Biology subjects

Debarnot, C.

Publications and source records attributed to Debarnot, C..

4 recordsLinked to original sources

bioRxiv2026

Hantaan virus polymerase has an in vitro Terminal Nucleotidyl Transferase activity that is conserved across the Bunyaviricetes class

Bunyaviricetes is a class of segmented negative strand RNA viruses (sNSV) that includes causative agents of severe zoonotic diseases resulting in hemorrhagic fever in humans and livestock. Hantaan virus (HTNV), family Hantaviridae, is an example of life-threatening bunyavirus transmitted by rodents which leads to Hantavirus hemorrhagic fever with renal syndrome in humans. Infection relies on the replication and transcription of their tripartite genomes through multifunctional RNA-dependent RNA polymerases (RdRp) in the context of ribonucleoproteins (RNPs) made of one viral RNA fragment, one RdRp and multiple copies of viral nucleoproteins. The RdRp, also known as L protein, performs replication by de novo initiation through a prime-and-realign mechanism while transcription initiates through a cap snatching mechanism, that engages a cap-binding domain and an endonuclease (EN) domain present in the L protein C and N terminal regions, respectively. In this work we show that HTNV L protein has a terminal nucleotidyl transferase (TNTase) activity over double-stranded RNA substrates (dsRNA) in vitro. We show that the TNTase activity is also present in other bunyaviral L proteins by comparing HTNV L protein activity with La Crosse (LACV, family Orthobunyaviridae) and Crimean Congo Hemorrhagic Fever Virus (CCHFV, family Nairoviridae) L proteins. Our results show that TNTase activity appears to be a general feature of Bunyaviruses and report the differences on substrate and nucleotide specificity for each family of virus.

biochemistry↗

bioRxiv2026

Structural and cellular insights into the inhibition of the drug efflux activity of the HEDGEHOG receptor PATCHED1

PTCH1, the receptor for the Sonic Hedgehog morphogen, mediates cholesterol transport across the plasma membrane by harnessing the proton motive force. In cancer, PTCH1 is frequently overexpressed and promotes chemoresistance by transporting drugs such as doxorubicin (dxr) out of cells. Among the inhibitors identified, PAH stands out for its ability to significantly enhance the efficacy of several chemotherapeutic drugs on melanoma and breast cancer cells. To investigate PTCH1s structure in complex with its inhibitor PAH, we overexpressed a construct spanning residues 1-619 and 720-1305 in HEK293 cells. The protein localized to the membrane, and transfected cells exhibited reduced sensitivity to dxr compared to control cells. Additionally, we observed a pH-dependent efflux of dxr, which was reversed by PAH, confirming that the PTCH1 construct used in this study functions as an active drug-efflux pump. In the structure of PTCH1 bound to PAH determined using cryo-electron microscopy, PAH occupies a hydrophobic cavity in an extracellular domain which is normally occupied by cholesterol in other PTCH1 structures, and engages in a key hydrogen bond via one of its hydroxyl groups, a feature previously established as essential for its inhibitory function. These findings not only clarify the molecular basis of PAHs action but also provide a structural roadmap for rational drug design, enabling the development of next-generation inhibitors with enhanced potency.

biochemistry↗

bioRxiv2025

Bidirectional control of a metabolic transition by the GID ubiquitin ligase

The GID/CTLH ubiquitin ligase is a multisubunit E3 conserved across eukaryotes. GID/CTLH has been implicated in a variety of processes, including metabolic regulation, cell proliferation, embryonic development and cell differentiation. However, our understanding of substrate recognition by GID/CTLH remains incomplete. Here, we characterize the regulation, specificity and functions of the putative substrate receptor Gid11 in budding yeast. We find that upon its expression during the switch of carbon source from glucose to ethanol, Gid11 associates with the core GID complex, likely forming a large ring assembly. Through systematic mutagenesis, we identify a negatively charged pocket on Gid11 that binds substrates carrying N-terminal degrons starting with threonine and define the Thr/N-degron motif. Such Thr/N-degrons are exposed upon co-translational removal of the initiator methionine, while escaping N-terminal acetylation. Finally, we show that during the switch to ethanol, GIDGid11 targets for degradation factors linked to glycolysis, opposing the established role of GID in the degradation of gluconeogenic enzymes during the reverse switch from ethanol to glucose. Taken together, our results establish Gid11 as a bona fide GID substrate receptor and highlight GID as a regulator of metabolic transitions.

molecular biology↗

bioRxiv2018

Phase transition and amyloid formation by a viral protein as an additional molecular mechanism of virus-induced cell toxicity

Henipaviruses are severe human pathogens responsible for severe encephalitis. Their V protein is a key player in the evasion of the host innate immune response. We have previously reported a biophysical characterization of the Henipavirus V proteins and shown that they interact with DDB1, a cellular protein that is a component of the ubiquitin ligase E3 complex. Here, we serendipitously discovered that the Hendra virus V protein undergoes a liquidhydrogel phase transition. By combining experimental and bioinformatics approaches, we have identified the V region responsible for this phenomenon. This region (referred to as PNT3), which falls within the long intrinsically disordered region of V, was further investigated using a combination of biophysical and structural approaches. ThioflavinT and Congo red binding assays, together with negative-staining electron microscopy studies, show that this region forms amyloid-like, {beta}-enriched structures. Such structures are also formed in mammal cells transfected to express PNT3. Those cells also exhibit a reduced viability in the presence of a stress agent. Interestingly, mammal cells expressing a rationally designed, non-amyloidogenic PNT3 variant (PNT33A), appear to be much less sensitive to the stress agent, thus enabling the establishment of a link between fibril formation and cell toxicity. The present findings therefore pinpoint a so far never reported possible mechanism of virus-induced cell toxicity.

biophysics↗