Biology subjects

Muyldermans, S.

Publications and source records attributed to Muyldermans, S..

6 recordsLinked to original sources

bioRxiv2024

Allosteric inhibition of trypanosomatid pyruvate kinases by a camelid single-domain antibody

African trypanosomes are the causative agents of neglected tropical diseases affecting both humans and livestock. Disease control is highly challenging due to an increasing number of drug treatment failures. African trypanosomes are extracellular, blood-borne parasites that mainly rely on glycolysis for their energy metabolism within the mammalian host. Trypanosomal glycolytic enzymes are therefore of interest for the development of trypanocidal drugs. Here, we report the serendipitous discovery of a camelid single-domain antibody (sdAb aka Nanobody) that selectively inhibits the enzymatic activity of trypanosomatid (but not host) pyruvate kinases through an allosteric mechanism. By combining enzyme kinetics, biophysics, structural biology, and transgenic parasite survival assays, we provide a proof-of-principle that the sdAb-mediated enzyme inhibition negatively impacts parasite fitness and growth.

biochemistry↗

bioRxiv2022

Sensitive protein detection using site-specifically oligonucleotide-conjugated nanobodies

High-quality affinity probes are critical for sensitive and specific protein detection, in particular for detection of protein biomarkers at early phases of disease development. Proximity extension assays (PEA) have been used for high-throughput multiplexed protein detection of up to a few thousand different proteins in one or a few microliters of plasma. Clonal affinity reagents can offer advantages over the commonly used polyclonal antibodies (pAbs) in terms of reproducibility and standardization of such assays. Here we explore nanobodies (Nb) as an alternative to pAbs as affinity reagents for PEA. We describe an efficient site-specific approach for preparing high-quality oligo-conjugated Nb probes via enzyme coupling using Sortase A (SrtA). The procedure allows convenient removal of unconjugated affinity reagents after conjugation. The purified high-grade Nb probes were used in PEA and the reactions provided an efficient means to select optimal pairs of binding reagents from a group of affinity reagents. We demonstrate that Nb-based PEA (nano-PEA) for interleukin-6 (IL6) detection can augment assay performance, compared to the use of pAb probes. We identify and validate Nb combinations capable of binding in pairs without competition for IL6 antigen detection by PEA.

bioengineering↗

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↗

bioRxiv2019

Structural basis of nanobody-recognition of grapevine fanleaf virus and of virus resistance loss

Grapevine fanleaf virus (GFLV) is a picorna-like plant virus transmitted by nematodes that affects vineyards worldwide. Nanobody (Nb)-mediated resistance against GFLV has been created recently and shown to be highly effective in plants including grapevine, but the underlying mechanism is unknown. Here we present the high-resolution cryo-EM structure of the GFLV-Nb23 complex which provides the basis for the molecular recognition by the nanobody. The structure reveals a composite binding site bridging over 3 domains of the capsid protein (CP) monomer. The structure provides a precise mapping of the Nb23 epitope on the GFLV capsid in which the antigen loop is accommodated through an induced fit mechanism. Moreover, we uncover and characterize several resistance-breaking GFLV isolates with amino acids mapping within this epitope, including C-terminal extensions of the CP, which would sterically interfere with Nb binding. Escape variants with such extended CP fail to be transmitted by nematodes linking Nb-mediated resistance to vector transmission. Together, these data provide insights into the molecular mechanism of Nb23-mediated recognition of GFLV and of virus resistance loss.\n\nSignificanceGrapevine fanleaf virus (GFLV) is a picorna-like plant virus that severely impacts vineyards worldwide. While Nanobodies (Nb) confer resistance to GFLV in plants the underlying molecular mechanism of action is unknown. Here we present the high-resolution cryo-EM structure of the GFLV-Nb complex. It uncovers the conformational epitope on the capsid surface which is a composite binding site into which the antigen loop is accommodated through an induced fit mechanism. Furthermore, we describe several resistance-breaking isolates of GFLV with reduced Nb binding capacity. Those that carry a C-terminal extension also fail to be transmitted by nematodes. Together, these data provide structure-function insights into the Nb-GFLV recognition and the molecular mechanism leading to loss of resistance.

biochemistry↗

bioRxiv2019

AAV mediated delivery of a novel anti-BACE1 VHH reduces Abeta in an Alzheimer’s disease mouse model

Single domain antibodies (VHH) are potentially disruptive therapeutics, with important biological value for treatment of several diseases, including neurological disorders. However, VHH have not been widely used in the central nervous system (CNS), as it is hard to reach therapeutic levels, both because of their restricted blood-brain-barrier penetration and their apparent rapid clearance from the parenchyma. Here, we propose a gene transfer strategy based on adeno-associated virus (AAV)-based vectors to deliver VHH directly into the CNS, ensuring continuous production at therapeutic levels. As a proof-of-concept, we explored the potential of AAV-delivered VHH to inhibit BACE1, a well-characterized target in Alzheimers disease. First, we generated a panel of VHHs targeting BACE1. One of them, VHH-B9, showed high selectivity for BACE1 and efficacy in lowering BACE1 activity in vitro. We then went on to demonstrate significant reductions in amyloid beta (A{beta}) levels after AAV-based delivery of VHH-B9 into the CNS of a mouse model of cerebral amyloidosis. These results constitute a novel therapeutic approach for neurodegenerative diseases, which is applicable to a range of CNS disease targets.

neuroscience↗

bioRxiv2019

ADSoluble aggregates present in cerebrospinal fluid change in size and mechanism of toxicity during Alzheimer's disease progression

Soluble aggregates of amyloid-{beta} (A{beta}) have been associated with neuronal and synaptic loss in Alzheimers disease (AD). However, despite significant recent progress, the mechanisms by which these aggregated species contribute to disease progression are not fully determined. As the analysis of human cerebrospinal fluid (CSF) provides an accessible window into the molecular changes associated with the disease progression, we studied the soluble A{beta} aggregates present in CSF samples from individuals with AD, mild cognitive impairment (MCI) and healthy controls. We found that these aggregates vary structurally and in their mechanisms of toxicity. More small aggregates of A{beta} that can cause membrane permeabilization already found in MCI; in established AD, the aggregates were larger and more prone to elicit a pro-inflammatory response in glial cells. These results suggest that different neurotoxic mechanisms are prevalent at different stages of AD.

neuroscience↗