Membrane Insertion Research Articles

Mechanistic Cooperation of the Two Pore-Forming Transmembrane Motifs Regulates the β-Barrel Pore Formation by Listeriolysin O.

Listeriolysin O (LLO) is a potent membrane-damaging pore-forming toxin (PFT) secreted by the bacterial pathogen Listeria monocytogenes. LLO belongs to the family of cholesterol-dependent cytolysins (CDCs), which specifically target cholesterol-containing cell membranes to form oligomeric pores and induce membrane damage. CDCs, including LLO, harbor designated pore-forming motifs. In the soluble monomeric state, these motifs are present as helical segments (two transmembrane helices (TMHs); TMH1 and TMH2), and in the course of oligomeric pore formation, they convert into transmembrane β-hairpins to form the β-barrel scaffold of the CDC pores. Despite their well-established role in forming the β-barrel pore scaffold, precise structural implications of the two distinct TMH motifs and their membrane-insertion mechanism still remain obscure. Here, we show that the two TMH motifs of LLO contribute differently to maintaining the structural integrity of the toxin. While the deletion of TMH1 imposed a more serious defect, truncation of TMH2 was found to have a less severe effect on the structural integrity. Despite showing membrane-binding and oligomerization ability, the TMH2-deleted LLO variant displayed drastically abrogated pore-forming activity, presumably due to compromised membrane-insertion efficacy of the pore-forming TMH motifs. When probed for the membrane-insertion mechanism, we found slower membrane-insertion kinetics for TMH2 than for TMH1. Interestingly, deletion of TMH2 arrested membrane insertion of TMH1, thus suggesting a stringent cooperation between the two TMH motifs in regulating the pore-formation mechanism of LLO. Taken together, our study provides new mechanistic insights regarding the membrane-damaging action of LLO, in the CDC family of PFTs.

Defective glycosylation and ELFN1 binding of mGluR6 congenital stationary night blindness mutants.

Synaptic transmission from photoreceptors to ON-bipolar cells (BCs) requires the postsynaptic metabotropic glutamate receptor mGluR6, located at BC dendritic tips. Binding of the neurotransmitter glutamate initiates G protein signaling that regulates the TRPM1 transduction channel. mGluR6 also interacts with presynaptic ELFN adhesion proteins, and these interactions are important for mGluR6 synaptic localization. The mechanisms of mGluR6 trafficking and synaptic targeting remain poorly understood. In this study, we investigated mGluR6 missense mutations from patients with congenital stationary night blindness (CSNB), which is associated with loss of synaptic transmission to ON-BCs. We found that multiple CSNB mutations in the extracellular ligand-binding domain of mGluR6 impart a trafficking defect leading to lack of complex N-glycosylation but efficient plasma membrane insertion, suggesting a Golgi bypass mechanism. These mutants fail to bind ELFN1, consistent with lack of a necessary modification normally acquired in the Golgi. The same mutants were mislocalized in bipolar cells, explaining the loss of function in CSNB. The results reveal a key role of Golgi trafficking in mGluR6 function, and suggest a role of the extracellular domain in Golgi sorting.

Insertion and Anchoring of the HIV-1 Fusion Peptide into a Complex Membrane Mimicking the Human T-Cell.

A fundamental understanding of how the HIV-1 envelope (Env) protein facilitates fusion is still lacking. The HIV-1 fusion peptide, consisting of 15 to 22 residues, is the N-terminus of the gp41 subunit of the Env protein. Further, this peptide, a promising vaccine candidate, initiates viral entry into target cells by inserting and anchoring into human immune cells. The influence of membrane lipid reorganization and the conformational changes of the fusion peptide during the membrane insertion and anchoring processes, which can significantly affect HIV-1 cell entry, remains largely unexplored due to the limitations of experimental measurements. In this work, we investigate the insertion of the fusion peptide into an immune cell membrane mimic through multiscale molecular dynamics simulations. We mimic the native T-cell by constructing a nine-lipid asymmetric membrane, along with geometrical restraints accounting for insertion in the context of gp41. To account for the slow time scale of lipid mixing while enabling conformational changes, we implement a protocol to go back and forth between atomistic and coarse-grained simulations. Our study provides a molecular understanding of the interactions between the HIV-1 fusion peptide and the T-cell membrane, highlighting the importance of the conformational flexibility of fusion peptides and local lipid reorganization in stabilizing the anchoring of gp41 into the targeted host membrane during the early events of HIV-1 cell entry. Importantly, we identify a motif within the fusion peptide critical for fusion that can be further manipulated in future immunological studies.

Excess shed mesothelin disrupts pancreatic cancer cell clustering to impair peritoneal colonization.

Peritoneum is the second most common site of metastasis in patients with pancreatic ductal adenocarcinoma (PDAC). Peritoneal colonization is impaired in PDAC cells with knockout (KO) of the cancer surface antigen mesothelin (MSLN) or by introducing Y318A mutation in MSLN to prevent binding to mucin-16 (MUC-16). MSLN has a membrane-bound form but is also shed to release soluble MSLN (sMSLN). Their individual roles in peritoneal metastasis are unknown. Here, a C-terminal truncated MSLN mutant (∆591) incapable of cell membrane insertion but proficient in secretion was engineered. Expression of ∆591 MSLN failed to rescue peritoneal metastasis in MSLN KO cells and inhibited peritoneal colonization when overexpressed in WT PDAC cells. Exposing PDAC cells to conditioned medium (CM) containing excess sMSLN impaired cancer cell clustering invitro and in peritoneal fluid invivo, while CM containing only Y318A sMSLN did not. These data demonstrate that interaction of membrane-bound MSLN with MUC-16 promotes cell clustering that is critical for efficient peritoneal metastasis. However, peritoneal colonization by MSLN KO cells was rescued by expression of ∆591 mutant MSLN bearing Y318A mutation, suggesting that sMSLN also has a MUC-16-independent role in peritoneal spread. Alterations in inflammatory signaling pathways occurred following KO cell exposure to CM containing sMSLN, and CM from cancer cells with intact peritoneal metastasis provoked increased KO cell secretion of IL-1α. While excess sMSLN inhibits cell clustering and peritoneal colonization, sMSLN may also promote PDAC peritoneal metastasis independent of MUC-16.

Very broad distribution of β sheet registries of the HIV gp41 fusion peptide supports mutational robustness for fusion and infection

HIV, like other membrane-enveloped viruses, has protein spikes that include a fusion peptide (Fp) segment that binds the host cell membrane and plays a critical role in fusion (joining) viral and cell membranes. The HIV Fp is the ~23 N-terminal residues of the gp41 spike protein. Fp adopts intermolecular antiparallel β sheet structure when lipid fraction cholesterol ≈0.3, which is comparable to host cells. Rotational-echo double-resonance NMR was applied to probe the registries (alignments) of adjacent Fp molecules in membrane-bound sheets. The data were fitted to determine quantitative populations, f(t)'s, of individual antiparallel registries indexed by t, the number of residues in the registry. Both wild-type (WT) and fusion-defective V2E Fp sheets have broad but very different registry distributions, each with at least eight populated registries with f(t) > 0.02, and [Formula: see text] and ⟨t⟩V2E = 18.5. The broad WT distribution likely improves mutational robustness for HIV, as Fp is a neutralization epitope of the immune system, and Fp mutations are required for immune evasion during chronic HIV infection. V2E fusion is reduced because longer Fp sheets increase separation between initially apposed membranes. The f(t)WT were well-fitted to free energies that were sums of contributions from sheet length, aligned leucines, and sidechain membrane insertion. The f(t)V2E's were similarly well-fitted except there wasn't the insertion contribution. Relative to V2E, WT fusion is enhanced by deeper membrane insertion of Fp with accompanying greater dislocation of neighboring lipids. This study provides a rare quantitative determination of broad molecular structural distributions by experiment.

Improving interlaminar fracture toughness and compression after impact strength of carbon fiber reinforced epoxy composites by interleaving electrospinning polyethersulfone nanofiber

AbstractThe effect of membrane thickness of electrospinning polyethersulfone (PES) nanofiber membranes on interlayer toughness and impact resistance of carbon fiber reinforced epoxy resin (CF/EP) composites are researched. Intercalation composites with different nanofiber membrane thicknesses, ranging from 10 to 50 μm, are tested at mode I and mode II interlaminar fracture toughness. The impact resistance of nanofiber membranes with different thicknesses is evaluated by the compression after impact (CAI) strength test. Moreover, nanofiber toughening contributions are investigated using the crack path and surface analysis. Results indicate that PES nanofibers increase the toughness of interlayer resin and change the interlaminar crack propagation path of CF/EP composites. The optimum nanofiber membrane thickness for mode I (GI = + 58%) and CAI (CAI strength = + 33%) is 50 μm, while for mode II is 30 μm (GII = + 16.4%). The storage modulus of CF/EP composites increases with the increase of nanofiber membrane thickness. The study also confirms the insertion of a nanofiber membrane slightly improves the tensile strength and interlaminar shear strength (ILSS) of the CF/EP composites.Highlights PES nanofiber membranes of different thicknesses were inserted into the CF/EP. CAI and GI improved by 33% and 58%, respectively of CF/EP composites. The relationship between interlayer fracture toughness and CAI was discussed.

Receptor interactions of protoxin and activated Vip3Aa structural conformations in Spodoptera exigua.

The Vip3Aa insecticidal protein, produced by Bacillus thuringiensis, has been effectively used in commercial Bt-crops to manage lepidopteran pests. Upon ingestion by larvae, the protoxin is processed by midgut proteases into the activated protein and binds specifically to its receptors in the midgut, leading to insect mortality. Cryo-EM resolution of the trypsin-processed Vip3Aa protein unveiled structural remodelling of the N-terminal region during the transition from protoxin to activated protein. This conformational change has been demonstrated to be crucial for toxicity against Spodoptera exigua larvae, a major global lepidopteran pest. In this study, we investigated the relevance of the structural remodelling for the specific binding to midgut receptors. We conducted in vitro binding assays with radiolabelled proteins and brush border membrane vesicles (BBMV) from S. exigua, employing structural mutants that lock the protein in either its protoxin or its activated conformation. Our results indicate that both structural stages of the protein share binding sites in the midgut epithelium. Moreover, in vivo competition assays revealed that Vip3Aa is able to bind to functional receptors in S. exigua larvae both as protoxin and as activated protein. Altogether, our findings point to both structural conformations contributing to receptor binding. In vivo, either spontaneous structural shift upon proteolytic cleavage or receptor-mediated remodelling could be occurring. However, the timing and context in which the conformational change occurs could influence membrane insertion and toxicity. Our results show the complex interplay between proteolytic processing, protein structure and receptor interactions in Vip3Aa's toxicity. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Insights into the structural changes that trigger receptor binding upon proteolytic activation of Bacillus thuringiensis Vip3Aa insecticidal protein.

Bacillus thuringiensis (Bt) bacteria produce different pore forming toxins with insecticidal activity, including Cry and Vip3 proteins. While both Cry and Vip3 cause insect death by forming pores in susceptible lepidopteran larval midgut cells, their mechanisms of action differ. The Vip3Aa protoxin adopts a tetramer-structure, where each monomer has five distinct domains. Upon proteolytic activation, the Vip3 tetramer undergoes a large conformational change forming a syringe like structure that is ready for membrane insertion and pore formation. Here we show that Vip3Aa protoxin had low binding to Spodoptera frugiperda brush border membrane vesicles (BBMV) unlike the activated toxin that bound specifically in a concentration dependent way, suggesting that a structural change upon Vip3Aa proteolytic activation is required for efficient receptor binding. Consistently, the Vip3Aa protoxin showed no toxicity to Sf9 cells compared to the activated toxin. In contrast, Cry1Fa protoxin and its activated toxin, were both highly toxic to Sf9 cells. To identify the region of Vip3 involved in binding to BBMV proteins, different overlapping peptides from Vip3Aa covering domains III, IV and V were expressed, and binding analysis were performed against BBMV, showing that domain III is the primary binding domain. Additionally, domains III, IV and V amino acid residues that become exposed upon activation of Vip3Aa were identified. Mutagenesis of these exposed residues revealed three amino acids (K385, K526 and V529) located in two structural adjacent loops, domain III loop β5-β6 and loop α11-β16 that connects domains III and IV, that are crucial for binding to the midguts of S. frugiperda larvae and for toxicity. Our results demonstrate that proteolytic activation of Vip3Aa exposes a receptor binding region essential for its toxicity.

Reversible Electrically Modulated Electroosmotic Flow Via Carbon Nanotube Membrane

Objectives The reversible electrical modulation of chemical delivery is crucial for various biomedical engineering applications, enabling a tailored drug delivery profile. Electroosmosis flow (EOF) represents a significant electrokinetic phenomenon where a bulk flow is induced through narrow channels including capillaries or microfluidic devices or porous medium, under the influence of an applied electric field (Fig. 1a). Since the velocity of EOF is directly proportional to the applied current density ( I ) and the EOF strength ( K eo ), a standardized indicator related to the zeta potential of the channel walls, it provides the possibility of controlling the direction and velocity of EOF by simply adjusting the surface zeta potential.[1] Herein, a novel concept of electrically modulated delivery utilizing EOF generated within the porous network of the carbon nanotube (CNT) membrane is reported. The charge sign and magnitude of the micropores inside the CNT membrane are altered by applying varying voltages, leading to corresponding changes in the direction and velocity of the EOF. Results and Discussion The multi-walled carbon nanotubes powered are first treated with ozone and dispersed in ethanol. Then, the suspension is formed into the random alignment and cohesive CNT membrane by vacuum-assisted filtration (Fig. 1b-c). The experimental setup consists of symmetrically positioned reservoirs at both ends and a CNT membrane placed between the two reservoirs filled with 0.1 M KCl solution. The electrically modulated system is comprised of two circuits: the main circuit that generates an axial electric field to drive the EOF and the sub-circuit that varies the sign and magnitude of charges on the CNT membrane. The controllability of the EOF direction and velocity is evaluated by observing the movement of the liquid column in the reservoirs (Fig. 1d -e). Based on the established experimental setup, the voltage at which EOF is unobserved is referred to as the neutral voltage (or bias voltage) and is used as the reference for electrical modulation. Significant differences in the direction and velocity of EOF can be observed and measured on the CNT membrane when a positive (or negative) voltage relative to the bias voltage is applied. The EOF under the main circuit of + 0.25 mA and the sub-circuit voltage of + 0.5 V and - 0.5 V relative to the bias voltage, indicating a mobility rate of - 0.781 μL/mm2·min and + 0.972 μL/mm2·min, respectively (Fig. 1f). The plus and minus means that opposite directions of EOF are generated with applied voltages. Next, to demonstrate the effect of applied voltages on the EOF rate, the sub-circuit voltage of + 0.5 V and + 0.25 V relative to the neutral voltage was applied (with the same main circuit of + 0.25 mA), generating a mobility rate of - 0.781 μL/mm2·min and - 0.431 μL/mm2·min. The difference in velocity is due to the differential surface zeta potential within the microchannels of the CNT membrane. Conclusion Here, this study introduces a novel concept of a chemical delivery system by focusing on the electrically modulated EOFs generated in the microchannels of the CNT membrane. According to the results of the experiments, it can be demonstrated that altering the direction and velocity of EOF through electrical modulation is achievable. Additionally, the electrical modulation on EOF is feasible under varying axial electric field strengths and concentration solution environments. Reference [1] Kusama, S., Sato, K., Matsui, Y., Kimura, N., Abe, H., Yoshida, S., & Nishizawa, M. (2021). Transdermal electroosmotic flow generated by a porous microneedle array patch. Nature communications , 12 (1), 658. Fig. 1 Experimental setup and electrical modulation performance. (a) The mechanism of EOF. (b) Optical and SEM images of CNT membrane. (c) Schematic illustration of EOF generated in the micropores of CNT membrane. (d) An experimental setup consisting of Franz Cells with insertion of CNT membrane and the EOF direction and velocity were analyzed by observing the liquid movement. (e) Electrical modulation. (f) The relationship between the applied voltages (relative to the neutral voltage) and flow rates. Figure 1

Anchorage of bacterial effector at plasma membrane via selective phosphatidic acid binding to modulate host cell signaling.

Binding phospholipid is a simple, yet flexible, strategy for anchorage of bacterial effectors at cell membrane to manipulate host signaling responses. Phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-biphosphate are the only two phospholipid species known to direct bacterial effectors to establish inner leaflet localization at the plasma membrane. Here, selectivity of phosphatidic acid (PA) by bacterial effectors for the plasma membrane anchorage and its molecular entity was identified. C-terminal BID domain of Bartonella T4SS effectors (Beps) directed the plasma membrane localization of Beps in host cells through binding with PA. A hydrophobic segment of the 'HOOK' subdomain from BID is inserted into the bilayer to enhance the interaction of positively charged residues with the lipid headgroups. Mutations of a conserved arginine facilitating the electrostatic interaction, a conserved glycine maintaining the stability of the PA binding groove, and hydrophobic residues determining membrane insertion, prevented the anchorage of Beps at the plasma membrane. Disassociation from plasma membrane to cytosol attenuated the BepC capacity to induce stress fiber formation and cell fragmentation in host cells. The substitution of alanine with aspartic acid at the -1 position preceding the conserved arginine residue hindered BepD anchoring at the plasma membrane, a vital prerequisite for its ability to elicit IL-10 secretion in host macrophages. In conclusion, our findings reveal the PA-binding properties of bacterial effectors to establish plasma membrane localization and will shed light on the intricate mechanisms employed by bacterial effectors within host cells.

Selective Recruitment of Antibodies to Cancer Cells and Immune Cell-mediated Killing via In Situ Click Chemistry.

Many current cancer immunotherapies function by redirecting immune system components to recognize cancer biomarkers and initiate a cytotoxic attack. The lack of a universal tumor biomarker limits the therapeutic potential of these approaches. However, one feature characteristic of nearly all solid tumors is extracellular acidity. This inherent acidity provides the basis for targeted drug delivery via the pH-low insertion peptide (pHLIP), which selectively accumulates in tumors in vivo due to a pH-dependent membrane insertion propensity. Previously, we established that we could selectively decorate cancer cells with antigen-pHLIP conjugates to facilitate antibody recruitment and subsequent killing by engineered effector cells via antibody-dependent cellular cytotoxicity (ADCC). Here, we present a novel strategy for opsonizing antibodies on target cell surfaces using click chemistry. We utilize pHLIP to facilitate selective tetrazine - trans-cyclooctene ligation of human IgGs to the cancer cell surface and induce ADCC. We demonstrate that our approach activates the primary ADCC signaling pathway via CD16a (FcγRIIIa) receptors on effector cells and induces the killing of cancer cell targets by engineered NK cells.

MRNA localization and thylakoid protein biogenesis in the filamentous heterocyst-forming cyanobacterium Anabaena sp. PCC 7120.

Heterocyst-forming cyanobacteria such as Anabaena (Nostoc) sp. PCC 7120 exhibit extensive remodeling of their thylakoid membranes during heterocyst differentiation. Here we investigate the sites of translation of thylakoid membrane proteins in Anabaena vegetative cells and developing heterocysts, using mRNA fluorescent in situ hybridization (FISH) to detect the location of specific mRNA species. We probed mRNAs encoding reaction center core components and the heterocyst-specific terminal oxidases Cox2 and Cox3. As in unicellular cyanobacteria, the mRNAs encoding membrane-integral thylakoid proteins are concentrated in patches at the inner face of the thylakoid membrane system, adjacent to the central cytoplasm. These patches mark the putative sites of translation and membrane insertion of these proteins. Oxidase activity in mature heterocysts is concentrated in the specialized "honeycomb" regions of the thylakoid membranes close to the cell poles. However, cox2 and cox3 mRNAs remain evenly distributed over the inner face of the thylakoids, implying that oxidase proteins migrate extensively after translation to reach their destination in the honeycomb membranes. The RNA-binding protein RbpG is the closest Anabaena homolog of Rbp3 in the unicellular cyanobacterium Synechocystis sp. PCC 6803, which we previously showed to be crucial for the correct location of photosynthetic mRNAs. An rbpG null mutant shows decreased cellular levels of photosynthetic mRNAs and photosynthetic complexes, coupled with perturbations to thylakoid membrane organization and lower efficiency of the Photosystem II repair cycle. This suggests that the chaperoning of photosynthetic mRNAs by RbpG is important for the correct coordination of thylakoid protein translation and assembly.IMPORTANCECyanobacteria have a complex thylakoid membrane system which is the site of the photosynthetic light reactions as well as most of the respiratory activity in the cell. Protein targeting to the thylakoids and the spatial organization of thylakoid protein biogenesis remain poorly understood. Further complexity is found in some filamentous cyanobacteria that produce heterocysts, specialized nitrogen-fixing cells in which the thylakoid membranes undergo extensive remodeling. Here we probe mRNA locations to reveal thylakoid translation sites in a heterocyst-forming cyanobacterium. We identify an RNA-binding protein important for the correct co-ordination of thylakoid protein translation and assembly, and we demonstrate the effectiveness of mRNA fluorescent in situ hybridization (FISH) as a way to probe cell-specific gene expression in multicellular cyanobacteria.

Driving DNA Nanopore Membrane Insertion through Dipolar Coupling.

DNA nanopores appear to be a plausible alternative to the use of transmembrane proteins. The specificity and programmability of DNA interactions allow the design of synthetic channels that insert into lipid bilayers and can regulate the ionic transport across them. In this Communication, we investigate the dependence of insertion capabilities on the electrostatic properties of the nanopore and show that the presence of a permanent electric dipole is an important factor for the nanopore to insert into the membrane. On the contrary, in the absence of such a dipole, most DNA nanopores bind to the bilayer without channel formation. We also show that this modification does not hinder the possibility of triggering a measurable conformational change with a single short oligonucleotide.

Exploring the Membrane-Active Interactions of Antimicrobial Long-Chain Fatty Acids Using a Supported Lipid Bilayer Model for Gram-Positive Bacterial Membranes.

The dynamic nature of bacterial lipid membranes significantly impacts the efficacy of antimicrobial therapies. However, traditional assay methods often fall short in replicating the complexity of these membranes, necessitating innovative approaches. Herein, we successfully fabricated model bacterially supported lipid bilayers (SLBs) that closely mimic the characteristics of Gram-positive bacteria using the solvent-assisted lipid bilayer (SALB) technique. By employing a quartz crystal microbalance with dissipation and fluorescence microscopy, we investigated the interactions between these bacterial mimetic membranes and long-chain unsaturated fatty acids. Specifically, linolenic acid (LNA) and linoleic acid (LLA) demonstrated interaction behaviors correlated with the critical micelle concentration (CMC) on Gram-positive membranes, resulting in membrane remodeling and removal at concentrations above their respective CMC values. In contrast, oleic acid (OA), while showing similar membrane remodeling patterns to LNA and LLA, exhibited membrane insertion and CMC-independent activity on the Gram-positive membranes. Particularly, LNA and LLA demonstrated bactericidal effects and promoted membrane permeability and ATP leakage in the bacterial membranes. OA, characterized by a CMC-independent activity profile, exhibited potent bactericidal effects due to its robust penetration into the SLBs, also enhancing membrane permeability and ATP leakage. These findings shed light on the intricate molecular mechanisms governing the interactions between long-chain unsaturated fatty acids and bacterial membranes. Importantly, this study underscores the potential of using biologically relevant model bacterial membrane systems to develop innovative strategies for combating bacterial infections and designing effective therapeutic agents.

CaMKIIα-TARPγ8 signaling mediates hippocampal synaptic impairment in aging.

Aging-related decline in memory and synaptic function are associated with the dysregulation of calcium homeostasis, attributed to the overexpression of voltage-gated calcium channels (VGCC). The membrane insertion of AMPAR governed by the AMPAR auxiliary proteins is essential for synaptic transmission and plasticity (LTP). In this study, we demonstrated the hippocampal expression of the transmembrane AMPAR regulatory proteins γ-8 (TARPγ8) was reduced in aged mice along with the reduced CaMKIIα activity and memory impairment. We further showed that TARPγ8 expression was dependent on CaMKIIα activity. Inhibition of CaMKIIα activity significantly reduced the hippocampal TARPγ8 expression and CA3-CA1 LTP in young mice to a similar level to that of the aged mice. Furthermore, the knockdown of hippocampal TARPγ8 impaired LTP and memory in young mice, which mimicked the aging-related changes. We confirmed the enhanced hippocampal VGCC (Cav-1.3) expression in aged mice and found that inhibition of VGCC activity largely increased both p-CaMKIIα and TARPγ8 expression in aged mice, whereas inhibition of NMDAR or Calpains had no effect. In addition, we found that the exogenous expression of human TARPγ8 in the hippocampus in aged mice restored LTP and memory function. Collectively, these results indicate that the synaptic and cognitive impairment in aging is associated with the downregulation of CaMKIIα-TARPγ8 signaling caused by VGCC activation. Our results suggest that TARPγ8 may be a key molecular biomarker for brain aging and that boosting CaMKIIα-TARPγ8 signaling may be critical for the restoration of synaptic plasticity of aging and aging-related diseases.

5143 Disulfide Bonds of Thyroid Peroxidase Are Critical Elements for Subcellular Localization, Proteasome-Dependent Degradation, and Enzyme Activity

Abstract Disclosure: H. Iwasaki: None. H. Suwanai: None. K. Kanekura: None. N. Satoshi: None. F. Yakou: None. H. Sakai: None. K. Ishii: None. N. hara: None. R. Suzuki: None. Background: Congenital hypothyroidism (CH) is caused by mutations in cysteine residues including Cys655 and Cys825 that form disulfide bonds in thyroid peroxidase (TPO). It is highly likely that disulfide bonds play an important role in TPO activity. However, no study has comprehensively analyzed cysteine mutations that form disulfide bonds in TPO. In this study, we induced mutations in cysteine residues involved in the formation of disulfide bonds and analyzed their effect on subcellular localization, degradation, and enzyme activities to evaluate the importance of disulfide bonds in TPO. Methods: Vector plasmid TPO mutants C655F and C825R in CH patients were transfected into HEK293 cells. TPO activity and protein expression levels were measured by Amplex red assay and western blotting. The same procedure was performed in the presence of proteasome inhibitor MG132. Subcellular localization was determined by immunocytochemistry and flow cytometry. The location of all disulfide bonds within TPO was predicted by in silico analysis. All TPO mutations associated with disulfide bonds were induced. TPO activity and protein expression levels were also measured in all TPO mutants associated with disulfide bonds using the Amplex red assay and western blotting. Results: C655F and C825R showed significantly decreased activity and protein expression compared to the wild-type (P < 0.05). In the presence of a proteasome inhibitor MG132, the protein expression level of TPO was increased to the level comparable with that of the wild-type but its activity did not. The degree of subcellular distribution of TPO to the cell surface in the mutant was lower than that in the wild-type TPO. Twenty-four cysteine residues were involved in the formation of 12 disulfide bonds in TPO, and all TPO mutants harboring an amino acid substitution in each cysteine showed significantly reduced TPO activity and protein expression levels. Furthermore, the differences in TPO activity depended on the position of the disulfide bond. Conclusions: All 12 disulfide bonds play an important role in the activity of TPO. Furthermore, the mutations lead to misfolding, degradation and membrane insertion. Presentation: 6/2/2024

Structural basis of α-latrotoxin transition to a cation-selective pore

The potent neurotoxic venom of the black widow spider contains a cocktail of seven phylum-specific latrotoxins (LTXs), but only one, α-LTX, targets vertebrates. This 130 kDa toxin binds to receptors at presynaptic nerve terminals and triggers a massive release of neurotransmitters. It is widely accepted that LTXs tetramerize and insert into the presynaptic membrane, thereby forming Ca2+-conductive pores, but the underlying mechanism remains poorly understood. LTXs are homologous and consist of an N-terminal region with three distinct domains, along with a C-terminal domain containing up to 22 consecutive ankyrin repeats. Here we report cryoEM structures of the vertebrate-specific α-LTX tetramer in its prepore and pore state. Our structures, in combination with AlphaFold2-based structural modeling and molecular dynamics simulations, reveal dramatic conformational changes in the N-terminal region of the complex. Four distinct helical bundles rearrange and together form a highly stable, 15 nm long, cation-impermeable coiled-coil stalk. This stalk, in turn, positions an N-terminal pair of helices within the membrane, thereby enabling the assembly of a cation-permeable channel. Taken together, these data give insight into a unique mechanism for membrane insertion and channel formation, characteristic of the LTX family, and provide the necessary framework for advancing novel therapeutics and biotechnological applications.

Biogenesis of mitochondrial β-barrel membrane proteins.

β-barrel membrane proteins in the mitochondrial outer membrane are crucial for mediating the metabolite exchange between the cytosol and the mitochondrial intermembrane space. In addition, the β-barrel membrane protein subunit Tom40 of the translocase of the outer membrane (TOM) is essential for the import of the vast majority of mitochondrial proteins encoded in the nucleus. The sorting and assembly machinery (SAM) in the outer membrane is required for the membrane insertion of mitochondrial β-barrel proteins. The core subunit Sam50, which has been conserved from bacteria to humans, is itself a β-barrel protein. The β-strands of β-barrel precursor proteins are assembled at the Sam50 lateral gate forming a Sam50-preprotein hybrid barrel. The assembled precursor β-barrel is finally released into the outer mitochondrial membrane by displacement of the nascent β-barrel, termed the β-barrel switching mechanism. SAM forms supercomplexes with TOM and forms a mitochondrial outer-to-inner membrane contact site with the mitochondrial contact site and cristae organizing system (MICOS) of the inner membrane. SAM shares subunits with the ER-mitochondria encounter structure (ERMES), which forms a membrane contact site between the mitochondrial outer membrane and the endoplasmic reticulum. Therefore, β-barrel membrane protein biogenesis is closely connected to general mitochondrial protein and lipid biogenesis and plays a central role in mitochondrial maintenance.

Antagonistic nanobodies implicate mechanism of GSDMD pore formation and potential therapeutic application

Inflammasome activation results in the cleavage of gasdermin D (GSDMD) by pro-inflammatory caspases. The N-terminal domains (GSDMDNT) oligomerize and assemble pores penetrating the target membrane. As methods to study pore formation in living cells are insufficient, the order of conformational changes, oligomerization, and membrane insertion remained unclear. We have raised nanobodies (VHHs) against human GSDMD and find that cytosolic expression of VHHGSDMD-1 and VHHGSDMD-2 prevents oligomerization of GSDMDNT and pyroptosis. The nanobody-stabilized GSDMDNT monomers partition into the plasma membrane, suggesting that membrane insertion precedes oligomerization. Inhibition of GSDMD pore formation switches cell death from pyroptosis to apoptosis, likely driven by the enhanced caspase-1 activity required to activate caspase-3. Recombinant antagonistic nanobodies added to the extracellular space prevent pyroptosis and exhibit unexpected therapeutic potential. They may thus be suitable to treat the ever-growing list of diseases caused by activation of (non-) canonical inflammasomes.

Upgrading In Vitro Digestion Protocols with Absorption Models

Intestinal digestion and absorption are complex processes; thus, it is a challenge to imitate them realistically. There are numerous approaches available, with different disadvantages and advantages. The simplest methods to mimic absorption are the non-cell-based transport models but these lack important characteristics of enterocytes of the intestine. Therefore, the most often used method is to measure absorption through viable mammalian cells (most commonly Caco-2 cells, cultured on membrane insert plates), which not only assures the incorporation of brush border enzymes (responsible for the final digestion of peptides and disaccharides), it also simulates the absorption process. This means that influx/efflux transporter-facilitated transport, carrier-mediated transport, endocytosis, and transcytosis is also imitated besides passive diffusion. Still, these also lack the complexity of intestinal epithelium. Organoids or ex vivo models are a better approach if we want to attain precision but the highest accuracy can be achieved with microfluidic systems (gut-on-a-chip models). We propose that more research is necessary, and food absorption should also be studied on gut-on-a-chips, especially with fragmented organoids. Our review supports the choices of a proper intestinal epithelium model, which may have a key role in functional food development, nutrition studies, and toxicity assessment.