Biology subjects

Pugniere, M.

Publications and source records attributed to Pugniere, M..

9 recordsLinked to original sources

bioRxiv2026

A new HIV-1 latency reversing agent activating HIV-Tat

Despite its efficiency to prevent viral multiplication, antiretroviral therapy does not affect HIV-1 latently-infected cells. These cells do not produce significant amounts of viruses and constitute HIV-1 reservoir. To purge this long-lived viral reservoir, the "shock and kill" strategy relies on the use of latency reversing agents (LRAs) to induce activation of latent cells. All LRAs developed until now target cellular proteins and are therefore not specific for HIV-infected cells. Here we present a new LRA that binds and activates HIV-1 Tat which is the key regulator for viral transcription and latency reversal. This molecule termed D10 was designed to bind to the major groove of the Tat protein, and found to activate Tat transcriptional activity by stabilizing the HIV transcription complex. This LRA induces strong HIV production by latent cell lines and latent cells from people living with HIV-1. On latent cells from PBMCs, D10 is active at [~]50 nM, the concentration required to stabilize HIV transcription complex. D10 is the first Tat activator available and the first LRA that targets an HIV protein.

microbiology↗

bioRxiv2026

Characterization of the p38α MAPK allosteric inhibition by a single chain Fv antibody

Most of the available p38 kinase inhibitors target the highly conserved ATP-binding site, thus explaining their off-target effects and toxicity. Here, we report the use of single-chain fragment variable (scFv) antibodies to identify new highly specific inhibitors. Using phage display, we selected five anti-p38 scFv antibodies among which one fully inhibited p38 kinase activity in vitro. We showed that this scFv did not affect ATP binding, while still acting as a competitive inhibitor of ATP hydrolysis. When expressed as intrabody in the nucleus of the THP-1 human monocytic cell line, the scFv inhibited the function of endogenous p38 and induced a significant decrease in lipopolysaccharide-induced tumor necrosis factor production. To gain insight into the mechanisms by which the scFv induced p38 inhibition, the scFv epitope was mapped by deep mutational scanning of p38 displayed on yeast surface and high-throughput selection by flow cytometry of p38 mutants no longer bound by the scFv. By this approach, we showed that the inhibitory scFv interacts with two helices within the C-terminal lobe of the kinase distant from the ATP-binding pocket and the docking groove. Altogether, our data show that the scFv antibody behaves as an allosteric inhibitor, constraining and maintaining p38 in an inactive form.

biochemistry↗

bioRxiv2024

Neutrophils in People Living With HIV-1 on antiretroviral treatment conserve their cytokine and chemokine secretion capacity and display enhanced expression of Fc receptors

Neutrophils are innate immune cells with key immunomodulatory functions. In a murine retroviral model, we previously showed their essential role in promoting protective immunity during antiviral antibody therapy via Fc-Fc{gamma} receptor (Fc{gamma}R) interactions. Here, we investigated the immunomodulatory properties of neutrophils in the context of HIV-1 infection and therapy through a comprehensive analysis of their functional activation and the regulation of Fc{gamma}R expression. Neutrophils from healthy donors (HD) and people living with HIV-1 (PLWH) were stimulated with TLR ligands, free HIV-1, immune complexes (ICs) formed with broadly neutralizing antibodies (bNAbs), or pro-inflammatory cytokines (TNF, IFN{gamma}). In response, they secreted various cytokines and chemokines that can recruit and activate immune cells in a stimulus-dependent manner. Compared to TLR agonist and cytokine activation, HD neutrophils showed limited cytokine production in response to free HIV-1 or ICs alone as well as a minimal Fc{gamma}R modulation. However, PLWH neutrophils showed heightened responsiveness to microbial stimuli linked to HIV-1 pathogenesis, secreting higher levels of IFN{gamma}, CXCL1, CCL2, CCL3, and CCL4. They also expressed higher levels of two activating Fc{gamma}Rs (Fc{gamma}RI and Fc{gamma}RIIIb), as well as CD11b, CD63, CXCR4, and PD-L1, indicating an altered activation state. These findings highlight the influence of the inflammatory milieu on neutrophil function and Fc{gamma}R regulation in HIV-1 infection and mAb-based therapies.

immunology↗

bioRxiv2022

A tripartite complex HIV-1 Tat-cyclophilin A-capsid protein enables Tat encapsidation that is required for HIV-1 infectivity

HIV-1 Tat is a key viral protein that stimulates several steps of viral gene expression. Tat is especially required for the transcription of viral genes but it is still not clear if and how Tat is incorporated into HIV-1 virions. Cyclophilin A (CypA) is a prolylisomerase that binds to HIV-1 capsid protein (CA) and is thereby encapsidated. Here we found that a Tat-CypA-CA tripartite complex assembles in HIV-1 infected cells. Biochemical and biophysical studies showed that high affinity interactions drive the assembly of this complex. Virions devoid of encapsidated Tat showed a 5-10 fold decrease in HIV-infectivity and, conversely, encapsidating Tat into {Delta}Tat viruses greatly enhanced infectivity. The absence of encapsidated Tat decreases the efficiency of retrotranscription by [~]50% and transcription by 99%. We thus identified a Tat-CypA-CA complex that enables Tat encapsidation and showed that encapsidated Tat is required to initiate robust HIV-1 infection and viral production.

microbiology↗

bioRxiv2021

Activation of peripheral neuronal FLT3 promotes exaggerated sensorial and emotional pain-related behaviors facilitating the transition from acute to chronic pain

Acute pain events have been associated with persistent pain sensitization of nociceptive pathways increasing the risk of transition from acute to chronic pain. However, it is unclear whether injuryinduced persistent pain sensitization can promote long-term mood disorders. The receptor tyrosine kinase FLT3 is causally required for pain chronification after peripheral nerve injury, questioning its role in the development of pain-induced mood alterations. Here, we evaluated the emotional and sensorial components of pain after a single (SI) or double paw incision (DI). We then investigated the role of FLT3 either by inhibition using transgenic knock-out mice and functional antibodies or by activation with FLT3 ligand (FL) administrations. DI mice showed significant anxiodepressive-like and spontaneous pain behaviors as opposed to SI mice. DI also promoted and extended mechanical pain hypersensitivity compared to SI. This emotional and sensorial pain exaggeration correlated with spinal changes especially by increased microglia activation after DI versus SI. Intrathecal minocycline, a microglial inhibitor, specifically reversed DI inducedmechanical hypersensitivity in males. Repeated treatment with the microglia proliferation inhibitor GW2580 not only eliminated the exaggerated pain hypersensitivity produced by DI but also prevented anxiodepressive-related behaviors in DI animals. Finally, FL injections in naive animals provoked mechanical allodynia and anxiodepressive-like disorders concomitant with a strong microglial activation while Flt3 silencing in a genetic mouse line or FLT3 blocking via functional antibodies, blunted the development of persistent pain and depression after DI. Altogether our results show that the repetition of peripheral lesions facilitate not only exaggerated nociceptive behaviors but also induced anxiodepressive disorders supported by spinal central changes. The inhibition of FLT3 could thus become a promising therapy in the management of pain sensitization and related mood alterations.

neuroscience↗

bioRxiv2021

Adenovirus-α-defensin complexes induce NLRP3-associated maturation of human phagocytes via TLR4 engagement

Intramuscular delivery of human adenovirus (HAdV)-based vaccines leads to rapid recruitment of neutrophils, which then release antimicrobial peptides/proteins (AMPs). How these AMPs influence vaccine efficacy over the subsequent 24 hr is poorly understood. In this study, we asked if human neutrophil protein 1 (HNP-1), an -defensin that influences the direct and indirect innate immune responses to a range of pathogens, impacts the innate response of human phagocytes to three HAdV species/types (HAdV-C5, -D26, -B35). We show that HNP-1 binds to the capsids, redirects HAdV-C5, -D26, -B35 to Toll-like receptor 4 (TLR4), which leads to internalization, an NLRP3-mediated inflammasome response, and IL-1{beta} release. Surprisingly, IL-1{beta} release was not associated with notable disruption of plasma membrane integrity. These data further our understanding of HAdV innate immunogenicity and may provide pathways to extend HAdV-based vaccines efficacy.

immunology↗

bioRxiv2021

Design of a new effector recognition specificity in a plant NLR immune receptor by molecular engineering of its integrated decoy domain

Plant nucleotide-binding and leucine-rich repeat domain proteins (NLRs) are immune sensors that specifically recognize pathogen effectors and induce immune responses. Designing artificial NLRs with new effector recognition specificities is a promising prospect for sustainable, knowledge-driven crop protection. However, such strategies are hampered by the complexity of NLR function. Here, we tested whether molecular engineering of the integrated decoy domain (ID) of an NLR could extend its recognition spectrum to a new effector. To this aim, we relied on the detailed molecular knowledge of the recognition of distinct Magnaporthe oryzae MAX (Magnaporthe AVRs and ToxB-like) effectors by the rice NLRs RGA5 and Pikp-1. For both NLRs, effector recognition involves physical binding to their HMA (Heavy Metal-Associated) IDs. However, AVR-PikD, the effector recognized by Pikp-1, binds to a completely different surface of the HMA domain compared to AVR-Pia and AVR1-CO39, recognized by RGA5. By introducing into the HMA domain of RGA5 the residues of the Pikp-1 HMA domain involved in AVR-PikD binding, we created a high-affinity binding surface for this new effector. In the Nicotiana benthamiana heterologous system, RGA5 variants carrying this engineered binding surface still recognize AVR-Pia and AVR1-CO39, but also perceive the new ligand, AVR-PikD, resulting in the activation of immune responses. Therefore, our study provides a proof of concept for the design of new effector recognition specificities in NLRs through molecular engineering of IDs. However, it pinpoints significant knowledge gaps that limit the full deployment of this NLR-ID engineering strategy and provides hypotheses for future research on this topic.

plant biology↗

bioRxiv2020

Lactoferrin retargets adenoviruses to TLR4 to induce an abortive NLRP3-associated pyroptotic response in human dendritic cells

Despite decades of investigations, we still poorly grasp the immunogenicity of human adenovirus (HAdV)-based vaccines in humans. In this study, we explored the role of lactoferrin, which belong to the alarmin subset of antimicrobial peptides that provide immediate direct and indirect activity against a range of pathogens following a breach in tissue homeostasis. Lactoferrin is a globular, iron-sequestering, glycoprotein that can increase HAdV infection and maturation of antigen-presenting cells. However, the mechanism by which HAdV-lactoferrin complexes induce maturation is unknown. We show that lactoferrin redirects HAdVs from species B, C, and D to toll-like receptor 4 (TLR4) complexes on human mononuclear phagocyte. TLR4-mediated internalization induces an abortive NLRP3-associated pyroptotic response inducing pro-inflammatory cytokine release and disrupting plasma membrane integrity without cell death. These data impact our understanding of the immunogenicity of HAdV-based vaccines and may provide ways to increase their efficacy.

immunology↗

bioRxiv2020

Development of Nanobodies as first-in-class inhibitors for the NEDP1 deNEDDylating enzyme

Protein NEDDylation emerges as an important post-translational modification and an attractive target for therapeutic intervention. Modification of NEDD8 onto substrates is finely balanced by the co-ordinated activity of conjugating and deconjugating enzymes. The NEDP1/DEN1/SENP8 protease is a NEDD8 specific processing and deconjugating enzyme that regulates the NEDDylation mainly of non-cullin substrates. Here, we report the development and characterisation of nanobodies as first-in-class inhibitors for NEDP1. The nanobodies display high-affinity (low nM) against NEDP1 and specifically inhibit NEDP1 processing activity in vitro and NEDP1 deconjugating activity in tissue-culture cells and in cell extracts. We also isolated nanobodies that bind to NEDP1 with high-affinity but do not affect NEDP1 activity. The developed nanobodies provide new tools to study the function of NEDP1 and to prevent deNEDDylation in cell extracts used in biochemical assays.

molecular biology↗