Grass carp reovirus(GCRV),the genus Aquareovirus in family Reoviridae,is viewed as the most pathogenic aquareovirus.To understand the molecular mechanism of how aquareovirus initiates productive infection,the roles of...Grass carp reovirus(GCRV),the genus Aquareovirus in family Reoviridae,is viewed as the most pathogenic aquareovirus.To understand the molecular mechanism of how aquareovirus initiates productive infection,the roles of endosome and microtubule in cell entry of GCRV are investigated by using quantum dots(QDs)-tracking in combination with biochemical approaches.We found that GCRV infection and viral protein synthesis were significantly inhibited by pretreating host cells with endosome acidification inhibitors NH4Cl,chloroquine and bafilomycin A1(Bafi).Confocal images indicated that GCRV particles could colocalize with Rab5,Rab7 and lysosomes in host cells.Further ultrastructural examination validated that viral particle was found in late endosomes.Moreover,disruption of microtubules with nocodazole clearly blocked GCRV entry,while no inhibitory effects were observed with cytochalasin D treated cells in viral infection,hinting that intracellular transportation of endocytic uptake in GCRV infected cells is via microtubules but not actin filament.Notably,viral particles were observed to transport along microtubules by using QD-labeled GCRV.Altogether,our results suggest that GCRV can use endosomes and microtubules to initiate productive infection.展开更多
Aim:Ligand-targeted therapeutics are experiencing increasing use for treatment of human diseases due to their ability to concentrate a desired drug at a pathologic site while reducing accumulation in healthy tissues.F...Aim:Ligand-targeted therapeutics are experiencing increasing use for treatment of human diseases due to their ability to concentrate a desired drug at a pathologic site while reducing accumulation in healthy tissues.For many ligand-targeted drug conjugates,a critical aspect of conjugate design lies in engineering release of the therapeutic payload to occur only after its internalization by targeted cells.Because disulfide bond reduction is frequently exploited to ensure intracellular drug release,an understanding of the redox properties of endocytic compartments can be critical to ligand-targeted drug design.While the redox properties of folate receptor trafficking endosomes have been previously reported,little is known about the trafficking of prostate-specific membrane antigen(PSMA),a receptor that is experiencing increasing use for drug targeting in humans.Methods:To obtain this information,we have constructed a PSMA-targeted fluorescence resonance energy transfer pair that reports on disulfide bond reduction by changing fluorescence from red to green.Results:We show here that this reporter exhibits rapid and selective uptake by PSMA-positive cells,and that reduction of its disulfide bond proceeds steadily but incompletely following internalization.The fact that maximal disulfide reduction reaches only~50%,even after 24 h incubation,suggests that roughly half of the conjugates must traffic through endosomes that display no reducing capacity.Conclusion:As the level of disulfide reduction differs between PSMA trafficked and previously published folate trafficked conjugates,it also follows that not all internalizing receptors are translocated through similar intracellular compartments.Taken together,these data suggest that the efficiency of disulfide bond reduction must be independently analyzed for each receptor trafficking pathway when disulfide bond reduction is exploited for intracellular drug release.展开更多
The potential of the skin immune system to generate immune responses is well established, and the skin is actively exploited as a vaccination site. Human skin contains several antigen-presenting cell subsets with spec...The potential of the skin immune system to generate immune responses is well established, and the skin is actively exploited as a vaccination site. Human skin contains several antigen-presenting cell subsets with specialized functions. In particular, the capacity to cross-present exogenous antigens to CD8^＋ T cells is of interest for the design of effective immunotherapies against viruses or cancer. Here, we show that primary human Langerhans cells （LCs） were able to cross-present a synthetic long peptide （SLP） to CD8^＋ T cells. In addition, modification of this SLP using antibodies against the receptor langerin, but not dectin-1, further enhanced the cross-presenting capacity of LCs through routing of internalized antigens to less proteolytic early endosome antigen 1 ^＋ early endosomes. The potency of LCs to enhance CD8^＋ T-cell responses could be further increased through activation of LCs with the toll-like receptor 3 ligand polyinosinic：polycytidylic acid （phC）. Altogether, the data provide evidence that human LCs are able to cross-present antigens after langerin-mediated internalization. Furthermore, the potential for antigen modification to target LCs specifically provides a rationale for generating effective anti-tumor or anti-viral cytotoxic T lymphocyte responses.展开更多
Cells producing cytokines often express the receptor for the same cytokine, which makes them prone to autocrine signaling. How cytokine release and signaling are regulated in the same cell is not understood? In this s...Cells producing cytokines often express the receptor for the same cytokine, which makes them prone to autocrine signaling. How cytokine release and signaling are regulated in the same cell is not understood? In this study, we demonstrate that signaling by exogenous and self-synthesized inflammatory cytokine interleukin-6 (IL-6) within endosomal compartments acts as a cellular brake that limits the synthesis of IL-6. Our data show that IL-6 is internalized by dendritic cells and signals from endosomal compartments containing the IL-6 receptor. Newly synthesized IL-6 also traffics via these endosomal compartments and signals in transit to the plasma membrane. This allows activation of STAT3 which in turn limits toll-like receptor 4 stimulant lipopolysaccharide (LPS) triggered transcription of IL-6. Long-term exposure to LPS removes this brake via inhibition of STAT3 by increased expression of suppressor of cytokine signaling 3 and results in fully fledged IL-6 production. This transient regulation could prevent excessive IL-6 production during early infections.展开更多
In eukaryotic cells,organelles in the secretory,lysosomal,and endocytic pathways actively exchange biological materials with each other through intracellular membrane trafficking,which is the process of transporting t...In eukaryotic cells,organelles in the secretory,lysosomal,and endocytic pathways actively exchange biological materials with each other through intracellular membrane trafficking,which is the process of transporting the cargo of proteins,lipids,and other molecules to appropriate compartments via transport vesicles or intermediates.These processes are strictly regulated by various small GTPases such as the RAS-like in rat brain(RAB)protein family,which is the largest subfamily of the RAS superfamily.Dysfunction of membrane trafficking affects tissue homeostasis and leads to a wide range of diseases,including neurological disorders and neurodegenerative diseases.Therefore,it is important to understand the physiological and pathological roles of RAB proteins in brain function.RAB35,a member of the RAB family,is an evolutionarily conserved protein in metazoans.A wide range of studies using cultured mammalian cells and model organisms have revealed that RAB35 mediates various processes such as cytokinesis,endocytic recycling,actin bundling,and cell migration.RAB35 is also involved in neurite outgrowth and turnover of synaptic vesicles.We generated brain-specific Rab35 knockout mice to study the physiological roles of RAB35 in brain development and function.These mice exhibited defects in anxiety-related behaviors and spatial memory.Strikingly,RAB35 is required for the precise positioning of pyramidal neurons during hippocampal development,and thereby for normal hippocampal lamination.In contrast,layer formation in the cerebral cortex occurred superficially,even in the absence of RAB35,suggesting a predominant role for RAB35 in hippocampal development rather than in cerebral cortex development.Recent studies have suggested an association between RAB35 and neurodegenerative diseases,including Parkinson's disease and Alzheimer's disease.In this review,we provide an overview of the current understanding of subcellular functions of RAB35.We also provide insights into the physiological role of RAB35 in mammalian brain development and function,and discuss the involvement of RAB35 dysfunction in neurodegenerative diseases.展开更多
Small interfering RNAs(siRNA)provide a novel and highly specific therapy due to their ability to effectively silence target genes,to date six siRNA therapeutics are approved for clinical use.Even so,some critical chal...Small interfering RNAs(siRNA)provide a novel and highly specific therapy due to their ability to effectively silence target genes,to date six siRNA therapeutics are approved for clinical use.Even so,some critical challenges remain to overcome in the therapeutic application of siRNAs,with delivery issues at the forefront.Among them,endo/lysosomal barrier is one of the important but often-neglected limitations hindering the delivery of siRNA therapeutics.In this review,we summarize the promising strategies that facilitate siRNAs overcoming endo/lysosomal barriers based on the cellular uptake and intracellular transport pathways,including promoting escape once endocytosis into the endo/lysosomes and bypassing lysosomes via endosome-Golgi-endoplasmic reticulum(ER)pathway or nonendocytosis pathway,and discuss the principal considerations and the future directions of promoting endo/lysosomal escape in the development of therapeutic siRNAs.展开更多
Efficient siRNA delivery is highly desirable for disease treatment.However,the application of conventional nanoparticles is limited by the inability to escape from endo-lysosomes.Herein,we report a strategy using smal...Efficient siRNA delivery is highly desirable for disease treatment.However,the application of conventional nanoparticles is limited by the inability to escape from endo-lysosomes.Herein,we report a strategy using small-molecule drugs to enhance siRNA endo-lysosomal release,addressing this challenge.We encapsulated gentamicin(GM)into the marketed Onpattro■ formulation to establish LNP-siRNA/GM nanoparticles that promote siRNA endo-lysosomal escape through endosomal disruption,mechanistically exhibiting unique functionality and synergistic effects of LNP-siRNA/GM to improve cancer therapy.Besides,GM induced reactive oxygen species(ROS)and phospholipids accumulation in endolysosomes,as well as the physical characteristics of lipid nanoparticles(LNPs)were preserved.We also revealed that GM causes endo-lysosomal swelling and disrupts the endosomal membrane to enable siRNA release,as confirmed by Galectin 3 recruitment and acridine orange release.This approach achieved∼81%mRNA-EGFR silencing,which is more than LNP-siEGFR(∼56.23%)by enhancing siRNA endo-lysosomal escape efficiency.Meanwhile,LNP-siEGFR/GM exhibited significant biological activities in HepG2 cells,driven by the synergistic effects of siEGFR and GM with the VEGF and CXCL12 downregulation of,and ROS and phospholipids upregulation.Furthermore,tumor growth was notably suppressed after intravenous injection of LNP-siEGFR/GM in tumor-bearing nude mice.The combination of EGFR-siRNA and GM could also greatly inhibit angiogenesis,be antiproliferative,and induce tumor cells apoptosis.Therefore,this GM and siRNA co-delivery system would provide an efficient strategy for siRNA endosomal escape,significantly improving knockdown in various LNPs based siRNA delivery systems and efficiently enhancing cancer therapy.展开更多
Conventional nutritional supplements frequently demonstrate limited clinical effectiveness due to the harsh milieu of the gastrointestinal tract,inefficient transepithelial transport,and rapid systemic clearance.Nanol...Conventional nutritional supplements frequently demonstrate limited clinical effectiveness due to the harsh milieu of the gastrointestinal tract,inefficient transepithelial transport,and rapid systemic clearance.Nanoliposomal delivery platforms-lipid bilayer vesicles on the nanometer scale-have attracted attention as an adaptive strategy to shield sensitive nutrients,navigate biological barriers,and deliver payloads directly to target tissues or even sub-cellular organelles.Despite a growing body of literature,a consolidated appraisal of design principles,targeting modalities,and translational hurdles is still needed to guide future nutraceutical innovation.We aim to:(1)Summarize the physicochemical foundations of nanoliposomal nutrient carriers;(2)Delineate state-of-the-art approaches for organ-specific and organelle-specific targeting,with particular emphasis on renal and mitochondrial delivery;(3)Evaluate current evidence supporting therapeutic benefits in cardiometabolic,neuroprotective,and renal-repair contexts;and(4)Map unresolved challenges-including manufacturing scale-up,cost,and regulatory oversight-to inform a roadmap for clinical translation.A systematic literature search was performed across PubMed,Web of Science,and Scopus through May 2025 using Boolean combinations of“nanoliposome”,“nutrient”,“targeted delivery”,“bioavailability”,and organ-specific terms(e.g.,“kidney”,“mitochondria”).Primary research articles,systematic reviews,and relevant meta-analyses written in English were included.Data were extracted on liposomal composition,particle size,surface modifications(e.g.,polyethylene glycol,ligand conjugation),in vitro and in vivo bio-distribution,efficacy outcomes,and safety profiles.Key design variables were mapped against reported biological performance to identify convergent principles.Sixty-four original studies and twenty-one reviews met inclusion criteria.Encapsulation within phosphatidylcholine-rich bilayers consistently enhanced nutrient stability in simulated gastric fluid and improved Caco-2 trans-epithelial transport two-fold to ten-fold compared with free compound controls.Ligand-mediated strategies-such as folate,lactoferrin,or peptide conjugation-achieved organ-specific accumulation,with kidney-directed liposomes demonstrating up to a four-fold increase in renal cortex uptake.Mitochondrial targeting using amphipathic peptides(e.g.,SS-31)or triphenylphosphonium moieties delivered antioxidant nutrients to the organelle,restoring mitochondrial membrane potential and reducing reactive oxygen species(ROS)in preclinical cardiomyopathy and neurodegeneration models.Endosomal escape was most effectively triggered by fusogenic lipids(e.g.,dioleoylphosphatidylethanolamine)or pH-responsive polymers.PEGylation prolonged circulation half-life by 3-6 hours but elicited anti-polyethylene glycol antibodies in approximately one-quarter of recipients;emerging natural sterol-mimetic or collagen-mimetic coatings showed comparable stealth behavior with superior biodegradability.Scalability remains limited:Only three studies reported pilot-scale(>10 L)batches with Good Manufacturing Practice-compliant reproducibility.Targeted nanoliposomal systems substantially improve nutrient stability,absorption,and tissue specificity,offering a credible route to transform supplement efficacy for cardiometabolic,renal,and neuroprotective indications.Optimization of lipid composition,escape mechanisms,and biocompatible surface chemistries can further enhance therapeutic indices.Nonetheless,industrial-scale manufacturing,cost containment,and immunogenicity mitigation remain critical obstacles.Addressing these gaps through standardized characterization protocols,head-to-head clinical trials,and biomaterial innovation will be essential to unlock the full potential of nanoliposomal nutraceuticals in routine healthcare practice.展开更多
With the identification of more than a dozen novel Hermansky-Pudlak Syndrome （HPS） proteins in vesicle trafficking in higher eukaryotes, a new class of trafficking pathways has been described. It mainly consists of ...With the identification of more than a dozen novel Hermansky-Pudlak Syndrome （HPS） proteins in vesicle trafficking in higher eukaryotes, a new class of trafficking pathways has been described. It mainly consists of three newly-defined protein com- plexes, BLOC-l, -2, and -3. Compelling evidence indicates that these complexes together with two other well-known complexes, AP3 and HOPS, play important roles in endosomal transport. The interactions between these complexes form a network in protein trafficking via endosomes and cytoskeleton. Each node of this network has intra-complex and extra-complex interactions. These complexes are connected by direct interactions between the subunits from different complexes or by indirect interactions through coupling nodes that interact with two or more subunits from different complexes. The dissection of this network facilitates the understanding of a dynamic but elaborate transport machinery in protein/membrane trafficking. The disruption of this network may lead to abnormal trafficking or defective organellar development as described in patients with Hermansky-Pudlak syndrome.展开更多
The internalization of essential nutrients,lipids and receptors is a crucial process for all eukaryotic cells.Accordingly,endocytosis is highly conserved across cell types and species.Once internalized,small cargocont...The internalization of essential nutrients,lipids and receptors is a crucial process for all eukaryotic cells.Accordingly,endocytosis is highly conserved across cell types and species.Once internalized,small cargocontaining vesicles fuse with early endosomes(also known as sorting endosomes),where they undergo segregation to distinct membrane regions and are sorted and transported on through the endocytic pathway.Although the mechanisms that regulate this sorting are still poorly understood,some receptors are directed to late endosomes and lysosomes for degradation,whereas other receptors are recycled back to the plasma membrane;either directly or through recycling endosomes.The Rab family of small GTP-binding proteins plays crucial roles in regulating these trafficking pathways.Rabs cycle from inactive GDP-bound cytoplasmic proteins to active GTP-bound membraneassociated proteins,as a consequence of the activity of multiple specific GTPase-activating proteins(GAPs) and GTP exchange factors(GEFs).Once bound to GTP,Rabs interact with a multitude of effector proteins that carry out Rab-specific functions.Recent studies have shown that some of these effectors are also interaction partners for the C-terminal Eps15 homology(EHD) proteins,which are also intimately involved in endocytic regulation.A particularly interesting example of common Rab-EHD interaction partners is the MICALlike protein,MICAL-L1.MICAL-L1 and its homolog,MICAL-L2,belong to the larger MICAL family of proteins,and both have been directly implicated in regulating endocytic recycling of cell surface receptors and junctional proteins,as well as controlling cytoskeletal rearrangement and neurite outgrowth.In this review,we summarize the functional roles of MICAL and Rab proteins,and focus on the significance of their interactions and the implications for endocytic transport.展开更多
The infection caused by porcine epidemic diarrhea virus(PEDV)is associated with high mortality in piglets worldwide.Host factors involved in the efficient replication of PEDV,however,remain largely unknown.Our recent ...The infection caused by porcine epidemic diarrhea virus(PEDV)is associated with high mortality in piglets worldwide.Host factors involved in the efficient replication of PEDV,however,remain largely unknown.Our recent proteomic study in the virus-host interaction network revealed a significant increase in the accumulation of CALML5(EF-hand protein calmodulin-like 5)following PEDV infection.A further study unveiled a biphasic increase of CALML5 in 2 and 12 h after viral infection.Similar trends were observed in the intestines of piglets in the early and late stages of the PEDV challenge.Moreover,CALML5 depletion reduced PEDV mRNA and protein levels,leading to a one-order-of-magnitude decrease in virus titer.At the early stage of PEDV infection,CALML5 affected the endosomal trafficking pathway by regulating the expression of endosomal sorting complex related cellular proteins.CALML5 depletion also suppressed IFN-βand IL-6 production in the PEDV-infected cells,thereby indicating its involvement in negatively regulating the innate immune response.Our study reveals the biological function of CALML5 in the virology field and offers new insights into the PEDV-host cell interaction.展开更多
Rabies virus(RABV) is a highly neurotropic virus that follows clathrin-mediated endocytosis and p H-dependent pathway for trafficking and invasion into endothelial cells. Early(Rab5, EEA1) and late(Rab7, LAMP1) endoso...Rabies virus(RABV) is a highly neurotropic virus that follows clathrin-mediated endocytosis and p H-dependent pathway for trafficking and invasion into endothelial cells. Early(Rab5, EEA1) and late(Rab7, LAMP1) endosomal proteins play critical roles in endosomal sorting, maturity and targeting various molecular cargoes, but their precise functions in the early stage of RABV neuronal infection remain elusive. In this study, the relationship between enigmatic entry of RABV with these endosomal proteins into neuronal and SH-SY5 Y cells was investigated.Immunofluorescence, TCID_(50) titers, electron microscopy and western blotting were carried out to determine the molecular interaction of the nucleoprotein(N) of RABV with early or late endosomal proteins in these cell lines. The expression of N was also determined by down-regulating Rab5 and Rab7 in both cell lines through RNA interference. The results were indicative that N proficiently colocalized with Rab5/EEA1 and Rab7/LAMP1 in both cell lines at 24 and 48 h post-infection, while N titers significantly decreased in early infection of RABV. Down-regulation of Rab5 and Rab7 did not inhibit N expression, but it prevented productive infection via blocking the normal trafficking of RABV in a low pH environment. Ultrathin sections of cells studied by electron microscope also verified the close association of RABV with Rab5 and Rab7 in neurons. From the data it was concluded that primary entry of RABV strongly correlates with the kinetics of Rab-proteins present on early and late vesicles, which provides helpful clues to explain the early events of RABV in nerve cells.展开更多
Endocytic tracers and marker enzyme of lysosomeswere used in the present study to analyze the processesof autophagocytosis and endocytosis, and the convergentpoint of these two pathways in Leydig cells. The endocytica...Endocytic tracers and marker enzyme of lysosomeswere used in the present study to analyze the processesof autophagocytosis and endocytosis, and the convergentpoint of these two pathways in Leydig cells. The endocyticand autophagic compartments call be easily identified inLeydig cells, which makes easier to define the stages of twopathways than was possible before. The evidences indicated that the late endosomes (dense MVBs) deliver theirendocytosed gold tracers together with lysosomal enzymesto the early autophagosomes and they are the convergentpoint of the two pathways. During this convergent process,the early autophagosomes transform into late autophagosomes and the late endosomes transform into mature lysosomes.展开更多
Transforming growth factor-β （TGF-β） signaling is tightly regulated to ensure its proper physiological functions in different cells and tissues. Like other cell surface receptors, TGF-β receptors are internalized...Transforming growth factor-β （TGF-β） signaling is tightly regulated to ensure its proper physiological functions in different cells and tissues. Like other cell surface receptors, TGF-β receptors are internalized into the cell, and this process plays an important regulatory role in TGF-β signaling. It is well documented that TGF-β receptors are endocytosed via clathrin-coated vesicles as TGF-β endocytosis can be blocked by potassium depletion and the GTPasedeficient dynamin K44A mutant. TGF-β receptors may also enter cells via cholesterol-rich membrane microdomain lipid rafts/caveolae and are found in caveolin-l-positive vesicles. Although receptor endocytosis is not essential for TGF-β signaling, clathrin-mediated endocytosis has been shown to promote TGF-β-induced Smad activation and transcriptional responses. Lipid rafts/caveolae are widely regarded as signaling centers for G protein-coupled recep- tors and tyrosine kinase receptors, but they are indicated to facilitate the degradation of TGF-β receptors and there- fore turnoff of TGF-β signaling. This review summarizes current understanding of TGF-β receptor endocytosis, the possible mechanisms underlying this process, and the role of endocytosis in modulation of TGF-β signaling.展开更多
Nanomaterials with multiple functions have become more and more popular in the domain of cancer research. MoS2 has a great potential in photothermal therapy, X-ray/CT imaging and drug delivery. In this study, a water ...Nanomaterials with multiple functions have become more and more popular in the domain of cancer research. MoS2 has a great potential in photothermal therapy, X-ray/CT imaging and drug delivery. In this study, a water soluble MoS2 nanosystem(MoS2-PEG) was synthesized and explored in drug delivery, photothermal therapy(PTT) and X-ray imaging.Doxorubicin(DOX) was loaded onto MoS2-PEG with a high drug loading efficiency(~69%)and obtained a multifunctional drug delivery system(MoS2-PEG/DOX). As the drug delivery, MoS2-PEG/DOX could efficiently cross the cell membranes, and escape from the endosome via NIR light irradiation, lead to more apoptosis in MCF-7 cells, and afford higher antitumor efficacy without obvious toxic effects to normal organs owing to its prolonged blood circulation and 11.6-fold higher DTX uptake of tumor than DOX. Besides, MoS2-PEG/DOX not only served as a drug delivery system, but also as a powerful PTT agent for thermal ablation of tumor and a strong X-ray contrast agent for tumor diagnosis. In the in vitro and in vivo studies, MoS2-PEG/DOX exhibited excellent tumor-targeting efficacy, outstanding synergistic anti-cancer effect of photothermal and chemotherapy and X-ray imaging property,demonstrating that MoS2-PEG/DOX had a great potential for simultaneous diagnosis and photothermal-chemotherapy in cancer treatment.展开更多
Cellular homeostasis requires a tightly controlled balance between protein synthesis, folding and degradation. Especially long-lived, post-mitotic cells such as neurons depend on an efficient proteostasis system to ma...Cellular homeostasis requires a tightly controlled balance between protein synthesis, folding and degradation. Especially long-lived, post-mitotic cells such as neurons depend on an efficient proteostasis system to maintain cellular health over decades. Thus, a functional decline of processes contributing to protein degradation such as autophagy and general lysosomal proteolytic capacity is connected to several age-associated neurodegenerative disorders, including Parkinson＇s, Alzheimer＇s and Huntington＇s diseases. These so called proteinopathies are characterized by the accumulation and misfolding of distinct proteins, subsequently driving cellular demise. We recently linked efficient lysosomal protein breakdown via the protease cathep- sin D to the Ca2＋/calmodulin-dependent phosphatase calcineurin. In a yeast model for Parkinson＇s disease, functional calcineurin was required for proper trafficking of cathepsin D to the lysosome and for recycling of its endosomal sorting receptor to allow further rounds of shuttling. Here, we discuss these findings in relation to present knowledge about the involvement of cathepsin D in proteinopathies in general and a possible connection between this protease, calcineurin signalling and endosomal sorting in particular. As dysregulation of Ca2＋ homeostasis as well as lysosomal impairment is connected to a plethora of neurode- generative disorders, this novel interplay might very well impact pathologies beyond Parkinson＇s disease.展开更多
Foot-and-mouth disease(FMD)is a highly contagious and economically important disease,which is caused by the FMD virus(FMDV).Although the cell receptor for FMDV has been identified,the specific mechanism of FMDV intern...Foot-and-mouth disease(FMD)is a highly contagious and economically important disease,which is caused by the FMD virus(FMDV).Although the cell receptor for FMDV has been identified,the specific mechanism of FMDV internalization after infection remains unknown.In this study,we found that kinesin family member 5B(KIF5B)plays a vital role during FMDV internalization.Moreover,we confirmed the interaction between KIF5B and FMDV structural protein VP1 by co-immunoprecipitation(Co-IP)and co-localization in FMDV-infected cells.In particular,the stalk[amino acids(aa)413–678]domain of KIF5B was indispensable for KIF5B-VP1 interaction.Moreover,overexpression of KIF5B dramatically enhanced FMDV replication;consistently,knockdown or knockout of KIF5B suppressed FMDV replication.Furthermore,we also demonstrated that KIF5B promotes the internalization of FMDV via regulating clathrin uncoating.KIF5B also promotes the transmission of viral particles to early and late endosomes during the early stages of infection.In conclusion,our results demonstrate that KIF5B promotes the internalization of FMDV via regulating clathrin uncoating and intracellular transport.This study may provide a new therapeutic target for developing FMDV antiviral drugs.展开更多
Objectives:Improper activation of Wnt/β-catenin signaling has been implicated in human diseases.Beyond the well-studied glycogen synthase kinase 3p(GSK3p)and casein kinase 1(CK1),other kinases affecting Wnt/β-cateni...Objectives:Improper activation of Wnt/β-catenin signaling has been implicated in human diseases.Beyond the well-studied glycogen synthase kinase 3p(GSK3p)and casein kinase 1(CK1),other kinases affecting Wnt/β-catenin signaling remain to be defined.Methods:To identify the kinases that modulate Wnt/β-catenin signaling,we applied a kinase small interfering RNA(siRNA)library screen approach.Luciferase assays,immunoblotting,and real-time polymerase chain reaction(PCR)were performed to confirm the regulation o f the Wnt/β-catenin signaling pathway by cyclin-dependent kinase 11(CDK11)and to investigate the underlying mechanism.Confocal immunofluorescence,coimmunoprecipitation(co-IP),and scratch wound assays were used to demonstrate colocalization,detect protein interactions,and explore the function of CDK11.Results:CDK11 was found to be a significant candidate kinase participating in the negative control of Wnt/P-catenin signaling.Down-regulation of CDK11 led to the accumulation of Wnt/β-catenin signaling receptor complexes,in a manner dependent on intact adenomatosis polyposis coli(APC)protein.Further analysis showed that CDK11 modulation of Wnt/P-catenin signaling engaged the endolysosomal machinery,and CDK11 knockdown enhanced the colocalization of Wnt/β-catenin signaling receptor complexes with early endosomes and decreased colocalization with lysosomes.Mechanistically,CDK11 was found to function in Wnt/β-catenin signaling by regulating microtubule stability.Depletion of CDK11 down-regulated acetyl-a-tubulin.Moreover,co-IP assays demonstrated that CDK11 interacts with the a-tubulin deacetylase SIRT2,whereas SIRT2 down-regulation in CDK11-depleted cells reversed the accumulation of Wnt/(3-catenin signaling receptor complexes.CDK11 was found to suppress cell migration through altered W nt/β-catenin signaling.Conclusions:CDK11 is a negative modulator of Wnt/β-catenin signaling that stabilizes microtubules,thus resulting in the dysregulation of receptor complex trafficking from early endosomes to lysosomes.展开更多
Currently, hepatitis B virus(HBV), upon attaching to human hepatocytes, is considered to interact first with heparan sulfate proteoglycan(HSPG) via an antigenic loop of HBV envelope S protein. Then, it is promptly tra...Currently, hepatitis B virus(HBV), upon attaching to human hepatocytes, is considered to interact first with heparan sulfate proteoglycan(HSPG) via an antigenic loop of HBV envelope S protein. Then, it is promptly transferred to the sodium taurocholate cotransporting polypeptide(NTCP) via the myristoylated N-terminal sequence of pre-S1 region(from Gly-2 to Gly-48, HBV genotype D), and it finally enters the cell by endocytosis. However, it is not clear how HSPG passes HBV to NTCP and how NTCP contributes to the cellular entry of HBV. Owing to the poor availability and the difficulty of manipulations, including fluorophore encapsulation, it has been nearly impossible to perform biochemical and cytochemical analyses using a substantial amount of HBV. A bio-nanocapsule(BNC), which is a hollow nanoparticle consisting of HBV envelope L protein, was efficiently synthesized in Saccharomyces cerevisiae. Since BNC could encapsulate payloads(drugs, genes, proteins) and specifically enter human hepatic cells utilizing HBV-derived infection machinery, it could be used as a model of HBV infection to elucidate the early infection machinery. Recently, it was demonstrated that the N-terminal sequence of pre-S1 region(from Asn-9 to Gly-24) possesses low p H-dependent fusogenic activity, which might play a crucial role in the endosomal escape of BNC payloads and in the uncoating process of HBV. In this minireview, we describe a model in which each domain of the HBV L protein contributes to attachment onto human hepatic cells through HSPG, initiation of endocytosis, interaction with NTCP in endosomes, and consequent provocation of membrane fusion followed by endosomal escape.展开更多
Evidence from genetics and from analyzing cellular and animal models have converged to suggest links between neurodegenerative disorders of early and late life.Here,we summarize emerging links between the most common ...Evidence from genetics and from analyzing cellular and animal models have converged to suggest links between neurodegenerative disorders of early and late life.Here,we summarize emerging links between the most common late life neurodegenerative disease,Alzheimer’s disease,and the most common early life neurodegenerative diseases,neuronal ceroid lipofuscinoses.Genetic studies reported an overlap of clinically diagnosed Alzheimer’s disease and mutations in genes known to cause neuronal ceroid lipofuscinoses.Accumulating data strongly suggest dysfunction of intracellular trafficking mechanisms and the autophagy-endolysosome system in both types of neurodegenerative disorders.This suggests shared cytopathological processes underlying these different types of neurodegenerative diseases.A better understanding of the common mechanisms underlying the different diseases is important as this might lead to the identification of novel targets for therapeutic concepts,the transfer of therapeutic strategies from one disease to the other and therapeutic approaches tailored to patients with specific mutations.Here,we review dysfunctions of the endolysosomal autophagy pathway in Alzheimer’s disease and neuronal ceroid lipofuscinoses and summarize emerging etiologic and genetic overlaps.展开更多
基金This work is supported in part by grants from the National Natural Science Foundation of China(31672693,31972838 and 31400139,31372565).
文摘Grass carp reovirus(GCRV),the genus Aquareovirus in family Reoviridae,is viewed as the most pathogenic aquareovirus.To understand the molecular mechanism of how aquareovirus initiates productive infection,the roles of endosome and microtubule in cell entry of GCRV are investigated by using quantum dots(QDs)-tracking in combination with biochemical approaches.We found that GCRV infection and viral protein synthesis were significantly inhibited by pretreating host cells with endosome acidification inhibitors NH4Cl,chloroquine and bafilomycin A1(Bafi).Confocal images indicated that GCRV particles could colocalize with Rab5,Rab7 and lysosomes in host cells.Further ultrastructural examination validated that viral particle was found in late endosomes.Moreover,disruption of microtubules with nocodazole clearly blocked GCRV entry,while no inhibitory effects were observed with cytochalasin D treated cells in viral infection,hinting that intracellular transportation of endocytic uptake in GCRV infected cells is via microtubules but not actin filament.Notably,viral particles were observed to transport along microtubules by using QD-labeled GCRV.Altogether,our results suggest that GCRV can use endosomes and microtubules to initiate productive infection.
文摘Aim:Ligand-targeted therapeutics are experiencing increasing use for treatment of human diseases due to their ability to concentrate a desired drug at a pathologic site while reducing accumulation in healthy tissues.For many ligand-targeted drug conjugates,a critical aspect of conjugate design lies in engineering release of the therapeutic payload to occur only after its internalization by targeted cells.Because disulfide bond reduction is frequently exploited to ensure intracellular drug release,an understanding of the redox properties of endocytic compartments can be critical to ligand-targeted drug design.While the redox properties of folate receptor trafficking endosomes have been previously reported,little is known about the trafficking of prostate-specific membrane antigen(PSMA),a receptor that is experiencing increasing use for drug targeting in humans.Methods:To obtain this information,we have constructed a PSMA-targeted fluorescence resonance energy transfer pair that reports on disulfide bond reduction by changing fluorescence from red to green.Results:We show here that this reporter exhibits rapid and selective uptake by PSMA-positive cells,and that reduction of its disulfide bond proceeds steadily but incompletely following internalization.The fact that maximal disulfide reduction reaches only~50%,even after 24 h incubation,suggests that roughly half of the conjugates must traffic through endosomes that display no reducing capacity.Conclusion:As the level of disulfide reduction differs between PSMA trafficked and previously published folate trafficked conjugates,it also follows that not all internalizing receptors are translocated through similar intracellular compartments.Taken together,these data suggest that the efficiency of disulfide bond reduction must be independently analyzed for each receptor trafficking pathway when disulfide bond reduction is exploited for intracellular drug release.
基金We would like to thank the personnel of the Bergman clinic in Bilthoven, the Netherlands for providing healthy donor skin. We would like to thank Tom O＇Toole for the technical assistance with imaging flow cytometry. The present work was funded by KWF （VU2009-2598）, the Dutch Science Foundation （NWO, VENI Grant NO 863.10.017）, the European Research Council （ERCAdvanced339977）, and NanoNext 3D01.
文摘The potential of the skin immune system to generate immune responses is well established, and the skin is actively exploited as a vaccination site. Human skin contains several antigen-presenting cell subsets with specialized functions. In particular, the capacity to cross-present exogenous antigens to CD8^＋ T cells is of interest for the design of effective immunotherapies against viruses or cancer. Here, we show that primary human Langerhans cells （LCs） were able to cross-present a synthetic long peptide （SLP） to CD8^＋ T cells. In addition, modification of this SLP using antibodies against the receptor langerin, but not dectin-1, further enhanced the cross-presenting capacity of LCs through routing of internalized antigens to less proteolytic early endosome antigen 1 ^＋ early endosomes. The potency of LCs to enhance CD8^＋ T-cell responses could be further increased through activation of LCs with the toll-like receptor 3 ligand polyinosinic：polycytidylic acid （phC）. Altogether, the data provide evidence that human LCs are able to cross-present antigens after langerin-mediated internalization. Furthermore, the potential for antigen modification to target LCs specifically provides a rationale for generating effective anti-tumor or anti-viral cytotoxic T lymphocyte responses.
文摘Cells producing cytokines often express the receptor for the same cytokine, which makes them prone to autocrine signaling. How cytokine release and signaling are regulated in the same cell is not understood? In this study, we demonstrate that signaling by exogenous and self-synthesized inflammatory cytokine interleukin-6 (IL-6) within endosomal compartments acts as a cellular brake that limits the synthesis of IL-6. Our data show that IL-6 is internalized by dendritic cells and signals from endosomal compartments containing the IL-6 receptor. Newly synthesized IL-6 also traffics via these endosomal compartments and signals in transit to the plasma membrane. This allows activation of STAT3 which in turn limits toll-like receptor 4 stimulant lipopolysaccharide (LPS) triggered transcription of IL-6. Long-term exposure to LPS removes this brake via inhibition of STAT3 by increased expression of suppressor of cytokine signaling 3 and results in fully fledged IL-6 production. This transient regulation could prevent excessive IL-6 production during early infections.
基金supported by the Japan Society for the Promotion of Science KAKENHI(grant Nos.23K05678 to IM,19H05711 and 20H00466 to KS)the Joint Research Program of Institute for Molecular and Cellular Regulation,Gunma University(to KS)。
文摘In eukaryotic cells,organelles in the secretory,lysosomal,and endocytic pathways actively exchange biological materials with each other through intracellular membrane trafficking,which is the process of transporting the cargo of proteins,lipids,and other molecules to appropriate compartments via transport vesicles or intermediates.These processes are strictly regulated by various small GTPases such as the RAS-like in rat brain(RAB)protein family,which is the largest subfamily of the RAS superfamily.Dysfunction of membrane trafficking affects tissue homeostasis and leads to a wide range of diseases,including neurological disorders and neurodegenerative diseases.Therefore,it is important to understand the physiological and pathological roles of RAB proteins in brain function.RAB35,a member of the RAB family,is an evolutionarily conserved protein in metazoans.A wide range of studies using cultured mammalian cells and model organisms have revealed that RAB35 mediates various processes such as cytokinesis,endocytic recycling,actin bundling,and cell migration.RAB35 is also involved in neurite outgrowth and turnover of synaptic vesicles.We generated brain-specific Rab35 knockout mice to study the physiological roles of RAB35 in brain development and function.These mice exhibited defects in anxiety-related behaviors and spatial memory.Strikingly,RAB35 is required for the precise positioning of pyramidal neurons during hippocampal development,and thereby for normal hippocampal lamination.In contrast,layer formation in the cerebral cortex occurred superficially,even in the absence of RAB35,suggesting a predominant role for RAB35 in hippocampal development rather than in cerebral cortex development.Recent studies have suggested an association between RAB35 and neurodegenerative diseases,including Parkinson's disease and Alzheimer's disease.In this review,we provide an overview of the current understanding of subcellular functions of RAB35.We also provide insights into the physiological role of RAB35 in mammalian brain development and function,and discuss the involvement of RAB35 dysfunction in neurodegenerative diseases.
基金supported by National Natural Science Foundation of China(No.82173769)the National Key R&D Program of China(No.2021YFE0106900)+1 种基金Applied Basic Research Multiinvestment Foundation of Tianjin(No.21JCYBJC01540)the Science&Technology Development Fund of Tianjin Education Commission for Higher Education(No.2023ZD019)。
文摘Small interfering RNAs(siRNA)provide a novel and highly specific therapy due to their ability to effectively silence target genes,to date six siRNA therapeutics are approved for clinical use.Even so,some critical challenges remain to overcome in the therapeutic application of siRNAs,with delivery issues at the forefront.Among them,endo/lysosomal barrier is one of the important but often-neglected limitations hindering the delivery of siRNA therapeutics.In this review,we summarize the promising strategies that facilitate siRNAs overcoming endo/lysosomal barriers based on the cellular uptake and intracellular transport pathways,including promoting escape once endocytosis into the endo/lysosomes and bypassing lysosomes via endosome-Golgi-endoplasmic reticulum(ER)pathway or nonendocytosis pathway,and discuss the principal considerations and the future directions of promoting endo/lysosomal escape in the development of therapeutic siRNAs.
基金supported by National Natural Science Foundation of China(81502688)Cooperation Research Funding of Capital Medical University(2020KJ000514)+1 种基金Cooperation Research Funding of Capital Medical University(2023KJ000814)R&D Program of Beijing Municipal Education Commission(KM202210025024).
文摘Efficient siRNA delivery is highly desirable for disease treatment.However,the application of conventional nanoparticles is limited by the inability to escape from endo-lysosomes.Herein,we report a strategy using small-molecule drugs to enhance siRNA endo-lysosomal release,addressing this challenge.We encapsulated gentamicin(GM)into the marketed Onpattro■ formulation to establish LNP-siRNA/GM nanoparticles that promote siRNA endo-lysosomal escape through endosomal disruption,mechanistically exhibiting unique functionality and synergistic effects of LNP-siRNA/GM to improve cancer therapy.Besides,GM induced reactive oxygen species(ROS)and phospholipids accumulation in endolysosomes,as well as the physical characteristics of lipid nanoparticles(LNPs)were preserved.We also revealed that GM causes endo-lysosomal swelling and disrupts the endosomal membrane to enable siRNA release,as confirmed by Galectin 3 recruitment and acridine orange release.This approach achieved∼81%mRNA-EGFR silencing,which is more than LNP-siEGFR(∼56.23%)by enhancing siRNA endo-lysosomal escape efficiency.Meanwhile,LNP-siEGFR/GM exhibited significant biological activities in HepG2 cells,driven by the synergistic effects of siEGFR and GM with the VEGF and CXCL12 downregulation of,and ROS and phospholipids upregulation.Furthermore,tumor growth was notably suppressed after intravenous injection of LNP-siEGFR/GM in tumor-bearing nude mice.The combination of EGFR-siRNA and GM could also greatly inhibit angiogenesis,be antiproliferative,and induce tumor cells apoptosis.Therefore,this GM and siRNA co-delivery system would provide an efficient strategy for siRNA endosomal escape,significantly improving knockdown in various LNPs based siRNA delivery systems and efficiently enhancing cancer therapy.
文摘Conventional nutritional supplements frequently demonstrate limited clinical effectiveness due to the harsh milieu of the gastrointestinal tract,inefficient transepithelial transport,and rapid systemic clearance.Nanoliposomal delivery platforms-lipid bilayer vesicles on the nanometer scale-have attracted attention as an adaptive strategy to shield sensitive nutrients,navigate biological barriers,and deliver payloads directly to target tissues or even sub-cellular organelles.Despite a growing body of literature,a consolidated appraisal of design principles,targeting modalities,and translational hurdles is still needed to guide future nutraceutical innovation.We aim to:(1)Summarize the physicochemical foundations of nanoliposomal nutrient carriers;(2)Delineate state-of-the-art approaches for organ-specific and organelle-specific targeting,with particular emphasis on renal and mitochondrial delivery;(3)Evaluate current evidence supporting therapeutic benefits in cardiometabolic,neuroprotective,and renal-repair contexts;and(4)Map unresolved challenges-including manufacturing scale-up,cost,and regulatory oversight-to inform a roadmap for clinical translation.A systematic literature search was performed across PubMed,Web of Science,and Scopus through May 2025 using Boolean combinations of“nanoliposome”,“nutrient”,“targeted delivery”,“bioavailability”,and organ-specific terms(e.g.,“kidney”,“mitochondria”).Primary research articles,systematic reviews,and relevant meta-analyses written in English were included.Data were extracted on liposomal composition,particle size,surface modifications(e.g.,polyethylene glycol,ligand conjugation),in vitro and in vivo bio-distribution,efficacy outcomes,and safety profiles.Key design variables were mapped against reported biological performance to identify convergent principles.Sixty-four original studies and twenty-one reviews met inclusion criteria.Encapsulation within phosphatidylcholine-rich bilayers consistently enhanced nutrient stability in simulated gastric fluid and improved Caco-2 trans-epithelial transport two-fold to ten-fold compared with free compound controls.Ligand-mediated strategies-such as folate,lactoferrin,or peptide conjugation-achieved organ-specific accumulation,with kidney-directed liposomes demonstrating up to a four-fold increase in renal cortex uptake.Mitochondrial targeting using amphipathic peptides(e.g.,SS-31)or triphenylphosphonium moieties delivered antioxidant nutrients to the organelle,restoring mitochondrial membrane potential and reducing reactive oxygen species(ROS)in preclinical cardiomyopathy and neurodegeneration models.Endosomal escape was most effectively triggered by fusogenic lipids(e.g.,dioleoylphosphatidylethanolamine)or pH-responsive polymers.PEGylation prolonged circulation half-life by 3-6 hours but elicited anti-polyethylene glycol antibodies in approximately one-quarter of recipients;emerging natural sterol-mimetic or collagen-mimetic coatings showed comparable stealth behavior with superior biodegradability.Scalability remains limited:Only three studies reported pilot-scale(>10 L)batches with Good Manufacturing Practice-compliant reproducibility.Targeted nanoliposomal systems substantially improve nutrient stability,absorption,and tissue specificity,offering a credible route to transform supplement efficacy for cardiometabolic,renal,and neuroprotective indications.Optimization of lipid composition,escape mechanisms,and biocompatible surface chemistries can further enhance therapeutic indices.Nonetheless,industrial-scale manufacturing,cost containment,and immunogenicity mitigation remain critical obstacles.Addressing these gaps through standardized characterization protocols,head-to-head clinical trials,and biomaterial innovation will be essential to unlock the full potential of nanoliposomal nutraceuticals in routine healthcare practice.
基金This work was supported in part by the National Science Fund for Distinguished Young Scholars (No. 30525007)National Basic Research Program of China (No. 2006CB504103+1 种基金 No. 2006CB500704)Hi-Tech Research and Development Program of China (No. 2006AA02Z322)
文摘With the identification of more than a dozen novel Hermansky-Pudlak Syndrome （HPS） proteins in vesicle trafficking in higher eukaryotes, a new class of trafficking pathways has been described. It mainly consists of three newly-defined protein com- plexes, BLOC-l, -2, and -3. Compelling evidence indicates that these complexes together with two other well-known complexes, AP3 and HOPS, play important roles in endosomal transport. The interactions between these complexes form a network in protein trafficking via endosomes and cytoskeleton. Each node of this network has intra-complex and extra-complex interactions. These complexes are connected by direct interactions between the subunits from different complexes or by indirect interactions through coupling nodes that interact with two or more subunits from different complexes. The dissection of this network facilitates the understanding of a dynamic but elaborate transport machinery in protein/membrane trafficking. The disruption of this network may lead to abnormal trafficking or defective organellar development as described in patients with Hermansky-Pudlak syndrome.
基金Supported by The National Institutes of Health grants R01GM074876 (Caplan S and Naslavsky N),R01GM087455 (Caplan S),the Nebraska Dept. of Health (Naslavsky N)P20 RR018759 from the National Center
文摘The internalization of essential nutrients,lipids and receptors is a crucial process for all eukaryotic cells.Accordingly,endocytosis is highly conserved across cell types and species.Once internalized,small cargocontaining vesicles fuse with early endosomes(also known as sorting endosomes),where they undergo segregation to distinct membrane regions and are sorted and transported on through the endocytic pathway.Although the mechanisms that regulate this sorting are still poorly understood,some receptors are directed to late endosomes and lysosomes for degradation,whereas other receptors are recycled back to the plasma membrane;either directly or through recycling endosomes.The Rab family of small GTP-binding proteins plays crucial roles in regulating these trafficking pathways.Rabs cycle from inactive GDP-bound cytoplasmic proteins to active GTP-bound membraneassociated proteins,as a consequence of the activity of multiple specific GTPase-activating proteins(GAPs) and GTP exchange factors(GEFs).Once bound to GTP,Rabs interact with a multitude of effector proteins that carry out Rab-specific functions.Recent studies have shown that some of these effectors are also interaction partners for the C-terminal Eps15 homology(EHD) proteins,which are also intimately involved in endocytic regulation.A particularly interesting example of common Rab-EHD interaction partners is the MICALlike protein,MICAL-L1.MICAL-L1 and its homolog,MICAL-L2,belong to the larger MICAL family of proteins,and both have been directly implicated in regulating endocytic recycling of cell surface receptors and junctional proteins,as well as controlling cytoskeletal rearrangement and neurite outgrowth.In this review,we summarize the functional roles of MICAL and Rab proteins,and focus on the significance of their interactions and the implications for endocytic transport.
基金supported by the National Key R&D Program of China(2023YFD1801100)the National Natural Science Foundation of China(32172821)a CAU-Grant for the Prevention and Control of Immunosuppressive Disease in Animals(CAU-G-PCIDA)of the China Agricultural University.
文摘The infection caused by porcine epidemic diarrhea virus(PEDV)is associated with high mortality in piglets worldwide.Host factors involved in the efficient replication of PEDV,however,remain largely unknown.Our recent proteomic study in the virus-host interaction network revealed a significant increase in the accumulation of CALML5(EF-hand protein calmodulin-like 5)following PEDV infection.A further study unveiled a biphasic increase of CALML5 in 2 and 12 h after viral infection.Similar trends were observed in the intestines of piglets in the early and late stages of the PEDV challenge.Moreover,CALML5 depletion reduced PEDV mRNA and protein levels,leading to a one-order-of-magnitude decrease in virus titer.At the early stage of PEDV infection,CALML5 affected the endosomal trafficking pathway by regulating the expression of endosomal sorting complex related cellular proteins.CALML5 depletion also suppressed IFN-βand IL-6 production in the PEDV-infected cells,thereby indicating its involvement in negatively regulating the innate immune response.Our study reveals the biological function of CALML5 in the virology field and offers new insights into the PEDV-host cell interaction.
基金supported by the National Key Research and Development Program of China(Grant No.216YFD0500402)Natural Science Foundation of China(Grants No.31272579 and 31472208)
文摘Rabies virus(RABV) is a highly neurotropic virus that follows clathrin-mediated endocytosis and p H-dependent pathway for trafficking and invasion into endothelial cells. Early(Rab5, EEA1) and late(Rab7, LAMP1) endosomal proteins play critical roles in endosomal sorting, maturity and targeting various molecular cargoes, but their precise functions in the early stage of RABV neuronal infection remain elusive. In this study, the relationship between enigmatic entry of RABV with these endosomal proteins into neuronal and SH-SY5 Y cells was investigated.Immunofluorescence, TCID_(50) titers, electron microscopy and western blotting were carried out to determine the molecular interaction of the nucleoprotein(N) of RABV with early or late endosomal proteins in these cell lines. The expression of N was also determined by down-regulating Rab5 and Rab7 in both cell lines through RNA interference. The results were indicative that N proficiently colocalized with Rab5/EEA1 and Rab7/LAMP1 in both cell lines at 24 and 48 h post-infection, while N titers significantly decreased in early infection of RABV. Down-regulation of Rab5 and Rab7 did not inhibit N expression, but it prevented productive infection via blocking the normal trafficking of RABV in a low pH environment. Ultrathin sections of cells studied by electron microscope also verified the close association of RABV with Rab5 and Rab7 in neurons. From the data it was concluded that primary entry of RABV strongly correlates with the kinetics of Rab-proteins present on early and late vesicles, which provides helpful clues to explain the early events of RABV in nerve cells.
文摘Endocytic tracers and marker enzyme of lysosomeswere used in the present study to analyze the processesof autophagocytosis and endocytosis, and the convergentpoint of these two pathways in Leydig cells. The endocyticand autophagic compartments call be easily identified inLeydig cells, which makes easier to define the stages of twopathways than was possible before. The evidences indicated that the late endosomes (dense MVBs) deliver theirendocytosed gold tracers together with lysosomal enzymesto the early autophagosomes and they are the convergentpoint of the two pathways. During this convergent process,the early autophagosomes transform into late autophagosomes and the late endosomes transform into mature lysosomes.
基金The work in Ye-Guang Chen＇s laboratory is supported by grants from the National Natural Science Foundation of China （30430360, 30671033） and the Ministry of Sciences and Technology of China 973 Program （2004CB720002, 2006CB943401, 2006CB910102） and 863 Program （2006AA02Z 172）.
文摘Transforming growth factor-β （TGF-β） signaling is tightly regulated to ensure its proper physiological functions in different cells and tissues. Like other cell surface receptors, TGF-β receptors are internalized into the cell, and this process plays an important regulatory role in TGF-β signaling. It is well documented that TGF-β receptors are endocytosed via clathrin-coated vesicles as TGF-β endocytosis can be blocked by potassium depletion and the GTPasedeficient dynamin K44A mutant. TGF-β receptors may also enter cells via cholesterol-rich membrane microdomain lipid rafts/caveolae and are found in caveolin-l-positive vesicles. Although receptor endocytosis is not essential for TGF-β signaling, clathrin-mediated endocytosis has been shown to promote TGF-β-induced Smad activation and transcriptional responses. Lipid rafts/caveolae are widely regarded as signaling centers for G protein-coupled recep- tors and tyrosine kinase receptors, but they are indicated to facilitate the degradation of TGF-β receptors and there- fore turnoff of TGF-β signaling. This review summarizes current understanding of TGF-β receptor endocytosis, the possible mechanisms underlying this process, and the role of endocytosis in modulation of TGF-β signaling.
基金supported by grants from the National Natural Science Foundation of China(Nos.81273451,81302717 and81101684)
文摘Nanomaterials with multiple functions have become more and more popular in the domain of cancer research. MoS2 has a great potential in photothermal therapy, X-ray/CT imaging and drug delivery. In this study, a water soluble MoS2 nanosystem(MoS2-PEG) was synthesized and explored in drug delivery, photothermal therapy(PTT) and X-ray imaging.Doxorubicin(DOX) was loaded onto MoS2-PEG with a high drug loading efficiency(~69%)and obtained a multifunctional drug delivery system(MoS2-PEG/DOX). As the drug delivery, MoS2-PEG/DOX could efficiently cross the cell membranes, and escape from the endosome via NIR light irradiation, lead to more apoptosis in MCF-7 cells, and afford higher antitumor efficacy without obvious toxic effects to normal organs owing to its prolonged blood circulation and 11.6-fold higher DTX uptake of tumor than DOX. Besides, MoS2-PEG/DOX not only served as a drug delivery system, but also as a powerful PTT agent for thermal ablation of tumor and a strong X-ray contrast agent for tumor diagnosis. In the in vitro and in vivo studies, MoS2-PEG/DOX exhibited excellent tumor-targeting efficacy, outstanding synergistic anti-cancer effect of photothermal and chemotherapy and X-ray imaging property,demonstrating that MoS2-PEG/DOX had a great potential for simultaneous diagnosis and photothermal-chemotherapy in cancer treatment.
基金supported by the Austrian Science Fund FWF(No.P27183-B24)the Swedish Research Council Vetenskapsradet(No.2015-05468)+2 种基金Ake Wiberg Stiftelse(No.M16-0130)Carl Trygger Stiftlese(No.CTS16:85)Goljes Stiftelse(No.LA2016-0123)
文摘Cellular homeostasis requires a tightly controlled balance between protein synthesis, folding and degradation. Especially long-lived, post-mitotic cells such as neurons depend on an efficient proteostasis system to maintain cellular health over decades. Thus, a functional decline of processes contributing to protein degradation such as autophagy and general lysosomal proteolytic capacity is connected to several age-associated neurodegenerative disorders, including Parkinson＇s, Alzheimer＇s and Huntington＇s diseases. These so called proteinopathies are characterized by the accumulation and misfolding of distinct proteins, subsequently driving cellular demise. We recently linked efficient lysosomal protein breakdown via the protease cathep- sin D to the Ca2＋/calmodulin-dependent phosphatase calcineurin. In a yeast model for Parkinson＇s disease, functional calcineurin was required for proper trafficking of cathepsin D to the lysosome and for recycling of its endosomal sorting receptor to allow further rounds of shuttling. Here, we discuss these findings in relation to present knowledge about the involvement of cathepsin D in proteinopathies in general and a possible connection between this protease, calcineurin signalling and endosomal sorting in particular. As dysregulation of Ca2＋ homeostasis as well as lysosomal impairment is connected to a plethora of neurode- generative disorders, this novel interplay might very well impact pathologies beyond Parkinson＇s disease.
基金supported by the National Natural Sciences Foundation of China(No.32102639 and 32072831)the National Key Research and Development Program of China(No.2021YFD1800300)+5 种基金the Gansu Science Foundation for Distinguished Young Scholars(No.21JR7RA026)the Earmarked Fund for CARS-35,the Strategic Priority Research Program of the National Center of Technology Innovation for Pigs(No.NCTIP-XD/C03)the Science and Technology Major Project of Gansu Province(No.22ZD6NA001)the Natural Science Foundation of Gansu Province(No.22JR5RA034 and 23JRRA549)the open competition program of top ten critical priorities of Agricultural Science and Technology Innovation for the 14th Five-Year Plan of Guangdong Province(No.2023SDZG02)the Fundamental Research Funds for the Central Universities(No.lzujbky-2022-ey20).
文摘Foot-and-mouth disease(FMD)is a highly contagious and economically important disease,which is caused by the FMD virus(FMDV).Although the cell receptor for FMDV has been identified,the specific mechanism of FMDV internalization after infection remains unknown.In this study,we found that kinesin family member 5B(KIF5B)plays a vital role during FMDV internalization.Moreover,we confirmed the interaction between KIF5B and FMDV structural protein VP1 by co-immunoprecipitation(Co-IP)and co-localization in FMDV-infected cells.In particular,the stalk[amino acids(aa)413–678]domain of KIF5B was indispensable for KIF5B-VP1 interaction.Moreover,overexpression of KIF5B dramatically enhanced FMDV replication;consistently,knockdown or knockout of KIF5B suppressed FMDV replication.Furthermore,we also demonstrated that KIF5B promotes the internalization of FMDV via regulating clathrin uncoating.KIF5B also promotes the transmission of viral particles to early and late endosomes during the early stages of infection.In conclusion,our results demonstrate that KIF5B promotes the internalization of FMDV via regulating clathrin uncoating and intracellular transport.This study may provide a new therapeutic target for developing FMDV antiviral drugs.
基金grants from the National Natural Science Foundation of China(Grant No.81530084,81874200,and 81572750)the Hunan Science and Technology Department(Grant No.2018RS3028)+1 种基金Central South University(Grant No.20170033010007)The Strategy-Orientated Special Project of Central South University(Grant No.ZLXD2017003).
文摘Objectives:Improper activation of Wnt/β-catenin signaling has been implicated in human diseases.Beyond the well-studied glycogen synthase kinase 3p(GSK3p)and casein kinase 1(CK1),other kinases affecting Wnt/β-catenin signaling remain to be defined.Methods:To identify the kinases that modulate Wnt/β-catenin signaling,we applied a kinase small interfering RNA(siRNA)library screen approach.Luciferase assays,immunoblotting,and real-time polymerase chain reaction(PCR)were performed to confirm the regulation o f the Wnt/β-catenin signaling pathway by cyclin-dependent kinase 11(CDK11)and to investigate the underlying mechanism.Confocal immunofluorescence,coimmunoprecipitation(co-IP),and scratch wound assays were used to demonstrate colocalization,detect protein interactions,and explore the function of CDK11.Results:CDK11 was found to be a significant candidate kinase participating in the negative control of Wnt/P-catenin signaling.Down-regulation of CDK11 led to the accumulation of Wnt/β-catenin signaling receptor complexes,in a manner dependent on intact adenomatosis polyposis coli(APC)protein.Further analysis showed that CDK11 modulation of Wnt/P-catenin signaling engaged the endolysosomal machinery,and CDK11 knockdown enhanced the colocalization of Wnt/β-catenin signaling receptor complexes with early endosomes and decreased colocalization with lysosomes.Mechanistically,CDK11 was found to function in Wnt/β-catenin signaling by regulating microtubule stability.Depletion of CDK11 down-regulated acetyl-a-tubulin.Moreover,co-IP assays demonstrated that CDK11 interacts with the a-tubulin deacetylase SIRT2,whereas SIRT2 down-regulation in CDK11-depleted cells reversed the accumulation of Wnt/(3-catenin signaling receptor complexes.CDK11 was found to suppress cell migration through altered W nt/β-catenin signaling.Conclusions:CDK11 is a negative modulator of Wnt/β-catenin signaling that stabilizes microtubules,thus resulting in the dysregulation of receptor complex trafficking from early endosomes to lysosomes.
文摘Currently, hepatitis B virus(HBV), upon attaching to human hepatocytes, is considered to interact first with heparan sulfate proteoglycan(HSPG) via an antigenic loop of HBV envelope S protein. Then, it is promptly transferred to the sodium taurocholate cotransporting polypeptide(NTCP) via the myristoylated N-terminal sequence of pre-S1 region(from Gly-2 to Gly-48, HBV genotype D), and it finally enters the cell by endocytosis. However, it is not clear how HSPG passes HBV to NTCP and how NTCP contributes to the cellular entry of HBV. Owing to the poor availability and the difficulty of manipulations, including fluorophore encapsulation, it has been nearly impossible to perform biochemical and cytochemical analyses using a substantial amount of HBV. A bio-nanocapsule(BNC), which is a hollow nanoparticle consisting of HBV envelope L protein, was efficiently synthesized in Saccharomyces cerevisiae. Since BNC could encapsulate payloads(drugs, genes, proteins) and specifically enter human hepatic cells utilizing HBV-derived infection machinery, it could be used as a model of HBV infection to elucidate the early infection machinery. Recently, it was demonstrated that the N-terminal sequence of pre-S1 region(from Asn-9 to Gly-24) possesses low p H-dependent fusogenic activity, which might play a crucial role in the endosomal escape of BNC payloads and in the uncoating process of HBV. In this minireview, we describe a model in which each domain of the HBV L protein contributes to attachment onto human hepatic cells through HSPG, initiation of endocytosis, interaction with NTCP in endosomes, and consequent provocation of membrane fusion followed by endosomal escape.
基金supported by the Deutsche Forschungsgemeinschaft(DFG,425373668,HE 3220/4-1)(to GH).
文摘Evidence from genetics and from analyzing cellular and animal models have converged to suggest links between neurodegenerative disorders of early and late life.Here,we summarize emerging links between the most common late life neurodegenerative disease,Alzheimer’s disease,and the most common early life neurodegenerative diseases,neuronal ceroid lipofuscinoses.Genetic studies reported an overlap of clinically diagnosed Alzheimer’s disease and mutations in genes known to cause neuronal ceroid lipofuscinoses.Accumulating data strongly suggest dysfunction of intracellular trafficking mechanisms and the autophagy-endolysosome system in both types of neurodegenerative disorders.This suggests shared cytopathological processes underlying these different types of neurodegenerative diseases.A better understanding of the common mechanisms underlying the different diseases is important as this might lead to the identification of novel targets for therapeutic concepts,the transfer of therapeutic strategies from one disease to the other and therapeutic approaches tailored to patients with specific mutations.Here,we review dysfunctions of the endolysosomal autophagy pathway in Alzheimer’s disease and neuronal ceroid lipofuscinoses and summarize emerging etiologic and genetic overlaps.
