Biology subjects

Aden, J.

Publications and source records attributed to Aden, J..

4 recordsLinked to original sources

bioRxiv2025

Mechanisms involved in the lipid membrane tubulation activity of the pH-responsive MakA protein from Vibrio cholerae

Bacterial pore-forming toxins (PFTs) are key virulence factors that disrupt host cell membranes. The Vibrio cholerae cytotoxin MakA, a member of the ClyA structural family of -PFTs, exhibits a unique, pH-dependent ability to induce lipid membrane tubulation. This study elucidates the molecular mechanisms underlying this process. We identify the N-terminal histidine residue (His30) of MakA as a critical pH sensor that is indispensable for membrane tubulation but not required for membrane binding and cell lysis. Substitution of His30 with lysine, MakAH30K , abrogated tubulation without affecting membrane insertion, instead triggering aberrant membrane fusion events. Circular dichroism spectroscopy analysis revealed that the MakAH30K substitution attenuates the robust -helical transition observed in wild-type MakA upon lipid membrane binding, providing a molecular explanation for its functional defect. Furthermore, we established that membrane cholesterol contributes to MakA-induced tubulation of lipid membranes, as its absence or mutation of a putative cholesterol-binding motif MakAI236D&I237D abolishes both membrane association and tubule formation. A key implication of our work is the mechanistic uncoupling of membrane tubulation from cytolysis, revealing that MakA can perturb membranes via distinct pathways. These findings define a sophisticated activation mechanism where His30 protonation in the acidic host environment and specific cholesterol binding synergistically drive the conformational change necessary for helical oligomerization and membrane tubulation. We discuss our findings from the viewpoint of possible convergent evolution, where bacterial PFTs like MakA partly may mimic ancient membrane remodelers involved in membrane repair pathways, such as ESCRT-III, but for bacterial fitness in host interactions and pathogenesis. Moreover, the present study provides a foundation for possible development of therapeutic strategies aimed at inhibiting these critical interactions.

biochemistry↗

bioRxiv2025

Bcl-2 Oligomerizes Bax on the Mitochondrial Membrane Surface Preventing the Initial Stages of Apoptosis

The Bcl-2 family of proteins control mitochondrial outer membrane (MOM) pore formation, crucial to cellular clearance via apoptosis. However, the molecular principles by which opposing family members inhibit, mediate or promote MOM perforation remain elusive. Here, we demonstrate that cell-protecting Bcl-2 directly sequesters cell-killing Bax into a protein-protein complex at the membrane interface preventing Bax forming apoptotic pores. Neutron reflectometry showed Bax association with Bcl-2 occurs through the formation of Bax protein oligomers on the membrane surface. Bax binding to the membrane surface was proportional to the membrane-embedded Bcl-2 suggestive of protein-protein complex formation through both Bcl-2/Bax and Bax/Bax interactions. Bcl-2/Bax sequestration to prevent perforation was observed in membrane models with and without pro-apoptotic cardiolipin present. Our findings shed fundamental new light on the communication of Bcl-2 with its cell-killing relatives at the mitochondrias membraneous exterior to prevent cells from undergoing apoptosis. TeaserMembrane-embedded Bcl-2 sequesters Bax, to prevent the perforation of mitochondrial membranes by Bax which would initiate apoptosis.

biophysics↗

bioRxiv2022

Bax Forms a Membrane Surface Protein-Lipid Complex as it Initiates Apoptosis

Cellular clearance by apoptosis is essential in life. In its intrinsic (mitochondrial) pathway apoptotic members of the Bcl-2 (B cell CLL/lymphoma-2) protein family, such as Bax (Bcl-2-associated X) protein, perforate the mitochondrial outer membrane (MOM), which causes release of apoptotic factors and final cell death. How those apoptotic proteins mechanistically exert their action at the membrane level still however remains elusive. Upon internal stress signals Bax is massively recruited to the MOM, where it oligomerizes and partially penetrates into the membrane. Using neutron reflectometry (NR) and attenuated total reflection Fourier-Transform infrared spectroscopy (ATR-FTIR) spectroscopy we unraveled key molecular steps of this membrane-affiliation process of Bax on a spatial and temporal scale. By titrating intact human Bax to MOM-like bilayers containing cardiolipin, essential for protein recruitment, we could identify different functional phases. Initially, there is a fast adsorption event to the membrane surface with high affinity. Thereafter, a kinetically slower (minutes to hours) event occurs with Bax penetration, thereby triggering a major reorganizing of the mitochondrial bilayer. Finally, a membrane-Bax complex is generated, with a minor Bax population remaining membrane-inserted, while the main population is relocated to the membrane surface upon lipid redistribution into a complex with Bax; a process enabling membrane perforation. We propose a comprehensive molecular model of mitochondrial membrane penetration by formation of complex Bax/lipid clusters; a concept which provides a new foundation to understand the cell-killing activity of Bax and its apoptotic relatives in human cells. Significance StatementThe apoptotic Bax protein is a key player in the mitochondrial apoptotic pathway. Here, neutron reflectometry (NR) unravels the mechanism by which Bax is targeting and perforating mitochondria to release apoptotic factors for final cell death. We found that this cardiolipin driven process of the outer mitochondrial membrane system has two main phases. Upon a fast (10-20 min) phase of membrane association Bax initiates the formation of pores by removing lipids and depositing them as Bax/lipid complexes on top of the bilayer on a time scale of several hours similar to in vivo apoptotic cell death. Our results provide a mechanistic rationale for cell-killing processes driven by apoptotic Bcl-2 proteins; and their molecular inhibition in many cancers.

biophysics↗

bioRxiv2021

Protein-lipid interaction at low pH induces oligomerisation of the MakA cytotoxin from Vibrio cholerae

Many pathogenic bacteria produce protein toxins that target and perturb host cell membranes. The secreted -pore-forming toxins (-PFTs) cause membrane damage via pore formation. This study demonstrates a remarkable, hitherto unknown mechanism by an -PFT protein from Vibrio cholerae. As part of the MakA/B/E tripartite toxin, MakA is involved in membrane pore formation similar to other -PFTs. In contrast, MakA protein alone induces tube-like structures in the acidic lysosomal host cell compartment. In vitro studies unravel the dynamics of tubular growth, which occur in a pH-, lipid- and concentration-dependent manner. A 3.7-[A] cryo-electron microscopy structure of MakA filaments reveals a unique protein-lipid superstructure. In its active -PFT conformation, MakA embeds its transmembrane helices into a thin annular lipid bilayer and spirals around a central cavity. Our study provides molecular insights into a novel tubulation mechanism of an -PFT protein, revealing a new mode of action by a secreted bacterial toxin.

cell biology↗