Reuter, J. | bioRxiv Science

bioRxiv2024

A missense variant effect map for the human tumour suppressor protein CHK2

The tumour suppressor CHEK2 encodes the serine/threonine protein kinase CHK2 which, upon DNA damage, is important for pausing the cell cycle, initiating DNA repair and inducing apoptosis. CHK2 phosphorylation of the tumour suppressor BRCA1 is also important for mitotic spindle assembly and chromosomal stability. Consistent with its cell cycle checkpoint role, both germline and somatic variants in CHEK2 have been linked to breast and multiple other cancer types. Over 90% of clinical germline CHEK2 missense variants are classified as variants of uncertain significance, complicating diagnosis of CHK2-dependent cancer. We therefore sought to test the functional impact of all possible missense variants in CHK2. Using a scalable multiplexed assay based on the ability of human CHK2 to complement DNA sensitivity of a S. cerevisiae lacking its ortholog RAD53, we generated a systematic missense variant effect map for CHEK2 missense variation. Map scores reflect known biochemical features of CHK2 and exhibit good performance in separating pathogenic from benign clinical missense variants. Thus, the missense variant effect map for CHK2 offers value in understanding both known and yet-to-be-observed CHK2 variants.

genetics↗

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↗