Biology subjects

Manon, S.

Publications and source records attributed to Manon, S..

8 recordsLinked to original sources

bioRxiv2026

Production of membrane-embedded Bcl-2 proteins - Use of cell-free synthesis in continuous exchange for co-translational insertion of Bcl-2 proteins in lipid bilayer nanodiscs

The BCL-2 family proteins are key regulators of apoptosis, functionally divided in pro- and anti-apoptotic proteins, with a third group acting as regulators. Their ability to partition between the cytosol and intra-cellular membranes (essentially the mitochondrial outer membrane) is a primary regulator of their functions. A second contributor is their ability to form homotypic complexes (pro-pro or anti-anti) or heterotypic complexes (pro-anti). If the structures of monomeric cytosolic members have largely been characterized, the functional and structural study of membrane-embedded proteins remains incomplete. Unlocking this knowledge is expected to enable evaluating new therapeutic strategies to either activate pro-apoptotic members, or inactivate anti-apoptotic ones. Lipid bilayer nanodiscs and improved cell-free protein synthesis have provided the technical breakthrough to achieve the description at the atomic level of conformations and higher order assemblies of these proteins in their membrane-associated states. Here we describe detailed and straightforward protocols for generating nanodisc-inserted members of the Bcl-2 family, through the example of anti-apoptotic Bcl-xL, and pro-apoptotic Bax and Bak. Full-length, untagged proteins are expressed from bacterial extracts in the presence of pre-assembled nanodiscs to allow co/post-translational insertion in lipid bilayer, followed by affinity chromatography purification. A more detailed characterization is presented for Bak, to exemplify structural and mechanistic studies enabled by these methods. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/745005v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@5da1d1org.highwire.dtl.DTLVardef@12aca96org.highwire.dtl.DTLVardef@5a3e73org.highwire.dtl.DTLVardef@ba009d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

VDAC2 stabilizes a membrane-inserted, primed intermediate of BAX activation

BAX, a major effector of mitochondrial apoptosis, is activated through a series of conformational transitions that lead to mitochondrial outer membrane permeabilization. Genetic studies have established VDAC2 as an essential regulator of BAX-mediated apoptosis, yet the molecular basis of this regulation remains unresolved. The absence of direct structural and biochemical characterization of VDAC2-BAX interactions has prevented mechanistic understanding of how VDAC2 influences BAX activation. Here, using complementary biochemical, biophysical and structural approaches, we reconstituted and characterized a stable VDAC2-BAX complex that was not observed with VDAC1, highlighting an isoform-specific role in BAX regulation. We show that VDAC2 captures and stabilizes a primed BAX conformation displaying the hallmarks of activation, including membrane insertion, BH3 exposure, and increased accessibility of the N-terminal activation region. By integrating AlphaFold3 predictions with molecular dynamics simulations, biochemical, biophysical and structural constraints, we derive an experimentally-supported structural model in which BAX is anchored through its 9 helix while its soluble domain partially extends over the VDAC2 pore. Together, our findings support a model in which VDAC2 facilitates BAX membrane insertion and stabilizes a membrane-inserted, activation- competent BAX intermediate. Rather than serving as a structural component of apoptotic pores, VDAC2 acts as a regulatory checkpoint in BAX activation. These results provide a molecular explanation for the emerging role of VDAC2 in mitochondrial apoptosis and establish structural basis for a previously inaccessible intermediate in the BAX activation pathway.

biochemistry↗

bioRxiv2026

Molecular determinants of Bcl-xL membrane insertion - Structural plasticity exploration in solution and in nanodiscs identifies IsoAsp deamidation as a loss-of function mechanism in vivo.

Multi-domain Bcl-2 family proteins share the ability to form dimers and oligomers, regardless of their pro- or anti-apoptotic activity. Homotypic interactions (pro-pro and anti-anti) and heterotypic interactions (pro-anti) are well-documented, but the role of higher-order organization in their survival/death functions and membrane interactions remains largely unresolved. Looking into anti-apoptotic Bcl-xL, essentially engineered/truncated proteoforms lacking the disordered loop and/or the hydrophobic C-terminal helix, have been used as proxies of the full-length (FL) protein, to elaborate on structural transitions and intermediate states between monomers and homooligomers prior to membrane insertion. Using a minimalist approach with recombinant FL-Bcl-xL (aa 1-233) and artificial nano-membranes, we demonstrate that both the loop and the C-terminal helix are potent contributors to Bcl-xL structural plasticity. Unlike 3D domain swapping (3DDS) dimers resolved with the C-terminal truncated protein, FL-Bcl-xL organized in solution as dimers bridging the unique Cys151 from two monomers. This spontaneous fold indicates that the C-terminal helix drives FL-Bcl-xL to explore different conformations than truncated Bcl-xL. Yet, dimerization was not a prerequisite for membrane insertion into nanodiscs and Cys151 did not contribute to Bcl-xL survival functions in cells. These data support monomeric Bcl-xL as the minimal functional unit in membranes. Further exploring the frequently deleted disordered loop, we discovered that deamidation of Asn52 and Asn66 in IsoAsp, but not in Asp, impairs membrane insertion into nanodiscs. Thus, this reductionist biochemical approach clarifies the loss of tumorigenic function we observed for deamidated Bcl-xL in xenograft experiments in vivo.

biochemistry↗

bioRxiv2024

The membrane insertion of the pro-apoptotic protein Bax is a Tom22-dependent multi-step process: a study in nanodiscs

Membrane insertion of the pro-apoptotic protein Bax was investigated by setting up cell-free synthesis of full-length Bax in the presence of pre-formed nanodiscs. While Bax was not spontaneously inserted in nanodiscs, co-synthesis with the mitochondrial receptor Tom22 promoted Bax membrane insertion. The initial interaction of Bax with the lipid bilayer exposed the hydrophobic GALLL motif in H1 leading to Bax precipitation through hydrophobic interactions. The same motif was recognized by Tom22, triggering conformational changes leading to the extrusion and the ensuing membrane insertion of the C-terminal hydrophobic H9. Tom22 was also required for Bax-membrane insertion after Bax was activated either by BH3-activators or by its release from Bcl-xL by WEHI-539. The effect of Tom22 was impaired by D154Y substitution in Bax-H7 and T174P substitution in Bax-H9, that are found in several tumors. Conversely, a R9E substitution promoted the spontaneous insertion of Bax in nanodiscs, in the absence of Tom22. Both Tom22-activated Bax and BaxR 9E alone permeabilized liposomes to dextran-10kDa and formed [~]5nm-diameter pores in nanodiscs. The concerted regulation of Bax membrane insertion by Tom22 and BH3-activators is discussed.

biochemistry↗

bioRxiv2022

Cell-free synthesis and reconstitution of Bax in nanodiscs: comparison between wild-type Bax and a constitutively active mutant

Bax is a major player in the mitochondrial pathway of apoptosis, by permeabilizing the Outer Mitochondrial Membrane (OMM) to various apoptogenic factors, including cytochrome c. In order to get further insight into the structure and function of Bax when it is inserted in the OMM, we attempted to reconstitute Bax in nanodiscs. Cell-free protein synthesis in the presence of nanodiscs did not allow to obtain Bax-containing nanodiscs, but it provided a simple way to purify full-length Bax without any tag. Purified wild-type Bax (BaxWT) and a constitutively active mutant (BaxP168A) displayed biochemical properties that were in line with previous characterizations following their expression in yeast and human cells followed by their reconstitution into liposomes. Both Bax variants were then reconstituted in nanodiscs. Size exclusion chromatography, dynamic light scattering and transmission electron microscopy showed that nanodiscs formed with BaxP168A were larger than nanodiscs formed with BaxWT. This was consistent with the hypothesis that BaxP168A was reconstituted in nanodiscs as an active oligomer.

biochemistry↗

bioRxiv2019

Pleiotropy of Bcl-2 family proteins is an ancient trait in the metazoan evolution

In the animal kingdom, proteins of the Bcl-2 family are widely recognized as regulators of mitochondrial outer membrane permeabilization (MOMP), leading to apoptotic cell death. These proteins were recently also shown to control IP3-dependent calcium fluxes at the level of the endoplasmic reticulum (ER). However, the origin and evolution of these pleiotropic functions remain elusive. Here, we molecularly characterized the four members of the Bcl-2 family (trBcl-2L1 to -2L4) in the most primitive metazoan, namely Trichoplax adhaerens. Primary structure and phylogenetic analyses demonstrated that all four trBcl-2 homologs belong to the multidomain Bcl-2 group and presented a conserved C-terminus transmembrane (TM) domain. TrBcl-2L1 and trBcl-2L2 are highly divergent proteins clustering with the anti-apoptotic Bcl-2 members, whereas trBcl-2L3 and trBcl-2L4 were homologous to the pro-apoptotic Bax (trBax) and Bak (trBak). Interestingly, at the functional level, trBak operates as a BH3 only sensitizer repressing the anti-apoptotic activities of trBcl-2L1 and trBcl-2L2, whereas trBax leads to MOMP, similarly to the well-known indirect model of Bax activation. Finally, we found that trBcl-2L1 had a dual ER and mitochondrial subcellular localization and was able to bind to IP3R. By generating two TM domain mutants we demonstrated that trBcl-2L1 targeted to the ER was able to control IP3-dependent calcium fluxes, whereas Mito-trBcl-2L1 represses trBax-dependent MOMP, suggesting that Bcl-2 pleiotropy appeared early and was conserved throughout metazoan evolution.

evolutionary biology↗

bioRxiv2019

Ubiquitination of the yeast receptor Atg32 modulates mitophagy

Mitophagy, the process that degrades mitochondria selectively through autophagy, is involved in the quality control of these organelles. In yeast, the presence of the Atg32 protein on the outer mitochondrial membrane allows for the recognition and targeting of superfluous or damaged mitochondria for degradation. Some posttranslational modifications, such as phosphorylation, are crucial for the execution of the mitophagy process. In our study, we showed that in the stationary phase of growth, and to a lesser extent during starvation, the Atg32 protein level decreases. The fact that a decline in Atg32 level can be prevented by inhibition of the proteolytic activity of proteasome may indicate that Atg32 is also ubiquitylated. In fact, mass spectrometry analysis of purified Atg32 protein showed ubiquitination of lysine residue in position 282. These different patterns of posttranslational modifications of Atg32 could allow cells to control the mitophagy process carefully.

cell biology↗

bioRxiv2018

Mitochondria-Associated Membranes (MAMs) are involved in Bax mitochondrial localization and cytochrome c release

The distribution of the pro-apoptotic protein Bax in the outer mitochondrial membrane (OMM) is a central point of regulation of apoptosis. It is now widely recognized that parts of the endoplasmic reticulum (ER) are closely associated to the OMM, and are actively involved in different signalling processes. We adressed a possible role of these domains, called Mitochondria-Associated Membranes (MAMs) in Bax localization and fonction, by expressing the human protein in a yeast mutant deleted of MDM34, a ERMES component (ER-Mitochondria Encounter Structure). By affecting MAMs stability, the deletion of MDM34 altered Bax mitochondrial localization, and decreased its capacity to release cytochrome c. Furthermore, the deletion of MDM34 decreased the size of an uncompletely released, MAMs-associated pool of cytochrome c.

biochemistry↗