Human salivary histatin 1 (Hst1) exhibits a series of cell-activating properties, such as promoting cell spreading, migration, and metabolic activity. We recently have shown that fluorescently labeled Hst1 (F-Hst1) targets and activates mitochondria, presenting an important molecular mechanism. However, its regulating signaling pathways remain to be elucidated. We investigated the influence of specific inhibitors of G protein-coupled receptors (GPCR), endocytosis pathways, extracellular signal-regulated kinases 1/2 (ERK1/2) signaling, p38 signaling, mitochondrial respiration and Na+/K+-ATPase activity on the uptake, mitochondria-targeting and -activating properties of F-Hst1. We performed a siRNA knockdown (KD) to assess the effect of Sigma-2 receptor (S2R) /Transmembrane Protein 97 (TMEM97)-a recently identified target protein of Hst1. We also adopted live cell imaging to monitor the whole intracellular trafficking process of F-Hst1. Our results showed that the inhibition of cellular respiration hindered the internalization of F-Hst1. The inhibitors of GPCR, ERK1/2, phagocytosis, and clathrin-mediated endocytosis (CME) as well as siRNA KD of S2R/TMEM97 significantly reduced the uptake, which was accompanied by the nullification of the promoting effect of F-Hst1 on cell metabolic activity. Only the inhibitor of CME and KD of S2R/TMEM97 significantly compromised the mitochondria-targeting of Hst1. We further showed the intracellular trafficking and targeting process of F-Hst1, in which early endosome plays an important role. Overall, phagocytosis, CME, GPCR, ERK signaling, and S2R/TMEM97 are involved in the internalization of Hst1, while only CME and S2R/TMEM97 are critical for its subcellular targeting. The inhibition of either internalization or mitochondria-targeting of Hst1 could significantly compromise its mitochondria-activating property.
Endocytosis/physiology* ; Histatins/pharmacology* ; Humans ; Membrane Proteins ; Mitochondria/metabolism* ; RNA, Small Interfering/pharmacology* ; Receptors, G-Protein-Coupled/metabolism* ; Receptors, sigma

As a member of the Ras superfamily, Rab proteins are small GTP-binding proteins. In the process of endocytosis of macromolecules and substances delivery between organelles, Rab proteins act on vesicle formation, transport, tethering and fusion by recruiting their effectors, therefore being key regulatory factors in vesicle trafficking. Disturbance of localizations and functions of Rab proteins and their effectors are involved in the pathogenesis of several diseases. This review focuses on the main functions of Rab proteins and their possible roles in the onset and progression of neurodegenerative diseases including Parkinson's disease, Alzheimer's disease, and Huntington's disease.
Cell Movement ; Endocytosis ; Humans ; Neurodegenerative Diseases ; Protein Transport ; rab GTP-Binding Proteins/metabolism*

OBJECTIVE: To evaluate the uptake of exosomes by stem cells from apical papilla (SCAP), thus to provide experimental basis for mechanism of the exosomes endocytosis by SCAP. METHODS: (1) Exosomes of dental pulp stem cells (DPSCs) were isolated by hypercentrifugation combined with ultrafiltration method. The exosomes were identified by transmission electron microscopy, nanoparticle tracking analysis and western blot. (2) PKH-26 membrane labeling technology was used to mark the DPSCs derived exosomes. The labeled exosomes were co-cultured with SCAP at 37 °C as positive control group, and co-cultured with SCAP at 4 °C as the low-temperature treatment group, while the negative control group was set up. (3) Using clathrin-mediated endocytosis inhibitor chlorpromazine (CPZ, 10 μmol /L) as CPZ group, caveolae-mediated endocytosis Genistein (200 μmol/L) as Genistein group, and macropinocytosis inhibitor LY294002 (50 μmol/L) as LY294002 group to treat the SCAP respectively. Solvent control group (DMSO group) was set. Immunofluorescence staining was used to detect the red fluorescence SCAP and flow cytometry was used to analyze the proportion of SCAP labeled with red fluorescence. RESULTS: (1) The bilayer membrane and cup-shaped appearance of representative exosomes were observed. The peak of the size of DPSCs-derived exosomes was at 144 nm. The exosomes expressed exosomal marker proteins TSG101 and CD63, but not GAPDH which was the cellular internal control protein. (2) Immunofluorescence staining showed that after being co-cultured at 37 °C for 6 hours, red fluorescence could be detected in SCAP but it could not be detected after being co-cultured at 4 °C for 6 hours. After endocytosis inhibition, the red fluorescence in SCAP was reduced. Flow cytometry showed that the proportion of SCAP labeled with red fluorescence in positive group was 35.0%, in negative control group was 0.5%, and in solvent control group was 29.7%, in CPZ group, Genistein group and Genistein group were reduced to 13.7%, 16.6%, and 20.9%, respectively. CONCLUSION SCAP could uptake the DPSCs derived exosomes, and low temperature could inhibit this process. The exosomes uptake of SCAP was mediated by the clathrin endocytosis pathway, caveolae-mediated endocytosis and macropinocytosis pathway.
Dental Papilla ; Endocytosis ; Epithelial Cells ; Exosomes ; Stem Cells

Diabetic nephropathy (DN) is currently the most common complication of diabetes. It is considered to be one of the leading causes of end-stage renal disease (ESRD) and affects many diabetic patients. The pathogenesis of DN is extremely complex and has not yet been clarified; however, in recent years, increasing evidence has shown the important role of innate immunity in DN pathogenesis. Pattern recognition receptors (PRRs) are important components of the innate immune system and have a significant impact on the occurrence and development of DN. In this review, we classify PRRs into secretory, endocytic, and signal transduction PRRs according to the relationship between the PRRs and subcellular compartments. PRRs can recognize related pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs), thus triggering a series of inflammatory responses, promoting renal fibrosis, and finally causing renal impairment. In this review, we describe the proposed role of each type of PRRs in the development and progression of DN.
Alarmins/physiology* ; C-Reactive Protein/physiology* ; Diabetic Nephropathies/etiology* ; Endocytosis ; Humans ; Immunity, Innate ; Mannose-Binding Lectin/physiology* ; Pathogen-Associated Molecular Pattern Molecules ; Receptors, Pattern Recognition/physiology* ; Serum Amyloid P-Component/physiology* ; Signal Transduction

β-amyloid precursor protein (APP) can be cleaved by α-, and γ-secretase at plasma membrane producing soluble ectodomain fragment (sAPPα). Alternatively, following endocytosis, APP is cleaved by β-, and γ-secretase at early endosomes generating β-amyloid (Aβ), the main culprit in Alzheimer's disease (AD). Thus, APP endocytosis is critical for Aβ production. Recently, we reported that Monsonia angustifolia, the indigenous vegetables consumed in Tanzania, improved cognitive function and decreased Aβ production. In this study, we examined the underlying mechanism of justicidin A, the active compound of M. angustifolia, on Aβ production. We found that justicidin A reduced endocytosis of APP, increasing sAPPα level, while decreasing Aβ level in HeLa cells overexpressing human APP with the Swedish mutation. The effect of justicidin A on Aβ production was blocked by endocytosis inhibitors, indicating that the decreased APP endocytosis by justicidin A is the underlying mechanism. Thus, justicidin A, the active compound of M. angustifolia, may be a novel agent for AD treatment.
Alzheimer Disease ; Cell Membrane ; Cognition ; Endocytosis ; Endosomes ; HeLa Cells ; Humans ; Tanzania ; Vegetables

Cell-penetrating peptides (CPPs) are short peptides that can penetrate the cell membrane or tissue barrier. CPPs can deliver a variety of biomacromolecules, such as proteins, RNA and DNA, into cells to produce intracellular functional effects. Endocytosis and direct penetration have been suggested as the two major uptake mechanisms for CPPs-mediated cargo delivery. Compared with other non-natural chemical molecules-based delivery reagents, the CPPs have better biocompatibility, lower cytotoxicity, are easily degraded after cargo delivery, and can be fused and recombined expressed with bioactive proteins. Because of these advantages, the CPPs have become an important potential tool for delivery of developing drugs which targets intracellular factors. As a novel delivery tool, the CPPs also show promising application prospects in biomedical researches. This review summarized recent advances regarding the classification characteristics, the cellular uptake mechanisms and therapeutic application potentials of CPPs.
Biological Transport ; Cell Membrane ; Cell-Penetrating Peptides ; metabolism ; Endocytosis

Target identification is an important prerequisite for the study of medicine action mechanism. Currently,drug target identification is mostly based on various cell models in vitro. However,the growth microenvironment,nutrition metabolism,biological properties as well as functions are quite different between in vitro cell culture and physiological environment in vivo; wherefore,it is a challenging scientific issue to establish an effective method for identifying drug targets in vivo condition. In this study,we successfully prepared a kind of magnetic nanoparticles( MNPs) which can be chemically modified by the hydroxyl structure of natural bioactive compound echinacoside( ECH) via the epoxy group label on the surface of MNPs. Therefore,organ-selective and recoverable nanoscale target-recognizing particles were prepared. We then intravenously injected the ECH-binding MNPs into rats and distributed them to specific organs in vivo. After cell endocytosis,ECH-binding MNPs captured target proteins in situ for further analysis. Based on this method,we discovered several potential target proteins in the spleen lysates for ECH,and preliminarily clarified the immuno-regulation mechanism of ECH. Collectively,our strategy developed a proof-of-concept technology using nanoparticles for in vivo target identification,and also provided a feasible approach for drug target prediction and pharmacological mechanism exploration.
Animals ; Drug Delivery Systems ; Endocytosis ; Glycosides ; analysis ; Magnetics ; Magnetite Nanoparticles ; Medicine, Chinese Traditional ; Proof of Concept Study ; Rats

OBJECTIVE: To evaluate the change of cell surface CXC chemokine receptor 4 (CXCR4) expression of stem cells from apical papilla (SCAP) after the inhibition of endocytotic pathway, thus to provide experimental basis for the mechanism of SCAP migration. METHODS: The immunofluorescence analysis was conducted to examine the co-expression of CXCR4 and endocytotic compartments, including early endosomes, recycling endosomes and lysosomes in SCAP. Several Rab proteins were applied as markers of organelles in the endocytotic pathway, including Rab5 for early endosomes, Rab11A for recycling endosomes, and Lamp1 for lysosomes. The co-localization of CXCR4 with these endodontic compartments was further observed by proximity ligation assay (PLA). SCAP was treated with two kinds of endocytotic inhibitors, Blebbistatin and Dynasore, at a concentration of 80 μmol/L, respectively. The conditioning time was 1 hour. Flow cytometry was carried out to evaluate the proportion of SCAP that expressed CXCR4 on cell surface. The data were analysed by analysis of variance (ANOVA). RESULTS: The red staining of CXCR4 on immunofluorescence confocal microscopy predominantly overlapped with the green staining of Rab5 and Rab11A, and partly overlapped with Lamp1. It indicated that most CXCR4 molecules were located in early endosomes and recycling endosomes, and some were located in lysosomes. The PLA results revealed that the co-localizaiton of CXCR4 with endocytotic compartments could be observed in early endosomes, recycling endosomes and lysosomes. According to the results of flow cytometry, the proportion of SCAP that expressed CXCR4 on cell surface was as low as 0.13%±0.10%. After the inhibition of endocytosis by pretreating the cells with the following two inhibitors, Blebbistatin and Dynasore, the percentage of SCAP that positively expressed CXCR4 on cell surface was significantly increased to 13.34%±1.31% in Blebbistatin group and 4.03%±0.92% in Dynasore group (F=16.721, P<0.001). Moreover, the number of SCAP that expressed CXCR4 on cell surface in Blebbistatin group was significantly higher than that in Dynasore group (P<0.001). CONCLUSION The inhibition of endocytotic pathway could increase the number of SCAP that expressed CXCR4 on cell surface, and provide potency for the migration of SCAP.
Endocytosis ; Endosomes ; Lysosomes ; Receptors, CXCR4 ; Stem Cells

Cell Membrane ; metabolism ; Clathrin ; metabolism ; DNA-(Apurinic or Apyrimidinic Site) Lyase ; metabolism ; Endocytosis ; HeLa Cells ; Humans ; Interleukin-2 Receptor alpha Subunit ; metabolism

Cell permeable peptide (CPP) is able to transport itself or conjugated molecules such as nucleotides, peptides, and proteins into cells. Since short peptide of human immunodeficiency virus-1 Tat has been discovered as CPP, it has been continuously studied for their ability to transport heterologous cargoes into cells. In this study, we have focused on the fusion protein of respiratory syncytial virus (RSV), which has six basic amino acids in multi basic furin-dependent cleavage site (MBFCS) required to be cationic CPP. To develop more efficient CPP, the sequence, which linked two MBFCS, was synthesized (called RS-CPP). To assess cell permeable efficiency of RS-CPP or MBFCS, the peptides was conjugated with fluorescein isothiocyanate, and cell permeable efficiency was measured by fluorescence-activated cell sorting. Cell permeability of RS-CPP or MBFCS was increased in a dose-dependent manner, but RS-CPP showed more efficient cell permeability than MBFCS in MDCK, HeLa, Vero E6, and A549 cells. To evaluate whether RS-CPP can transport its conjugated functional peptide (VIVIT) in CD8+ T cell, it was confirmed that IL-2 and β-galactosidase expression were significantly inhibited through selective block of nuclear factor activated T-cell. To investigate endocytic pathways, Cre-mediated DNA recombination (loxP-STOP-loxP-LacZ reporter system) was investigated with divergent endocytosis inhibitors in TE671 cells, and RS-CPP endocytosis is occurred via binding cell surface glycosaminoglycan and clathrin-mediated endocytosis, or macropinocytosis. These results indicated that RS-CPP could be a novel cationic CPP, and it would help understanding for delivery of biologically functional molecules based on viral basic amino acids.
Amino Acids, Basic ; DNA ; Endocytosis ; Flow Cytometry ; Fluorescein ; Humans ; Interleukin-2 ; Nucleotides ; Peptides ; Permeability ; Recombination, Genetic ; Respiratory Syncytial Viruses* ; T-Lymphocytes