Biology subjects

Bozhkov, P.

Publications and source records attributed to Bozhkov, P..

5 recordsLinked to original sources

bioRxiv2026

A family of metabolite damage-control phosphatases modulates ROS-induced autophagy and plant stress resilience

Autophagy is an intracellular recycling pathway with profound impacts on development, growth, and stress tolerance in eukaryotes. Therefore, unravelling signalling mechanisms that modulate the process has broad applicability. Here, we characterised a small organic molecule as an enhancer of autophagy across diverse plant lineages. Through an in vivo photoaffinity proteomics approach in Arabidopsis thaliana seedlings, we identified Domain of Unknown Function 89 (DUF89) proteins as molecular targets. Hereafter, we refer to these proteins as Reactive Metabolite Damage-Control Phosphatases (RMDPs), which are metabolite-repair phosphatases that clear reactive products. The new autophagy modulator is called RMDP inhbitor-1 (RMDPi-1) due to its demonstrated inhibitory effect on AtRMDP1 activity in vitro. Binding was further confirmed through co-incubation that thermostabilised the protein. Crystal structures of AtRMDP1 were obtained via X-ray diffraction, confirming binding of RMDPi-1 in the catalytic site and revealing a flexible region near the binding pocket that was absent in previous models. Molecular docking predicted two possible binding modes consistent with the observed electron density. RMDPi-1 treatment and the phosphatase gene knockout increased autophagic flux in Arabidopsis and Chlamydomonas reinhardtii, establishing a conserved effect. The observed autophagy response is linked to a spike in reactive oxygen species (ROS). AtRMDP knockout also results in higher ROS tolerance and greater biomass in Arabidopsis seedlings. Targeted and untargeted metabolomic experiments revealed that both pharmacological and genetic suppression of RMDP leads to accumulation of glycating agents and phosphate sugars, associating loss of RMDP activity to the ROS stress that induces alternate ROS-scavenging mechanisms, and autophagy. Taken together, our integrated chemical genetics approach reveals enhanced autophagy via inhibition of RMDP family members, which impacts on plant stress tolerance via conserved metabolite damage-control functions.

plant biology↗

bioRxiv2026

Starvation-induced autophagy occurs independently of the ATG1 complex in Chlamydomonas

The survival of eukaryotes during starvation depends on effective nutrient recycling via autophagy. Accordingly, loss of autophagy-related (ATG) proteins, including the nutrient-sensing ATG1 kinase complex, typically results in reduced fitness or lethality under nutrient limitation. The green alga Chlamydomonas reinhardtii provides a tractable model for autophagy studies, as its ATG repertoire is encoded by single-copy genes. Here, we generated a comprehensive library of ATG deletion mutants and examined their growth and autophagy during starvation. Surprisingly, starvation-induced autophagy occurred in the absence of ATG1 complex components (ATG1, ATG11, ATG13, and ATG101), revealing ATG1-independent autophagy and challenging the canonical model for autophagy initiation.

molecular biology↗

bioRxiv2023

ATG8 delipidation is dispensable for plant autophagy

Intracellular recycling via autophagy is governed by post-translational modifications of the autophagy-related (ATG) proteins. One notable example is ATG4-dependent delipidation of ATG8, a process that plays critical but distinct roles in autophagosome formation in yeast and mammals. Here, we aimed to elucidate the specific contribution of this process to autophagosome formation in species representative of evolutionary distant green plant lineages: unicellular green alga Chlamydomonas reinhardtii, with a relatively simple set of ATG genes, and a vascular plant Arabidopsis thaliana, harboring expanded ATG gene families. Remarkably, the more complex autophagy machinery of Arabidopsis rendered ATG8 delipidation entirely dispensable for the maturation of autophagosomes, autophagic flux and related stress tolerance; whereas autophagy in Chlamydomonas strictly depended on the ATG4-mediated delipidation of ATG8. Importantly, we uncovered the distinct impact of different Arabidopsis ATG8 orthologs on autophagosome formation, especially prevalent under nitrogen depletion, providing a new insight into potential drivers behind the expansion of the ATG8 family in higher plants. Our findings underscore the evolutionary diversification of the molecular mechanism governing the maturation of autophagosomes in eukaryotic lineages and highlight how this conserved pathway is tailored to diverse organisms.

plant biology↗

bioRxiv2023

Thermoprotection by a cell membrane-localized metacaspase in a green alga

Caspases are restricted to animals, while other organisms, including plants possess metacaspases (MCAs), a more ancient and broader class of structurally-related yet biochemically distinct proteases. Our current understanding of plant MCAs is derived from studies in streptophytes, and mostly in Arabidopsis expressing nine MCAs with partly redundant activities. In contrast to streptophytes, most chlorophytes contain only one or two hitherto uncharacterized MCAs, providing an excellent platform for MCA research. Here we investigate CrMCA-II, a single type II MCA from a model chlorophyte Chlamydomonas reinhardtii. Surprisingly, unlike other studied MCAs and similar to caspases, CrMCA-II dimerizes both in vitro and in vivo. Furthermore, activation of CrMCA-II in vivo correlates with the dimerization. Most of CrMCA-II in the cell is present as a zymogen attached to the plasma membrane (PM). Deletion of CrMCA-II by CRISPR/Cas9 compromises thermotolerance leading to increased cell death under heat stress. Adding back either wild-type or catalytically dead CrMCA-II restores thermoprotection, suggesting that its proteolytic activity is dispensable for this effect. Finally, we link the non-proteolytic role of CrMCA-II in thermotolerance to the ability to modulate PM fluidity. Our study reveals an ancient, MCA-dependent thermotolerance mechanism retained by Chlamydomonas and probably lost during the evolution of multicellularity.

plant biology↗

bioRxiv2017

The Proteolytic Landscape Of An Arabidopsis Separase-Deficient Mutant Reveals Novel Substrates Associated With Plant Development

Digestive proteolysis executed by the proteasome plays an important role in plant development. Yet, the role of limited proteolysis in this process is still obscured due to the absence of studies. Previously, we showed that limited proteolysis by the caspase-related protease separase (EXTRA SPINDLE POLES [ESP]) modulates development in plants through the cleavage of unknown substrates. Here we used a modified version of the positional proteomics method COmbined FRActional DIagonal Chromatography (COFRADIC) to survey the proteolytic landscape of wild-type and separase mutant RADIALLY SWOLLEN 4 (rsw4) root tip cells, as an attempt to identify targets of separase. We have discovered that proteins involved in the establishment of pH homeostasis and sensing, and lipid signalling in wild-type cells, suggesting novel potential roles for separase. We also observed significant accumulation of the protease PRX34 in rsw4 which negatively impacts growth. Furthermore, we observed an increased acetylation of N-termini of rsw4 proteins which usually comprise degrons identified by the ubiquitin-proteasome system, suggesting that separase intersects with additional proteolytic networks. Our results hint to potential pathways by which separase could regulate development suggesting also novel proteolytic functions.

plant biology↗