Domain Peptide Research Articles

Inhibitory Potential and Binding Thermodynamics of Scyllatoxin-Based BH3 Domain Mimetics Targeting Repressor BCL2 Proteins.

The B-cell lymphoma 2 (BCL2) proteins are a class of apoptosis regulators that control the release of apoptogenic factors from mitochondria. Under normal physiological conditions, apoptosis is inhibited through the actions of anti-apoptotic (repressor) BCL2 proteins that bind semi-indiscriminately to the helical BH3 domains of pro-apoptotic (effector) BCL2 proteins. In this work, we developed a series of BH3 domain mimetics by grafting residues from the effector BCL2 protein Bax onto the α-helix of scyllatoxin (ScTx). These so-called "ScTx-Bax" constructs were then used to gain insight into the physicochemical nature of repressor/effector BCL2 interactions. Specifically, we utilized competitive binding and isothermal titration calorimetry (ITC) to investigate the inhibitory potential and binding thermodynamics of ScTx-Bax structural variants that target the repressor protein Bcl-2 (proper) invitro. Our data show that ScTx-Bax mimetics compete with isolated Bax BH3 domain peptides for Bcl-2 with IC50 values in the mid-nanomolar range and that greater flexibility within the ScTx-Bax BH3 domain correlates with more effective inhibition. Furthermore, ITC experiments revealed that unstructured ScTx-Bax variants target Bcl-2 with greater entropic, but lower enthalpic, efficiencies than structured ScTx-Bax peptides. These results suggest that entropic contributions to binding Bcl-2 are more favorable for flexible BH3 domains; however, this enhancement is counterbalanced by a moderate enthalpic penalty. Overall, this study improves understanding of how structural properties of effector BH3 domains influence the promiscuous binding patterns of BCL2 proteins and expands the utility of ScTx-based BH3 domain mimetics as molecular tools to study discrete recognition elements that facilitate repressor/effector BCL2 interactions.

In Vitro Selection of Collagen-Binding Vascular Endothelial Growth Factor Containing Genetically Encoded Mussel-Inspired Adhesive Amino Acids.

Protein immobilization technology is important in medical and industrial applications. We previously reported all-in-one in vitro selection, wherein a collagen-binding vascular endothelial growth factor (CB-VEGF) was identified from a fusion library of random and VEGF sequences. However, its interaction chemistry is mainly limited to the interaction established by the 20 canonical amino acids. Herein, we incorporated an adhesive non-natural amino acid found in marine mussels, L-3,4-dihydroxyphenylalanine (DOPA), into the library for all-in-one in vitro selection. After selection, we identified DOPA-containing CB-VEGF. CB-VEGF binds to collagen with an apparent dissociation constant of 2 nM; naïve VEGF does not bind to collagen. The collagen-binding peptide domain of CB-VEGF (CB-peptide) exhibited stronger binding to collagen than a mutant peptide (substitution from DOPA to tyrosine), indicating the importance of DOPA to collagen binding. The collagen-binding affinity of CB-VEGF is 10-fold higher than that of CB-peptide, suggesting that the collagen-binding ability of CB-VEGF is not due to the additive function of CB-peptide to VEGF, but is synergistic. Furthermore, increased cell growth was observed on CB-VEGF-treated collagen surfaces, not VEGF-treated collagen surfaces. Thus, integrating all-in-one in vitro selection and DOPA incorporation shows promise in generating adhesive proteins on solid supports.

Development of Tools for Evaluating the Matrix Metalloproteinase 14 Cytoplasmic Domain in the Tumor Microenvironment

BackgroundMembrane type 1 matrix metalloproteinase/matrix metalloproteinase 14 (MT1-MMP-MMP-14) is associated with various stages of cancer, particularly metastasis. The intracellular cytoplasmic domain of MMP-14 has recently been recognized for the ability to regulate cellular behaviors, particularly under conditions of low pH and/or hypoxia, which can occur within the tumor microenvironment.PurposeThe present work has focused on developing tools to evaluate changes that occur to the cytoplasmic domain of MMP-14 under the aforementioned conditions.MethodsVariants of the cytoplasmic domain containing posttranslational modifications have been synthesized and characterized, and flow cytometric analysis has been examined as an approach to quantify extracellular and intracellular MMP-14 in pancreatic cancer cell lines under normal and hypoxic conditions.ResultsFour variants of the MMP-14 cytoplasmic domain were synthesized, where the variants contained no modifications, phosphorylation, palmitoylation, or monoubiquitination. The structure of each variant was impacted when the pH was lowered. For quantification of intracellular MMP-14 flow cytometry was effective under both normoxic and hypoxic conditions.ConclusionThe synthesized cytoplasmic domain peptides can be used to raise antibodies for flow cytometric evaluation of the state of the cytoplasmic domain in cell spheroids under tumor microenvironment (low pH, hypoxic) conditions.

Minimal domain peptides derived from enterocins exhibit potent antifungal activity

Minimal domain peptides derived from enterocins exhibit potent antifungal activity

Functional Characterization of FgAsp, a Gene Coding an Aspartic Acid Protease in Fusarium graminearum.

Aspartic proteases (APs), hydrolases with aspartic acid residues as catalytic active sites, are closely associated with processes such as plant growth and development and fungal and bacterial pathogenesis. F. graminearum is the dominant pathogenic fungus that causes Fusarium head blight (FHB) in wheat. However, the relationship of APs to the growth, development, and pathogenesis of F. graminearum is not clear. Therefore, we selected the FGSG_09558 gene, whose function annotation is aspartate protease, for further study. In this study, FGSG_09558 was found to contain a conserved structural domain and signal peptide sequence of aspartic acid protease and was therefore named FgAsp. The function of FgAsp in F. graminearum was investigated by constructing the knockout and complementation mutants of this gene. The results showed that with respect to the wild type (PH-1), the knockout mutant showed a significant reduction in mycelial growth, asexual spore production, and sexual spore formation, highlighting the key role of FgAsp in the growth and development of F. graminearum. In addition, the mutants showed a significant reduction in the virulence and accumulation level of deoxynivalenol (DON) content on maize whiskers, wheat germ sheaths, and wheat ears. DON, as a key factor of virulence, plays an important role in the F. graminearum infection of wheat ears, suggesting that FgAsp is involved in the regulation of F. graminearum pathogenicity by affecting the accumulation of the DON toxin. FgAsp had a significant effect on the ability of F. graminearum to utilize various sugars, especially arabinose. In response to the stress, hydrogen peroxide inhibited the growth of the mutant most significantly, indicating the important function of FgAsp in the strain's response to environmental stress. Finally, FgAsp plays a key role in the regulation of F. graminearum growth and development, pathogenicity, and environmental stress response.

Conjugation with S4 protein transduction domain enhances the immunogenicity of the peptide vaccine against breast cancer.

Although peptide vaccines offer a novel venue for cancer immunotherapy, clinical success has been rather limited. Cell-penetrating peptides, due to their ability to translocate through the cell membrane, could be conjugated to the peptide vaccine to2 enhance therapeutic efficiency. The S4 transduction domain of the shaker-potassium channel was conjugated to mammaglobin-A (MamA) immunodominant epitope (MamA2.1) to verify its anticancer immunogenicity. S4-MamA2.1 peptide has demonstrated significantly higher epitope loading and stable membrane expression of HLA-A2 antigen-presenting molecules on T2 cell lines. Further, these S4-MamA2.1 treated T2 cells were able to activate naïve CD8+ T cells to induce MamA-specific cytotoxicity against breast cancer cells. Conjugation of the S4 domain has also demonstrated a slight increase in immunogenicity of lesser immunodominant MamA epitopes. The conjugation of the S4 domain to N-terminus of MamA2.1 demonstrated significantly higher immunogenicity over C-terminus conjugation. Taken together, the results of the present study suggest that conjugation of the S4 cell-penetrating peptide domain to MamA2.1 epitope enhances the peptide vaccine immunogenicity against MamA-expressing breast cancers.

FVIII peptides presented on HLA-DP and identification of an A3 domain peptide binding with high affinity to the commonly expressed HLA-DP4.

The development of neutralizing antibodies (inhibitors) against coagulation factor VIII (FVIII) poses a major challenge in hemophilia A (HA) treatment. The formation of FVIII inhibitors is a CD4+ T-cell dependent mechanism which includes antigen presenting cells (APCs), B- and T-helper lymphocytes. APCs present FVIII derived peptides on major histocompatibility complex class II (MHC-II) to CD4+ Tcells. We previously established a mass spectrometry based approach to delineate the FVIII repertoire presented on HLA-DR and HLA-DQ. In this study, specific attention was directed towards the identification of FVIII peptides presented on HLA-DP. A data-set of naturally processed FVIII peptides was generated by incubating human FVIII with immature monocytes-derived dendritic cells (moDCs) from HLA-typed healthy donors. Using this method, we identified 176 to 1352 different HLA-DP presented peptides per donor, including 26 different FVIII derived peptides. The most frequently presented peptides derived from the A3, and C2 domains of FVIII. Comparison of the FVIII repertoire presented on HLA-DP with that presented on HLA-DR revealed considerable overlap but also suggested preferential presentation of specific peptides on either HLA-DR or HLA-DP. Fourteen FVIII peptides presented on HLA-DP were synthesized and evaluated for their binding ability to the commonly expressed HLA-DP4 molecule which is highly prevalent in the Caucasian population. Peptide binding studies showed that 7 of 14 peptides competed with a reference peptide to HLADP4. Interestingly, an A3 domain derived peptide bound with high affinity to HLA-DP4 positioning this peptide as a prime candidate for the development of novel peptide-based tolerogenic strategies for FVIII inhibitors.

Computer-guided design of Z domain peptides with improved inhibition of VEGF.

Computational protein design is becoming increasingly helpful in the development of new protein therapeutics with enhanced efficacy, specificity, and minimal side effects, for precise modulation of biological pathways. In vascular biology, the interaction between vascular endothelial growth factor A (VEGFA) and its receptors (VEGFR1-R3) is a pivotal process underlying blood vessel growth. Dysregulation of this pathway contributes to diseases such as cancer and diabetic retinopathy. Existing VEGFA inhibitors are effective but have limitations, driving interest in peptide-based therapeutics. Peptide inhibitors offer advantages, including reduced toxicity, improved formulation flexibility, and enhanced stability. This study leverages computational tools, particularly ProteinMPNN and Rosetta, to design optimized peptide-based VEGFA inhibitors. Building on the existing peptide templates mini-Z-1 and Z-1-2, new sequences were computationally predicted and experimentally validated. A novel peptide with improved affinity (KD = 6.2 µM) compared to mini-Z-1 (KD = 9.3 µM) was found, requiring only one round of design and testing. The integration of ProteinMPNN and Rosetta enabled a rapid and cost-effective pipeline for designing potent VEGFA inhibitors, underscoring the potential of computational peptide design in developing next-generation therapeutics targeting angiogenesis-dependent diseases.

Functional Characterization of Plant Peptide-Containing Sulfated Tyrosine (PSY) Family in Wheat (Triticum aestivum L.).

The plant peptide-containing sulfated tyrosine (PSY) family plays critical roles in plant cell proliferation and stress responses. However, the functional characterization of the PSY peptide family in wheat remains unclear. This study systematically identified a total of 29 TaPSY genes at the genome-wide level, classifying them into six subgroups based on PSY-like motifs. These peptides contain a highly conserved active peptide domain, closely resembling the Arabidopsis AtPSY1 motif. All TaPSY homologs are predicted to have a sulfated tyrosine catalyzed by plant tyrosylprotein sulfotransferase (TPST). The TaPSY genes displayed distinct expression patterns across various tissues, with most genes showing higher expression levels in roots and stems. Synthetic sulfated TaPSY peptides enhanced root growth in both wild-type Arabidopsis and the tpst-1 mutant plants. In wheat, exogenous application of TaPSY peptides also promoted root growth, with the synthetic TaPSY5 peptide affecting reactive oxygen species levels in wheat taproots to stimulate primary root growth. Furthermore, transgenic Arabidopsis plants overexpressing TaPSY10 exhibited longer primary roots and increased lateral root numbers. These findings provide insights into the physiological roles of TaPSY peptides in regulating wheat root growth.

Comparison of Cell-Penetrating and Fusogenic TAT-HA2 Peptide Performance in Peptideplex, Multicomponent, and Conjugate siRNA Delivery Systems.

In this study, the performance of the cell-penetrating and fusogenic peptide, TAT-HA2, which consists of a cell-permeable HIV trans-activator of transcription (TAT) protein transduction domain and a pH-responsive influenza A virus hemagglutinin protein (HA2) domain, was comparatively evaluated for the first time in peptideplex, multicomponent, and conjugate siRNA delivery systems. TAT-HA2 in all three systems protected siRNA from degradation, except in the conjugate system with a low Peptide/siRNA ratio. The synergistic effect of different peptide domains enhanced the transfection efficiency of multicomponent and conjugate systems compared to that of peptideplexes, which was attributed to the surface configuration of TAT-HA2 peptides depending on the nature of attachment. Particularly, the multicomponent system showed better cellular uptake and endosomal escape than the peptideplexes, resulting in enhanced siRNA delivery in the cytoplasm. In addition, the presence of cleavable disulfide bonds in multicomponent and conjugate systems promoted the effective siRNA delivery in the cytoplasm, resulting in improved gene silencing activity. The multicomponent system reduced the level of luciferase expression in SKOV3 cells to 45% (±4). In contrast, the conjugate system and the commercially available siRNA transfection agent, Lipofectamine RNAiMax, caused luciferase suppression down to 55% (±2) at a siRNA dose of 100 nM. For the same dose, the peptideplex system could only reduce the luciferase expression to 65% (±5). None of the developed systems showed significant toxicity at any dose. Overall, the TAT-HA2 peptide is promising as a siRNA delivery vector; however, its performance depends on the nature of attachment and, as a result, its surface configuration on the developed delivery system.

Rapid prediction of key residues for foldability by machine learning model enables the design of highly functional libraries with hyperstable constrained peptide scaffolds.

Peptides are an emerging modality for developing therapeutics that can either agonize or antagonize cellular pathways associated with disease, yet peptides often suffer from poor chemical and physical stability, which limits their potential. However, naturally occurring disulfide-constrained peptides (DCPs) and de novo designed Hyperstable Constrained Peptides (HCPs) exhibiting highly stable and drug-like scaffolds, making them attractive therapeutic modalities. Previously, we established a robust platform for discovering peptide therapeutics by utilizing multiple DCPs as scaffolds. However, we realized that those libraries could be further improved by considering the foldability of peptide scaffolds for library design. We hypothesized that specific sequence patterns within the peptide scaffolds played a crucial role in spontaneous folding into a stable topology, and thus, these sequences should not be subject to randomization in the original library design. Therefore, we developed a method for designing highly diverse DCP libraries while preserving the inherent foldability of each scaffold. To achieve this, we first generated a large-scale dataset from yeast surface display (YSD) combined with shotgun alanine scan experiments to train a machine-learning (ML) model based on techniques used for natural language understanding. Then we validated the ML model with experiments, showing that it is able to not only predict the foldability of peptides with high accuracy across a broad range of sequences but also pinpoint residues critical for foldability. Using the insights gained from the alanine scanning experiment as well as prediction model, we designed a new peptide library based on a de novo-designed HCP, which was optimized for enhanced folding efficiency. Subsequent panning trials using this library yielded promising hits having good folding properties. In summary, this work advances peptide or small protein domain library design practices. These findings could pave the way for the efficient development of peptide-based therapeutics in the future.

The molecular basis for functional divergence of duplicated SOX factors controlling endoderm formation and left-right patterning in zebrafish.

Endoderm, one of three primary germ layers of vertebrate embryos, makes major contributions to the respiratory and gastrointestinal tracts and associated organs, including liver and pancreas. In mammals, the transcription factor SOX17 is vital for endoderm organ formation and can induce endoderm progenitor identity. Duplication of ancestral sox17 in the teleost lineage produced the paralogues sox32 and sox17 in zebrafish. Sox32 is required for specification of endoderm and progenitors of the left-right organiser (Kupffer's Vesicle, KV), with Sox17 a downstream target of Sox32 that is implicated in further KV development. Phenotypic evidence therefore suggests functional similarities between zebrafish Sox32 and Sox17 and mammalian SOX17. Here, we directly compare these orthologues and paralogues, using the early zebrafish embryo as a biological platform for functional testing. Our results indicate that, unlike Sox32, human SOX17 cannot induce endoderm specification in zebrafish. Furthermore, using hybrid protein functional analyses, we show that Sox32 specificity for the endoderm gene regulatory network is linked to evolutionary divergence in its DNA-binding HMG domain from its paralogue Sox17. Additionally, changes in the C-terminal regions of Sox32 and Sox17 underpin their differing target specificities. Finally, we establish that specific conserved peptides in the C-terminal domain are essential for the role of Sox17 in establishing correct organ asymmetry. Overall, our results illuminate the molecular basis for functional divergence of Sox32 and Sox17 in vertebrate endoderm development and left-right patterning, and the evolution of SoxF transcription factor function.

Bioengineering approach for the design of magnetic bacterial cellulose membranes

Biopolymer research has led to the development of novel products through innovative strategies. Their functionalization is typically achieved by physical/chemical methods that require harsh chemicals or mechanical treatments. These functionalities could be alternatively achieved by employing bioengineering design methods. We demonstrate, a bioengineered dual-microbial approach to create functional bacterial cellulose from microbial workhorses. Komagataeibacter hansenii ATCC 53582 is used to produce bacterial cellulose and engineered E. coli is used to functionalize the matrix with a recombinant fibrous protein. The E. coli harbours synthetic genes for the secretion of amyloid curli protein subunit (CsgA) tagged with short functional M6A peptide domains. The incorporation of M6A-functionalized amyloid proteins into bacterial cellulose facilitates magnetite nanoparticle nucleation. We achieved a saturation magnetization of 40 emu g−1, a three-fold increase compared to existing strategies. The magnetic bacterial cellulose films demonstrate cytocompatibility and accelerate cell migration in the presence of magnetic field.

Bone-Targeted Fluoropeptide Nanoparticle Inhibits NF-κB Signaling to Treat Osteosarcoma and Tumor-Induced Bone Destruction.

Osteosarcoma is a malignant bone cancer usually characterized by symptoms of bone loss due to pathologically enhanced osteoclast activity. Activated osteoclasts enhance bone resorption and promote osteosarcoma cell progression by secreting various cytokines. Intercepting the detrimental interplay between osteoclasts and osteosarcoma cells is considered as an option for osteosarcoma treatment. Here, a bone-targeted fluoropeptide nanoparticle that can inhibit the nuclear factor kappa B (NF-κB) signaling in both osteoclasts and osteosarcoma to address the above issue is developed. The NF-κB essential modulator binding domain (NBD) peptide is conjugated with a fluorous tag to improve its proteolytic stability and intracellular penetration. The NBD peptide is efficiently delivered into cells after fluorination to induce apoptosis of osteocarcoma cells, and inhibits osteoclasts differentiation. The fluorous-tagged NBD peptide is further co-assembled with an oligo (aspartic acid) terminated fluoropeptide to form bone-targeted peptide nanoparticles for osteosarcoma treatment. The targeted nanoparticles efficiently inhibited tumor progression and osteosarcoma-induced bone destruction in vivo. This co-assembled fluoropeptide nanoplatform proposed in this study offers a promising approach for targeted and intracellular delivery of peptide therapeutics in the treatment of various diseases.

Mitochondrial HKDC1 suppresses oxidative stress and apoptosis by regulating mitochondrial function in goose fatty liver

Different from human non-alcoholic fatty liver disease (NAFLD), goose fatty liver is physiological with no inflammation. Consistently, mitochondrial dysfunction, oxidative stress and apoptosis are rarely seen in goose fatty liver. Hexokinase domain-containing protein 1 (HKDC1) is involved in maintaining systemic glucose homeostasis, and its absence causes mitochondrial dysfunction. Here, we demonstrated that mitochondrial outer membrane-bound HKDC1 (mHKDC1) had an expression pattern different from that of whole-cell HKDC1 (wHKDC1). Data indicated that the protein level of whole-cell HKDC1 (wHKDC1) was increased but mHKDC1 was decreased in mouse fatty liver. Interestingly, both the protein levels of wHKDC1 and mHKDC1 were significantly increased in goose fatty liver. Treatment of goose or mouse hepatocytes with fatty liver-related factors could influence the expression of wHKDC1 and mHKDC1, but the influence on wHKDC1 was not identical to mHKDC1. HKDC1 overexpression in goose hepatocytes increased wHKDC1 and mHKDC1 expression, mitochondrial membrane potential (MMP), mitochondrial respiratory chain activity, and suppressed reactive oxygen species (ROS) generation, apoptosis and cytokine-cytokine receptor signaling pathway. In addition, mutations in mitochondrial signal peptide or activation domain of HKDC1 altered MMP or ROS levels. In conclusion, HKDC1, particularly mHKDC1, may protect goose fatty liver by regulating mitochondrial function, ROS generation, apoptosis, and inflammation-related pathways.

Catalytic peptide-based coacervates for enhanced function through structural organization and substrate specificity

Liquid-liquid phase separation (LLPS) in living cells provides innovative pathways for synthetic compartmentalized catalytic systems. While LLPS has been explored for enhancing enzyme catalysis, its potential application to catalytic peptides remains unexplored. Here, we demonstrate the use of coacervation, a key LLPS feature, to constrain the conformational flexibility of catalytic peptides, resulting in structured domains that enhance peptide catalysis. Using the flexible catalytic peptide P7 as a model, we induce reversible biomolecular coacervates with structured peptide domains proficient in hydrolyzing phosphate ester molecules and selectively sequestering phosphorylated proteins. Remarkably, these coacervate-based microreactors exhibit a 15,000-fold increase in catalytic efficiency compared to soluble peptides. Our findings highlight the potential of a single peptide to induce coacervate formation, selectively recruit substrates, and mediate catalysis, enabling a simple design for low-complexity, single peptide-based compartments with broad implications. Moreover, LLPS emerges as a fundamental mechanism in the evolution of chemical functions, effectively managing conformational heterogeneity in short peptides and providing valuable insights into the evolution of enzyme activity and catalysis in prebiotic chemistry.

Collagen VI and endotrophin: potential extracellular matrix targets to attenuate adverse cardiac remodeling post-infarction

Abstract Background The extracellular matrix (ECM) is a key component of cardiac repair after myocardial infarction (post-MI). After tissue damage following myocardial ischaemia, the ECM becomes enriched in proteases that are known to induce partial enzymatic cleavage and fragmentation of native structural and fibrillar macromolecules such as collagens. While some of the resulting protein fragments (matrikines) may have beneficial effects by mediating an active scar formation, other such as the collagen VI (COL6) proteolytic component "endothrophin" has been related to deleterious evolution and fibrosis, particularly in the presence of cardiovascular risk factors. Purpose We aimed to investigate the collagen profile in the structural fraction of ECM (enriched in fibrillar and structural proteins) of post-MI hearts at late evolution stages after MI. Methods The ECM protein profile was investigated in the ischemic (ISCH) and non-ischemic (NISCH) zones of the post-MI myocardium in pigs. MI was induced by closed-chest balloon occlusion of the LAD for 90 minutes. Studies were performed in normo- and hypercholesterolemic (diet induced) animals. Myocardial tissue was obtained 30d after MI. ECM proteins were selectively extracted, identified by LC/MS-MS and validated by Western Blot. Plasma levels of endothrophin were quantified by ELISA. Myocardial tissue from the same areas was characterized by immunohistochemistry. Results The ECM in the ISCH zone showed a differential signature of 39 proteins (14 upregulated / 25 downregulated) 30 days after MI compared to the NISCH area of the same animals. In the ISCH region, significant changes in collagen referring to COL6A3 and COL6A1 (increase) and COL6A2 and COL6A6 (decrease) were detected. COL6A3 protein content was 1.8fold higher (p=0.01) in the ISCH-ECM. A similar increase was found in normo- and hypercholesterolemic animals. A higher trend in COL6A3 levels was found in the ISCH- vs, the NISCH-zone in the myocardial cellular fraction suggesting that the COL6 ECM-related increase in ISCH is produced at a transcript level. Importantly, the COL6A3 found in the ECM did not contain the protein peptide (C5 domain) corresponding to endotrophin matrikine, suggesting its cleavage and release. Using a specific ELISA, hypercholesterolemic animals showed significantly higher endothrophin plasma levels than normolipemic controls (p=0.003) even in the absence of MI. Conclusion Our results evidence an increase in COL6A3 content in the ECM of the ischemic myocardium at 30 days post-MI. COL6A3 is depleted in a peptide of the C5 domain corresponding to endotrophin, found to be enhanced in hypercholesterolemia. These findings support a role for collagen type VI in adverse cardiac remodeling post-MI.

Coiled Coil Peptide Tiles (CCPTs): Expanding the Peptide Building Block Design with Multivalent Peptide Macrocycles.

Owing to their synthetic accessibility and protein-mimetic features, peptides represent an attractive biomolecular building block for the fabrication of artificial biomimetic materials with emergent properties and functions. Here, we expand the peptide building block design space through unveiling the design, synthesis, and characterization of novel, multivalent peptide macrocycles (96mers), termed coiled coil peptide tiles (CCPTs). CCPTs comprise multiple orthogonal coiled coil peptide domains that are separated by flexible linkers. The constraints, imposed by cyclization, confer CCPTs with the ability to direct programmable, multidirectional interactions between coiled coil-forming "edge" domains of CCPTs and their free peptide binding partners. These fully synthetic constructs are assembled using a convergent synthetic strategy via a combination of native chemical ligation and Sortase A-mediated cyclization. Circular dichroism (CD) studies reveal the increased helical stability associated with cyclization and subsequent coiled coil formation along the CCPT edges. Size-exclusion chromatography (SEC), analytical high-performance liquid chromatography (HPLC), and fluorescence quenching assays provide a comprehensive biophysical characterization of various assembled CCPT complexes and confirm the orthogonal colocalization between coiled coil domains within CCPTs and their designed on-target free peptide partners. Lastly, we employ molecular dynamics (MD) simulations, which provide molecular-level insights into experimental results, as a supporting method for understanding the structural dynamics of CCPTs and their complexes. MD analysis of the simulated CCPT architectures reveals the rigidification and expansion of CCPTs upon complexation, i.e., coiled coil formation with their designed binding partners, and provides insights for guiding the designs of future generations of CCPTs. The addition of CCPTs into the repertoire of coiled coil-based building blocks has the potential for expanding the coiled coil assembly landscape by unlocking new topologies having designable intermolecular interfaces.

Characterization of Kunitz-Domain Anticoagulation Peptides Derived from Acinetobacter baumannii Exotoxin Protein F6W77.

Recent studies have revealed that the coagulation system plays a role in mammalian innate defense by entrapping bacteria in clots and generating antibacterial peptides. So, it is very important for the survival of bacteria to defend against the host coagulation system, which suggests that bacterial exotoxins might be a new source of anticoagulants. In this study, we analyzed the genomic sequences of Acinetobacter baumannii and a new bacterial exotoxin protein, F6W77, with five Kunitz-domains, KABP1-5, was identified. Each Kunitz-type domain features a classical six-cysteine framework reticulated by three conserved disulfide bridges, which was obviously similar to animal Kunitz-domain peptides but different from plant Kunitz-domain peptides. Anticoagulation function evaluation showed that towards the intrinsic coagulation pathway, KABP1 and KABP5 had apparently inhibitory activity, KABP4 had weak inhibitory activity, and KBAP2 and KABP3 had no effect even at a high concentration of 20 μg/mL. All five Kunitz-domain peptides, KABP1-5, had no inhibitory activity towards the extrinsic coagulation pathway. Enzyme-inhibitor experiments showed that the high-activity anticoagulant peptide KABP1 had apparently inhibitory activity towards two key coagulation factors, Xa and XIa, which was further confirmed by pull-down experiments that showed that KABP1 can bind to coagulation factors Xa and XIa directly. Structure-function relationship analyses of five Kunitz-type domain peptides showed that the arginine of the P1 site of three new bacterial anticoagulants, KABP1, KABP4 and KABP5, might be the key residue for their anticoagulation activity. In conclusion, with bioinformatics analyses, peptide recombination, and functional evaluation, we firstly found bacterial-exotoxin-derived Kunitz-type serine protease inhibitors with selectively inhibiting activity towards intrinsic coagulation pathways, and highlighted a new interaction between pathogenic bacteria and the human coagulation system.

Revealing the Diversity of Sequences, Structures, and Targets of Peptides from South China Sea Macrodactyla doreensis Based on Transcriptomics.

The South China Sea is rich in sea anemone resources, and the protein and peptide components from sea anemone toxins comprise an important treasure trove for researchers to search for leading compounds. This study conducted a comprehensive transcriptomic analysis of the tentacles and column of Macrodactyla doreensis and explored the distribution and diversity of proteins and peptides in depth using bioinformatics, initially constructing a putative protein and peptide database. In this database, typical peptide families are identified through amino acid sequence analysis, and their 3D structures and potential biological activities are revealed through AlphaFold2 modeling and molecular docking. A total of 4239 transcripts were identified, of which the putative protein accounted for 81.53%. The highest content comprised immunoglobulin and a variety of proteases, mainly distributed in the column and related to biological functions. Importantly, the putative peptide accounted for 18.47%, containing ShK domain and Kunitz-type peptides, mainly distributed in the tentacles and related to offensive predatory behavior. Interestingly, 40 putative peptides belonging to eight typical peptide families were identified, and their structures and targets were predicted. This study reveals the diversity and complexity of Macrodactyla doreensis toxins and predicts their structure and targets based on amino acid sequences, providing a feasible approach for research regarding the discovery of peptides with potentially high activity.