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Viruses Oct 2014Virus entry is a complex process characterized by a sequence of events. Since the discovery of KSHV in 1994, tremendous progress has been made in our understanding of... (Review)
Review
Virus entry is a complex process characterized by a sequence of events. Since the discovery of KSHV in 1994, tremendous progress has been made in our understanding of KSHV entry into its in vitro target cells. KSHV entry is a complex multistep process involving viral envelope glycoproteins and several cell surface molecules that is utilized by KSHV for its attachment and entry. KSHV has a broad cell tropism and the attachment and receptor engagement on target cells have an important role in determining the cell type-specific mode of entry. KSHV utilizes heparan sulfate, integrins and EphrinA2 molecules as receptors which results in the activation of host cell pre-existing signal pathways that facilitate the subsequent cascade of events resulting in the rapid entry of virus particles, trafficking towards the nucleus followed by viral and host gene expression. KSHV enters human fibroblast cells by dynamin dependant clathrin mediated endocytosis and by dynamin independent macropinocytosis in dermal endothelial cells. Once internalized into endosomes, fusion of the viral envelope with the endosomal membranes in an acidification dependent manner results in the release of capsids which subsequently reaches the nuclear pore vicinity leading to the delivery of viral DNA into the nucleus. In this review, we discuss the principal mechanisms that enable KSHV to interact with the host cell surface receptors as well as the mechanisms that are required to modulate cell signaling machinery for a successful entry.
Topics: Cell Nucleus; DNA, Viral; Endocytosis; Endosomes; Endothelial Cells; Fibroblasts; Gene Expression Regulation, Viral; Herpesvirus 8, Human; Host-Pathogen Interactions; Humans; Pinocytosis; Receptors, Cell Surface; Signal Transduction; Virus Internalization
PubMed: 25341665
DOI: 10.3390/v6104024 -
Journal of Extracellular Vesicles Apr 2024Besides participating in diverse pathological and physiological processes, extracellular vesicles (EVs) are also excellent drug-delivery vehicles. However, clinical...
Besides participating in diverse pathological and physiological processes, extracellular vesicles (EVs) are also excellent drug-delivery vehicles. However, clinical drugs modulating EV levels are still lacking. Here, we show that proton pump inhibitors (PPIs) reduce EVs by enhancing macropinocytosis-mediated EV uptake. PPIs accelerate intestinal cell endocytosis of autocrine immunosuppressive EVs through macropinocytosis, thereby aggravating inflammatory bowel disease. PPI-induced macropinocytosis facilitates the clearance of immunosuppressive EVs from tumour cells, improving antitumor immunity. PPI-induced macropinocytosis also increases doxorubicin and antisense oligonucleotides of microRNA-155 delivery efficiency by EVs, leading to enhanced therapeutic effects of drug-loaded EVs on tumours and acute liver failure. Mechanistically, PPIs reduce cytosolic pH, promote ATP6V1A (v-ATPase subunit) disassembly from the vacuolar membrane and enhance the assembly of plasma membrane v-ATPases, thereby inducing macropinocytosis. Altogether, our results reveal a mechanism for macropinocytic regulation and PPIs as potential modulators of EV levels, thus regulating their functions.
Topics: Proton Pump Inhibitors; Extracellular Vesicles; Endocytosis; Pinocytosis; Adenosine Triphosphatases
PubMed: 38532609
DOI: 10.1002/jev2.12426 -
Toxins Feb 2018Plant Ribosome-inactivating proteins (RIPs) including the type I RIP Saporin have been used for the construction of Immunotoxins (ITxs) obtained via chemical conjugation... (Review)
Review
Plant Ribosome-inactivating proteins (RIPs) including the type I RIP Saporin have been used for the construction of Immunotoxins (ITxs) obtained via chemical conjugation of the toxic domain to whole antibodies or by generating genetic fusions to antibody fragments/targeting domains able to direct the chimeric toxin against a desired sub-population of cancer cells. The high enzymatic activity, stability and resistance to conjugation procedures and especially the possibility to express recombinant fusions in yeast, make Saporin a well-suited tool for anti-cancer therapy approaches. Previous clinical work on RIPs-based Immunotoxins (including Saporin) has shown that several critical issues must be taken into deeper consideration to fully exploit their therapeutic potential. This review focuses on possible combinatorial strategies (chemical and genetic) to augment Saporin-targeted toxin efficacy. Combinatorial approaches may facilitate RIP escape into the cytosolic compartment (where target ribosomes are), while genetic manipulations may minimize potential adverse effects such as vascular-leak syndrome or may identify T/B cell epitopes in order to decrease the immunogenicity following similar strategies as those used in the case of bacterial toxins such as Pseudomonas Exotoxin A or as for Type I RIP Bouganin. This review will further focus on strategies to improve recombinant production of Saporin-based chimeric toxins.
Topics: Animals; Genetic Therapy; Humans; Immunotoxins; Phototherapy; Pinocytosis; Saporins
PubMed: 29438358
DOI: 10.3390/toxins10020082 -
Molecular Biology of the Cell Jun 2019Macropinocytosis is an actin-driven form of clathrin-independent endocytosis that generates an enlarged structure, the macropinosome. Although many studies focus on...
Macropinocytosis is an actin-driven form of clathrin-independent endocytosis that generates an enlarged structure, the macropinosome. Although many studies focus on signaling molecules and phosphoinositides involved in initiating macropinocytosis, the commitment to forming a macropinosome and the handling of that membrane have not been studied in detail. Here we show in HT1080 cells, a human fibrosarcoma cell line, a requirement for microtubules, dynein, the JIP3 microtubule motor scaffold protein, and Arf6, a JIP3 interacting protein, for the formation and inward movement of the macropinosome. While actin and myosin II also play critical roles in the formation of ruffling membrane, microtubules provide an important tract for initiation, sealing, and transport of the macropinosome through the actin- and myosin-rich lamellar region.
Topics: ADP-Ribosylation Factor 6; ADP-Ribosylation Factors; Actins; Adaptor Proteins, Signal Transducing; Cell Line, Tumor; Clathrin; Dyneins; Humans; Microtubules; Models, Biological; Mutation; Nerve Tissue Proteins; Pinocytosis
PubMed: 30969891
DOI: 10.1091/mbc.E19-01-0022 -
Autophagy May 2021Poorly vascularized tumors embedded within a thick desmoplastic stroma, like pancreatic ductal adenocarcinoma (PDAC), are nutritionally stressed. Such tumors are also...
Poorly vascularized tumors embedded within a thick desmoplastic stroma, like pancreatic ductal adenocarcinoma (PDAC), are nutritionally stressed. Such tumors are also hypoxic and rely on a number of adaptive responses, including macroautophagy/autophagy and macropinocytosis (MP), to support their bioenergetic needs. Whereas autophagy enables starved cells to recycle intracellular macromolecules via lysosomal degradation and use the liberated amino acids (AA) to fuel their metabolism, MP allows cells to take up extracellular proteins via fluid-phase endocytosis and use them as an energy source. However, how any MP-enabled organism, including the prototypical cancer cell, coordinately regulates and balances autophagy and MP is not fully understood. We recently found that inhibition of autophagy results in upregulation of MP, which enables cancer cells to overcome autophagy deficiency and continue to support their bioenergetic demands. The NFE2L2/NRF2-driven induction of MP-related genes (MRGs) is responsible for the upregulation of MP in autophagy inhibited, hypoxic, and oxidatively stressed-exposed cancer cells. Concurrent autophagy and MP blockade effectively cuts off the cancer cell's nutrient and supplies, leading to rapid tumor regression. These findings suggest MP to be an important target in cancer treatment and that shutting off the energy spigot is a promising therapeutic strategy. AA: amino acids; ADCs: autophagy deficient-cells; AI: autophagy inhibition; ALB: albumin; CHUK/IKKα: component of inhibitor of nuclear factor kappa B kinase complex; CQ: chloroquine; ECM: extracellular matrix; HCQ: hydroxychloroquine; MI: MP inhibition; MP: macropinocytosis; MRGs: MP-related genes; MRPs: MP-related proteins; PDAC: pancreatic ductal adenocarcinoma.
Topics: Autophagy; Carcinoma, Pancreatic Ductal; Humans; NF-E2-Related Factor 2; Pancreatic Neoplasms; Pinocytosis
PubMed: 33879021
DOI: 10.1080/15548627.2021.1919969 -
Microbiome Feb 2021Sponges are increasingly recognised as key ecosystem engineers in many aquatic habitats. They play an important role in nutrient cycling due to their unrivalled capacity...
BACKGROUND
Sponges are increasingly recognised as key ecosystem engineers in many aquatic habitats. They play an important role in nutrient cycling due to their unrivalled capacity for processing both dissolved and particulate organic matter (DOM and POM) and the exceptional metabolic repertoire of their diverse and abundant microbial communities. Functional studies determining the role of host and microbiome in organic nutrient uptake and exchange, however, are limited. Therefore, we coupled pulse-chase isotopic tracer techniques with nanoscale secondary ion mass spectrometry (NanoSIMS) to visualise the uptake and translocation of C- and N-labelled dissolved and particulate organic food at subcellular level in the high microbial abundance sponge Plakortis angulospiculatus and the low microbial abundance sponge Halisarca caerulea.
RESULTS
The two sponge species showed significant enrichment of DOM- and POM-derived C and N into their tissue over time. Microbial symbionts were actively involved in the assimilation of DOM, but host filtering cells (choanocytes) appeared to be the primary site of DOM and POM uptake in both sponge species overall, via pinocytosis and phagocytosis, respectively. Translocation of carbon and nitrogen from choanocytes to microbial symbionts occurred over time, irrespective of microbial abundance, reflecting recycling of host waste products by the microbiome.
CONCLUSIONS
Here, we provide empirical evidence indicating that the prokaryotic communities of a high and a low microbial abundance sponge obtain nutritional benefits from their host-associated lifestyle. The metabolic interaction between the highly efficient filter-feeding host and its microbial symbionts likely provides a competitive advantage to the sponge holobiont in the oligotrophic environments in which they thrive, by retaining and recycling limiting nutrients. Sponges present a unique model to link nutritional symbiotic interactions to holobiont function, and, via cascading effects, ecosystem functioning, in one of the earliest metazoan-microbe symbioses. Video abstract.
Topics: Animals; Carbon; Microbiota; Nitrogen; Nutrients; Porifera; Symbiosis
PubMed: 33583434
DOI: 10.1186/s40168-020-00984-w -
Antimicrobial Agents and Chemotherapy Feb 2020There is an urgent need for safe, efficacious, affordable, and field-adapted drugs for the treatment of cutaneous leishmaniasis, which newly affects around 1.5 million...
There is an urgent need for safe, efficacious, affordable, and field-adapted drugs for the treatment of cutaneous leishmaniasis, which newly affects around 1.5 million people worldwide annually. Chitosan, a biodegradable cationic polysaccharide, has previously been reported to have antimicrobial, antileishmanial, and immunostimulatory activities. We investigated the activity of chitosan and several of its derivatives and showed that the pH of the culture medium plays a critical role in antileishmanial activity of chitosan against both extracellular promastigotes and intracellular amastigotes of and Chitosan and its derivatives were approximately 7 to 20 times more active at pH 6.5 than at pH 7.5, with high-molecular-weight chitosan being the most potent. High-molecular-weight chitosan stimulated the production of nitric oxide and reactive oxygen species by uninfected and -infected macrophages in a time- and dose-dependent manner at pH 6.5. Despite the activation of bone marrow macrophages by chitosan to produce nitric oxide and reactive oxygen species, we showed that the antileishmanial activity of chitosan was not mediated by these metabolites. Finally, we showed that rhodamine-labeled chitosan is taken up by pinocytosis and accumulates in the parasitophorous vacuole of -infected macrophages.
Topics: Amphotericin B; Animals; Antiprotozoal Agents; Chitosan; Culture Media; Dose-Response Relationship, Drug; Female; Humans; Hydrogen-Ion Concentration; Leishmania major; Leishmania mexicana; Life Cycle Stages; Macrophages; Macrophages, Peritoneal; Mice; Mice, Inbred BALB C; Molecular Weight; Nitric Oxide; Parasitic Sensitivity Tests; Pinocytosis; Primary Cell Culture; Reactive Oxygen Species; THP-1 Cells; Tumor Necrosis Factor-alpha
PubMed: 31871082
DOI: 10.1128/AAC.01772-19 -
Frontiers in Nutrition 2022The Mediterranean diet is regarded as one of the most healthful dietary patterns in the world, owing to a combination of foods high in antioxidants and anticancer...
The Mediterranean diet is regarded as one of the most healthful dietary patterns in the world, owing to a combination of foods high in antioxidants and anticancer constituents. Barley bran is one of the components of the Mediterranean diet. It has nutritional and beneficial effects in different pathological conditions. Many studies were achieved to assess the nutritious values of barley bran, but there is no research indicating immunomodulatory and anticancer activities of barley bran grown in Jordan. The present study aims to examine and assess the potential immunomodulatory and anti-tumor activities of ethanol, -hexane, aqueous/methanol, and water extracts obtained from barley bran. The Maceration method was utilized to prepare ethanol, -hexane, aqueous/methanol, and water extracts. Various phytochemical groups were determined by using qualitative phytochemical tests. The antiproliferative activity of extracts was determined against MCF-7, HCT-116, A549, and EMT6/p by the MTT assay. The Folin-Ciocalteu reagent was used to detect the total phenolic content in extracts. Furthermore, immunomodulatory activity was assessed by determining the effect of extracts on splenocytes proliferation in the presence and absence of mitogens. The nitro blue tetrazolium assay and the neutral red method were used to assess the effect of each extract on the phagocytic activity of macrophages and pinocytosis, respectively. For the part, three different concentrations (10, 20, and 30% w/v) of barley bran were used to test the prophylactic effect in four Balb/C mice groups inoculated with EMT6/p cell-line subcutaneously. Also, serum samples were collected to assess the effect on cytokines (IFN-gamma, IL-2, IL-4, and IL-10). Barley bran extracts inhibited cancer cell proliferation. According to immunoassays, hexane and aqueous/methanol extracts could significantly rise lymphocyte proliferation and pinocytosis activity of macrophages. The activity of phagocytosis was increased by hexane and ethanol extracts. For the part, the average tumor size and weight of mice given the 30% barley bran group was significantly reduced ( < 0.05) compared with the control group. During our study, higher levels of TH1 cytokines (IFN- γ, IL-2) and lower levels of TH2 cytokine (IL-4) and T regulatory cytokine (IL-10) were obtained due to consumption of barley bran in food. Barley bran can be used as a prophylactic agent because it has anti-cancer and immunomodulatory activities.
PubMed: 35662936
DOI: 10.3389/fnut.2022.838373 -
JCI Insight Jun 2021Patients with chronic kidney disease (CKD) and end-stage renal disease suffer from increased cardiovascular events and cardiac mortality. Prior studies have demonstrated...
Patients with chronic kidney disease (CKD) and end-stage renal disease suffer from increased cardiovascular events and cardiac mortality. Prior studies have demonstrated that a portion of this enhanced risk can be attributed to the accumulation of microbiota-derived toxic metabolites, with most studies focusing on the sulfonated form of p-cresol (PCS). However, unconjugated p-cresol (uPC) itself was never assessed due to rapid and extensive first-pass metabolism that results in negligible serum concentrations of uPC. These reports thus failed to consider the host exposure to uPC prior to hepatic metabolism. In the current study, not only did we measure the effect of altering the intestinal microbiota on lipid accumulation in coronary arteries, but we also examined macrophage lipid uptake and handling pathways in response to uPC. We found that atherosclerosis-prone mice fed a high-fat diet exhibited significantly higher coronary artery lipid deposits upon receiving fecal material from CKD mice. Furthermore, treatment with uPC increased total cholesterol, triglycerides, and hepatic and aortic fatty deposits in non-CKD mice. Studies employing an in vitro macrophage model demonstrated that uPC exposure increased apoptosis whereas PCS did not. Additionally, uPC exhibited higher potency than PCS to stimulate LDL uptake and only uPC induced endocytosis- and pinocytosis-related genes. Pharmacological inhibition of varying cholesterol influx and efflux systems indicated that uPC increased macrophage LDL uptake by activating macropinocytosis. Overall, these findings indicate that uPC itself had a distinct effect on macrophage biology that might have contributed to increased cardiovascular risk in patients with CKD.
Topics: Animals; Aorta; Cholesterol; Cholesterol, LDL; Coronary Artery Disease; Coronary Vessels; Cresols; Diet, High-Fat; Fecal Microbiota Transplantation; Gastrointestinal Microbiome; Kidney Failure, Chronic; Liver; Macrophages; Mice; Pinocytosis; Renal Insufficiency, Chronic; Triglycerides
PubMed: 33914709
DOI: 10.1172/jci.insight.144410 -
Philosophical Transactions of the Royal... Feb 2019Nucleic acids are a rapidly emerging therapeutic modality with the potential to become the third major drug modality alongside antibodies and small molecules. Owing to... (Review)
Review
Nucleic acids are a rapidly emerging therapeutic modality with the potential to become the third major drug modality alongside antibodies and small molecules. Owing to the unfavourable physico-chemical characteristics of nucleic acids, such as large size and negative charge, intracellular delivery remains a fundamental challenge to realizing this potential. Delivery technologies such as lipids, polymers and peptides have been used to facilitate delivery, with many of the most successful technologies using macropinocytosis to gain cellular entry; mostly by default rather than design. Fundamental knowledge of macropinocytosis is rapidly growing, presenting opportunities to better tailor design strategies to target this pathway. Furthermore, certain types of tumour cells have been observed to have high levels of macropinocytic activity and traffic cargo to favourable destinations within the cell for endosomal release, providing unique opportunities to further use this entry route for drug delivery. In this article, we review the delivery systems reported to be taken up by macropinocytosis and what is known about the mechanisms for regulating macropinocytosis in tumour cells. From this analysis, we identify new opportunities for exploiting this pathway for the intracellular delivery of nucleic acids to tumour cells. This article is part of the Theo Murphy meeting issue 'Macropinocytosis'.
Topics: Drug Delivery Systems; Nucleic Acids; Pinocytosis; Tumor Cells, Cultured
PubMed: 30967005
DOI: 10.1098/rstb.2018.0156