Early Endosome Antigen Research Articles

Human Coronavirus 229E Uses Clathrin-Mediated Endocytosis as a Route of Entry in Huh-7 Cells.

Human coronavirus 229E (HCoV-229E) is an endemic coronavirus responsible for approximately one-third of "common cold" cases. To infect target cells, HCoV-229E first binds to its receptor on the cell surface and then can follow different pathways, entering by direct fusion or by taking advantage of host cell mechanisms such as endocytosis. Based on the role of clathrin, the process can be classified into clathrin-dependent or -independent endocytosis. This study characterizes the role of clathrin-mediated endocytosis (CME) in HCoV-229E infection of the human hepatoma cell line Huh-7. Using specific CME inhibitory drugs, we demonstrated that blocking CME significantly reduces HCoV-229E infection. Additionally, CRISPR/Cas9-mediated knockout of the µ subunit of adaptor protein complex 2 (AP-2) further corroborated the role of CME, as KOs showed over a 50% reduction in viral infection. AP-2 plays an important role in clathrin recruitment and the maturation of clathrin-coated vesicles. Our study also confirmed that in Huh-7 cells, HCoV-229E requires endosomal acidification for successful entry, as viral entry decreased when treated with lysomotropic agents. Furthermore, the colocalization of HCoV-229E with early endosome antigen 1 (EEA-1), only present in early endosomes, suggested that the virus uses an endosomal route for entry. These findings highlight, for the first time, the role of CME in HCoV-229E infection and confirm previous data of the use of the endosomal route at a low pH in the experimental cell model Huh-7. Our results provide new insights into the mechanisms of entry of HCoV-229E and provide a new basis for the development of targeted antiviral therapies.

Xiaowugui decoction alleviates experimental rheumatoid arthritis by suppressing Rab5a-mediated TLR4 internalization in macrophages

BackgroundRheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by exacerbated synovial inflammation and joint destruction. Recent studies suggest toll-like receptor 4 (TLR4) internalization facilitate inflammatory response of macrophage. The role of TLR4 internalization in the pathogenesis of RA is unknown. PurposeTo investigate the role and mechanism of TLR4 internalization in macrophage inflammatory response of RA and explore whether TLR4 internalization mediates the anti-arthritic effect of Xiaowugui (XWG) decoction, a patented herbal formula used in China. MethodsThe co-expression of TLR4 and the internalization marker, early endosome antigen 1 (EEA1), in the synovial samples of RA patients and joint tissue of collagen-induced arthritis (CIA) mice, were evaluated using immunofluorescence. The effect of Rab5a-mediated early internalization of TLR4 on the activation induced by lipopolysaccharide (LPS) in RAW264.7 cells was investigated using small interfering RNAs that act against Rab5a. CIA was induced in Rab5a−/− mice to evaluate the role of Rab5a in vivo. The disease progression and expression of Rab5a and TLR4 in the joint tissue were evaluated in CIA mice treated with XWG. Inflammatory factors production, TLR4 internalization, and activation of downstream signaling pathways were examined in RAW264.7 cells treated with XWG in vitro. ResultsThe co-expression and co-localization of TLR4 and EEA1 were elevated in the synovial samples of RA patients and joint tissue of CIA mice. Pharmaceutical inhibition of TLR4 internalization reduced macrophages inflammatory responses induced by LPS. The co-expression and co-localization of Rab5a and TLR4 were significantly increased in macrophages treated with LPS. Silencing Rab5a reduced LPS-induced TLR4 internalization, inflammatory factors production, and phosphorylation of Jun N-terminal kinases (JNK) and p65. Genetic deletion of Rab5a inhibited TLR4 internalization and the development of arthritis in vivo. The co-expression of TLR4 and Rab5a was also elevated in the synovial samples of RA patients. XWG treatment of mice with CIA alleviated arthritis and reduced the co-expression of Rab5a and TLR4 in the joint tissue. XWG treatment of macrophage inhibited LPS-induced IL-6 and TNF-α production, co-expression of Rab5a and TLR4, and phosphorylation of JNK and p65. ConclusionsOur findings highlight the pathogenic role of TLR4 internalization in patients with RA and identify a novel Rab5a-dependent internalization pathway that promotes macrophage inflammatory response. XWG treatment demonstrated outstanding therapeutic effects in experimental arthritis, and targeting the Rab5a-mediated internalization of TLR4 may be the main underlying mechanism.

Septin-coated microtubules promote maturation of multivesicular bodies by inhibiting their motility.

The microtubule cytoskeleton consists of microtubule subsets with distinct compositions of microtubule-associated proteins, which instruct the position and traffic of subcellular organelles. In the endocytic pathway, these microtubule-associated cues are poorly understood. Here, we report that in MDCK cells, endosomes with multivesicular body (MVB) and late endosome (LE) markers localize preferentially to microtubules coated with septin GTPases. Compared with early endosomes, CD63-containing MVBs/LEs are largely immotile on septin-coated microtubules. In vitro reconstitution assays revealed that the motility of isolated GFP-CD63 endosomes is directly inhibited by microtubule-associated septins. Quantification of CD63-positive endosomes containing the early endosome antigen (EEA1), the Rab7 effector and dynein adaptor RILP or Rab27a, showed that intermediary EEA1- and RILP-positive GFP-CD63 preferentially associate with septin-coated microtubules. Septin knockdown enhanced GFP-CD63 motility and decreased the percentage of CD63-positive MVBs/LEs with lysobiphosphatidic acid without impacting the fraction of EEA1-positive CD63. These results suggest that MVB maturation involves immobilization on septin-coated microtubules, which may facilitate multivesiculation and/or organelle-organelle contacts.

The role of Phafin proteins in cell signaling pathways and diseases.

Membrane-associated proteins are important membrane readers that mediate and facilitate the signaling and trafficking pathways in eukaryotic membrane-bound compartments. The protein members in the Phafin family are membrane readers containing two phosphoinositide recognition domains: the Pleckstrin Homology domain and the FYVE (Fab1, YOTB, Vac1, and early endosome antigen 1) domain. Phafin proteins, categorized into two subfamilies, Phafin1 and Phafin2, associate with cellular membranes through interactions involving membrane-embedded phosphoinositides and phosphoinositide-binding domains. These membrane-associated Phafin proteins play pivotal roles by recruiting binding partners and forming complexes, which contribute significantly to apoptotic, autophagic, and macropinocytotic pathways. Elevated expression levels of Phafin1 and Phafin2 are observed in various cancers. A recent study highlights a significant increase in Phafin1 protein levels in the lungs of idiopathic pulmonary fibrosis patients compared to normal subjects, suggesting a crucial role for Phafin1 in the pathogenesis of pulmonary fibrosis. Additionally, phosphatidylinositol-3-phosphate-binding 2 (Pib2), a close relative of the Phafin1 protein, functions as an amino acid sensor activating the TOCR1 pathway in yeasts. This review focuses on delineating the involvement of Phafin proteins in cellular signaling and their implications in diseases and briefly discusses the latest research findings concerning Pib2.

Actin cytoskeleton remodeling disrupts physical barriers to infection and presents entry receptors to respiratory syncytial virus.

RSV is the leading cause of infant hospitalizations and a significant cause of paediatric and geriatric morbidity worldwide. Recently, we reported that insulin-like growth factor 1 receptor (IGF1R) was a receptor for respiratory syncytial virus (RSV) in airway epithelial cells and that activation of IGF1R recruited the coreceptor, nucleolin (NCL), to the cell surface. Cilia and mucus that line the airways pose a significant barrier to viral and bacterial infection. The cortical actin cytoskeleton has been shown by others to mediate RSV entry, so we studied the roles of the RSV receptors and actin remodelling during virus entry. We found that IGF1R expression and phosphorylation were associated with the ability of RSV to infect cells. Confocal immunofluorescence imaging showed that actin projections, a hallmark of macropinocytosis, formed around viral particles 30 min after infection. Consistent with prior reports we also found that virus particles were internalized into early endosome antigen-1 positive endosomes within 90 min. Inhibiting actin polymerization significantly reduced viral titre by approximately ten-fold. Inhibiting PI3 kinase and PKCζ in stratified air-liquid interface (ALI) models of the airway epithelium had similar effects on reducing the actin remodelling observed during infection and attenuating viral entry. Actin projections were associated with NCL interacting with RSV particles resting on apical cilia of the ALIs. We conclude that macropinocytosis-like actin projections protrude through normally protective cilia and mucus layers of stratified airway epithelium that helps present the IGF1R receptor and the NCL coreceptor to RSV particles waiting at the surface.

Adenosine A2A Receptor Activation Regulates Niemann-Pick C1 Expression and Localization in Macrophages.

Adenosine plays an important role in modulating immune cell function, particularly T cells and myeloid cells, such as macrophages and dendritic cells. Cell surface adenosine A2A receptors (A2AR) regulate the production of pro-inflammatory cytokines and chemokines, as well as the proliferation, differentiation, and migration of immune cells. In the present study, we expanded the A2AR interactome and provided evidence for the interaction between the receptor and the Niemann-Pick type C intracellular cholesterol transporter 1 (NPC1) protein. The NPC1 protein was identified to interact with the C-terminal tail of A2AR in RAW 264.7 and IPMФ cells by two independent and parallel proteomic approaches. The interaction between the NPC1 protein and the full-length A2AR was further validated in HEK-293 cells that permanently express the receptor and RAW264.7 cells that endogenously express A2AR. A2AR activation reduces the expression of NPC1 mRNA and protein density in LPS-activated mouse IPMФ cells. Additionally, stimulation of A2AR negatively regulates the cell surface expression of NPC1 in LPS-stimulated macrophages. Furthermore, stimulation of A2AR also altered the density of lysosome-associated membrane protein 2 (LAMP2) and early endosome antigen 1 (EEA1), two endosomal markers associated with the NPC1 protein. Collectively, these results suggested a putative A2AR-mediated regulation of NPC1 protein function in macrophages, potentially relevant for the Niemann-Pick type C disease when mutations in NPC1 protein result in the accumulation of cholesterol and other lipids in lysosomes.

SORL1 stabilizes ABCB1 to promote cisplatin resistance in ovarian cancer.

Ovarian cancer (OC) has the worst prognosis among gynecological malignancies. Cisplatin (CDDP) is one of the most commonly used treatments for OC, but recurrence and metastasis are common due to endogenous or acquired resistance. High expression of ATP-binding cassette (ABC) transporters is an important mechanism of resistance to OC chemotherapy, but targeting ABC transporters in OC therapy remains a challenge. The expression of sortilin-related receptor 1 (SORL1; SorLA) in the response of OC to CDDP was determined by analysis of TCGA and GEO public datasets. Immunohistochemistry and western blotting were utilized to evaluate the expression levels of SORL1 in OC tissues and cells that were sensitive or resistant to CDDP treatment. The in vitro effect of SORL1 on OC cisplatin resistance was proven by CCK-8 and cell apoptosis assays. The subcutaneous xenotransplantation model verified the in vivo significance of SORL1 in OC. Finally, the molecular mechanism by which SORL1 regulates OC cisplatin resistance was revealed by coimmunoprecipitation, gene set enrichment analysis and immunofluorescence analysis. This study demonstrated that SORL1 is closely related to CDDP resistance and predicts a poor prognosis in OC. In vivo xenograft experiments showed that SORL1 knockdown significantly enhanced the effect of CDDP on CDDP-resistant OC cells. Mechanistically, silencing of SORL1 inhibits the early endosomal antigen 1 (EEA1) pathway, which impedes the stability of ATP-binding cassette B subfamily member 1 (ABCB1), sensitizing CDDP-resistant OC cells to CDDP. The findings of this study suggest that targeting SORL1 may represent a promising therapeutic approach for overcoming CDDP resistance in OC.

Fluorescent visualization and evaluation of NPC1L1-mediated vesicular endocytosis during intestinal cholesterol absorption in mice

Abstract Excessive cholesterol absorption from intestinal lumen contributes to the pathogenesis of hypercholesterolemia, which is an independent risk factor for atherosclerotic cardiovascular disease. Niemann-Pick C1-like 1 (NPC1L1) is a major membrane protein responsible for cholesterol absorption, in which the physiological role of vesicular endocytosis is still controversial, and it lacks a feasible tool to visualize and evaluate the endocytosis of NPC1L1 vesicles in vivo. Here, we genetically labeled endogenous NPC1L1 protein with EGFP in a knock-in mouse model, and demonstrated fluorescent visualization and evaluation of the endocytic vesicles of NPC1L1-cago during intestinal cholesterol absorption. The homozygous NPC1L1-EGFP mice have normal NPC1L1 expression pattern as well as cholesterol homeostasis on chow or high-cholesterol diets. The fluorescence of NPC1L1-EGFP fusion protein localizes at the brush border membrane of small intestine, and EGFP-positive vesicles is visualized beneath the membrane as early as 5 min post oral gavage of cholesterol. Of note, the vesicles colocalize with the early endosomal marker early endosome antigen 1 (EEA1) and the filipin-stained free cholesterol. Pretreatment with NPC1L1 inhibitor ezetimibe inhibits the formation of these cholesterol-induced endocytic vesicles. Our data support the notion that NPC1L1-mediated cholesterol absorption is a vesicular endocytic process. NPC1L1-EGFP mice are a useful model for visualizing cellular NPC1L1-cargo vesicle itineraries and for evaluating NPC1L1 activity in vivo in response to diverse pharmacological agents and nutrients.

Intestinal Barrier in Post-Campylobacter jejuni Irritable Bowel Syndrome.

Campylobacter jejuni (C. jejuni) is one of the most common causes of bacterial gastroenteritis worldwide. One sequela of this infection is the development of post-infectious irritable bowel syndrome (PI-IBS). It has been suggested that a dysfunctional intestinal barrier may promote IBS development. We aimed to test this hypothesis against the background of the leaky gut concept for low-grade inflammation in PI-IBS. We identified patients with persistent PI-IBS symptoms after C. jejuni infection. During sigmoidoscopy, forceps biopsies were obtained for electrophysiological measurements of epithelial transport and barrier function in miniaturized Ussing devices. C. jejuni absence was checked by PCR and cytokine production with immunohistochemistry. In PI-IBS, the epithelial resistance of the colon epithelium was unaltered, reflecting an intact paracellular pathway. In contrast, temperature-dependent horseradish peroxidase (HRP, 44 kDa) permeation increased. Short-circuit current (Isc) reflecting active anion secretion and ENaC-dependent electrogenic sodium absorption was unaffected. Early endosome antigen-1 (EEA1) and IL-4 levels increased. C. jejuni is not incorporated into the resident microbiota of the colon mucosa in PI-IBS. In PI-IBS after C. jejuni infection, macromolecule uptake via endocytosis was enhanced, leading to low-grade inflammation with pro-inflammatory cytokine release. The findings will allow C. jejuni-induced pathomechanisms to be targeted during infection and, thereafter to reduce sequelae such as PI-IBS.

Modifications of the endosomal compartment in fibroblasts from sporadic Alzheimer’s disease patients are associated with cognitive impairment

Morphological alterations of the endosomal compartment have been widely described in post-mortem brains from Alzheimer’s disease (AD) patients and subjects with Down syndrome (DS) who are at high risk for AD. Immunostaining with antibodies against endosomal markers such as Early Endosome Antigen 1 (EEA1) revealed increased size of EEA1-positive puncta. In DS, peripheral cells such as peripheral blood mononuclear cells (PBMCs) and fibroblasts, share similar phenotype even in the absence of AD. We previously found that PBMCs from AD patients have larger EEA1-positive puncta, correlating with brain amyloid load. Here we analysed the endosomal compartment of fibroblasts from a very well characterised cohort of AD patients (IMABio3) who underwent thorough clinical, imaging and biomarkers assessments. Twenty-one subjects were included (7 AD with mild cognitive impairment (AD-MCI), 7 AD with dementia (AD-D) and 7 controls) who had amyloid-PET at baseline (PiB) and neuropsychological tests at baseline and close to skin biopsy. Fibroblasts isolated from skin biopsies were immunostained with anti-EEA1 antibody and imaged using a spinning disk microscope. Endosomal compartment ultrastructure was also analysed by electron microscopy. All fibroblast lines were genotyped and their AD risk factors identified. Our results show a trend to an increased EEA1-positive puncta volume in fibroblasts from AD-D as compared to controls (p.adj = 0.12) and reveal enhanced endosome area in fibroblasts from AD-MCI and AD-AD versus controls. Larger puncta size correlated with PiB retention in different brain areas and with worse cognitive scores at the time of biopsy as well as faster decline from baseline to the time of biopsy. Finally, we identified three genetic risk factors for AD (ABCA1, COX7C and MYO15A) that were associated with larger EEA1 puncta volume. In conclusion, the endosomal compartment in fibroblasts could be used as cellular peripheral biomarker for both amyloid deposition and cognitive decline in AD patients.

Airway Smooth Muscle Cell Mitochondria Damage and Mitophagy in COPD via ERK1/2 MAPK.

Chronic obstructive pulmonary disease (COPD) is characterized by irreversible deterioration of the airway wall. Cigarette smoking is the major trigger, and in vitro studies showed that cigarette smoke extract (CSE) induced mitophagy in airway epithelial cells via oxidative stress, but this mechanism was not studied in airway smooth muscle cells (ASMCs). Primary ASMCs isolated from COPD patients or non-disease donors were investigated for CSE-induced remodeling and mitochondria structure. Proteins were assessed by Western blots for remodeling: collagen type-I, α-smooth muscle actin (α-SMA) and fibronectin; autophagy: beclin-1, protein62 (p62), light chain (LC)3A/B; mitochondria activity: mitochondrially encoded cytochrome c oxidase II & -IV (MTCO2, MTCO4), peroxisome proliferator activated receptor gamma coactivator 1α (PGC-1α); lysosomes: early endosome antigen 1, lysosome activated membrane protein 1; and cell signaling: extracellular signal regulated kinase (ERK1/2). Lysotracker and Mitotracker were used to monitor mitochondria morphology and organelle co-localization. Compared with controls, untreated COPD ASMCs showed lower collagen type-I and α-SMA expressions, but increased fibronectin levels. CSE further downregulated collagen type-I and α-SMA expression, but upregulated fibronectin. CSE decreased PGC-1α, MTCO2, and MTCO4, but increased beclin-1, p62, and LC3. CSE upregulated mitophagy and lysosomes activity via ERK1/2 phosphorylation. In vitro, cigarette smoke induced the deterioration of ASMCs, which might explain the tissue loss and structural remodeling in COPD bronchi. The results suggest that preventing exceeded mitophagy in ASMCs might present a novel therapeutic target for COPD.

AKAP12 Modulates β‐arrestin2 Recruitment to the β2‐Adrenergic Receptors

BackgroundG‐Protein Coupled Receptors (GPCRs) are central mediators of signaling cascades in many biological systems. GPCRs internalization rates vary based on the presence/absence of its ligand and the type of the receptor. β2‐Adrenergic Receptor (β2AR) is a prototypical GPCR, reaching its maximum in internalization within ~10 minutes after ligand treatment. One regulator of β2AR internalization is β‐arrestin2. GPCR–β‐arrestin binding is biphasic where the phosphorylated carboxyl terminus of GPCRs docks to the N‐domain of β‐arrestin first, and then the seven transmembrane core of the receptor engages with β‐arrestin. However, it has been discovered that the core interaction is dispensable for receptor endocytosis. AKAP12 is a large scaffolding protein that was reported to be essential in agonist‐induced internalization and resensitization of β2AR. Our central hypothesis is that higher expression levels of AKAP12 may enhance β‐arrestin2 recruitment to the β2AR and subsequently increase β2AR internalization.MethodsHEK293 cells were grouped into three groups: [1] Controls: endogenous AKAP12 expression, [2] Overexpression (OX): transiently overexpressing human AKAP12, and [3] Knockdown (KD): treated with human AKAP12 siRNA. β‐arrestin2 recruitment to the receptor and β2AR internalization was evaluated using the Nanobit luciferase system. For each assay, we tested four β2AR‐β‐arrestin2 or Early Endosome‐β2AR orientations. Orientation pairs with the highest luminescence signals were used for the experiments to obtain maximum sensitivity. At 48 hours post‐transfection, Nanobit assays were performed by recording baseline luminescence for 10 minutes. Next, 10µM epinephrine was added and luminescence measurements were recorded immediately and then once every minute for 1 hour. Results are represented as Luminescence Fold Induction (LFI). Receptor trafficking to early endosomes post epinephrine treatment was further confirmed using the Proximity Ligation Assay (PLA).ResultsHEK cells, OX AKAP12 [9 fold higher] had a significantly lower β‐arrestin2 recruitment to the β2AR (2.26±0.39, LFI) as compared to controls (7.47±1.12, LFI; p=0.0004). AKAP12 KD [AKAP12 ~80% silenced] had a significantly higher β‐arrestin2 recruitment to the β2AR (10.35±0.56, LFI) as compared to both control and OX AKAP12 groups (p=0.035 and p<0.0001), respectively. At 15 minutes post epinephrine addition, β2AR trafficking to early endosomes was significantly lower in the OX AKAP12 group (1.82±0.22, LFI), as compared to the control (2.47±0.09, LFI; p=0.01), while the KD AKAP12 group had a significantly higher internalization (2.98±0.14, LFI), as compared to control and OX AKAP12 groups (p=0.005 and p=0.0003), respectively. Our PLA data further confirmed that the OX AKAP12 group displayed significantly lower β2AR trafficking as defined by its decreased interaction with Early Endosome Antigen 1 (EEA1), as compared to the control (p= 0.0451).ConclusionsContrary to previously thought, higher AKAP12 expression levels may interfere with β2AR trafficking to early endosomes partially through inhibiting β‐arrestin2 recruitment to the β2AR.

Crinum latifolium extract inhibits lipopolysaccharide-induced inflammation in human macrophages

Abstract Introduction: Crinum latifolium is a natural plant showing the anti-inflammatory effects. We aimed to evaluate the effects of Crinum latifolium extract on proinflammatory cytokine production and maturation of early and late endosomes in the lipopolysaccharide (LPS)-induced human macrophages at light microscopic and biochemical levels. Material and Methods: The effects of Crinum latifolium extract in human macrophages stimulated with LPS was evaluated to optic densities of the lysosome-associated membrane protein-1 (LAMP-1) and early endo-some antigen 1 (EEA1) by immunohistochemistry staining and to level of the proinflammatory cytokine interleukin (IL)-6 by enzyme-linked immunosorbent assay (ELISA). Results: Crinum latifolium group exhibited a significant decreased in the levels of IL-6 in the supernatant as compared to the LPS group for 2 and 72 hours (p<0.0001). Crinum latifolium group exhibited a significant increased in the optic density of the EEA1 and LAMP-1 in coverslips as compared to the control and lipopolysaccharide groups after 2 and 72 hours (p<0.0001). Conclusions: Crinum latifolium may a therapeutic plant in the inflammatory diseases, such as sepsis, through anti-inflammatory effects, such as decrease in production of the proinflammatory cytokine and increase in maturations of the early and late endosomes in macrophages.

Identification of Alternative Splicing Events Associated with Paratuberculosis in Dairy Cattle Using Multi-Tissue RNA Sequencing Data.

Paratuberculosis is a major endemic disease caused by Mycobacterium avium subspecies paratuberculosis (MAP) infection and leads to huge economic loss in the dairy sector worldwide. Alternative splicing (AS) events, playing indispensable regulatory roles in many protein functions and biological pathways, are shown to be associated with complex traits and diseases. In this study, by integrating the RNA sequencing (RNA-seq) data of 24 samples from three tissues (peripheral blood, jejunum and salivary gland) of Holstein cows, we obtained 2,706,541,696 uniquely mapped reads in total that represented 12,870 expressed genes, and detected 4285 differentially expressed genes (DEGs) between MAP-infected and healthy cows (p < 0.05). Of them, 92 differentially expressed splicing factors (DESFs) were included. Further, 119, 150 and 68 differential alternative splicing (DAS) events between MAP-infected and healthy cows were identified in peripheral blood, jejunum and salivary glands, respectively. Of note, six DAS events were highly and significantly correlated with the DESFs (R2 > 0.9; p < 0.01), and their corresponding genes (COPI coat complex subunit gamma 2 gene (COPG2), kinesin family member 2C gene (KIF2C), exocyst complex component 7 (EXOC7), Rab9 effector protein with kelch motifs gene (RABEPK), deoxyribonuclease 1 gene (DNASE1) and early endosome antigen 1 gene (EEA1)) were significantly enriched in immune response such as vesicle-mediated transport, regulation of acute inflammatory response and tuberculosis through gene ontology (GO) and KEGG analysis. KS test showed that the DAS events in the EXOC7 and KIF2C genes indeed displayed differences between MAP-infected cows and healthy cows. The DAS in EXOC7 might produce a new protein sequence with lack of 23 amino acids, and the DAS in KIF2C induced a stop codon of premature occurrence and resulted in a lack of functional domain. In summary, this study identified the DAS events and corresponding genes related to MAP-infection base on the RNA-seq data from multiple tissues of Holstein cows, providing novel insights into the regulatory mechanisms underpinning paratuberculosis in dairy cattle.

Cadmium induces endosomal/lysosomal enlargement and blocks autophagy flux in rat hepatocytes by damaging microtubules

Acute exposure to cadmium (Cd) causes vacuolar degeneration in buffalo rat liver 3 A (BRL 3 A) cells. The present study aimed to determine the relationship between Cd-induced microtubule damage and intracellular vacuolar degeneration. Western blotting results showed that Cd damaged the microtubule network and downregulated the expression of microtubule-associated proteins—kinesin-1 heavy chain (KIF5B), γ-tubulin, and acetylated α-tubulin in BRL 3 A cells. Immunofluorescence staining revealed that Cd inhibited interactions between α-tubulin and microtubule-associated protein 4 (MAP4) as well as KIF5B. Increasing Cd concentrations decreased the levels of the lipid kinase, PIKfyve, which regulates the activity of endosome-lysosome fission. Immunofluorescence and transmission electron microscopy revealed vacuole-like organelles that were late endosomes and lysosomes. The PIKfyve inhibitor, YM201636, and the microtubule depolymerizer, nocodazole, aggravated Cd-induced endosome-lysosome enlargement. Knocking down the kif5b gene that encodes KIF5B intensified the enlargement of endosome-lysosomes and expression of early endosome antigen 1 (EEA1), Ras-related protein Rab-7a (RAB7), and lysosome-associated membrane glycoprotein 2 (LAMP2). Nocodazole, YM201636, and the knockdown of kif5b blocked autophagic flux. We concluded that Cd-induced damage to the microtubule network is the main reason for endosome-lysosome enlargement and autophagic flux blockage in BRL 3 A cells, and kinesin-1 plays a critical role in this process.

Thermal fluctuations assist mechanical signal propagation in coiled-coil proteins.

Recently, it has been shown that the long coiled-coil membrane tether protein early endosome antigen 1 (EEA1) switches from a rigid to a flexible conformation upon binding of a signaling protein to its free end. This flexibility switch represents a motorlike activity, allowing EEA1 to generate a force that moves vesicles closer to the membrane they will fuse with. It was hypothesized that the binding-induced signal could propagate along the coiled coil and lead to conformational changes through the localized domains of the protein chain that deviate from a perfect coiled-coil structure. To elucidate, if upon binding of a single protein the corresponding mechanical signal could propagate through the whole 200-nm-long chain, we propose a simplified description of the coiled coil as a one-dimensional Frenkel-Kontorova chain. Using numerical simulations, we find that an initial perturbation of the chain can propagate along its whole length in the presence of thermal fluctuations. This may enable the change of the configuration of the entire molecule and thereby affect its stiffness. Our work sheds light on intramolecular communication and force generation in long coiled-coil proteins.

Inhibition of Sphingosine-1-Phosphate Receptor 2 by JTE013 Promoted Osteogenesis by Increasing Vesicle Trafficking, Wnt/Ca2+, and BMP/Smad Signaling.

Sphingosine-1-phosphate receptor 2 (S1PR2) is a G protein-coupled receptor that regulates various immune responses. Herein, we determine the effects of a S1PR2 antagonist (JTE013) or a S1PR2 shRNA on osteogenesis by culturing murine bone marrow stromal cells (BMSCs) in osteogenic media with JTE013, dimethylsulfoxide (DMSO), a S1PR2 shRNA, or a control shRNA. Treatment with JTE013 or the S1PR2 shRNA increased alkaline phosphatase and alizarin red s staining, and enhanced alkaline phosphatase, RUNX2, osteocalcin, and osterix mRNA levels in BMSCs compared with the controls. Protein analysis revealed that a high dose of JTE013 (4 or 8 μM) increased vesicle trafficking-associated proteins (F-actin, clathrin, Early Endosome Antigen 1 (EEA1), and syntaxin 6) and Wnt/Ca2+ signaling. On the other hand, a low dose of JTE013 (1 to 2 μM) increased BMP/Smad signaling. In contrast, the S1PR2 shRNA reduced vesicle trafficking-associated proteins and attenuated Wnts and BMP/Smad signaling, but enhanced p-CaMKII compared with the control, suggesting that the S1PR2 shRNA influenced osteogenesis via different signaling pathways. Moreover, inhibiting protein trafficking by brefeldin A in BMSCs suppressed Wnts and BMPRs expressions. These data supported that enhanced osteogenesis in JTE013-treated BMSCs is associated with increased vesicle trafficking, which promotes the synthesis and transport of osteogenic protein and matrix vesicles and enhances matrix mineralization.

SdhA blocks disruption of the Legionella-containing vacuole by hijacking the OCRL phosphatase.

SUMMARYLegionella pneumophila grows intracellularly within a replication vacuole via action of Icm/Dot-secreted proteins. One such protein, SdhA, maintains the integrity of the vacuolar membrane, thereby preventing cytoplasmic degradation of bacteria. We show here that SdhA binds and blocks the action of OCRL (OculoCerebroRenal syndrome of Lowe), an inositol 5-phosphatase pivotal for controlling endosomal dynamics. OCRL depletion results in enhanced vacuole integrity and intracellular growth of a sdhA mutant, consistent with OCRL participating in vacuole disruption. Overexpressed SdhA alters OCRL function, enlarging endosomes, driving endosomal accumulation of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), and interfering with endosomal trafficking. SdhA interrupts Rab guanosine triphosphatase (GTPase)-OCRL interactions by binding to the OCRL ASPM-SPD2-Hydin (ASH) domain, without directly altering OCRL 5-phosphatase activity. The Legionella vacuole encompassing the sdhA mutant accumulates OCRL and endosomal antigen EEA1 (Early Endosome Antigen 1), consistent with SdhA blocking accumulation of OCRL-containing endosomal vesicles. Therefore, SdhA hijacking of OCRL is associated with blocking trafficking events that disrupt the pathogen vacuole.

Fibroblast growth factor 23 leads to endolysosomal routing of the renal phosphate cotransporters NaPi-IIa and NaPi-IIc in vivo.

Na+-dependent phosphate cotransporters NaPi-IIa and NaPi-IIc, located at the brush-border membrane of renal proximal tubules, are regulated by numerous factors, including fibroblast growth factor 23 (FGF23). FGF23 downregulates NaPi-IIa and NaPi-IIc abundance after activating a signaling pathway involving phosphorylation of ERK1/2 (phospho-ERK1/2). FGF23 also downregulates expression of renal 1-α-hydroxylase (Cyp27b1) and upregulates 24-hydroxylase (Cyp24a1), thus reducing plasma calcitriol levels. Here, we examined the time course of FGF23-induced internalization of NaPi-IIa and NaPi-IIc and their intracellular pathway toward degradation in vivo. Mice were injected intraperitoneally with recombinant human (rh)FGF23 in the absence (biochemical analysis) or presence (immunohistochemistry) of leupeptin, an inhibitor of lysosomal proteases. Phosphorylation of ERK1/2 was enhanced 60 min after rhFGF23 administration, and increased phosphorylation was still detected 480 min after injection. Colocalization of phospho-ERK1/2 with NaPi-IIa was seen at 60 and 120 min and partly at 480 min. The abundance of both cotransporters was reduced 240 min after rhFGF23 administration, with a further reduction at 480 min. NaPi-IIa and NaPi-IIc were found to colocalize with clathrin and early endosomal antigen 1 as early as 120 min after rhFGF23 injection. Both cotransporters partially colocalized with cathepsin B and lysosomal-associated membrane protein-1, markers of lysosomes, 120 min after rhFGF23 injection. Thus, NaPi-IIa and NaPi-IIc are internalized within 2 h upon rhFGF23 injection. Both cotransporters share the pathway of clathrin-mediated endocytosis that leads first to early endosomes, finally resulting in trafficking toward the lysosome as early as 120 min after rhFGF23 administration.NEW & NOTEWORTHY The hormone fibroblast growth factor 23 (FGF23) controls phosphate homeostasis by regulating renal phosphate excretion. FGF23 acts on several phosphate transporters in the kidney. Here, we define the time course of this action and demonstrate how phosphate transporters NaPi-IIa and NaPi-IIc are internalized.

Membrane-binding mechanism of the EEA1 FYVE domain revealed by multi-scale molecular dynamics simulations.

Early Endosomal Antigen 1 (EEA1) is a key protein in endosomal trafficking and is implicated in both autoimmune and neurological diseases. The C-terminal FYVE domain of EEA1 binds endosomal membranes, which contain phosphatidylinositol-3-phosphate (PI(3)P). Although it is known that FYVE binds PI(3)P specifically, it has not previously been described of how FYVE attaches and binds to endosomal membranes. In this study, we employed both coarse-grained (CG) and atomistic (AT) molecular dynamics (MD) simulations to determine how FYVE binds to PI(3)P-containing membranes. CG-MD showed that the dominant membrane binding mode resembles the crystal structure of EEA1 FYVE domain in complex with inositol-1,3-diphospate (PDB ID 1JOC). FYVE, which is a homodimer, binds the membrane via a hinge mechanism, where the C-terminus of one monomer first attaches to the membrane, followed by the C-terminus of the other monomer. The estimated total binding energy is ~70 kJ/mol, of which 50-60 kJ/mol stems from specific PI(3)P-interactions. By AT-MD, we could partition the binding mode into two types: (i) adhesion by electrostatic FYVE-PI(3)P interaction, and (ii) insertion of amphipathic loops. The AT simulations also demonstrated flexibility within the FYVE homodimer between the C-terminal heads and coiled-coil stem. This leads to a dynamic model whereby the 200 nm long coiled coil attached to the FYVE domain dimer can amplify local hinge-bending motions such that the Rab5-binding domain at the other end of the coiled coil can explore an area of 0.1 μm2 in the search for a second endosome with which to interact.