Biology subjects

Plavec, J.

Publications and source records attributed to Plavec, J..

4 recordsLinked to original sources

bioRxiv2025

Fast MAS NMR Spectroscopy Can Identify G-Quartets and Double-Stranded Structures in Aggregates Formed by GGGGCC RNA Repeats

The expansion of GGGGCC repeats within the C9orf72 gene has been linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In neurons from patients with expanded repeats, C9orf72 GGGGCC repeat RNA predominantly forms nuclear foci, and in vitro, repeat-containing RNA can self-aggregate. Two structural motifs have been proposed to provide the interstrand interactions that drive aggregation: G-quartet (G4) structures and double-strand interactions with GG mismatches. Using in vitro transcribed RNA with pathologically relevant number of repeats, we were able to form gel-like aggregates suitable for investingation using fast MAS NMR spectroscopy. This approach enabled us to characterize the dominant interstrand interactions within the RNA gels. Both Watson-Crick and Hoogsteen base pairs were identified in RNA gels formed by RNA with 48 GGGGCC repeats. Their relative abundance shifted upon reconstitution in the presence of different divalent cations or nuclear extracts, underscoring the dynamic equilibrium between G-quadruplex and duplex interactions in GGGGCC RNA aggregation.

biophysics↗

bioRxiv2024

Lipid polymorphism of the marginal region of plant thylakoid membranes

Thylakoid membranes (TMs) of oxygenic photosynthetic organisms are flat membrane vesicles, which form highly organized, interconnected membrane networks. In vascular plants, they are differentiated into stacked and unstacked regions, the grana and stroma lamellae, respectively; they are densely packed with protein complexes performing the light reactions of photosynthesis and generating a proton motive force (pmf). The maintenance of pmf and its utilizations for ATP synthesis require sealing the TMs at their highly curved regions (CRs). These regions are devoid of chlorophyll-containing proteins but contain the curvature inducing CURVATURE THYLAKOID1 (CURT1) proteins, and are enriched in lipids. Because of the highly curved nature of this region, at the margins of grana and stroma TMs, the molecular organization of lipid molecules is likely to possess distinct features compared to those in the major TM domains. To clarify this question, we isolated CR fractions from Spinacia oleracea and, using BN-PAGE and western blot analysis, verified that they are enriched in CURT1 proteins and in lipids. The lipid phase behavior of these fractions was fingerprinted with 31P-NMR spectroscopy, which revealed that the bulk lipid molecules assume a non-bilayer, isotropic lipid phase. This finding underpins the importance of the main, non-bilayer lipid species, monogalactosyldiacylglycerol, of TMs in their self-assembly and functional activity.

plant biology↗

bioRxiv2023

Resolving the pharmacological redox-sensitivity of SARS-CoV-2 PLpro in drug repurposing screening enabled identification of the competitive GRL-0617 binding site inhibitor CPI-169

The SARS CoV-2 Papain-Like protease has multiple roles in the viral replication cycle, related to both its polypeptide cleavage function and its capacity to antagonize host immune response. Targeting PLpro function is recognized as a promising mechanism to modulate viral replication whilst supporting host immune responses. However, development of PLpro specific inhibitors remains challenging. Upcoming studies revealed the limitation of reported inhibitors by profiling them through a pipeline of enzymatic, binding and cellular activity assays showing unspecific activity. GRL-0617 remained the only validated molecule with demonstrated anti-viral activity in cells. In this study we refer to the pitfalls of redox-sensitivity of PLpro. Using a screening-based approach to identify inhibitors of PLpro proteolytic activity, we made extensive efforts to validate the active compounds over a range of conditions and readouts, emphasising the need for comprehensive orthogonal data when profiling putative PLpro inhibitors. The remaining active compound CPI-169, showed to compete with GRL-0617 in NMR-based experiments, suggesting to share a similar binding mode, opening novel design opportunities for further developments as antiviral agents. Author summaryThe increasing knowledge about SARS-CoV-2 allowed the development of multiple strategies to contain the spread of COVID-19 infection. Nevertheless, effective antiviral pharmacological treatments are still rare and viral evolution allowed a fast adaptation and escape from available containment methods. The papain like protease (PLpro) has now become the next most promising SARS-CoV-2 therapeutic due to its multiple functions in virus replication cycle and antagonization of host immune response. However, due to inherent flexibility and sensitivity of this enzyme specific inhibitors are rare. Here we report on a screening strategy using repurposing of known drugs that takes into account PLpro characteristics to identify new inhibitors, showing the success of the approach by identifying CPI-169 that competitive targets the well described GRL-0617 inhibitor binding pocket of PLpro and helping to design further antiviral agents.

molecular biology↗

bioRxiv2023

Effects of protein G-quadruplex interactions on phase transitions and protein aggregation

The SERF family of proteins were originally discovered for their ability to accelerate amyloid formation. Znf706 is an uncharacterized protein whose N-terminus is homologous to SERF proteins. We show here that human Znf706 can promote protein aggregation and amyloid formation. Unexpectedly, Znf706 specifically interacts with stable, non-canonical nucleic acid structures known as G-quadruplexes. G-quadruplexes can affect gene regulation and suppress protein aggregation; however, it is unknown if and how these two activities are linked. We find Znf706 binds preferentially to parallel G-quadruplexes with low micromolar affinity, primarily using its N-terminus, and upon interaction, its dynamics are constrained. G-quadruplex binding suppresses Znf706s ability to promote protein aggregation. Znf706 in conjunction with G-quadruplexes therefore may play a role in regulating protein folding. RNAseq analysis shows that Znf706 depletion specifically impacts the mRNA abundance of genes that are predicted to contain high G-quadruplex density. Our studies give insight into how proteins and G-quadruplexes interact, and how these interactions affect both partners and lead to the modulation of protein aggregation and cellular mRNA levels. These observations suggest that the SERF family of proteins, in conjunction with G-quadruplexes, may have a broader role in regulating protein folding and gene expression than previously appreciated.

biochemistry↗