Hydroxamate groups provide a unique opportunity to promote amide-bond formation between unprotected peptide fragments through intramolecular O-acyl capture and O→N acyl transfer. Here, we report a general ligation strategy that exploits the reactivity of backbone N-hydroxy peptide (NHP) fragments to achieve efficient couplings with peptide thioesters in aqueous buffer. NHP ligation is broadly compatible with β-branched, aromatic, charged, and N-methylated residues at the nucleophilic N-terminus. Preparative-scale ligation and subsequent N─O bond reduction furnish products with a restored native peptide bond. The versatility of this method is demonstrated through the convergent synthesis of two miniproteins whose folding and thermal stability are modulated by backbone N-hydroxylation, and in the synthesis of the macrocyclic NHP natural product talarolide A. These studies establish backbone NHP ligation as a robust and widely applicable strategy for peptide and protein synthesis.

Background: Age-related macular degeneration (AMD) is a leading cause of vision loss and is strongly associated with mitochondrial dysfunction in retinal pigment epithelial cells. Mitochondrial-derived peptides, including Humanin and its analogs, have demonstrated cytoprotective effects in AMD-related cellular models. However, the effects of shorter Humanin-derived fragments in disease-specific mitochondrial models remain incompletely characterized. Methods: Transmitochondrial retinal pigment epithelial cybrid cell lines containing mitochondria from AMD patients or age-matched normal donors were treated with HNF14, a 14-amino acid Humanin fragment peptide. Cellular metabolic activity, cytotoxicity, oxidative stress, apoptotic signaling, inflammatory markers, angiogenic factor expression, and amyloid-β1-42-induced apoptosis were evaluated using biochemical assays, protein analyses, and live-cell imaging approaches. Results: HNF14 treatment was associated with improved metabolic activity and reduced cytotoxicity in AMD cybrids, with minimal effects in normal cybrids. HNF14 significantly reduced intracellular and mitochondrial oxidative stress, suppressed apoptotic and inflammatory markers, and decreased VEGF-A protein expression in AMD cybrids. In addition, HNF14 attenuated amyloid-β1-42-induced apoptotic signaling in AMD cybrids. These effects were selective for cybrids containing AMD-derived mitochondria. Conclusions: This study demonstrates that HNF14 mitigates mitochondrial and cellular stress responses in AMD transmitochondrial cybrid cells. The findings indicate that a short Humanin-derived fragment retains cytoprotective activity in a disease-specific mitochondrial context and support further investigation of mitochondrial-derived peptides as modulators of mitochondrial dysfunction relevant to AMD pathophysiology.

Sactipeptides area class of natural product peptides with remarkableantibiotic properties that are defined by the presence of thioaminoketalsin their structure. Recently, we reported the first total synthesisof a sactipeptide in our synthesis of enteropeptin A. The key to oursynthesis involved the use of a dithiophosphoric acid catalyzed Markovnikovhydrothiolation of dehydroamino acids. With this reaction, thioaminoketalsfound in sactipeptides can be prepared directly from a dehydroaminoacid and a cysteine residue. This article summarizes our initial approachtoward enteropeptin synthesis and the evolution of our strategy thatultimately enabled the synthesis of these peptide natural products.Our first strategy involved late-stage Markovnikov hydrothiolationof an 8-mer peptide containing a dehydroamino acid and a cysteineresidue that was unsuccessful. The second strategy involved an annulationreaction between a methyl ester of a dehydroamino acid and a cysteinewith an unprotected amine that forged the central thiomorpholine ringalbeit in low yield. The third strategy involved a divergent synthesisof the enteropeptins by early stage formation of the thiomorpholinering by Markovnikov hydrothiolation followed by amidative couplingof the N- and C-terminal peptide fragments. This modular strategyenabled the unified synthesis of the enteropeptin sactipeptides.

Effective glycopeptide identification with tandem mass spectrometry (MS/MS) often relies on both low mass-to-charge (m/z) ions derived from glycan-specific oxonium ions and higher m/z peptide fragment ions that retain glycan modifications. Thus, glycoproteomic experiments benefit from a wider MS/MS scan range, i.e., the breadth of m/z values measured in fragmentation spectra, than those typically used in nonmodified peptide analyses. Here, we explore the implications of breaking a common axiom for scan range settings called the "5-10-15 rule" used to maximize transmission of ion populations of interest. This 5-10-15 rule, which defines the upper m/z value for a scan as a multiple of the first m/z value, comes from fundamental requirements for stable ion trajectories, where voltage settings must balance retention of low m/z ions while also generating effective pseudopotential wells to trap high m/z ions. Adhering to this calculation for MS/MS scan range settings can reduce glycopeptide ion coverage by excluding the analysis of either low m/z oxonium ions or high m/z fragment ions. We use a quadrupole-Orbitrap-linear ion trap Tribrid MS system (Orbitrap Ascend) to investigate the implications of following or breaking the 5-10-15 rule in MS/MS scans for glycopeptide characterization with higher-energy collisional dissociation (HCD), electron-transfer dissociation (ETD), and electron-transfer/higher-energy collision dissociation (EThcD). For scans with a first m/z value around m/z 120 (i.e., capturing most common glycan-specific oxonium ions), we show that breaking the 5-10-15 rule does not lead to a significant loss of fragment ion transmission at either extreme of the m/z range. We use this case study to discuss the concepts important to using the 5-10-15 rule wisely and when it can be practically ignored, such as using large scan ranges to improve glycopeptide characterization.

Aromaticity, hydrogen bonding, and metal cofactors are fundamental interactions governing the structure, stability, and function of biomolecular and catalytic systems. Their accurate computational representation remains a major challenge due to the combined influence of electron delocalization, polarization effects, and complex quantum mechanical behavior, particularly in transition-metal environments. Classical molecular mechanics force fields, while computationally efficient, fail to capture these phenomena reliably, motivating the development of quantum mechanical (QM), hybrid QM/MM, and machine-learning (ML) enhanced approaches. This article systematically reviews recent advances in the modelling of aromatic stabilization, hydrogen-bonding dynamics, and metal–ligand coordination using density functional theory (DFT), multi-scale QM/MM simulations, and modern ML potentials. Benchmark systems including aromatic hydrocarbons, hydrogen-bonded clusters, peptide fragments, and biologically relevant metal complexes were analyzed using dispersion-corrected DFT functionals and ML-based force fields trained on high-level QM datasets. Validation metrics such as interaction energies, geometric parameters, aromaticity indices, hydrogen-bond lifetimes, and metal-coordination stability were employed to assess predictive performance. The results demonstrate that modern DFT methods accurately reproduce electronic delocalization and interaction energetics, while QM/MM techniques effectively capture environmental effects in large biomolecular systems. Machine-learning potentials achieve near-QM accuracy at substantially reduced computational cost, showing strong performance for aromatic systems and hydrogen-bond networks, though challenges remain for redox-active metal centers and multi-reference electronic states. Overall, the study highlights that no single modelling strategy is universally optimal. Instead, integrated hybrid frameworks combining QM accuracy, ML efficiency, and classical scalability offer the most promising pathway toward predictive and interpretable simulations. Future progress will depend on metal-inclusive training datasets, physics-informed ML architectures, and improved treatment of polarization and electronic correlation to enable robust modeling across complex chemical space.

Peptide therapeutics are becoming an increasingly important class of pharmaceuticals due to their attractive therapeutic properties. However, their development remains challenging because of inherent structural complexity, requiring advanced analytical techniques. Peptide mapping is a key method for comprehensive identification of a peptide's primary structure, and FDA and EMA recommend its use to demonstrate structural "sameness." In this study, we describe a multienzyme peptide-mapping workflow designed to improve sequence coverage and structural verification. The protocol integrates multienzymatic digestion with complementary specificity, enabling generation of alternative peptide maps. Waters XBridge Peptide BEH C18 column (300 Å, 2.5 μm, 4.6 × 150 mm) with gradient separation program was used for separation. Further, LC-HRMS (Orbitrap) was employed to obtain high-resolution MS/MS data, achieving precise peptide fragment identification with mass accuracy within < 5 ppm. Two antidiabetic peptides, exenatide and H-GLP-1, were selected as model systems. Full sequence coverage of both peptides was achieved by combining peptide maps produced with trypsin, Glu-C, and chymotrypsin. Application of the workflow to a GLP-1 analog provided > 95% sequence coverage and accurate intact-mass confirmation. Overall, the described method offers a reliable and stepwise approach for peptide-mapping-based structural verification, enabling confident assessment of the primary structure of exenatide and H-GLP-1.

Hypertension is a globally prevalent chronic cardiovascular disease, with angiotensin-converting enzyme (ACE) serving as a key target for therapeutic intervention. Synthetic ACE inhibitors have side effects, making natural food-derived ACE-inhibitory peptides a research hotspot owing to safety advantages. Softshell turtle (Pelodiscus sinensis) bone collagen (STBC) has potential bioactivity, but its ACE-inhibitory peptides have not been systematically investigated. This study used computer-aided screening: STBC α1(I) (K7FHL1) and α2(I) (K7G8R1) sequences from UniProt were processed via SignalP 5.0. BIOPEP-UWM analysis showed ACE-inhibitory peptide frequencies of 0.8947 and 0.9261 in the two chains, confirming STBC as a high-quality precursor. Papain-ficin was selected as the optimal enzymatic system via simulation; 105 potential novel peptides were obtained after toxicity/allergenicity prediction. Twenty-seven highly active peptide fragments were screened out via pLM4ACE, and four peptide fragments with relatively high binding energy (QICVCDS, DVWK, IIEY, APMDVG) were identified through molecular docking. These peptides (molecular weight: 536.6-766.9 Da) possessed excellent physicochemical properties and pharmacokinetic characteristics, while bioinformatics analysis revealed that they could target and regulate SRC/HSP90AA1 to modulate the renin-angiotensin system (RAS). This study provides an efficient strategy for the high-value utilization of softshell turtle resources and the development of food-derived ACE-inhibitory peptides.

Interactions between proteins and metal cations are central to biochemical processes and shape protein structures. SilE, an intrinsically disordered protein involved in bacterial silver resistance, folds into α-helices upon binding Ag+ ions. Focusing on the B1 peptide fragment from SilE, we investigate the mechanism of Ag+-induced folding with atomistic simulations and experiments. Guided by Mass Spectrometry and NMR, we prepare a structural model of Ag+-bound B1, which we parametrize using DFT. Then, with replica-exchange simulations and deep learning, we map B1's folding landscape and how it is shaped by Ag+. Specifically, Ag+ binding promotes folding by lowering the entropy of the disordered state and stabilizing the folded state. We also describe how Ag+ alters the folding pathways. Overall, we improve our understanding of metal-induced protein folding and lay the groundwork for further computational investigations of the bacterial silver-resistance machinery.

Accurate disease diagnosis requires analyzing multiple biomarkers in a single sample to improve diagnostic precision and guide treatment. However, the simultaneous detection of three or more proteases in a sample, especially with a single probe, remains a major challenge. Herein, we developed a triple-response peptide probe for monitoring three proteases via a single aerolysin nanopore. The T-shaped probe contains three independent arms each with a target-specific cleavage sequence. Upon protease recognition, short peptide fragments are released and generate characteristic current signatures during nanopore translocation. The enzymes themselves are sterically excluded, ensuring signals originate solely from cleavage products. Each peptide yields a distinct signature, enabling simultaneous, label- and amplification-free quantification with detection limits extending to the femtomolar range. Integrated with a machine learning classifier, the system achieves 97.7% accuracy in signal discrimination. This strategy enables accurate and multiplexed quantification of multiple enzymes in complex biological fluids, such as serum, which is important for disease diagnosis based on liquid biopsies.

Cyclic peptide-polymer conjugates offer a unique biocompatible system with many advantages but come at the cost of being analytically challenging. Developing further analytical techniques of complex polymer-conjugate systems is key to understanding synthetic and medicinal properties. In this contribution, a synthetic cyclic peptide-polymer conjugate is analyzed using electron capture dissociation (ECD), infrared multiphoton absorption dissociation (IRMPD), and 193 nm ultraviolet photodissociation (UVPD) on the same mass spectrometry system. IRMPD and UVPD were shown to effectively characterize unconjugated cyclic peptide species. ECD was less informative during cyclic peptide analysis due to the production of multiple sequence scrambling fragments and radical side chain losses. ECD was shown to produce extensive fragmentation and enable the characterization of conjugated side chains of cyclic species. ECD and IRMPD thus provided complementary data, enabling the target analysis of conjugated systems. UVPD effectively characterized both the cyclic peptide and the conjugating polymer in one experiment, being able to produce complete cyclic peptide fragmentation via b/y fragment pathways and polymer fragmentation via a/x poly(2-ethyl-2-oxazoline) fragment pathways.

RNF4, a RING-type E3 ubiquitin ligase, targets polySUMOylated proteins for ubiquitination and subsequent proteasomal degradation. The ability to chemically synthesize RNF4 will enable future studies of its structure and biological function, particularly its role in degrading the oncoprotein PML-RARα in acute promyelocytic leukemia. To achieve this, we performed a total chemical synthesis of RNF4 using sequential native chemical ligation. The presence of nine cysteine residues enables stepwise ligation of five peptide fragments to assemble the full-length protein. Two synthetic strategies were explored: the first employed a convergent C-to-N ligation, while the second used an N-to-C ligation. In the convergent C-to-N approach, cysteine residues were protected with acetamidomethyl groups to prevent side reactions during ligation, although this required multiple deprotection and purification steps. Conversely, the N-to-C synthesis method proceeded efficiently without cysteine protection, thereby simplifying the workflow and reducing the number of purification steps. This research presents a reliable and accessible method for the complete chemical synthesis of RNF4, addressing significant challenges in synthesizing large proteins and opening up new opportunities for future biological research.

BackgroundClinically, phosphorylation of Tau protein at threonine 181 (p-Tau181) acts as a crucial biomarker for AD detection. However, the mechanisms through which Tau phosphorylation at threonine (Thr)181 site leads to Tau aggregation and corresponding neuropathological changes remain unclear.ObjectiveTo investigate the effect of the phosphorylated tau peptide at Thr181 on tau aggregation, synaptic and cognitive impairments.MethodsWe synthesized the phosphorylated tau peptide Tau-pT181 and the non-phosphorylated tau peptide Tau-nT181, and verified their effects on the aggregation of the Tau repeat domain R3 fragment peptide and Tau pathology.ResultsThioflavin S assay showed that Tau-pT181 significantly promoted the aggregation of R3, whereas Tau-nT181 did not induce R3 aggregation. Moreover, Tau-pT181 not Tau-nT181 led to the aggregation of Tau protein in 293/tau cells and decreased synapse-associated proteins in primary hippocampal neurons. One month after injecting Tau-pT181 and Tau-nT181 respectively into the rat CA1 hippocampal region, we found that exclusively the phosphorylated peptide Tau-pT181 induced endogenous Tau aggregation, synaptic damage, neuronal loss, while Tau-pT181 group exhibited significant cognitive impairment compared with the normal saline rats. Transcriptome analysis of neurons differentiated from iPSCs treated with Tau-pT181/Tau-nT181 suggest that phosphorylated peptides had a greater impact on axonogenesis, neuronal development, and Wnt signaling pathway.ConclusionsThe present study offers the first direct evidence that Tau phosphorylation at Thr181 induces Tau aggregation, and Tau-pT181 directly leads to neuropathological alterations and cognitive impairments, and establishes a new theoretical foundation for Tau Thr181 phosphorylation site as a core diagnostic marker and therapeutic target for AD.

Structured Entropy Analysis (SEA): A computational framework for latent biomolecular insights beyond conventional descriptors in molecular dynamics simulations.

Rational design of peptide-based programmed cell death 1 immune checkpoint inhibitors using advanced integrated computational approach.

Feather waste was valorised into bioactive peptides by extracting high-purity L-Cys-urea solution, followed by enzymatic hydrolysis with commercial proteases selected for their catalytic efficiency. The extraction reduced the α-helix content in favor of the β-sheet leading to less ordered crystal structures as ATR FT-IR and the XRD analyses revealed. These structural changes decreased keratin's thermal stability, resulting in its slower yet more gradual decomposition, as TGA and DSC results showed. The hydrolysis with trypsin, chymotrypsin, pepsin and subtilisin altered the secondary structure and thermal stability of extracted keratin, with changes depending on enzyme specificity. All hydrolysates showed the preserved amide features but reduced α-helix and β-sheet content and crystallinity. Subtilisin induced the most profound structural and molecular transformations, including the secondary structure loss, carboxylate group formation, and short peptide fragment generation, resulting in reduced structural order but enhanced thermal stability attributed to the stabilizing effect of sodium carboxylates.

COG133 ameliorates depression-like behaviors in mice by inhibiting p38 MAPK-mediated neuroinflammation.

Gluten protein generates an immunogenic 33-mer peptide upon incomplete digestion, leading to induced cytotoxic and inflammatory responses in intestinal epithelial cells of susceptible individuals with Celiac disease (CeD). The present study investigates the protective effects of gluten hydrolyzing probiotic strains-Bacillus subtilis AJG10 and Bacillus tequilensis AJG23 on gliadin and 33-mer peptide in the Caco-2 cell model. The effect of probiotics on further degradation of 33-mer peptide and pepsin-trypsin digested gliadin was analysed using SDS-PAGE and mass spectrometry. The effect of all fractions on Caco-2 cell viability, nitric oxide (NO) production, and expression of pro-inflammatory cytokines was evaluated. A significant degradation of immunogenic peptide fragments after probiotic treatments resulted in 44%-53% reduction in NO production and a decrease of 27%-37% in cytokine expression and overall improvement in cell viability. The results suggested the potential of probiotic interventions in reducing the toxic effects of digested gliadin and immunogenic peptides.

Parasporin PS2Aa1, recently designated as Mpp46Aa1, is recognized for its selective anticancer activity against various human cell lines. In this study, specific regions of the native protein were fragmented, and targeted amino acid substitutions were introduced to improve cytotoxic selectivity and potency. The modified fragments were evaluated individually and in combination with curcumin, a polyphenol with well-documented anticancer properties, and Sacha inchi-derived matrices, known for their antioxidant and antiproliferative activities. Experimental results demonstrated that the substituted variant designated T104L-G108W exhibited superior anticancer activity compared to the native peptide P102-K11. Synergism assays revealed that curcumin-bioconjugated peptides were more effective against the tested cell lines, whereas combinations with Sacha inchi reduced cytotoxicity, suggesting possible interference in the mechanisms of action. Functional assays, including caspase 3/7 and 9 activation, Annexin V-Cy3 staining, and cell viability analysis with 6-CFDA, confirmed increased sensitivity in SiHa and HeLa cell lines, particularly for peptide T104L-G108W. Collectively, these findings support the effectiveness of a substitution-based strategy in improving parasporin fragments and underscore the therapeutic potential of peptide T104L-G108W as a novel anticancer candidate. Furthermore, this study provides preliminary evidence that natural biomolecules can be optimized through targeted modifications and rational combinations, establishing a framework for the development of sustainable and selective therapeutic approaches in cancer treatment.

ABSTRACT Bacterial collagen‐like proteins (CLPs) are composed of tandem Gly‐ X ‐ Y sequence repeats, but, unlike metazoan collagens, their X‐ and Y‐ positions are enriched in hydrophobic and polar residues rather than proline. This distinctive sequence bias suggests that CLPs may harbor sequence features that could promote amyloid‐like assembly. Using bioinformatic screening of CLPs, we identified amyloidogenic motifs enriched in alanine, isoleucine, leucine, or valine at the X‐ position and threonine at the Y‐ position. Guided by these findings, we designed a focused library of peptides incorporating Gly‐ X ‐Thr triplet and the highly abundant GATGVT sextet repeats. Peptides containing Gly‐ X ‐Thr produced insoluble fibers, restricting exploration of material properties. In contrast, peptides containing GATGVT repeats formed micrometer‐long fibers that physically crosslinked into a robust hydrogel upon centrifugation. Circular dichroism (CD) and Fourier Transform Infrared Spectroscopy (FTIR) revealed length‐dependent variations in secondary structure. Imaging of the higher‐order structures confirmed densely packed fiber networks while rheological measurements indicated sequence‐length‐dependent viscoelastic behavior. Notably, a 30 residue GATGVT peptide hydrogel supported high in vitro cell viability of fibroblasts. Overall, these findings suggest that CLPs are an underexplored reservoir of sequence motifs with promising biomaterial potential.

Polycystic ovary syndrome (PCOS) is a complex and heterogeneous metabolic disorder that affects 6-20% of women of reproductive age. However, research on the lactylation-modified proteome in PCOS remains limited. This study included 30 patients with PCOS and 30 control subjects, all of whom underwent intracytoplasmic sperm injection or in vitro fertilization-embryo transfer treatments at the fertility center between October 2022 and May 2023. A 4-dimensional label-free proteomic quantitation method was applied to analyze enzymatically digested peptide fragments of granulosa cell proteins. Liquid chromatography-mass spectrometry was used for protein identification and quantification. Bioinformatics analysis of differentially expressed proteins (DEPs) and differentially lactylated proteins identified 1057 DEPs between the two groups. Among these, 478 proteins were upregulated, and 579 were downregulated in the PCOS group. Regarding lactylation modifications, 668 proteins exhibited increased lactylation levels, while 1059 proteins indicated decreased lactylation levels in the PCOS group. Additionally, site-level analysis revealed 1041 upregulated and 2143 downregulated lactylation sites in the PCOS group. This study provides a comprehensive quantitative overview of proteomic and lactylation-modified proteomic expression profiles in granulosa cells from patients with PCOS, offering novel insights into PCOS research. Further research is needed to clarify the specific roles of protein lactylation in PCOS pathogenesis.