Biology subjects

Laeremans, T.

Publications and source records attributed to Laeremans, T..

5 recordsLinked to original sources

bioRxiv2024

Proteasome inhibition enhances latency reversal and boosts NK cell-mediated elimination of HIV-1 infected cells through HLA-E downregulation

The shock and kill strategy primarily depends on using latency reversal agents (LRAs) to reactivate the dormant viral reservoir, rendering it visible for recognition and subsequent elimination by the hosts immune system. While this approach has shown high efficacy in vitro and ex vivo, its in vivo application has yet to show significant delays in time to viral rebound. This lack of in vivo efficacy is most likely due to the insufficient elimination of reactivated reservoir cells by the hosts immune effector cells, including natural killer (NK) cells. Given the pivotal role of NK cells in antiviral immune responses, we hypothesized that they are crucial players in pursuing a functional cure against HIV-1. However, the inhibitory interaction between NKG2A and HLA-E diminishes their effectiveness. Notably, proteasome inhibition has been effective in reducing HLA-E expression on various tumor cell types, thereby enhancing NK-cell mediated killing. However, its impact on HIV-1 latency remains unexplored. We found that the proteasome inhibition could reverse the latent state of J-Lat cells while substantially reducing HLA-E expression. Additionally, a reduced expression of NKGA on primary NK cells was observed which led to an increase in NK-cell cytotoxicity. These results suggest that disrupting the NKG2A/HLA-E interaction could potentially augment the effectiveness of the shock and kill strategy by improving NK cell-mediated clearance of reactivated cells. ImportanceDespite promising in vitro results, purging the viral reservoir using LRAs has yet to demonstrate clinical benefits. A significant challenge lies in the inadequate activation of immune effector cells, such as CD8+ T cells and NK cells. Therefore, developing therapeutic strategies to address these challenges could enhance the effectiveness of the shock and kill strategy. This study highlights the need for therapeutic interventions to overcome these hurdles. Our findings show that proteasome inhibition not only triggers latency reversal but also enhances NK-cell mediated elimination of latently infected cells in vitro by downregulating HLA-E. This suggests that targeting the proteasome could be a novel therapeutic approach in the shock and kill strategy, potentially improving clinical outcomes.

immunology↗

bioRxiv2024

Structures of native SV2A reveal the binding mode for tetanus neurotoxin and anti-epileptic racetams

The synaptic vesicle glycoprotein 2A (SV2A) is a bona fide synaptic vesicle (SV) constituent of controversial function with homology to the major facilitator superfamily (MFS) and essential in vertebrate neurotransmission. Despite its high medical relevance as the target of the anti-epileptic drug Levetiracetam (LEV) and as receptor for clostridial neurotoxins (CNTs), among them several botulinum neurotoxin (BoNT) serotypes and potentially tetanus neurotoxin (TeNT), we lack detailed insight about these molecular interactions. We purified native SV2A from brain and subjected it to a structural analysis to advance our understanding of drug-binding to this enigmatic protein and explore structurally uncharacterized toxin-SV2A interfaces. Our analysis uncovered that TeNT binds SV2 proteins strikingly different from BoNT/A and delivers visual evidence for the dual receptor hypothesis through structurally resolved, co-purified gangliosides in the complex. The structures provide compelling support for SV2A as the protein receptor for TeNT in central neurons, recapitulate the geometry of CNT binding to dipartite SV2-ganglioside receptors on neuronal surfaces in a membrane-like constellation and have implications for toxin-engineering through a previously unknown SV2A-toxin interface. Further, a LEV-bound structure of SV2A reveals the drug-interacting residues and delineates a putative substrate pocket in SV2A. Our work provides an explanation for the SV2-isoform-specificity of LEV and its derivatives and paves the way for improved design of anti-convulsant drugs in epilepsy treatment.

biochemistry↗

bioRxiv2023

Structural Basis of μ-Opioid Receptor-Targeting by a Nanobody Antagonist

The -opioid receptor (OR), a prototypical member of the G protein-coupled receptor (GPCR) family, is the molecular target of opioid analgesics such as morphine and fentanyl. Due to the limitations and severe side effects of currently available opioid drugs, there is considerable interest in developing novel modulators of OR function. Most GPCR ligands today are small molecules, however biologics, including antibodies and nanobodies, are emerging as alternative therapeutics with clear advantages such as affinity and target selectivity. Here, we describe the nanobody NbE, which selectively binds to the OR and acts as an antagonist. We functionally characterize NbE as an extracellular and genetically encoded {micro}OR ligand and uncover the molecular basis for {micro}OR antagonism by solving the cryo-EM structure of the NbE-{micro}OR complex. NbE displays a unique ligand binding mode and achieves {micro}OR selectivity by interactions with the orthosteric pocket and extracellular receptor loops. Based on a {beta}-hairpin loop formed by NbE that deeply inserts into the {micro}OR and centers most binding contacts, we design short peptide analogues that retain {micro}OR antagonism. The work illustrates the potential of nanobodies to uniquely engage with GPCRs and describes novel OR ligands that can serve as a basis for therapeutic developments.

molecular biology↗

bioRxiv2022

Natural Killer activating Multimeric Immunotherapeutic compleXes (NaMiX) induce cytotoxic activity and killing of HIV-1 infected cells

HIV-1 persists in viral reservoirs of latently infected CD4+ T cells containing integrated replication-competent viral DNA. Combined Antiretroviral Therapy (cART) does not eradicate HIV-1 reservoirs and treatment interruption will ultimately lead to viral load rebound. HIV-1 infection dramatically reduces the proportion of functional NK cell subsets and increases the expression of the checkpoint inhibitors NKG2A and KIR2DL. In this regard, we developed novel recombinant molecules combining multimers of the IL-15/IL-15R complex with the single-chain fragment variables (scFvs) of NKG2A or KIR2DL, and named them as Natural killer activating Multimeric immunotherapeutic compleXes (NaMiX). NaMiX significantly improved the cytotoxic activity of NK cells against HIV-1 positive ACH-2 cells and resistant Raji cancer cells by increasing their degranulation capacity, release of granzyme B, perforin and IFN-{gamma} expression. Targeting the NKG2A receptor had a stronger effect compared to the targeting of the KIR2DL receptor due to its higher expression on NK cells. In a viral inhibition assay using CD4+ T cells from HIV-1 positive patients under cART, NaMiX initially increased viral replication which was subsequently inhibited by stimulated NK cells. In humanized NSG tg-huIL-15 mice showing functional NK cells, we observed enhanced activation, degranulation and killing by NK cells from the spleen of mice treated with anti-NKG2A NaMiX compared to the cells of control mice previously infected with HIV-1 and treated with cART. Although NaMiX did not delay viral load rebound after treatment interruption in a first attempt, it tend to decrease total HIV-1 DNA in the lungs of the mice. Blocking the inhibitory receptor NKG2A in combination with targeted multimers of IL-15 on NK cells could therefore be a promising immunotherapeutic strategy towards HIV-1 functional cure.

immunology↗

bioRxiv2018

A genetically encoded biosensor reveals location bias of opioid drug action

Opioid receptors (ORs) precisely modulate behavior when activated by native peptide ligands but distort behaviors to produce pathology when activated by non-peptide drugs. A fundamental question is how drugs differ from peptides in their actions on target neurons. One way that drugs can differ is by imposing selective effects on the conformational equilibrium of individual ORs. We wondered if drugs can also impose selective effects on the location of OR activation in individual OR-expressing neurons. Here we develop a genetically encoded biosensor that directly localizes ligand-induced activation and deactivation of ORs in living cells, and use it to generate the first real-time map of the spatiotemporal organization of - and {delta}-OR activation in neurons. Peptide agonists produce a characteristic activation pattern initiated in the plasma membrane and propagating to endosomes after receptor internalization. Drugs produce a different activation pattern by uniquely driving OR activation in the somatic Golgi apparatus and extending throughout the dendritic arbor in Golgi outposts. These results demonstrate a new approach to probe the cellular basis of neuromodulation and reveal that drugs profoundly distort the spatiotemporal landscape of neuronal OR activation.

neuroscience↗