Instruct Instruct PartnerThe latest webinar in the Instruct-ERIC Structure Meets Function series this month includes speakers who have accessed cutting-edge structural biology services through Instruct. The webinar series offers an insight into the very cutting edge of structural biology research, utilising the latest techniques available through Instruct-ERIC facilities and centres.
On 17 June, the webinar will feature speakers from Instruct-Slovenia. Slovenia joined as a member in 2023, and hosted a mini-symposium showcasing the latest research carried out in the country in 2024.
The speakers this month include Peter Podbevšek, outlining their research at the National Institute of Chemistry on guanine oxidation, as well as Nika Žibrat, completing their PhD in Department of Molecular Biology and Nanobiotechnology, also at the Kemijski Inštitut.
The webinar will begin at 11:00 CEST on 17 June 2025. Register for the webinar here.
Moderator: Marjetka Podobnik, Kemijski Inštitut
Speaker: Nika Žibrat - Department of Molecular Biology and Nanobiotechnology, Kemijski Inštitut
Talk Title: Membrane Binding and Pore Formation by NLPs from Phylogenetically Distinct Plant Pathogens
Abstract: Necrosis- and ethylene-inducing 1-like proteins (NLPs) form a large superfamily of proteins present across various phyla of plant-associated microorganisms. Many NLPs exhibit cytotoxicity and aid infection in a wide variety of crops. Their known target on the plant plasma membrane is the sphingolipid glycosyl inositol phosphoceramide (GIPC). Previous studies on NLPPya, an NLP from the oomycete Pythium aphanidermatum, revealed a unique mechanism of membrane disruption: upon binding, the protein oligomerizes on the membrane surface, forming transient, heterogeneous pores that allow the passage of small molecules. Despite the identification of over 1,800 nlp genes in the genomes of more than 500 bacteria, fungi, and oomycetes, the majority of NLPs remain uncharacterized in terms of their membrane-binding properties and pore-forming activity. Furthermore, it is still unclear whether NLP-induced pore formation affects both dicot and monocot plants. In this study, we investigated the pore-forming activities of two phylogenetically unrelated NLPs, MpNEP2 from the fungus Moniliophthora perniciosa and NLPPya from the oomycete P. aphanidermatum. Using artificial plant membrane models containing GIPCs extracted from tobacco (a dicot) and leek (a monocot), we demonstrated with two independent systems that both NLPs are capable of forming pores in membranes derived from both plant groups. This indicates a conserved mechanism of action among NLPs from evolutionarily distant plant pathogens. Additionally, functional and structural characterization of a third NLP, NLPPp from the notorious plant pathogen Phytophthora parasitica, revealed subtle but important differences in key structural regions. These variations likely contribute to differences in GIPC binding affinity and pore-forming efficiency in plant plasma membranes. Our findings provide new insight into the structural diversity and functional versatility of NLPs in plant pathogenesis.
Speaker: Peter Podbevšek - Slovenian NMR Centre, Kemijski Inštitut
Talk Title: Oxidative Damage Modulates G-Quadruplex Folding and Enzyme Bypass
Abstract: Guanine has the lowest redox potential among the four DNA nucleobases, making it especially prone to oxidative damage. This susceptibility is heightened in guanine-rich tracts, which are key components of G-quadruplex structures. Among the various products of guanine oxidation, 8-oxo-7,8-dihydroguanine (8-oxoG) is one of the most prevalent and widely used biomarkers of oxidative stress. Many DNA-processing enzymes translocate along DNA strands but are hindered by stable secondary structures such as G-quadruplexes. To explore how oxidative lesions affect enzyme progression, we employed a polymerase stop assay to investigate the impact of structural features in human telomeric and BCL2 promoter G-quadruplexes on the activity of the Klenow fragment. Primer extension profiles revealed that G-quartets (planar arrangements of four guanines) pose strong barriers to enzyme progression, whereas auxiliary base pairs are more easily bypassed. To assess the influence of oxidative damage, we introduced 8-oxoG lesions into G-rich regions. While these lesions occasionally impaired enzyme bypass efficiency, they generally destabilized the G-quadruplex structure, facilitating polymerase progression. Although our study focused on the Klenow fragment, the observed trends likely extend to other DNA-processing enzymes that utilize a clamp-like mechanism for DNA traversal. Notably, we also identified a biologically relevant G-quadruplex structure that was significantly stabilized by oxidative lesions, adding a new layer of complexity to the folding landscape of telomeric and promoter G-rich regions. These unique structural effects may have implications for the recognition, repair, and biological consequences of oxidative lesions in the human genome.
