furthermore, Leu217, Gln377, Glu378, Pro474 and Cys475 were postulated as the key binding sites of TMC4. These findings establish a robust theoretical foundation for salt reduction strategies in food and provide novel insights into the potential applications of yeast proteins.

A Module for Analyzing Interactomes via APEX-MS Integrated into PatternLab for Proteomics.

Proximity labeling techniques, such as APEX-MS, provide valuable insights into proximal interactome mapping; however, the verification of biotinylated peptides is not straightforward. With this as motivation, we present a new module integrated into PatternLab for proteomics to enable APEX-MS data interpretation by targeting diagnostic fragment ions associated with APEX modifications. We reanalyzed a previously published APEX-MS data set and report a significant number of biotinylated peptides and, consequently, a confident set of proximal proteins. As the module is part of the widely adopted PatternLab for proteomics software suite, it offers users a comprehensive, easy, and integrated solution for data analysis. Given the broad utility of the APEX-MS technique in various biological contexts, we anticipate that our module will be a valuable asset to researchers, facilitating and enhancing interactome studies. PatternLab's APEX, including a usage protocol, is available at http://patternlabforproteomics.org/apex.