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Bone Research May 2024Mature osteoclasts degrade bone matrix by exocytosis of active proteases from secretory lysosomes through a ruffled border. However, the molecular mechanisms underlying...
Mature osteoclasts degrade bone matrix by exocytosis of active proteases from secretory lysosomes through a ruffled border. However, the molecular mechanisms underlying lysosomal trafficking and secretion in osteoclasts remain largely unknown. Here, we show with GeneChip analysis that RUN and FYVE domain-containing protein 4 (RUFY4) is strongly upregulated during osteoclastogenesis. Mice lacking Rufy4 exhibited a high trabecular bone mass phenotype with abnormalities in osteoclast function in vivo. Furthermore, deleting Rufy4 did not affect osteoclast differentiation, but inhibited bone-resorbing activity due to disruption in the acidic maturation of secondary lysosomes, their trafficking to the membrane, and their secretion of cathepsin K into the extracellular space. Mechanistically, RUFY4 promotes late endosome-lysosome fusion by acting as an adaptor protein between Rab7 on late endosomes and LAMP2 on primary lysosomes. Consequently, Rufy4-deficient mice were highly protected from lipopolysaccharide- and ovariectomy-induced bone loss. Thus, RUFY4 plays as a new regulator in osteoclast activity by mediating endo-lysosomal trafficking and have a potential to be specific target for therapies against bone-loss diseases such as osteoporosis.
Topics: Animals; Osteoclasts; Lysosomes; Endosomes; Mice; Mice, Knockout; Bone Resorption; Protein Transport; Mice, Inbred C57BL; rab GTP-Binding Proteins; Cell Differentiation; Gene Deletion; Cathepsin K; Female; rab7 GTP-Binding Proteins
PubMed: 38744829
DOI: 10.1038/s41413-024-00326-8 -
Antimicrobial Agents and Chemotherapy May 2024Cell culture-based screening of a chemical library identified diphenoxylate as an antiviral agent against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)....
Cell culture-based screening of a chemical library identified diphenoxylate as an antiviral agent against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The observed 50% effective concentrations ranged between 1.4 and 4.9 µM against the original wild-type strain and its variants. Time-of-addition experiments indicated that diphenoxylate is an entry blocker targeting a host factor involved in viral infection. Fluorescence microscopic analysis visualized that diphenoxylate prevented SARS-CoV-2 particles from penetrating the cell membrane and also impaired endo-lysosomal acidification. Diphenoxylate exhibited a synergistic inhibitory effect on SARS-CoV-2 infection in human lung epithelial Calu-3 cells when combined with a transmembrane serine protease 2 (TMPRSS2) inhibitor, nafamostat. This synergy suggested that efficient antiviral activity is achieved by blocking both TMPRSS2-mediated early and endosome-mediated late SARS-CoV-2 entry pathways. The antiviral efficacy of diphenoxylate against SARS-CoV-2 was reproducible in a human tonsil organoids system. In a transgenic mouse model expressing the obligate SARS-CoV-2 receptor, human angiotensin-converting enzyme 2, intranasal administration of diphenoxylate (10 mg/kg/day) significantly reduced the viral RNA copy number in the lungs by 70% on day 3. This study underscores that diphenoxylate represents a promising core scaffold, warranting further exploration for chemical modifications aimed at developing a new class of clinically effective antiviral drugs against SARS-CoV-2.
PubMed: 38742905
DOI: 10.1128/aac.00341-24 -
Bio-protocol May 2024The cation-independent mannose 6-phosphate receptors (CI-M6PR) bind newly synthesized mannose 6-phosphate (Man-6-P)-tagged enzymes in the Golgi and transport them to...
The cation-independent mannose 6-phosphate receptors (CI-M6PR) bind newly synthesized mannose 6-phosphate (Man-6-P)-tagged enzymes in the Golgi and transport them to late endosomes/lysosomes, providing them with degradative functions. Following the cargo delivery, empty receptors are recycled via early/recycling endosomes back to the trans-Golgi network (TGN) retrogradely in a dynein-dependent motion. One of the most widely used methods for studying the retrograde trafficking of CI-M6PR involves employing the CD8α-CI-M6PR chimera. This chimera, comprising a CD8 ectodomain fused with the cytoplasmic tail of the CI-M6PR receptor, allows for labeling at the plasma membrane, followed by trafficking only in a retrograde direction. Previous studies utilizing the CD8α-CI-M6PR chimera have focused mainly on colocalization studies with various endocytic markers under steady-state conditions. This protocol extends the application of the CD8α-CI-M6PR chimera to live cell imaging, followed by a quantitative analysis of its motion towards the Golgi. Additionally, we present an approach to quantify parameters such as speed and track lengths associated with the motility of CD8α-CI-M6PR endosomes using the Fiji plugin TrackMate. Key features • This assay is adapted from the methodology by Prof. Matthew Seaman for studying the retrograde trafficking of CI-M6PR by expressing CD8α-CI-M6PR chimera in HeLa cells. • The experiments include live-cell imaging of surface-labeled CD8α-CI-M6PR molecules, followed by a chase in cells. • Allows the monitoring of real-time motion of CD8α-CI-M6PR endosomes and facilitates calculation of kinetic parameters associated with endosome trajectories, e.g., speed and distance (run lengths).
PubMed: 38737505
DOI: 10.21769/BioProtoc.4979 -
Scientific Reports May 2024The closely related endolysosomal tethering complexes HOPS and CORVET play pivotal roles in the homo- and heterotypic fusion of early and late endosomes, respectively,...
The closely related endolysosomal tethering complexes HOPS and CORVET play pivotal roles in the homo- and heterotypic fusion of early and late endosomes, respectively, and HOPS also mediates the fusion of lysosomes with incoming vesicles including late endosomes and autophagosomes. These heterohexameric complexes share their four core subunits that assemble with additional two, complex-specific subunits. These features and the similar structure of the complexes could allow the formation of hybrid complexes, and the complex specific subunits may compete for binding to the core. Indeed, our biochemical analyses revealed the overlap of binding sites for HOPS-specific VPS41 and CORVET-specific VPS8 on the shared core subunit VPS18. We found that the overexpression of CORVET-specific VPS8 or Tgfbrap1 decreased the amount of core proteins VPS11 and VPS18 that are assembled with HOPS-specific subunits VPS41 or VPS39, indicating reduced amount of assembled HOPS. In line with this, we observed the elevation of both lipidated, autophagosome-associated LC3 protein and the autophagic cargo p62 in these cells, suggesting impaired autophagosome-lysosome fusion. In contrast, overexpression of HOPS-specific VPS39 or VPS41 did not affect the level of assembled CORVET or autophagy. VPS8 or Tgfbrap1 overexpression also induced Cathepsin D accumulation, suggesting that HOPS-dependent biosynthetic delivery of lysosomal hydrolases is perturbed, too. These indicate that CORVET-specific subunit levels fine-tune HOPS assembly and activity in vivo.
Topics: Endosomes; Humans; Vesicular Transport Proteins; Lysosomes; Protein Subunits; Autophagy; Autophagosomes; HeLa Cells; Protein Binding
PubMed: 38698024
DOI: 10.1038/s41598-024-59775-0 -
Neuron Apr 2024Genome-wide association studies (GWASs) have uncovered over 75 genomic loci associated with risk for late-onset Alzheimer's disease (LOAD), but identification of the...
Genome-wide association studies (GWASs) have uncovered over 75 genomic loci associated with risk for late-onset Alzheimer's disease (LOAD), but identification of the underlying causal genes remains challenging. Studies of induced pluripotent stem cell (iPSC)-derived neurons from LOAD patients have demonstrated the existence of neuronal cell-intrinsic functional defects. Here, we searched for genetic contributions to neuronal dysfunction in LOAD using an integrative systems approach that incorporated multi-evidence-based gene mapping and network-analysis-based prioritization. A systematic perturbation screening of candidate risk genes in Caenorhabditis elegans (C. elegans) revealed that neuronal knockdown of the LOAD risk gene orthologs vha-10 (ATP6V1G2), cmd-1 (CALM3), amph-1 (BIN1), ephx-1 (NGEF), and pho-5 (ACP2) alters short-/intermediate-term memory function, the cognitive domain affected earliest during LOAD progression. These results highlight the impact of LOAD risk genes on evolutionarily conserved memory function, as mediated through neuronal endosomal dysfunction, and identify new targets for further mechanistic interrogation.
PubMed: 38692279
DOI: 10.1016/j.neuron.2024.04.009 -
Frontiers in Microbiology 2024Bovine respiratory syncytial virus (BRSV) is an RNA virus with envelope that causes acute, febrile, and highly infectious respiratory diseases in cattle. However, the...
Bovine respiratory syncytial virus (BRSV) is an RNA virus with envelope that causes acute, febrile, and highly infectious respiratory diseases in cattle. However, the manner and mechanism of BRSV entry into cells remain unclear. In this study, we aimed to explore the entry manner of BRSV into MDBK cells and its regulatory mechanism. Our findings, based on virus titer, virus copies, western blot and IFA analysis, indicate that BRSV enters MDBK cells through endocytosis, relying on dynamin, specifically via clathrin-mediated endocytosis rather than caveolin-mediated endocytosis and micropinocytosis. We observed that the entered BRSV initially localizes in early endosomes and subsequently localizes in late endosomes. Additionally, our results of western blot, virus titer and virus copies demonstrate that BRSV entry through clathrin-mediated endocytosis is regulated by PI3K-Akt and Src-JNK signaling pathways. Overall, our study suggests that BRSV enters MDBK cells through clathrin-mediated endocytosis, entered BRSV is trafficked to late endosome via early endosome, BRSV entry through clathrin-mediated endocytosis is regulated by PI3K-Akt and Src-JNK signaling pathways.
PubMed: 38690369
DOI: 10.3389/fmicb.2024.1393127 -
Viruses Apr 2024Lymphocytic choriomeningitis virus (LCMV) and Lassa virus (LASV) share many genetic and biological features including subtle differences between pathogenic and...
Lymphocytic choriomeningitis virus (LCMV) and Lassa virus (LASV) share many genetic and biological features including subtle differences between pathogenic and apathogenic strains. Despite remarkable genetic similarity, the viscerotropic WE strain of LCMV causes a fatal LASV fever-like hepatitis in non-human primates (NHPs) while the mouse-adapted Armstrong (ARM) strain of LCMV is deeply attenuated in NHPs and can vaccinate against LCMV-WE challenge. Here, we demonstrate that internalization of WE is more sensitive to the depletion of membrane cholesterol than ARM infection while ARM infection is more reliant on endosomal acidification. LCMV-ARM induces robust NF-κB and interferon response factor (IRF) activation while LCMV-WE seems to avoid early innate sensing and failed to induce strong NF-κB and IRF responses in dual-reporter monocyte and epithelial cells. Toll-like receptor 2 (TLR-2) signaling appears to play a critical role in NF-κB activation and the silencing of TLR-2 shuts down IL-6 production in ARM but not in WE-infected cells. Pathogenic LCMV-WE infection is poorly recognized in early endosomes and failed to induce TLR-2/Mal-dependent pro-inflammatory cytokines. Following infection, Interleukin-1 receptor-associated kinase 1 (IRAK-1) expression is diminished in LCMV-ARM- but not LCMV-WE-infected cells, which indicates it is likely involved in the LCMV-ARM NF-κB activation. By confocal microscopy, ARM and WE strains have similar intracellular trafficking although LCMV-ARM infection appears to coincide with greater co-localization of early endosome marker EEA1 with TLR-2. Both strains co-localize with Rab-7, a late endosome marker, but the interaction with LCMV-WE seems to be more prolonged. These findings suggest that LCMV-ARM's intracellular trafficking pathway may facilitate interaction with innate immune sensors, which promotes the induction of effective innate and adaptive immune responses.
Topics: Lymphocytic choriomeningitis virus; Immunity, Innate; Animals; Virus Internalization; Humans; Mice; Toll-Like Receptor 2; Endosomes; NF-kappa B; Signal Transduction; Cell Line; Lymphocytic Choriomeningitis; Epithelial Cells
PubMed: 38675975
DOI: 10.3390/v16040635 -
BioRxiv : the Preprint Server For... Apr 2024Lipid nanoparticles (LNPs) have emerged as the dominant platform for RNA delivery, based on their success in the COVID-19 vaccines and late-stage clinical studies in...
Lipid nanoparticles (LNPs) have emerged as the dominant platform for RNA delivery, based on their success in the COVID-19 vaccines and late-stage clinical studies in other indications. However, we and others have shown that LNPs induce severe inflammation, and massively aggravate pre-existing inflammation. Here, using structure-function screening of lipids and analyses of signaling pathways, we elucidate the mechanisms of LNP-associated inflammation and demonstrate solutions. We show that LNPs' hallmark feature, endosomal escape, which is necessary for RNA expression, also directly triggers inflammation by causing endosomal membrane damage. Large, irreparable, endosomal holes are recognized by cytosolic proteins called galectins, which bind to sugars on the inner endosomal membrane and then regulate downstream inflammation. We find that inhibition of galectins abrogates LNP-associated inflammation, both and . We show that rapidly biodegradable ionizable lipids can preferentially create endosomal holes that are smaller in size and reparable by the endosomal sorting complex required for transport (ESCRT) pathway. Ionizable lipids producing such ESCRT-recruiting endosomal holes can produce high expression from cargo mRNA with minimal inflammation. Finally, we show that both routes to non-inflammatory LNPs, either galectin inhibition or ESCRT-recruiting ionizable lipids, are compatible with therapeutic mRNAs that ameliorate inflammation in disease models. LNPs without galectin inhibition or biodegradable ionizable lipids lead to severe exacerbation of inflammation in these models. In summary, endosomal escape induces endosomal membrane damage that can lead to inflammation. However, the inflammation can be controlled by inhibiting galectins (large hole detectors) or by using biodegradable lipids, which create smaller holes that are reparable by the ESCRT pathway. These strategies should lead to generally safer LNPs that can be used to treat inflammatory diseases.
PubMed: 38659905
DOI: 10.1101/2024.04.16.589801 -
Biotechnology For Biofuels and... Apr 2024The saprophytic filamentous fungus Trichoderma reesei represents one of the most prolific cellulase producers. The bulk production of lignocellulolytic enzymes by T....
BACKGROUND
The saprophytic filamentous fungus Trichoderma reesei represents one of the most prolific cellulase producers. The bulk production of lignocellulolytic enzymes by T. reesei not only relies on the efficient transcription of cellulase genes but also their efficient secretion after being translated. However, little attention has been paid to the functional roles of the involved secretory pathway in the high-level production of cellulases in T. reesei. Rab GTPases are key regulators in coordinating various vesicle trafficking associated with the eukaryotic secretory pathway. Specifically, Rab7 is a representative GTPase regulating the transition of the early endosome to the late endosome followed by its fusion to the vacuole as well as homotypic vacuole fusion. Although crosstalk between the endosomal/vacuolar pathway and the secretion pathway has been reported, the functional role of Rab7 in cellulase production in T. reesei remains unknown.
RESULTS
A TrRab7 was identified and characterized in T. reesei. TrRab7 was shown to play important roles in T. reesei vegetative growth and vacuole morphology. Whereas knock-down of Trrab7 significantly compromised the induced production of T. reesei cellulases, overexpression of the key transcriptional activator, Xyr1, restored the production of cellulases in the Trrab7 knock-down strain (Ptcu-rab7) on glucose, indicating that the observed defective cellulase biosynthesis results from the compromised cellulase gene transcription. Down-regulation of Trrab7 was also found to make T. reesei more sensitive to various stresses including carbon starvation. Interestingly, overexpression of Snf1, a serine/threonine protein kinase known as an energetic sensor, partially restored the cellulase production of Ptcu-rab7 on Avicel, implicating that TrRab7 is involved in an energetic adaptation to carbon starvation which contributes to the successful cellulase gene expression when T. reesei is transferred from glucose to cellulose.
CONCLUSIONS
TrRab7 was shown to play important roles in T. reesei development and a stress response to carbon starvation resulting from nutrient shift. This adaptation may allow T. reesei to successfully initiate the inducing process leading to efficient cellulase production. The present study provides useful insights into the functional involvement of the endosomal/vacuolar pathway in T. reesei development and hydrolytic enzyme production.
PubMed: 38643207
DOI: 10.1186/s13068-024-02504-6 -
The EMBO Journal May 2024Phosphatidylinositol (PI) is the precursor lipid for the minor phosphoinositides (PPIns), which are critical for multiple functions in all eukaryotic cells. It is poorly...
Phosphatidylinositol (PI) is the precursor lipid for the minor phosphoinositides (PPIns), which are critical for multiple functions in all eukaryotic cells. It is poorly understood how phosphatidylinositol, which is synthesized in the ER, reaches those membranes where PPIns are formed. Here, we used VT01454, a recently identified inhibitor of class I PI transfer proteins (PITPs), to unravel their roles in lipid metabolism, and solved the structure of inhibitor-bound PITPNA to gain insight into the mode of inhibition. We found that class I PITPs not only distribute PI for PPIns production in various organelles such as the plasma membrane (PM) and late endosomes/lysosomes, but that their inhibition also significantly reduced the levels of phosphatidylserine, di- and triacylglycerols, and other lipids, and caused prominent increases in phosphatidic acid. While VT01454 did not inhibit Golgi PI4P formation nor reduce resting PM PI(4,5)P levels, the recovery of the PM pool of PI(4,5)P after receptor-mediated hydrolysis required both class I and class II PITPs. Overall, these studies show that class I PITPs differentially regulate phosphoinositide pools and affect the overall cellular lipid landscape.
Topics: Humans; Phosphatidylinositols; Phospholipid Transfer Proteins; Lipid Metabolism; Cell Membrane; HeLa Cells; Organelles; Endosomes; Animals
PubMed: 38627600
DOI: 10.1038/s44318-024-00096-3