N-terminal acetylation modulates peptide stability, fibrillogenesis and MMP-7 processing

Molecular mechanisms governing peptide nanodisc assembly and stability.

Apolipoprotein A-I mimetic 4F, an 18-residue amphipathic α-helix, can self-assemble with lipids to form peptide nanodiscs, yet the molecular determinants governing their assembly and stability remain poorly understood. Here, using coarse-grained molecular dynamics (CG-MD), we capture the de novo formation of 4F nanodiscs with DMPC and reveal a multistep assembly pathway involving nucleation, fusion, and ellipse-to-disc maturation. All-atom back-mapping shows that the nanodisc rim is structurally heterogeneous and stabilized by aromatic-acyl interactions, Lys headgroup anchoring, and inter-peptide electrostatic contacts. Lipid composition and temperature critically regulate nanodisc integrity: DMPC supports continuous peptide belts and long-term stability, whereas DPPC below its main phase transition temperature suppresses fusion and yields fragmented, non-uniform rims. These findings validate the ability of CG-MD to resolve nanodisc assembly mechanisms. Experimental measurements corroborate the simulations, demonstrating that 4F nanodiscs exhibit lower thermal resilience than MSP nanodiscs while retaining structural integrity at moderate temperatures. As a functional benchmark, MSP nanodiscs suppress the amyloid-binding thioflavin-T fluorescence signal associated with Aβ (1-40) fibrillar assembly, consistent with our previously reported findings for 4F nanodiscs and supporting the ability of amphipathic nanodisc rims to delay Aβ (1-40) aggregation. Together, these results establish a mechanistic framework and design principles for single-helix peptide nanodiscs and delineate the conditions under which they converge with or diverge from MSP-based scaffolds.

Purification and characterization of recombinant human type III collagen (COL3A1) fragment (C9).

Collagen, a key structural component of the extracellular matrix (ECM), plays a critical role in tissue repair and regeneration. Compared to traditional animal-derived collagen, recombinant collagen presents enhanced safety and uniformity, avoiding immunogenicity and pathogen transmission. Here, we engineered a recombinant COL3A1-derived fragment, designated C9, composed of nine tandem repeats of the Gly228-Pro281 fragment from human COL3A1. C9 was successfully overexpressed in Escherichia coli under optimized conditions (initial OD600 of 0.8, 0.5 mM IPTG, 10 h, 25 °C). C9 was purified by nickel-affinity chromatography, yielding a final concentration of 3.7 mg/mL. In vitro assays revealed that C9 exhibits substantial antioxidant activity, efficiently scavenging DPPH and ABTS radicals. Cellular assays further demonstrated that C9 exhibited low cytotoxicity and favorable cytocompatibility. In addition, C9 significantly promoted NIH/3T3 cell proliferation, adhesion, and migration. Collectively, these findings highlight the preliminary biological activity of C9 and support its further investigation as a recombinant collagen-derived biomaterial.

Prolyl 4-hydroxylases in cancer: Molecular mechanisms and therapeutic perspectives

Prolyl 4-hydroxylases (P4Hs) are important regulators of the extracellular matrix in cancer by promoting collagen deposition. These enzymes catalyze the hydroxylation of proline residues in collagen, facilitating the formation of its triple-helical structure and enhancing thermal stability. However, several collagen-independent mechanisms of P4Hs have recently been identified in tumor pathogenesis. The P4H family includes catalytic P4HA isoforms and the chaperone protein P4HB/protein disulfide isomerase, each exhibiting distinct tumor type-specific expression patterns. For instance, P4HA1 has been reported to promote glycolytic reprogramming, at least partly through indirect stabilization of hypoxia-inducible factor 1α. P4Hs can hydroxylate a variety of proteins, thereby modulating their functions. Additionally, P4H isoforms may also interact with post-translational regulatory networks, both as modifiers of selected substrates and as proteins that are subject to regulatory modifications themselves. Furthermore, these enzymes are involved in regulating cell death, including ferroptosis and cuproptosis, as well as modulating endoplasmic reticulum stress responses. The activity of P4Hs is tightly regulated by complex networks of upstream transcription factors and downstream signaling pathways. This review summarizes both collagen-dependent and collagen-independent mechanisms of P4H-related proteins and evaluates their potential associations with tumor prognosis and immunotherapy response based on current preclinical and translational evidence. Finally, the therapeutic potential of targeting P4H-related pathways is discussed, together with key limitations, including limited isoform selectivity, off-target effects, disruption of normal collagen homeostasis, and insufficient clinical validation. Emerging targeted-delivery and microenvironment-responsive strategies are also considered as potential approaches to improve spatial specificity and therapeutic safety.

Beyond proteolysis: rational modification of mucin-derived peptidomimetics with enhanced metal-mediated antimicrobial activity

Mucin-derived peptides constitute attractive antimicrobial candidates, but their clinical application is restricted by limited stability and moderate efficacy. To address these limitations, we modified d-amino-acid-containing peptidomimetics and investigated their Cu(ii) and Zn(ii) complexes with respect to coordination chemistry, structure, proteolytic resistance, and antimicrobial activity. Potentiometric, spectroscopic, and DFT studies revealed that metal binding donor sets are analogous to those of the native peptide, producing only minor local conformational effects without significant global structural rearrangement, as confirmed by circular dichroism analysis. In contrast to the modest structural changes, biological activity was strongly influenced by chirality and metal coordination. The fully d-configured analogue displayed the highest antimicrobial potency, particularly at pH 5.5, and its Zn(ii) and Cu(ii) complexes showed enhanced antibacterial and antifungal effects relative to the native system. Proteolytic assays demonstrated rapid plasma degradation of the native peptide and the partially modified analogue, whereas the fully d-substituted peptidomimetic remained largely intact after 2 h. All compounds exhibited minimal hemolytic and cytotoxic effects. These findings demonstrate that d-amino-acid incorporation combined with metal coordination significantly improves both enzymatic stability and antimicrobial performance of mucin-derived peptides.

Structural Fine-Tuning and Gel-Forming Properties of Peptides Containing Aromatic Amino Acid Residues

Rational Tuning of Intracellular Release Kinetics in PROTAC–Peptide Conjugates via Positional Modulation of Functional Peptide Domains

Proteolysis-targeting chimeras (PROTACs) can induce degradation of otherwise challenging proteins, but their large size and unfavorable physicochemical properties often limit cellular delivery. We designed PROTAC–peptide conjugates containing a nona-arginine cell-penetrating peptide (R9), a redox-sensitive disulfide linker, and an aromatic-rich peptide sequence previously described as an endosomal escape domain (EED). The EED sequence was positioned at either the N- or C-terminus to examine how peptide-domain arrangement affects intracellular fluorescence, payload release, and BRD4 degradation. At 10 and 100 nM, the C-terminal variant MZ1-R9-EED produced a greater reduction in BRD4 abundance at 24 h than MZ1-R9 and MZ1-EED-R9, although the two-concentration comparison did not permit determination of DC50, Dmax, or hook effects. FAM-based flow cytometry yielded pH-sensitive cell-associated fluorescence signals that could not be interpreted as an absolute measure of total cellular uptake. Qualitative confocal microscopy showed punctate fluorescence with apparent LysoTracker overlap but did not provide quantitative evidence of endosomal escape. Under a defined cell-free reducing condition, MZ1-R9-EED underwent faster apparent disulfide cleavage, with a half-life of 0.21 h versus 0.36–0.47 h for the other conjugates, and yielded higher apparent intracellular free MZ1 levels under identical analytical conditions. Square-wave voltammetry revealed no substantial differences in reduction potential, whereas molecular dynamics simulations suggested a possible contribution of local steric accessibility. These findings associate peptide-domain positioning with cleavage kinetics, free-payload levels, and BRD4 reduction, identifying domain arrangement as a design variable for disulfide-linked PROTAC–peptide conjugates without establishing cleavage as the rate-limiting or causal mechanism.