Biology subjects

Bubeck, D.

Publications and source records attributed to Bubeck, D..

11 recordsLinked to original sources

bioRxiv2026

Bacteroidales Secreted Antimicrobial Protein-1 receptor recognition is coupled to protease-activation to form bactericidal pores.

Bacteroidales secreted antimicrobial proteins (BSAPs) are diffusible MACPF-domain toxins that mediate intra-species antagonism in the gut microbiota. Here we define the mechanism of action of BSAP-1 from Bacteroides fragilis, showing how target specificity encoded within the N- and C-terminal domains is coordinated with pore-forming activity of the MACPF. We show that specificity of the toxin for its receptor is mediated by an extended interface comprised of the BSAP-1 C-terminal domain and residues on the receptor that differ from the orthologous protein of BSAP-1 producing strains. On the surface of susceptible cells, BSAP-1 undergoes proteolytic cleavage of an N-terminal regulatory domain, triggering its assembly into oligomeric pores. Cryo-electron microscopy of membrane-inserted BSAP-1 reveals a 13-subunit transmembrane {beta}-barrel pore formed through canonical MACPF rearrangements. Comparative modelling supports a conserved oligomerization mechanism across the BSAP family despite diversification of receptor-binding domains that target either proteins or glycan receptors. Together, these findings establish BSAP-1 as a receptor-targeted, protease-activated antibacterial MACPF toxin and provide a framework for understanding how gut Bacteroidales spatially restrict toxin activation to shape strain-level competition.

microbiology↗

bioRxiv2026

Internalized Components of Membrane Attack Complexes Disrupt Proteostasis and Acquire Alarmin-Like Properties

Immune effects of membrane attack complexes (MAC) have been widely attributed to their abilities to cause cell death. Here, we show that the MAC component, C9, forms non-cytolytic aggregates with pro-inflammatory effects. Intracellular aggregates of C9 are detected within inflamed tissues of patients in association with endothelial cell (EC) activation but not increased cell death. We identify NUMBL as a Rab35 effector that directly binds surface-bound C9 to promote C9 internalization and entry into the endolysosomal pathway. Within acidified endolysosomes, C9 forms insoluble aggregates that are targeted for degradative aggrephagy in a process that activates NF-{kappa}B. For C9 aggrephagy to occur, ZFYVE21, a Rab5 effector, complexes with RNF34 to bridge C9 aggregates to LC3B+ aggresome membranes. We detect C9 aggregates in vivo, and we show that a ZFYVE21-RNF34 signaling axis is required for C9 aggrephagy and NF-{kappa}B -dependent EC activation in three separate mouse models. Mice with conditional loss of ZFYVE21 in ECs show reduced aggregraphy, resulting in attenuated systemic inflammation and reduced tissue injury following skin transplantation. Our data show that the C9 component of MACs forms intracellular aggregates with alarmin-like properties.

biochemistry↗

bioRxiv2024

An oomycete effector co-opts a host RabGAP protein to remodel pathogen interface and subvert defense-related secretion

Pathogens have evolved sophisticated mechanisms to manipulate host cell membrane dynamics, a crucial adaptation to survive in hostile environments shaped by innate immune responses. Plant- derived membrane interfaces, engulfing invasive hyphal projections of fungal and oomycete pathogens, are prominent junctures dictating infection outcomes. Understanding how pathogens transform these host-pathogen interfaces to their advantage remains a key biological question. Here, we identified a conserved effector, secreted by plant pathogenic oomycetes, that co-opts a host Rab GTPase-activating protein (RabGAP), TBC1D15L, to remodel the host-pathogen interface. The effector, PiE354, hijacks TBC1D15L as a susceptibility factor to usurp its GAP activity on Rab8a--a key Rab GTPase crucial for defense-related secretion. By hijacking TBC1D15L, PiE354 purges Rab8a from the plasma membrane, diverting Rab8a-mediated immune trafficking away from the pathogen interface. This mechanism signifies an uncanny evolutionary adaptation of a pathogen effector in co- opting a host regulatory component to subvert defense-related secretion, thereby providing unprecedented mechanistic insights into the reprogramming of host membrane dynamics by pathogens.

plant biology↗

bioRxiv2022

AF2-multimer guided high accuracy prediction of typical and atypical ATG8 binding motifs

Macroautophagy/autophagy is an intracellular degradation process central to cellular homeostasis and defense against pathogens in eukaryotic cells. Regulation of autophagy relies on hierarchical binding of autophagy cargo receptors and adaptors to ATG8/LC3 protein family members. Interactions with ATG8/LC3 are typically facilitated by a conserved, short linear sequence, referred to as the ATG8/LC3 interacting motif/region (AIM/LIR), present in autophagy adaptors and receptors as well as pathogen virulence factors targeting host autophagy machinery. Since the canonical AIM/LIR sequence can be found in many proteins, identifying functional AIM/LIR motifs has proven challenging. Here we show that protein modelling using Alphafold-Multimer (AF2-multimer) identifies both canonical and atypical AIM/LIR motifs with a high level of accuracy. AF2-multimer can be modified to detect additional functional AIM/LIR motifs by using protein sequences with mutations in primary AIM/LIR residues. By combining protein modelling data from AF2-multimer with phylogenetic analysis of protein sequences and protein-protein interaction assays, we demonstrate that AF2-multimer predicts the physiologically relevant AIM motif in the ATG8-interacting protein 2 (ATI-2) as well as the previously uncharacterized non-canonical AIM motif in ATG3 from potato (Solanum tuberosum). AF2-multimer also identified the AIM/LIR motifs in pathogen-encoded virulence factors that target ATG8 members in their plant and human hosts, revealing that cross-kingdom ATG8-LIR/AIM associations can also be predicted by AF2-multimer. We conclude that the AF2-guided discovery of autophagy adaptors/receptors will substantially accelerate our understanding of the molecular basis of autophagy in all biological kingdoms.

molecular biology↗

bioRxiv2022

Structural basis for membrane attack complex inhibition by CD59

CD59 is an abundant immuno-regulatory receptor that protects human cells from damage during complement activation. Here we show how the receptor binds complement proteins C8 and C9 at the membrane to prevent insertion and polymerization of membrane attack complex (MAC) pores. We present cryoelectron microscopy structures of two inhibited MAC precursors known as C5b8 and C5b9. We discover that in both complexes, CD59 binds the pore-forming {square}-hairpins of C8 to form an intermolecular {square}-sheet that prevents membrane perforation. While bound to C8, CD59 deflects the cascading C9 {square}-hairpins, rerouting their trajectory into the membrane. Preventing insertion of C9 restricts structural transitions of subsequent monomers and indirectly halts MAC polymerization. We combine our structural data with cellular assays and molecular dynamics simulations to explain how the membrane environment impacts the dual roles of CD59 in controlling pore formation of MAC, and as a target of bacterial virulence factors which hijack CD59 to lyse human cells.

immunology↗

bioRxiv2022

Structural insights into the assembly and activation of IL-27 signalling complex

Interleukin 27 (IL-27) is a heterodimeric cytokine that elicits potent immuno-suppressive responses. Comprised of EBI3 and p28 subunits, IL-27 binds GP130 and IL-27R receptor chains to activate the JAK/STAT signalling cascade. However, how these receptors recognize IL-27 and form a complex capable of phosphorylating JAK proteins remains unclear. Here, we used cryo electron microscopy (cryoEM) to solve the structure of the IL-27 receptor recognition complex. Our data show how IL-27 serves as a bridge connecting IL-27R with GP130 to initiate signalling. While both receptors weakly bind the p28 component of the heterodimeric cytokine, EBI3 stabilizes the complex by binding a positively charged surface of IL-27R. We find that assembly of the IL-27 receptor recognition complex is distinct from both IL-12 and IL-6 cytokine families and provides a mechanistic blueprint for tuning IL-27 pleiotropic actions.

immunology↗

bioRxiv2021

Structural basis for how sMAC is packaged for clearance

Unregulated complement activation causes inflammatory and immunological pathologies with consequences for human disease. To prevent bystander damage during an immune response, extracellular chaperones (clusterin and vitronectin) capture and clear soluble precursors to the membrane attack complex (sMAC). However, how these chaperones block further polymerization of MAC and prevent the complex from binding target membranes remains unclear. Here, we address that question by combining cryo electron microscopy (cryoEM) and cross-linking mass spectrometry (XL-MS) to solve the structure of sMAC. Together our data reveal how clusterin recognizes and inhibits polymerizing complement proteins by binding a negatively charged surface of sMAC. Furthermore, we show that the pore-forming C9 protein is trapped in an intermediate conformation whereby only one of its two transmembrane {beta}-hairpins has unfurled. This structure provides molecular details for immune pore formation and helps explain a complement control mechanism that has potential implications for how cell clearance pathways mediate immune homeostasis.

biophysics↗

bioRxiv2020

Structural basis for tuning activity and membrane specificity of bacterial cytolysins

Cholesterol-dependent cytolysins (CDCs) form protein nanopores to lyse cells. They target eukaryotic cells using different mechanisms, but all require the presence of cholesterol to pierce lipid bilayers. How CDCs use cholesterol to selectively lyse cells is essential for understanding virulence strategies of several pathogenic bacteria, and for repurposing CDCs to kill new cellular targets. Here we address that question by trapping an early state of pore formation for the CDC intermedilysin, bound to the human immune receptor CD59 in a nanodisc model membrane. Our cryo-electron microscopy map reveals structural transitions required for oligomerization, which include the lateral movement of a key amphipathic helix. We demonstrate that the charge of this helix is crucial for tuning lytic activity of CDCs. Furthermore, we discover modifications that overcome the requirement of cholesterol for membrane rupture, which will facilitate engineering the target-cell specificity of pore-forming proteins.

biochemistry↗

bioRxiv2018

Single-molecule kinetics of pore assembly by the membrane attack complex

The membrane attack complex (MAC) is a hetero-oligomeric protein assembly that kills pathogens by perforating their cell envelopes. The MAC is formed by sequential assembly of soluble complement proteins C5b, C6, C7, C8 and C9, but little is known about the rate-limiting steps in this process. Here, we use rapid atomic force microscopy (AFM) imaging to show that MAC proteins oligomerize within the membrane, unlike structurally homologous bacterial pore-forming toxins. C5b6 interacts with the lipid bilayer prior to recruiting C7 and C8. We discover that incorporation of the first C9 is the kinetic bottleneck of MAC formation, after which rapid C9 oligomerization completes the pore. This defines the kinetic basis for MAC assembly and provides insight into how human cells are protected from bystander damage by the cell surface receptor CD59, which is offered a maximum temporal window to halt the assembly at the point of C9 insertion.

biophysics↗

bioRxiv2018

Structural basis of light-induced redox regulation in the Calvin cycle

In plants, carbon dioxide is fixed via the Calvin cycle in a tightly regulated process. Key to this regulation is the conditionally disordered protein CP12. CP12 forms a complex with two Calvin cycle enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK), inhibiting their activities. The mode of CP12 action was unknown. By solving crystal structures of CP12 bound to GAPDH, and the ternary GAPDH-CP12-PRK complex by electron cryo-microscopy, we reveal that formation of the N-terminal disulfide pre-orders CP12 prior to binding the PRK active site. We find that CP12 binding to GAPDH influences substrate accessibility of all GAPDH active sites in the binary and ternary inhibited complexes. Our model explains how CP12 integrates responses from both redox state and nicotinamide dinucleotide availability to regulate carbon fixation.\n\nOne Sentence SummaryHow plants turn off carbon fixation in the dark.

biochemistry↗

bioRxiv2018

CryoEM reveals how the complement membrane attack complex ruptures lipid bilayers

The membrane attack complex (MAC) is one of the immune systems first responders. Complement proteins assemble on target membranes to form pores that lyse pathogens and impact tissue homeostasis of self-cells. How MAC disrupts the membrane barrier remains unclear. Here we use electron cryo-microscopy and flicker spectroscopy to show that MAC interacts with lipid bilayers in two distinct ways. Whereas C6 and C7 associate with the outer leaflet and reduce the energy for membrane bending, C8 and C9 traverse the bilayer increasing membrane rigidity. CryoEM reconstructions reveal plasticity of the MAC pore and demonstrate how C5b6 acts as a platform, directing assembly of a giant {beta}-barrel whose structure is supported by a glycan scaffold. Our work provides a structural basis for understanding how {beta}-pore forming proteins breach the membrane and reveals a mechanism for how MAC kills pathogens and regulates cell functions.

biophysics↗