Biology subjects

Sieber, S. A.

Publications and source records attributed to Sieber, S. A..

7 recordsLinked to original sources

bioRxiv2026

Mitochondrial protein import couples proteostasis failure to mitochondrial permeabilization

Proteostasis failure is a hallmark of stress and disease, yet how it compromises mitochondrial integrity remains unclear. Here, we identify mitochondrial protein import as a critical pathway linking proteostasis failure to mitochondrial injury. We show that Raptinal, previously characterized as a rapid inducer of apoptosis, impairs the folding of newly synthesized proteins rather than directly disrupting mitochondrial membranes. The resulting proteotoxic stress drives mitochondrial outer membrane permeabilization and intrinsic apoptosis independently of BCL-2 family pore-forming proteins. VBIT4, a compound commonly used to maintain mitochondrial integrity, inhibited this pathway, and chemical proteomics with a photoaffinity analogue implicated the TIM23 import machinery. Genetic or pharmacological inhibition of the TIM23-PAM axis suppressed mitochondrial permeabilization without affecting canonical BAX-BAK-dependent apoptosis. These findings establish that mitochondrial protein import couples translation-associated proteotoxic stress to mitochondrial injury and identify regulation of import flux as a determinant of mitochondrial integrity during proteostasis failure.

cell biology↗

bioRxiv2026

Decoding antibiotic modes of action from multimodal cellular responses

Antibiotic resistance continues to rise, yet most new drug candidates act through long-established targets. Faster mode of action (MoA) assessment would enable more effective prioritization of screening hits and help identify compounds with novel mechanisms. In this study, we aimed to develop a scalable framework for MoA inference from antibiotic-induced cellular response profiles in Escherichia coli. We generated a multimodal dataset spanning more than 50 antibiotics, including proteome profiles, chemical structure descriptors, inhibitory concentrations and growth dynamics, and used it to build MAPPER (Mode of Action Prediction via Proteomics-Enhanced Representation), a framework comprising a fixed multimodal predictor and an uncertainty module. MAPPER accurately classified antibiotics across nine mechanistic classes, flagged compounds with likely novel mechanisms and retained predictive power in proteomics-only transfer experiments across mass spectrometry platforms and external data. Together, these results establish MAPPER as an innovative tool for MoA prediction and novelty detection, enabling prioritization of antibacterial candidates with distinct mechanisms.

bioinformatics↗

bioRxiv2024

Cystobactamid off-target profiling reveals favorable safety, superoxide reduction, and SCARB1 inhibition in eukaryotes

Antimicrobial resistance (AMR) poses a fundamental global threat, necessitating new strategies for effective therapies. Cystobactamids (CYS), a class of antibacterial agents targeting bacterial gyrase and topoisomerase IV, represent a non-traditional chemical scaffold with broad-spectrum activity. For toxicological de-risking, we performed a comprehensive profiling on eukaryotic cells, focusing on cytotoxicity, genotoxicity, and mitochondrial toxicity, demonstrating cellular safety and superoxide scavenging properties. Studies in zebrafish embryos assessed developmental, cardiovascular, and hepatic toxicity, indicating a favorable in vivo safety profile. Metabolism studies revealed glucuronidation and amide bond hydrolysis as key pathways, whereby CYS metabolic stability substantially improved by cobicistat co-treatment. Affinity-based protein profiling identified the cholesterol- and HCV-receptor scavenger receptor class B member 1 (SCARB1) as a primary eukaryotic off-target protein, with cystobactamids shown to inhibit SCARB1s function, preventing hepatitis C virus pseudoparticle entry into cells. These findings suggest a high therapeutic potential for cystobactamids and highlight SCARB1 as a primary eukaryotic target.

pharmacology and toxicology↗

bioRxiv2024

Helicobacter pylori gamma-glutamyltransferase relates to proteomic adaptions important for gastric colonization

Helicobacter pylori {gamma}-glutamyltransferase (gGT) is a virulence factor that promotes bacterial colonization and immune tolerance. Although some studies addressed potential functional mechanisms, the supportive role of gGT for in-vivo colonization remains unclear. Additionally, it is unknown how different gGT expression levels may lead to compensatory mechanisms ensuring infection and persistence. Hence, it is crucial to unravel the in-vivo function of gGT. We assessed acid survival under conditions mimicking the human gastric fluid and elevated the pH in the murine stomach prior to H. pylori infection to link gGT-mediated acid resistance to colonization. By comparing proteomes of gGT-proficient and -deficient isolates before and after infecting mice, we investigated proteomic adaptations of gGT-deficient bacteria during infection. Our data indicate that gGT is crucial to sustain urease activity in acidic environments, thereby supporting survival and successful colonization. Absence of gGT triggers expression of proteins involved in the nitrogen and iron metabolism and boosts the expression of adhesins and flagellar proteins during infection, resulting in increased motility and adhesion capacity. In summary, gGT-dependent mechanisms confer a growth advantage to the bacterium in the gastric environment, which renders gGT a valuable target for the development of new treatments against H. pylori infection. Author SummaryH. pylori {gamma}-glutamyltransferase (gGT) is a virulence factor that strongly supports bacterial colonization. Despite considerable research on the function of gGT, the exact role of this enzyme in ensuring in-vivo infection remained elusive. We developed a novel system that allowed us to selectively inhibit gGT-activity and used this model to assess the function of gGT in the gastric environment. We found that gGT sustains urease activity in acidic environments thereby facilitating survival and effective colonization. In addition, we identified several compensatory mechanisms triggered by the loss of gGT which ensure colonization and persistence. These mechanisms included increased flagellar motility, adhesion capacity and expression of proteins involved in the nitrogen and iron metabolism. These findings unraveled novel functional roles of gGT important for bacterial colonization and thereby confirmed gGT as a promising target for novel treatments against H. pylori infection. By comprehensively addressing the compensatory mechanisms resulting from the loss of gGT-activity, the success of such new treatments can be improved.

microbiology↗

bioRxiv2022

Antibiotic acyldepsipeptides stimulate the Streptomyces Clp-ATPase/ClpP complex for accelerated proteolysis

Clp proteases consist of a proteolytic, tetradecameric core ClpP and AAA+ Clp-ATPases. Streptomycetes, producers of a plethora of secondary metabolites, encode up to five different ClpP homologs and the composition of their unusually complex Clp protease machinery has remained unsolved. Here, we report on the composition of the house-keeping Clp protease in Streptomyces, consisting of a hetero-tetradecameric core built of ClpP1, ClpP2 and the cognate Clp-ATPases ClpX, ClpC1 or ClpC2, all interacting with ClpP2 only. ADEP antibiotics dysregulate the Clp protease for unregulated proteolysis. We observed that ADEP binds Streptomyces ClpP1, but not ClpP2, thereby not only triggering the degradation of non-native protein substrates but also accelerating Clp-ATPase-dependent proteolysis. The explanation is the concomitant binding of ADEP and Clp-ATPases to opposite sides of the ClpP1P2 barrel, hence revealing a third, so far unknown mechanism of ADEP action, i.e., the accelerated proteolysis of native protein substrates by the Clp protease. SignificanceClp proteases are antibiotic and anti-cancer drug targets. Composed of the proteolytic core ClpP and a regulatory Clp-ATPase, the protease machinery is important for protein homeostasis and regulatory proteolysis. The acyldepsipeptide antibiotic ADEP targets ClpP and has shown promise for treating multi-resistant and persistent bacterial infections. The molecular mechanism of ADEP is multi-layered. Here, we present a new way how ADEP can deregulate the Clp protease system. Clp-ATPases and ADEP bind to opposite sides of Streptomyces ClpP, accelerating the degradation of natural Clp protease substrates. We also demonstrate the composition of the major Streptomyces Clp protease complex, a heteromeric ClpP1P2 core with the Clp-ATPases ClpX, ClpC1 or ClpC2 exclusively bound to ClpP2, and the killing mechanism of ADEP in Streptomyces.

microbiology↗

bioRxiv2021

Listeria monocytogenes utilizes the ClpP1/2 proteolytic machinery for fine-tuned substrate degradation under heat stress

Listeria monocytogenes exhibits two ClpP isoforms (ClpP1/ClpP2) which assemble into a heterooligomeric complex with enhanced proteolytic activity. Herein, we demonstrate that the formation of this complex depends on temperature and reaches a maximum ratio of about 1:1 at heat shock conditions, while almost no complex formation occurred below 4{degrees}C. In order to decipher the role of the two isoforms at elevated temperatures, we constructed L. monocytogenes ClpP1, ClpP2 and ClpP1/2 knockout strains and analyzed their protein regulation in comparison to the wild type (WT) strain via whole proteome mass-spectrometry (MS) at 37 {degrees}C and 42 {degrees}C. While {Delta}clpP1 strain only altered the expression of very few proteins, {Delta}clpP2 and {Delta}clpP1/2 strains revealed the dysregulation of many proteins at both temperatures. These effects were corroborated by crosslinking co-immunoprecipitation MS analysis. Thus, while ClpP1 serves as a mere enhancer of protein degradation in the heterocomplex, ClpP2 is essential for ClpX binding and thus functions as a gatekeeper for substrate entry. Applying an integrated proteomic approach combining whole proteome and co-immunoprecipitation datasets, several putative ClpP2 substrates were identified in the context of different temperatures and discussed with regards to their function in cellular pathways such as the SOS response.

biochemistry↗

bioRxiv2019

FICD activity and AMPylation remodelling modulate human neurogenesis.

Posttranslational modification (PTM) of proteins represents an important cellular mechanism for controlling diverse functions such as signalling, localisation or protein-protein interactions1. AMPylation (also termed adenylylation) has recently been discovered as a prevalent PTM for regulating protein activity2. In human cells AMPylation has been exclusively studied with the FICD protein3-6. Here we investigate the role of AMPylation in human neurogenesis by introducing a cell-permeable propargyl adenosine pronucleotide probe to infiltrate cellular AMPylation pathways and report distinct modifications in intact cancer cell lines, human-derived stem cells, neural progenitor cells (NPCs), neurons and cerebral organoids (COs) via LC-MS/MS as well as imaging methods. A total of 162 AMP modified proteins were identified. FICD-dependent AMPylation remodelling accelerates differentiation of neural progenitor cells into mature neurons in COs, demonstrating a so far unknown trigger of human neurogenesis.

biochemistry↗