Biology subjects

Dumoulin, M.

Publications and source records attributed to Dumoulin, M..

3 recordsLinked to original sources

bioRxiv2026

Structure of PCPE-2 in complex with the BMP-1 metalloprotease reveals the molecular basis of its mechanism of inhibition

PCPE-2 (procollagen C-proteinase enhancer-2) is an extracellular glycoprotein playing dual functions in the regulation of BMP-1/tolloid-like proteinases. Like its homologue PCPE-1, PCPE-2 can enhance the proteolytic maturation of fibrillar procollagens, but it also acts as a potent and specific inhibitor of BMP-1 through formation of a high-affinity complex with the protease. Here, we investigated the molecular basis of this inhibitory interaction using complementary biochemical, biophysical and structural approaches. We show that the two CUB domains of PCPE-2 must be covalently linked for efficient BMP-1 binding and inhibition, consistent with cooperative engagement of the protease. Cryo-electron microscopy combined with density-guided structural modelling and hydrogen/deuterium-exchange mass spectrometry supports a bipartite interaction complex in which the CUB1 domain of PCPE-2 engages the catalytic domain of BMP-1, while the CUB2 domains of both proteins interact together. The interaction involves the calcium-binding surfaces of the PCPE-2 CUB domains that also participate in procollagen recognition, providing a structural framework for understanding the mutually exclusive interactions of PCPE-2 with BMP-1 and procollagen. Allosteric inhibition is supported by the binding of PCPE-2 CUB1 opposite to the active site cleft, leading to the shielding of the residues surrounding the S1-prime pocket, as observed by HDX-MS. Together, these findings define the architecture of the inhibitory PCPE-2/BMP-1 complex and provide the molecular basis for understanding the distinct regulatory activities of the two procollagen C-proteinase enhancers.

biochemistry↗

bioRxiv2022

Development of nanobodies as theranostic agents against CMY-2-like class C β-lactamases

Soluble single-domain fragments derived from the unique variable region of camelid heavy-chain antibodies (VHHs) against enzymes may behave as potent inhibitors. The immunization of alpacas with the CMY-2 {beta}-lactamase led to the isolation of three VHHs that specifically recognized and inhibited CMY-2. The structure of the complex VHH cAbCMY-2(254)/CMY-2 was determined by X-ray crystallography. We showed that the epitope is close to the active site and that the CDR3 of the VHH protrudes in the catalytic site. The {beta}-lactamase inhibition was found to follow a mixed profile with a predominant non-competitive component. The three isolated VHHs recognized overlapping epitopes since they behaved as competitive binder. Our study identified a binding site that can be targeted by a new class of {beta}-lactamases inhibitors designed with the help of a peptidomimetic approach. Furthermore, the use of mono or bivalent VHH and rabbit polyclonal anti-CMY-2 antibodies enable the development of the first generation of ELISA test for the detection of CMY-2 produced by resistant bacteria. IMPORTANCEThe still increasing antimicrobial resistance in human clinic or veterinary medicine is a major threat for modern chemotherapy. Beside the major caution in the use of current antibiotics, it is important to develop new classes of antibiotics. This work was focused on {beta}-lactamases that are the enzymes involved in the hydrolysis of the major class of antibiotics, the {beta}-lactam compounds. We selected camelid antibodies that inhibit CMY-2, a class C {beta}-lactamase produced by bacteria isolated from the veterinary and human settings. We characterized the conformational epitope present in CMY-2 in order to create a new family of inhibitors based on the paratope of the antibody. Finally, we designed a primary version of a detection system based on an ELISA using VHH and polyclonal antibodies.

biochemistry↗

bioRxiv2021

A β-secretase modulator decreases Tau pathology and preserves short-term memory in a mouse model of neurofibrillary degeneration

A structure-activity relationship has enabled us to identify two molecules, MAGS02-14 and PEL24-199, sharing a {beta}-secretase modulatory effect but having or not a lysosomotropic activity, respectively. More importantly, MAGS02-14 and PEL24-199 only differ from each other by a single nitrogen atom. However, which of the lysosomotropic and/or {beta}-secretase modulating activities is necessary for the pharmacological effect in vivo remains ill-defined. To address this question, the THY-Tau22 transgenic model of NFD was treated for 6 weeks in a curative paradigm and short-term memory, Tau burden, and inflammatory processes were studied. PEL24-199, possessing only the {beta}-secretase modulatory activity, was shown to restore the short-term memory and to reduce NFD. This effect was associated with reduced phosphorylation of Tau, increased phosphatase expression, and a decrease of astrogliosis. Our results therefore suggest that the lysosomotropic activity may be dispensable for the effect on both A{beta} and Tau pathologies.

pharmacology and toxicology↗