Biology subjects

Otten, C.

Publications and source records attributed to Otten, C..

3 recordsLinked to original sources

bioRxiv2023

ACRC/GCNA is an essential protease for the repair of DNA-protein crosslinks during vertebrate development

DNA-protein crosslinks (DPCs) are toxic DNA lesions that block all DNA transactions including replication and transcription, and the consequences of impaired DNA-Protein Crosslink Repair (DPCR) are severe. At the cellular level, impaired DPCR leads to the formation of double strand breaks, genomic instability and cell death, while at the organismal level, it is associated with cancer, aging and neurodegeneration. Despite its importance, the mechanisms of DPCR at the organismal level are largely unknown. Proteases play a central role in DPCR, as they remove proteinaceous part of the DPCs, while the peptide remnant crosslinked to DNA is subsequently removed by other repair factors. We characterized the role of putative protease ACRC/GCNA (ACidic Repeat Containing/Germ Cell Nuclear Antigen) in DPCR at the organismal level. For this purpose, we have created new animal models with CRISPR/Cas system: two zebrafish lines with inactive Acrc. We were able to overcome the early embryonic lethality caused by Acrc inactivation by injecting Acrc-WT mRNA and have created a viable animal model to study the role of Acrc in adult tissues. We identified histone H3, topoisomerases 1 and 2, Dnmt1, Parp1, Polr3a and Mcm2 as DPC substrates of Acrc. We have proven that Acrc is absolutely essential for vertebrate development, and that the mechanism behind it is DPC proteolysis.

molecular biology↗

bioRxiv2023

Tyrosyl-DNA phosphodiesterase 1 (TDP1) and SPRTN protease repair histone 3 and topoisomerase 1 DNA-protein crosslinks in vivo

DNA-protein crosslinks (DPCs) are frequent and damaging DNA lesions that affect all DNA transactions, which in turn can lead to the formation of DSBs, genomic instability and cell death. At the organismal level, impaired DPC repair (DPCR) is associated with cancer, aging, and neurodegeneration. Despite the severe consequences of DPCs, little is known about the processes underlying repair pathways at the organism level. SPRTN is a protease that removes most cellular DPCs during replication, whereas tyrosyl-DNA phosphodiesterase 1 repairs one of the most abundant enzymatic DPCs, topoisomerase 1-DPC (TOP1-DPC). How these two enzymes repair DPCs at the organism level is currently unknown. We perform phylogenetic, syntenic, structural and expression analysis to compare TDP1 orthologs between human, mouse and zebrafish. Using the zebrafish animal model and human cells, we demonstrate that TDP1 and SPRTN repair endogenous, camptothecin- and formaldehyde-induced DPCs, including histone H3- and TOP1-DPCs. We show that resolution of H3-DNA crosslinks depends on upstream proteolysis by SPRTN and subsequent peptide removal by TDP1 in RPE1 cells and zebrafish embryos, whereas SPRTN and TDP1 function in different pathways in the repair of endogenous TOP1-DPCs and total DPCs. Furthermore, our results suggest that TDP2 could potentially compensate for the impairment of TDP1 function in vivo and in human cells. Understanding the role of TDP1 in DPC repair at the cellular and organismal levels could provide an impetus for the development of new drugs and combination therapies with TOP1-DPC inducing drugs.

cell biology↗

bioRxiv2022

An NlpC/P60 protein catalyzes a key step in peptidoglycan recycling at the intersection of energy recovery, cell division and immune evasion in the intracellular pathogen Chlamydia trachomatis

The obligate intracellular Chlamydiaceae do not need to resist osmotic challenges and thus lost their cell wall in the course of evolution. Nevertheless, these pathogens maintain a rudimentary peptidoglycan machinery for cell division. They build a transient peptidoglycan ring, which is remodeled during the process of cell division and degraded afterwards. Uncontrolled degradation of peptidoglycan poses risks to the chlamydial cell, as essential building blocks might get lost or trigger host immune response upon release into the host cell. Here, we provide evidence that a primordial enzyme class prevents energy intensive de novo synthesis and uncontrolled release of immunogenic peptidoglycan subunits in Chlamydia trachomatis. Our data indicate that the homolog of a Bacillus NlpC/P60 protein is widely conserved among Chlamydiales. We show that the enzyme is tailored to hydrolyze peptidoglycan-derived peptides, does not interfere with peptidoglycan precursor biosynthesis, and is targeted by cysteine protease inhibitors in vitro and in cell culture. The peptidase plays a key role in the underexplored process of chlamydial peptidoglycan recycling. Our study suggests that chlamydiae orchestrate a closed-loop system of peptidoglycan ring biosynthesis, remodeling, and recycling to support cell division and maintain long-term residence inside the host. Operating at the intersection of energy recovery, cell division and immune evasion, the peptidoglycan recycling NlpC/P60 peptidase could be a promising target for the development of drugs that combine features of classical antibiotics and anti-virulence drugs. Author SummaryFree-living bacteria are wrapped by a peptidoglycan cell wall. This envelope provides shape and protects from osmotic stress, while also triggering human immune defense. Chlamydial pathogens live inside human cells and dispensed with a cell wall in their isotonic niche. Instead, they produce a transient peptidoglycan ring that is essentially involved in cell division. Recycling of peptidoglycan is likely important for chlamydiae to maintain a complete cycle of peptidoglycan ring synthesis and cell division. Moreover, the process may help to save energy and to protect from recognition through the immune system. Despite a potentially central role in cell viability and pathogenicity almost nothing is known about recycling of peptidoglycan in chlamydiae. The minimalist organisms lack all enzymes known to catalyze the process in model organism Escherichia coli. Here, we found an NlpC/P60 enzyme to serve a critical step in decomposing peptidoglycan ring derived peptides in Chlamydia trachomatis. Our data indicate that the peptidase recycles energy cost intensive components and breaks down the minimal recognition motif of innate immune factor NOD1. We also identified a natural lead compound to inhibit the NlpC/P60 enzyme opening the way for innovative anti-chlamydial drug development at the intersection of energy recovery, cell division, and immune evasion.

microbiology↗