Ines Neundorf

Backbone rigidification is frequently used to enhance both function and stability of membrane-active peptides. However, while it does reduce the backbone flexibility, its impact depends heavily on the chosen method. In this study, we used a recently developed membrane-active peptide as candidate to evaluate ease of synthesis, impact on secondary structure, and biological activity of triazolyl- and m-xylene-bridging, and disulfide bond formation. Structural confinement was achieved solely through the triazolyl-bridge, yet the cells responded similarly to the m-xylene-bridged peptide. Our results highlight the complex relationship between the peptide, cellular membranes, and chemical derivatization.

Proteins in the mitochondrial intermembrane space (IMS) play essential roles in respiratory chain assembly, metabolism, signaling, and organelle dynamics. Their stability and functionality often depend on structural disulfide bonds introduced by the mitochondrial disulfide relay, mediated by MIA40 and ALR. In this system, the sulfhydryl oxidase ALR reoxidizes MIA40, which in turn oxidizes incoming substrate proteins. Although evidence has suggested that ALR can also act independently of MIA40, its endogenous substrates have remained unknown. In this study, we captured proteins directly oxidized by ALR. Among these, we found coproporphyrinogen III oxidase (CPOX), a key enzyme in heme biosynthesis. We show that ALR-mediated disulfide bond formation is crucial for maintaining CPOX stability in the IMS, thereby ensuring effective heme biosynthesis and mitochondrial functionality. Notably, while disulfide-deficient CPOX failed to rescue CPOX loss when localized to the IMS, it retained functionality when redirected to the cytosol. However, this bypass compromised pathway efficiency, leading to the accumulation of protoporphyrinogen IX, a highly hydrophobic and redox-active intermediate that sensitized cells to cell death. Together, our findings reveal that ALR has functions beyond the MIA pathway and highlight that oxidative protein folding in the IMS relies not only on a relay mechanism but also on a broader disulfide-introducing network of enzymes.

Before human genome sequencing, a genome-wide study of sibling centenarian pairs identified a longevity-associated locus on chromosome 4. Here, we mapped the genes in this locus and identified a collagen gene, COL25A1. Introducing an SNP linked to longevity that changes a serine predicted to be phosphorylated to leucine in COL25A1, into col-99, the C. elegans ortholog, extended lifespan. These col-99(gk694263[S106L]) SNP-mutants exhibited enhanced innate immune-related transcriptional responses, and their lifespan extension was abolished by inhibiting the p38 MAPK pathway. YAP-1, a transcriptional co-activator responsive to extracellular matrix changes, was essential for this longevity. Mechanistically, we find that this SNP modifies furin-mediated cleavage of this transmembrane collagen in vitro, and expressing the cleaved extracellular domain of COL-99 alone was sufficient to prolong C. elegans’ lifespan. These findings reveal a potential mechanism by which a human centenarian-associated SNP in COL25A1 influences furin cleavage and shedding of the collagen ectodomain to promote healthy longevity.

Background: The role of vitamin D in Coronavirus Disease 2019 (COVID-19) outcomes remains debated, but emerging evidence suggests it may enhance recovery by strengthening immune responses. Vitamin D upregulates LL-37, an antimicrobial peptide with broad antiviral activity, including potential benefits against SARS-CoV-2. LL-37’s interactions with viral proteins, however, remain incompletely understood.Methods: We investigated LL-37’s interactions with the SARS-CoV-2 Spike glycoprotein and the accessory proteins ORF7a and ORF8 using surface plasmon resonance and negative-stain electron microscopy. These approaches were employed to assess LL-37’s binding capabilities and potential impact on viral infectivity.Results: LL-37 bound multiple domains of the Spike protein and inhibited its interaction with the human angiotensin-converting enzyme 2 (hACE2) receptor in vitro. Up to seven LL-37 molecules were observed surrounding Spike, forming a halo-like structure that may block receptor engagement. LL-37 also bound to ORF7a and ORF8, potentially impairing their ability to disrupt host cell processes. Notably, LL-37’s interaction with ORF7a may prevent degradation of SNAP29, restoring autophagy and promoting viral clearance.Conclusions: LL-37 disrupts key viral-host interactions by binding to Spike, ORF7a, and ORF8, thereby reducing SARS-CoV-2 infectivity. These findings highlight LL-37’s potential as a therapeutic agent in COVID-19 and provide mechanistic insight into its antiviral actions.

The increase in antibacterial resistance is one of the greatest challenges in modern medicine, driving an urgent need to develop new drugs to combat resistant pathogens. Peptides represent a promising class of molecules that can be efficiently designed to exhibit high antimicrobial efficacy. Recently, we have highlighted how prestructuring by a triazolyl-bridge significantly enhances the activity of an antimicrobial peptide. To learn more from these findings, the aim of this study is to relate the NMR-based structure of a triazolyl-bridged peptide to its antimicrobial activity against Gram-positive and Gram-negative bacteria in comparison to its linear analogues. As we show, the triazole modification indeed induces a well-defined α-helical structure, resulting in an improved positive electrostatic surface potential on one side of the peptide and clustering of hydrophilic and hydrophobic residues on opposite surface areas of the molecule. Systematic alanine substitution further suggested that the side chains of arginine 3 and 7 and of asparagine 11 have a stronger productive impact on antimicrobial activity than those of lysine 4, 8, and 12. As shown by micelle-bound peptide structures determined by NMR, we identify arginine 3 and asparagine 11 as presumable membrane-interacting residues. Collectively, our NMR-based analysis provides evidence that an α-helical structure enhances antimicrobial activity by creating positively charged and hydrophilic, and hydrophobic areas as molecular surface signatures, potentially promoting the interaction of the peptide with the cellular target membrane.

Prenyltransferases add a lipid group to the cysteine of a CaaX motif of proteins. This posttranslational modification enables proteins to attach to membranes where they are essential hubs for signaling, trafficking, and apoptosis. Recently, cell‐permeable CaaX‐peptides are developed as possible tools to interfere with the prenylation machinery. These peptides cause cytotoxic effects, particularly in KRas mutant pancreatic cancer cells (PANC‐1) in which they also alter downstream signaling of Ras proteins. Herein, the aim is to get more clues about the relevance of the mutational status of KRas. Therefore, the activity of CaaX‐peptides in KRas wildtype BxPC‐3 and KRas mutated PANC‐1 cells is compared. CaaX‐peptides differently influence these two cell lines, although they internalize pretty much to the same extent. Indeed, an altered KRas plasma membrane localization in PANC‐1 cells is observed, probably induced by disturbed KRas prenylation based on the presence of CaaX‐peptides. The impact of CaaX‐peptides on KRas signaling is likely dependent on the KRas mutation in PANC‐1 cells in which they further trigger effects on KRas‐dependent regulators, e.g., Neurofibromin −1 (NF1) and son of sevenless homolog 1 (SOS1). All in all, CaaX peptides are identified as promising tools for studying and manipulating the function of therapeutically important prenylated proteins.

Protein S‐palmitoylation is a reversible posttranslational modification transferring the 16‐carbon fatty acid palmitate to cysteines. It plays a critical role in many cellular processes by influencing protein function, localization, stability, and protein–protein interactions and has a significant impact on various physiological and pathological conditions. This emphasizes the need to develop new technologies to study and treat diseases associated with aberrant palmitoylation. To address these challenges, cell‐permeable peptides containing an Asp–His–His–Cys (DHHC) palmitoylation motif are presented aiming to affect intracellular protein S‐palmitoylation. A small library of peptides is generated and screened for cellular uptake and cell compatibility. Interestingly, the newly designed peptides internalize to high extent into different cell lines and human breast cell spheroids dependent on their palmitoylation motif. In addition, out of this screen, DC‐2 is identified as very potent and this peptide is investigated in more detail concerning its impact on palmitoylated proteins that are connected to cancer progression. These initial explorations highlight that DC‐2 affected the localization of HRas and altered S‐palmitoylation‐related signaling cascades of epidermal growth factor receptor. These findings suggest a peptide‐driven impact on proteins having palmitoylation sites and highlight cell‐permeable DHHC peptides as a potential tool to be further evolved in the context of palmitoylation and cancer.

Recently, we presented cell‐permeable CaaX peptides as versatile tools to study intracellular prenylation of proteins. These peptides consist of a cell‐penetrating peptide (CPP) and a C‐terminal CaaX motif derived from Ras proteins and demonstrated high cellular accumulation and the ability to influence Ras signaling in cancerous cells. Here, we aimed to gain a deeper insight into how such cell‐permeable CaaX peptides, particularly the KRas4B‐derived CaaX‐1 peptide, interact with farnesyltransferase (FTase) and likely influence further intracellular processes. We show that CaaX‐1 is farnesylated by FTase ex cellulo and that an intact CaaX motif is required for modification. A competition experiment revealed a slower farnesylation of CaaX‐1 by FTase compared to a CaaX motif‐containing control peptide. CaaX‐1 inhibited farnesylation of this control peptide at considerably lower concentrations; thus, a higher affinity for FTase is hypothesized. Notably, AlphaFold3 not only predicted interactions between CaaX‐1 and FTase but also suggested interactions between the peptide and geranylgeranyltransferase type I. This finding encourages further investigation, as cross‐prenylation is a well‐known drawback of FTase inhibitors. Our results are further evidence for the usefulness of CaaX peptides as tools to study and manipulate the prenylation of proteins. They offer real potential for the development of novel inhibitors targeting the prenylation pathway.

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