Endosomal Research Articles

Dengue Virus Fusion Peptide Promotes Hemifusion Formation by Disordering the Interfacial Region of the Membrane.

Membrane fusion is the first step in the infection process of the enveloped viruses. Enveloped viruses fuse either at the cell surface or enter the cell through endocytosis and transfer their internal genetic materials by fusing with the endosomal membrane at acidic pH. In this work, we have evaluated the effect of the Dengue virus fusion peptide (DENVFP) on the polyethylene glycol (PEG)-mediated lipid mixing of vesicles (hemifusion formation) at pH 5 and pH 7.4 with varying cholesterol concentrations. We have demonstrated that the DENVFP promotes hemifusion formation during the fusion of small unilamellar vesicles (SUVs) mainly at pH 5.0. Moreover, the fusion process demonstrates a strong correlation between fusogenicity and the amount of membrane cholesterol. We have further evaluated the partitioning ability of the peptide in three different membranes at pH 5.0 and pH 7.4. The fusogenic ability of the peptide at pH 5.0 is associated with the composition-dependent binding affinity of the peptide to the membrane. The depth-dependent fluorescence probes are used to evaluate membrane organization and dynamics utilizing steady-state and time-resolved fluorescence spectroscopic techniques. Our results show that the DENV FP promotes hemifusion formation by fluidizing the interfacial region of the membrane.

Comprehensive Analysis of the NHX Gene Family and Its Regulation Under Salt and Drought Stress in Quinoa (Chenopodium quinoa Willd.)

Background/Objectives: Abiotic stresses such as salinity and drought significantly constrain crop cultivation and affect productivity. Quinoa (Chenopodium quinoa Willd.), a facultative halophyte, exhibits remarkable tolerance to drought and salinity stresses, making it a valued model for understanding stress adaptation mechanisms. The objective of this study was to identify and characterize Sodium/Hydrogen antiporter (NHX) genes from the quinoa genome and study their role in stress tolerance. Methods: We identified and characterized 10 NHX genes from the quinoa genome, which belong to the monovalent cation/proton antiporter 1 (CPA1) superfamily. Comprehensive analysis, including phylogenetic relationships, motif patterns, and structural characteristics, was performed to classify these genes into three subfamilies. Physicochemical properties such as isoelectric point (pI), GRAVY, and transmembrane domains were examined. Promoter analysis was conducted to identify cis-elements linked to abiotic stress responses, phytohormone signalling, and light regulation. qPCR analysis was used to assess the differential expression patterns of CqNHX genes under salt and drought stress. Results: The analysis revealed that the NHX genes were divided into three subfamilies localized to vacuolar, plasma, and endosomal membranes. These genes exhibited structural and functional diversity. Promoter analysis indicated the presence of cis-elements associated with abiotic stress responses, phytohormone signalling, and light regulation, suggesting diverse regulatory roles. qPCR analysis revealed differential expression patterns of CqNHX genes under salt and drought stress, with vacuolar NHXs showing higher induction in leaf tissues under salinity. This underscores their critical role in sodium sequestration and ion homeostasis. Evolutionary analysis indicated a high degree of conservation within subfamilies, alongside evidence of purifying selection. Conclusions: The findings enhance our understanding of the molecular basis of stress tolerance in quinoa and provide valuable targets for genetic engineering to improve crop resilience to environmental challenges.

Developing peptide-based fusion inhibitors as an antiviral strategy utilizing coronin 1 as a template.

Enveloped viruses enter the host cells by endocytosis and subsequently fuse with the endosomal membranes, or fuse with the plasma membrane at the cell surface. The crucial stage of viral infection, regardless of the route taken to enter the host cell, is membrane fusion. The present work aims to develop a peptide-based fusion inhibitor that prevents membrane fusion by modifying the properties of the participating membranes, without targeting a protein. This would allow us to develop a fusion inhibitor that might work against a larger spectrum of enveloped viruses as it does not target any specific viral fusion protein. With this goal in mind, we have designed a novel peptide by modifying a native sequence derived from coronin 1, a phagosomal protein, that helps to avoid lysosomal degradation of mycobacterium-loaded phagosomes. The designed peptide, mTG-23, inhibits ∼30-40% fusion between small unilamellar vesicles containing varying amounts of cholesterol by modulating the biophysical properties of the participating bilayers. As a proof of principle, we have further demonstrated that the mTG-23 inhibits Influenza A virus infection in A549 and MDCK cells (with ∼EC50 of 20.45 μM and 21.55 μM, respectively), where viral envelope and endosomal membrane fusion is a crucial step. Through a gamut of biophysical and biochemical methods, we surmise that mTG-23 inhibits viral infection by inhibiting viral envelope and endosomal membrane fusion. We envisage that the proposed antiviral strategy can be extended to other viruses that employ a similar modus operandi, providing a novel pan-antiviral approach.

Development of a StIW111C-based bioresponsive pore-forming conjugate for permeabilizing the endosomal membrane.

Gene expression manipulation is pivotal in therapeutic approaches for various diseases. Non-viral delivery systems present a safer alternative to viral vectors, with reduced immunogenicity and toxicity. However, their effectiveness in promoting endosomal escape, a crucial step in gene transfer, remains limited. To address this drawback, we developed a reducible conjugate combining the StIW111C mutant of Sticholysin I, a pore-forming protein, with a polylysine peptide. This conjugate aims to enhance plasmid DNA (pDNA) release from endosomes, thereby improving gene expression. A 16-polylysine peptide was attached to StIW111C via a disulfide bridge to block its membrane-binding site, enabling controlled modulation of pore-forming activity in response to a reductive environment. This modification also enhances the conjugate's positive charge, facilitating binding to nucleic acids. Formation of positively charged nanometric complexes was achieved by mixing pDNA with the bio-responsive StIW111C conjugate and polylysine peptide. In vitro assays showed the conjugate could permeabilize endosomes, but reporter gene expression was limited, likely due to the largest complexes or aggregates that reduced conjugate entry and blocked nucleic acid release. CryoTEM imaging revealed the presence of small aggregate fraction, highlighting the need for further redesign to prevent aggregation and optimize endosomal release of non-viral systems for enhanced gene expression.

Mechanistic insights into endosomal escape by sodium oleate-modified liposomes

Endosomal entrapment significantly limits the efficacy of drug delivery systems. This study investigates sodium oleate-modified liposomes (SO-Lipo) as an innovative strategy to enhance endosomal escape and improve cytosolic delivery in 4T1 triple-negative breast cancer cells. We aimed to elucidate the mechanistic role of sodium oleate in promoting endosomal escape and compared the performance of SO-Lipo with unmodified liposomes (Unmodified-Lipo) and Aurein 1.2-modified liposomes (AUR-Lipo). Liposomes were prepared using the thin-film hydration method, resulting in Unmodified-Lipo, SO-Lipo, and AUR-Lipo formulations. The particle sizes were 102.2 ± 3.30 nm for Unmodified-Lipo, 109.6 ± 7.65 nm for SO-Lipo, and 151.9 ± 5.88 nm for AUR-Lipo, with polydispersity indices below 0.25, indicating uniform size distribution. Endosomal escape efficiency was evaluated through confocal microscopy by measuring the colocalization of labeled liposomes with lysosomal markers, quantified using Pearson’s correlation coefficient. Lipid mixing assays assessed the potential fusogenic effect, and molecular dynamics (MD) simulations explored the interactions of protonated sodium oleate (SO) with the endosomal membrane. Results demonstrated that SO-Lipo exhibited superior endosomal escape compared to Unmodified-Lipo, as evidenced by reduced colocalization with lysosomal markers, and achieved comparable efficacy to AUR-Lipo with lower cytotoxicity. Lipid mixing assays confirmed the potential fusogenic effect of SO with endosomal membrane models. MD simulations revealed that under acidic endosomal conditions, SO is protonated to oleic acid, which integrates into the membrane, enhancing fluidity and promoting fusion events essential for cytosolic release. SO-Lipo enhance endosomal escape through a fusogenic mechanism, facilitating cytosolic delivery with reduced cytotoxicity. This approach offers a safer and more effective option for targeted drug delivery applications.

Organelle-Level Trafficking and Metabolism Kinetics for Redox-Responsive Paclitaxel Prodrug Nanoparticles Characterized by Experimental and Modeling Analysis.

Redox-responsive self-assembled prodrug nanoparticles have received extensive attention for their high loading efficiency and environmentally responsive properties. However, the intracellular metabolism and transportation kinetics were poorly understood, which limited the rational design and development of this delivery system. Herein, tetraphenylporphyrin-paclitaxel (TxP) prodrugs with thioether, disulfide, and dicarbon linkers (TsP, TssP, and TccP) were synthesized and self-assembled as nanoparticles. The redox responsiveness was investigated both in the simulated medium and in tumor cells via mass spectrometry. TxP NP and PTX concentrations in 4T1 whole cells, endosomal systems, and cytoplasm over time were quantified by UPLC-MS/MS and modeled using the nonlinear mixed effect (NLME) approach. Cytotoxicity was studied in 4T1 and MCF-7 cell lines, and antitumor efficacy was analyzed in 4T1 tumor-bearing mice. Mass spectrometry identified both oxidative and reductive metabolites in redox simulants for TssP NPs and TsP NPs. In 4T1 cells, only reductive metabolites for TssP NPs were detected, while both oxidative and reductive metabolites for TsP NPs were detected. The developed subcellular pharmacokinetic model suggested that the estimated metabolism rates of TxP NPs in endosomal systems were 10 to 27 times of the rates in cytoplasm, indicating that endosomal systems were the dominant place for intracellular metabolism. These rates were numerically higher for TsP NPs than TssP NPs in endosomal systems (1.7-fold) and the cytoplasm (2.5-fold). The internalization of nanoparticles was identified to be slow (kmax,int, 0.015 h-1) and saturable. The transportation rate constant across the endosomal membranes was fast for PTX (27.1 h-1) and slow for TxP NPs (0.098 h-1). TsP and TssP NPs had comparable in vivo antitumor efficacy, which was higher than that of TccP NPs. This study quantified the organelle-level transportation and metabolism kinetics for three prodrug nanoparticles with redox-responsive or inert linkers using a combined experimental and modeling approach. These findings and the modeling framework might inform future studies for redox-responsive prodrug design and drug delivery systems.

PH-Responsive Peptide Nanoparticles Deliver Macromolecules to Cells via Endosomal Membrane Nanoporation.

The synthetically evolved pHD family of peptides is known to self-assemble into macromolecule-sized nanopores of 2-10 nm diameter in synthetic lipid bilayers, but only when the pH is below ∼6. Here, we show that a representative family member, pHD108, has the same pH-responsive nanopore-forming activity in the endosomal membranes of living human cells, which is triggered by endosomal acidification. This enables the cytosolic delivery of endocytosed proteins and other macromolecules. Acylation of either peptide terminus significantly decreases the concentration of peptide required for macromolecule delivery to the cell cytosol while not causing any measurable cytotoxicity. Longer acyl chains are more effective. The N-terminal palmitoylated C16-pHD108 is the most potent of all of the acyl-pHD108 variants and readily delivers a cytotoxic enzyme, fluorescent proteins, and a dye-labeled dextran to the cell cytosol. C16-pHD108 forms stable monodisperse micellar nanoparticles in a buffer at pH 7 with an average diameter of around 120 nm. These nanoparticles are not cytolytic or cytotoxic because the acylated pHD peptide does not partition from the nanoparticles into cell membranes at pH 7. At pH 5, the nanoparticles are unstable, driving acylated pHD108 to bind strongly to membranes. We hypothesize that passive endocytosis of macromolecular cargo and stable peptide nanoparticles, followed by endosomal acidification-dependent destabilization of the nanoparticles, triggers the nanopore-forming activity of acylated pHD peptides in the endosomal membrane, enabling internalized macromolecules to be delivered to the cytosol.

Selective cargo and membrane recognition by SNX17 regulates its interaction with Retriever.

The Retriever complex recycles a wide range of transmembrane proteins from endosomes to the plasma membrane. The cargo adapter protein SNX17 has been implicated in recruiting the Retriever complex to endosomal membranes, yet the details of this interaction have remained elusive. Through biophysical and structural model-guided mutagenesis studies with recombinant proteins and liposomes, we have gained a deeper understanding of this process. Here, we demonstrate a direct interaction between SNX17 and Retriever, specifically between the C-terminal region of SNX17 and the interface of the Retriever subunits VPS35L and VPS26C. This interaction is enhanced upon the binding of SNX17 to its cargo in solution, due to the disruption of an intramolecular autoinhibitory interaction between the C-terminal region of SNX17 and the cargo binding pocket. In addition, SNX17 binding to membranes containing phosphatidylinositol-3-phosphate also promotes Retriever recruitment in a cargo-independent manner. Therefore, this work provides evidence of the dual activation mechanisms by which SNX17 modulates Retriever recruitment to the proximity of cargo and membranes, offering significant insights into the regulatory mechanisms of protein recycling at endosomes.

A Bioinspired Nanovaccine for Personalized Cancer Immunotherapy.

Poly I:C (pIC) can act on endosomal and cytosolic pathogen recognition receptors to enhance T cell immunity. However, the poor cytosolic delivery of pIC and lack of facile methods for codelivery with antigens limit its efficacy. Inspired by the structure of a virus, we developed a liponanogel (LNG) consisting of a nanogel core and lipid shell to address these challenges. An LNG-based vaccine increases the endosomal membrane permeability in a nanogel core-dependent manner, thus enhancing cytosolic sensing of pIC. LNG induces 44.9-fold stronger CD8+ T cell responses than soluble pIC or Hiltonol adjuvanted vaccines and even induces stronger CD8+ T cell responses than state-of-the-art lipid nanoparticle adjuvanted vaccines. Remarkably, the LNG vaccine regresses 100% TC1 tumors and even regresses 60% aggressive B16F10 tumors upon combination with αPD-L1. Our study provides a safe and effective strategy for enhancing T cell immunity and may inspire new approaches for cancer immunotherapy.

The actomyosin system is essential for the integrity of the endosomal system in bloodstream form Trypanosoma brucei.

The actin cytoskeleton is a ubiquitous feature of eukaryotic cells, yet its complexity varies across different taxa. In the parasitic protist Trypanosoma brucei, a rudimentary actomyosin system consisting of one actin gene and two myosin genes has been retained despite significant investment in the microtubule cytoskeleton. The functions of this highly simplified actomyosin system remain unclear, but appear to centre on the endomembrane system. Here, advanced light and electron microscopy imaging techniques, together with biochemical and biophysical assays, were used to explore the relationship between the actomyosin and endomembrane systems. The class I myosin (TbMyo1) had a large cytosolic pool and its ability to translocate actin filaments in vitro was shown here for the first time. TbMyo1 exhibited strong association with the endosomal system and was additionally found on glycosomes. At the endosomal membranes, TbMyo1 colocalised with markers for early and late endosomes (TbRab5A and TbRab7, respectively), but not with the marker associated with recycling endosomes (TbRab11). Actin and myosin were simultaneously visualised for the first time in trypanosomes using an anti-actin chromobody. Disruption of the actomyosin system using the actin-depolymerising drug latrunculin A resulted in a delocalisation of both the actin chromobody signal and an endosomal marker, and was accompanied by a specific loss of endosomal structure. This suggests that the actomyosin system is required for maintaining endosomal integrity in T. brucei.

Endosome rupture enables enteroviruses from the family Picornaviridae to infect cells

Membrane penetration by non-enveloped viruses is diverse and generally not well understood. Enteroviruses, one of the largest groups of non-enveloped viruses, cause diseases ranging from the common cold to life-threatening encephalitis. Enteroviruses enter cells by receptor-mediated endocytosis. However, how enterovirus particles or RNA genomes cross the endosome membrane into the cytoplasm remains unknown. Here we used cryo-electron tomography of infected cells to show that endosomes containing enteroviruses deform, rupture, and release the virus particles into the cytoplasm. Blocking endosome acidification with bafilomycin A1 reduced the number of particles that released their genomes, but did not prevent them from reaching the cytoplasm. Inhibiting post-endocytic membrane remodeling with wiskostatin promoted abortive enterovirus genome release in endosomes. The rupture of endosomes also occurs in control cells and after the endocytosis of very low-density lipoprotein. In summary, our results show that cellular membrane remodeling disrupts enterovirus-containing endosomes and thus releases the virus particles into the cytoplasm to initiate infection. Since the studied enteroviruses employ different receptors for cell entry but are delivered into the cytoplasm by cell-mediated endosome disruption, it is likely that most if not all enteroviruses, and probably numerous other viruses from the family Picornaviridae, can utilize endosome rupture to infect cells.

Membrane fusion by dengue virus: The first step

Flaviviruses include important human pathogens such as Dengue, Zika, West Nile, Yellow fever, Japanese encephalitis, and Tick-borne encephalitis viruses as well as some emerging viruses that affect millions of people worldwide. They fuse their membrane with the late endosomal one in a pH-dependent way and therefore the merging of the membranes is one of the main goals for obtaining new antivirals. The envelope E protein, a membrane fusion protein, is accountable for fusion and encompasses different domains involved in the fusion mechanism, including the fusion peptide segment. In this work we have used molecular dynamics to study the interaction of the distal end of domain II of the DENV envelope E protein with a membrane like the late endosomal membrane in order to observe the initiation of membrane fusion carried out by a number of trimers of the DENV envelope E protein interacting with a complex biomembrane and demonstrate its feasibility. Our results demonstrate the likelihood of membrane disorganization and pore formation by trimer complex organization, the amino acids responsible for such condition and the secondary structure arrangements needed for such fundamental process. At the same time, we define new targets of the envelope E protein sequence which could permit designing potent antiviral bioactive molecules.

The endosomal-vacuolar transport system acts as a docking platform for the Pmk1 MAP kinase signaling pathway in Magnaporthe oryzae.

In Magnaporthe oryzae, the Pmk1 MAP kinase signaling pathway regulates appressorium formation, plant penetration, effector secretion, and invasive growth. While the Mst11-Mst7-Pmk1 cascade was characterized two decades ago, knowledge of its signaling in the intracellular network remains limited. In this study, we demonstrate that the endosomal surface scaffolds Pmk1 MAPK signaling and Msb2 activates Ras2 on endosomes in M. oryzae. Protein colocalization demonstrated that Msb2, Ras2, Cap1, Mst50, Mst11, Mst7, and Pmk1 attach to late endosomal membranes. Damage to the endosome-vacuole transport system influences Pmk1 phosphorylation. When Msb2 senses a plant signal, it internalizes and activates Ras2 on endosome membrane surfaces, transmitting the signal to Pmk1 via Mst11 and Mst7. Signal-sensing and delivery proteins are ubiquitinated and sorted for degradation in late endosomes and vacuoles, terminating signaling. Plant penetration and lowered intracellular turgor are required for the transition from late endosomes to vacuoles in appressoria. Our findings uncover an effective mechanism that scaffolds and controls Pmk1 MAPK signaling through endosomal-vacuolar transport, offering new knowledge for the cytological and molecular mechanisms by which the Pmk1 MAPK pathway modulates development and pathogenicity in M. oryzae.

Deletion of nephrocystin 4 exacerbates cardiac function and survival rate in a mouse model of cardio-renal syndrome

Abstract Background Cardiorenal syndrome (CRS), characterized by cardiac and renal dysfunction, is an increasing health problem associated with high mortality rate. Nephrocystin 4 (NPHP4) gene is the major cause of end-stage renal disease in children. We have reported NPHP4 single nucleotide polymorphism is the genetic risk for CRS and subsequent cardiovascular events in general population. However, the functional role of Nphp4 in the development of CRS has never been examined yet. Methods and Results We generated systemic Nphp4 knockout (KO) mice deleting exon 3 to 8 using CRISPR-Cas9 system. RNA sequence analysis of heart and kidney tissues demonstrated that gene-sets related to mitochondrial function were significantly downregulated in Nphp4 KO mice compared to their wild-type (WT) littermates. Nphp4 KO mice showed less cardiac hypertrophy, however, exacerbated cardiac function and survival compared to WT mice in a CRS model induced by thoracic transverse aortic constriction. Nphp4 located in endosome and cell membrane in H9C2 cardiomyocytes. Immunoprecipitation confirmed protein interaction between Nphp4 and HECT-type E3 ligase Itch. Furthermore, we found that Itch targeted Lats2 and insulin-like growth factor-1 receptor in cardiomyocytes. Knockdown of Nphp4 activated Hippo signaling and worsened mitochondrial function in H9C2 cardiomyocytes through the inhibition of Itch-dependent Lats2 degradation. Knockdown of Nphp4 inhibited insulin-like growth factor-1 signaling through the disturbance of Itch-mediated endosome function. Conclusions Deletion of Nphp4 impaired compensatory cardiac hypertrophy and exacerbated cardiac function and survival in a mouse CRS model through mitochondrial dysfunction. Nphp4 mediated Hippo signaling and insulin-like growth factor-1 signaling through the interaction with Itch. NPHP4 could be the novel therapeutic target in CRS.figure1figure2

Drought Tolerance in Plants: Physiological and Molecular Responses.

Drought, a significant environmental challenge, presents a substantial risk to worldwide agriculture and the security of food supplies. In response, plants can perceive stimuli from their environment and activate defense pathways via various modulating networks to cope with stress. Drought tolerance, a multifaceted attribute, can be dissected into distinct contributing mechanisms and factors. Osmotic stress, dehydration stress, dysfunction of plasma and endosome membranes, loss of cellular turgidity, inhibition of metabolite synthesis, cellular energy depletion, impaired chloroplast function, and oxidative stress are among the most critical consequences of drought on plant cells. Understanding the intricate interplay of these physiological and molecular responses provides insights into the adaptive strategies plants employ to navigate through drought stress. Plant cells express various mechanisms to withstand and reverse the cellular effects of drought stress. These mechanisms include osmotic adjustment to preserve cellular turgor, synthesis of protective proteins like dehydrins, and triggering antioxidant systems to counterbalance oxidative stress. A better understanding of drought tolerance is crucial for devising specific methods to improve crop resilience and promote sustainable agricultural practices in environments with limited water resources. This review explores the physiological and molecular responses employed by plants to address the challenges of drought stress.

Endosomal membrane budding patterns in plants

Multivesicular endosomes (MVEs) sequester membrane proteins destined for degradation within intralumenal vesicles (ILVs), a process mediated by the membrane-remodeling action of Endosomal Sorting Complex Required for Transport (ESCRT) proteins. In Arabidopsis, endosomal membrane constriction and scission are uncoupled, resulting in the formation of extensive concatenated ILV networks and enhancing cargo sequestration efficiency. Here, we used a combination of electron tomography, computer simulations, and mathematical modeling to address the questions of when concatenated ILV networks evolved in plants and what drives their formation. Through morphometric analyses of tomographic reconstructions of endosomes across yeast, algae, and various land plants, we have found that ILV concatenation is widespread within plant species, but only prevalent in seed plants, especially in flowering plants. Multiple budding sites that require the formation of pores in the limiting membrane were only identified in hornworts and seed plants, suggesting that this mechanism has evolved independently in both plant lineages. To identify the conditions under which these multiple budding sites can arise, we used particle-based molecular dynamics simulations and found that changes in ESCRT filament properties, such as filament curvature and membrane binding energy, can generate the membrane shapes observed in multiple budding sites. To understand the relationship between membrane budding activity and ILV network topology, we performed computational simulations and identified a set of membrane remodeling parameters that can recapitulate our tomographic datasets.

PePalA/B/C are required for virulence and patulin biosynthesis by regulating the PePacC-processing proteolytic activity in Penicillium expansum

Abstract Background The Pal/Rim signaling pathway is crucial for fungal responses to ambient pH conditions. It comprises the transcription factor PacC/Rim101 and six upstream Pal proteins. While the role of PacC has been extensively studied, there is limited information on the functions of the upstream Pal proteins. Results In the genome of Penicillium expansum, genes encoding the endosomal membrane complex (PalA/B/C) were identified. Among these, only the expression of PePalB, not PePalA or PePalC, is pH-dependent. Subcellular localization and functional analyses showed that PePalA/B/C are localized to the cytosol and peripheral punctate structures. Deletion of PePalA/B/C resulted in reduced growth and conidiation of P. expansum across various pH conditions. The virulence of the ΔPePalA/B/C mutant was significantly reduced in pear and apple fruits. Additionally, the mutants exhibited a loss of patulin production under both acidic and alkaline conditions and the down-regulation of genes in the patulin biosynthetic cluster. pH shift experiments further demonstrated that PePalA/B/C are essential for both the PePacC expression and the pH-dependent proteolytic activation of PePacC. Conclusions These findings highlight the significant roles of PePalA/B/C in regulating growth, conidiation, virulence, and patulin production in P. expansum, thereby enhancing our understanding of the regulatory mechanisms underlying the Pal/Rim signaling pathway.

Insights into the distinct membrane targeting mechanisms of WDR91 family proteins

WDR91 and SORF1, members of the WD repeat-containing protein 91 family, control phosphoinositide conversion by inhibiting phosphatidylinositol 3-kinase activity on endosomes, which promotes endosome maturation. Here, we report the crystal structure of the human WDR91 WD40 domain complexed with Rab7 that has an unusual interface at the C-terminus of the Rab7 switch II region. WDR91 is highly selective for Rab7 among the tested GTPases. A LIS1 homology (LisH) motif within the WDR91 N-terminal domain (NTD) mediates self-association and may contribute partly to the augmented interaction between full-length WDR91 and Rab7. Both the Rab7 binding site and the LisH motif are indispensable for WDR91 function in endocytic trafficking. For the WDR91 orthologue SORF1 lacking the C-terminal WD40 domain, a C-terminal amphipathic helix (AH) mediates strong interactions with liposomes containing acidic lipids. During evolution the human WDR91 ancestor gene might have acquired a WD40 domain to replace the AH for endosomal membrane targeting.

Amplification of Protein Expression by Self-Amplifying mRNA Delivered in Lipid Nanoparticles Containing a β-Aminoester Ionizable Lipid Correlates with Reduced Innate Immune Activation.

Self-amplifying mRNA (saRNA) is witnessing increased interest as a platform technology for protein replacement therapy, gene editing, immunotherapy, and vaccination. saRNA can replicate itself inside cells, leading to a higher and more sustained production of the desired protein at a lower dose. Controlling innate immune activation, however, is crucial to suppress unwanted inflammation upon delivery and self-replication of RNA in vivo. In this study, we report on a class of β-aminoester lipids (βAELs) synthesized through the Michael addition of an acrylate to diethanolamine, followed by esterification with fatty acids. These lipids possessed one or two ionizable amines, depending on the use of nonionic or amine-containing acrylates. We utilized βAELs for encapsulating saRNA in lipid nanoparticles (LNPs) and evaluated their transfection efficiency in vitro and in vivo in mice, while comparing them to LNPs containing ALC-0315 as an ionizable lipid reference. Among the tested lipids, OC7, which comprises two unsaturated oleoyl alkyl chains and an ionizable azepanyl motif, emerged as a βAEL with low cytotoxicity and immunogenicity relative to ALC-0315. Interestingly, saRNA delivered via the OC7 LNP exhibited a distinct in vivo transfection profile. Initially, intramuscular injection of OC7 LNP resulted in low protein expression shortly after administration, followed by a gradual increase over a period of up to 7 days. This pattern is indicative of successful self-amplification of saRNA. In contrast, saRNA delivered via ALC-0315 LNP demonstrated high protein translation initially, which gradually declined over time and lacked the amplification seen with OC7 LNP. We observed that, in contrast to saRNA OC7 LNP, saRNA ALC-0315 LNP induced potent innate immune activation by triggering cytoplasmic RIG-I-like receptors (RLRs), likely due to the highly efficient endosomal membrane rupturing properties of ALC-0315 LNP. Consequently, the massive production of type I interferons quickly hindered the amplification of the saRNA. Our findings highlight the critical role of the choice of ionizable lipid for saRNA formulation in LNPs, particularly in shaping the qualitative profile of protein expression. For applications where minimizing inflammation is desired, the use of ionizable lipids, such as the βAEL reported in this study, that elicit a low type I interferon response in saRNA LNP is crucial.

The p. S178L mutation in Tbc1d24 disrupts endosome-mediated synaptic vesicle trafficking of cochlear hair cells and leads to hearing impairment in mice.

The ribbon synapses of cochlear inner hair cells (IHCs) employ efficient vesicle resupply to enable fast and sustained release rates. However, the molecular mechanisms of these physiological activities remain unelucidated. Previous studies showed that the RAB-specific GTPase-activating protein TBC1D24 controls the endosomal trafficking of the synaptic vesicles (SVs) in Drosophila and mammalian neurons, and mutations in TBC1D24 may lead to non-syndromic hearing loss or hearing loss associated with the DOORS syndrome in humans. In this study, we generated a knock-in mouse model for the p. S178L mutation in TBC1D24, which leads to autosomal dominant non-syndromic hearing loss (DFNA65). The p.S178L mutant mice show mild hearing loss and progressively declined wave I amplitude of the auditory brainstem responses. Despite the normal gross and cellular morphology of the cochlea, transmission electron microscopy reveals accumulation of endosome-like vacuoles and a lower-than-normal number of SVs directly associated with the ribbons in the IHCs. Consistently, patch clamp of the IHCs shows reduced exocytosis under prolonged stimulus. ARF6, a TBC1D24-interacting protein also involved in endosomal membrane trafficking, was underexpressed in the cochleae of the mutant mouse and has weakened in vitro interaction with the p.S178L mutant TBC1D24. Our results suggest an important role of TBC1D24 in maintaining endosomal-mediated vesicle recycling and sustained exocytosis of hair cell ribbon synapses.