ABSTRACT
Among the infectious diseases caused by pathogenic microorganisms such as bacteria, viruses, parasites, or fungi, the most prevalent ones today are malaria, tuberculosis, influenza, HIV/AIDS, Ebola, dengue fever, and methicillin-resistant Staphylococcus aureus (MRSA) infection, and most recently Covid-19 (SARS-CoV2). Others with a rather devastating history and high fatality rates such as plague, cholera, or typhus seem less threatening today but have not been eradicated, and with a declining efficacy of current antibiotics they ought to be watched carefully. Another emerging issue in this context is health-care associated infection. About 100,000 hospitalized patients in the USA ( www.cdc.gov ) and 33,000 in Europe ( https://www.ecdc.europa.eu ) die each year as a direct consequence of an infection caused by bacteria resistant to antibiotics. Among viral infections, influenza is responsible for about 3-5 million cases of severe illness, and about 250,000 to 500,000 deaths annually ( www.who.int ). About 37 million people are currently living with HIV infection and about one million die from it each year. Coronaviruses such as MERS-CoV, SARS-CoV, but in particular the recent outbreak of Covid-19 (caused by SARS-CoV2) have resulted in large numbers of infections worldwide with an estimated several hundred thousand deaths (anticipated fatality rate: <5%). With a comparatively low mortality rate dengue virus causes between 50 and 100 million infections every year, leading to 50,000 deaths. In contrast, Ebola virus is the causative agent for one of the deadliest viral diseases. The Ebola outbreak in West Africa in 2014 is considered the largest outbreak in history with more than 11,000 deaths. Many of the deadliest pathogens such as Ebola virus, influenza virus, mycobacterium tuberculosis, dengue virus, and cholera exploit the endo-lysosomal trafficking system of host cells for penetration into the cytosol and replication. Defects in endo-lysosomal maturation, trafficking, fusion, or pH homeostasis can efficiently reduce the cytotoxicity caused by these pathogens. Most of these functions critically depend on endo-lysosomal membrane proteins such as transporters and ion channels. In particular, cation channels such as the mucolipins (TRPMLs) or the two-pore channels (TPCs) are involved in all of these aspects of endo-lysosomal integrity. In this review we will discuss the correlations between pathogen toxicity and endo-lysosomal cation channel function, and their potential as drug targets for infectious disease therapy.
Subject(s)
COVID-19 , Cholera , Ebolavirus , HIV Infections , Hemorrhagic Fever, Ebola , Influenza, Human , Methicillin-Resistant Staphylococcus aureus , Humans , COVID-19/metabolism , Hemorrhagic Fever, Ebola/metabolism , Influenza, Human/metabolism , Cholera/metabolism , HIV Infections/metabolism , RNA, Viral/metabolism , SARS-CoV-2 , Lysosomes/metabolism , Cations/metabolismABSTRACT
Liver cancers, including hepatocellular carcinoma (HCC), are the second leading cause of cancer death worldwide, and novel therapeutic strategies are still highly needed. Recently, the endolysosomal cation channel TRPML1 (also known as MCOLN1) has gained focus in cancer research because it represents an interesting novel target. We utilized the recently developed isoform-selective TRPML1 activator ML1-SA1 and the CRISPR/Cas9 system to generate tools for overactivation and loss-of-function studies on TRPML1 in HCC. After verification of our tools, we investigated the role of TRPML1 in HCC by studying proliferation, apoptosis and proteomic alterations. Furthermore, we analyzed mitochondrial function in detail by performing confocal and transmission electron microscopy combined with SeahorseTM and Oroboros® functional analysis. We report that TRPML1 overactivation mediated by a novel, isoform-selective small-molecule activator induces apoptosis by impairing mitochondrial function in a Ca2+-dependent manner. Additionally, TRPML1 loss-of-function deregulates mitochondrial renewal, which leads to proliferation impairment. Thus, our study reveals a novel role for TRPML1 as regulator of mitochondrial function and its modulators as promising molecules for novel therapeutic options in HCC therapy.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Transient Receptor Potential Channels , Calcium/metabolism , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Humans , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Lysosomes/metabolism , Mitochondria/metabolism , Proteomics , Transient Receptor Potential Channels/genetics , Transient Receptor Potential Channels/metabolismABSTRACT
The endo-lysosomal two-pore channel (TPC2) has been established as an intracellular cation channel of significant physiological and pathophysiological relevance in recent years. For example, TPC2-/- mice show defects in cholesterol degradation, leading to hypercholesterinemia; TPC2 absence also results in mature-onset obesity, and a role in glucagon secretion and diabetes has been proposed. Infections with bacterial toxins or viruses e.g., cholera toxin or Ebola virus result in reduced infectivity rates in the absence of TPC2 or after pharmacological blockage, and TPC2-/- cancer cells lose their ability to migrate and metastasize efficiently. Finally, melanin production is affected by changes in hTPC2 activity, resulting in pigmentation defects and hair color variation. Here, we analyzed several publicly available genome variation data sets and identified multiple variations in the TPC2 protein in distinct human populations. Surprisingly, one variation, L564P, was found to be the predominant TPC2 isoform on a global scale. By applying endo-lysosomal patch-clamp electrophysiology, we found that L564P is a prerequisite for the previously described M484L gain-of-function effect that is associated with blond hair. Additionally, other gain-of-function variants with distinct geographical and ethnic distribution were discovered and functionally characterized. A meta-analysis of genome-wide association studies was performed, finding the polymorphisms to be associated with both distinct and overlapping traits. In sum, we present the first systematic analysis of variations in TPC2. We functionally characterized the most common variations and assessed their association with various disease traits. With TPC2 emerging as a novel drug target for the treatment of various diseases, this study provides valuable insights into ethnic and geographical distribution of TPC2 polymorphisms and their effects on channel activity.
Subject(s)
Calcium Channels/genetics , Genome-Wide Association Study , Hair Color/genetics , Animals , Fibroblasts/metabolism , Gain of Function Mutation/genetics , Genome, Human/genetics , Humans , Lysosomes/genetics , Mice , Mice, Knockout , NADP/genetics , Pigmentation/genetics , Polymorphism, Single Nucleotide/genetics , Signal Transduction/geneticsABSTRACT
Intestinal handling of dietary proteins usually prevents local inflammatory and immune responses and promotes oral tolerance. However, in ~ 1% of the world population, gluten proteins from wheat and related cereals trigger an HLA DQ2/8-restricted TH1 immune and antibody response leading to celiac disease. Prior epithelial stress and innate immune activation are essential for breaking oral tolerance to the gluten component gliadin. How gliadin subverts host intestinal mucosal defenses remains elusive. Here, we show that the α-gliadin-derived LGQQQPFPPQQPY peptide (P31-43) inhibits the function of cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel pivotal for epithelial adaptation to cell-autonomous or environmental stress. P31-43 binds to, and reduces ATPase activity of, the nucleotide-binding domain-1 (NBD1) of CFTR, thus impairing CFTR function. This generates epithelial stress, tissue transglutaminase and inflammasome activation, NF-κB nuclear translocation and IL-15 production, that all can be prevented by potentiators of CFTR channel gating. The CFTR potentiator VX-770 attenuates gliadin-induced inflammation and promotes a tolerogenic response in gluten-sensitive mice and cells from celiac patients. Our results unveil a primordial role for CFTR as a central hub orchestrating gliadin activities and identify a novel therapeutic option for celiac disease.
Subject(s)
Celiac Disease/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Gliadin/pharmacology , Peptide Fragments/pharmacology , Adolescent , Aminophenols/administration & dosage , Aminophenols/pharmacology , Animals , Caco-2 Cells , Celiac Disease/drug therapy , Celiac Disease/genetics , Cell Line , Child , Cystic Fibrosis Transmembrane Conductance Regulator/chemistry , Disease Models, Animal , Down-Regulation , Female , Humans , Male , Mice , Protein Binding/drug effects , Protein Conformation , Protein Domains , Quinolones/administration & dosage , Quinolones/pharmacology , Young AdultABSTRACT
Two-pore channels (TPCs) are endolysosomal cation channels. Two members exist in humans, TPC1 and TPC2. Functional roles associated with the ubiquitously expressed TPCs include VEGF-induced neoangiogenesis, LDL-cholesterol trafficking and degradation, physical endurance under fasting conditions, autophagy regulation, the acrosome reaction in sperm, cancer cell migration, and intracellular trafficking of pathogens such as Ebola virus or bacterial toxins (e.g., cholera toxin). In a genome-wide association study for variants associated with human pigmentation characteristics two coding variants of TPC2, rs35264875 (encoding M484L) and rs3829241 (encoding G734E), have been found to be associated with a shift from brown to blond hair color. In two recent follow-up studies a role for TPC2 in pigmentation has been further confirmed. However, these human polymorphic variants have not been functionally characterized until now. The development of endolysosomal patch-clamp techniques has made it possible to investigate directly ion channel activities and characteristics in isolated endolysosomal organelles. We applied this technique here to scrutinize channel characteristics of the polymorphic TPC2 variants in direct comparison with WT. We found that both polymorphisms lead to a gain of channel function by independent mechanisms. We next conducted a clinical study with more than 100 blond- and brown/black-haired individuals. We performed a genotype/phenotype analysis and subsequently isolated fibroblasts from WT and polymorphic variant carriers for endolysosomal patch-clamp experimentation to confirm key in vitro findings.
Subject(s)
Calcium Channels/genetics , Hair/chemistry , Pigmentation/genetics , Polymorphism, Genetic , Calcium Channels/physiology , Genome-Wide Association Study , HEK293 Cells , Hair/metabolism , Humans , Patch-Clamp Techniques , PhenotypeABSTRACT
G-Quadruplex (G4) DNA structures are important regulatory elements in central biological processes. Small molecules that selectively bind and stabilize G4 structures have therapeutic potential, and there are currently >1000 known G4 ligands. Despite this, only two G4 ligands ever made it to clinical trials. In this work, we synthesized several heterocyclic G4 ligands and studied their interactions with G4s (e.g., G4s from the c-MYC, c-KIT, and BCL-2 promoters) using biochemical assays. We further studied the effect of selected compounds on cell viability, the effect on the number of G4s in cells, and their pharmacokinetic properties. This identified potent G4 ligands with suitable properties and further revealed that the dispersion component in arene-arene interactions in combination with electron-deficient electrostatics is central for the ligand to bind with the G4 efficiently. The presented design strategy can be applied in the further development of G4-ligands with suitable properties to explore G4s as therapeutic targets.
Subject(s)
DNA , G-Quadruplexes , Ligands , Static Electricity , DNA/metabolism , Promoter Regions, GeneticABSTRACT
The gut microbiome and its metabolites are increasingly implicated in several cardiovascular diseases, but their role in human myocardial infarction (MI) injury responses have yet to be established. To address this, we examined stool samples from 77 ST-elevation MI (STEMI) patients using 16 S V3-V4 next-generation sequencing, metagenomics and machine learning. Our analysis identified an enriched population of butyrate-producing bacteria. These findings were then validated using a controlled ischemia/reperfusion model using eight nonhuman primates. To elucidate mechanisms, we inoculated gnotobiotic mice with these bacteria and found that they can produce beta-hydroxybutyrate, supporting cardiac function post-MI. This was further confirmed using HMGCS2-deficient mice which lack endogenous ketogenesis and have poor outcomes after MI. Inoculation increased plasma ketone levels and provided significant improvements in cardiac function post-MI. Together, this demonstrates a previously unknown role of gut butyrate-producers in the post-MI response.
Subject(s)
Myocardial Infarction , ST Elevation Myocardial Infarction , Humans , Animals , Mice , Butyrates/metabolism , Heart , Ketone BodiesABSTRACT
The human immunodeficiency virus type 1 (HIV-1)-encoded RNA-binding protein Tat is known to play an essential role in viral gene expression. In the search for novel compounds to inhibit Tat transactivity, one coumarin derivative, BPRHIV001, was identified, with a 50% effective concentration (EC(50)) against HIV-1 at 1.3 nM. BPRHIV001 is likely to exert its effects at the stage after initiation of RNAPII elongation since Tat protein expression and the assembly of the Tat/P-TEFb complex remained unchanged. Next, a reduction of the p300 protein level, known to modulate Tat function through acetylation, was observed upon BPRHIV001 treatment, while the p300 mRNA level was unaffected. A concordant reduction of phosphorylated Akt, which was shown to be closely related to p300 stability, was observed in the presence of BPRHIV001 and was accompanied by a decrease of phosphorylated PDPK1, a well-known Akt activator. Furthermore, the docking analysis revealed that the reduced PDPK1 phosphorylation likely resulted from the allosteric effect of interaction between BPRHIV001 and PDPK1. With strong synergistic effects with current reverse transcriptase inhibitors, BPRHIV001 has the potential to become a promising lead compound for the development of a novel therapeutic agent against HIV-1 infection.
Subject(s)
Anti-HIV Agents/pharmacology , Coumarins/pharmacology , HIV-1/drug effects , HIV-1/physiology , Oncogene Protein v-akt/metabolism , Transcription, Genetic/drug effects , tat Gene Products, Human Immunodeficiency Virus/antagonists & inhibitors , Cell Line , Humans , Microbial Sensitivity Tests , Phosphorylation , p300-CBP Transcription Factors/metabolismABSTRACT
Despite novel therapy regimens and extensive research, chemoresistance remains a challenge in leukemia treatment. Of note, recent studies revealed lysosomes as regulators of cell death and chemotherapy response, suggesting this organelle is a novel target for chemosensitization. Interestingly, drug-resistant VCR-R CEM acute lymphoblastic leukemia (ALL) cells have an increased expression of the lysosomal cation channel Two-Pore-Channel 2 (TPC2) compared to drug-naïve CCRF-CEM ALL cells. Concurrently, knockout (KO) of TPC2 sensitized drug-resistant VCR-R CEM cells to treatment with cytostatics. The chemosensitizing effect could be confirmed in several cell lines as well as in heterogeneous, patient-derived xenograft ALL cells, using the pharmacological TPC2 inhibitors naringenin and tetrandrine. We reveal that a dual mechanism of action mediates chemo sensitization by loss of lysosomal TPC2 function. First, because of increased lysosomal pH, lysosomal drug sequestration is impaired, leading to an increased nuclear accumulation of doxorubicin and hence increased DNA damage. Second, lysosomes of TPC2 KO cells are more prone to lysosomal damage as a result of morphological changes and dysregulation of proteins influencing lysosomal stability. This leads to induction of lysosomal cell death (LCD), evident by increased cathepsin B levels in the cytosol, truncation of pro-apoptotic Bid, as well as the reversibility of cell death by co-treatment with the cathepsin B inhibitor CA-074Me in TPC2 KO cells. In summary, this study establishes TPC2 as a novel, promising, druggable target for combination therapy approaches in ALL to overcome chemoresistance, which could be exploited in the clinic in the future. Additionally, it unravels LCD signaling as an important death-inducing component upon loss of TPC2 function.
Subject(s)
Calcium Channels , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Calcium Channels/metabolism , Cathepsin B/metabolism , Humans , Lysosomes/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Signal TransductionABSTRACT
Background: The gut microbiota plays a vital role in maintaining tissue homeostasis and regulating disease pathophysiology; however, the underlying mechanisms remain to be elucidated. We previously showed that mice depleted of gut microbiota with antibiotics (ABX mice) were more prone to cardiac rupture after infarction, suggesting that the gut microbiota impacts cardiac structural remodeling following injury. Here, we aimed to determine whether the gut microbiota is required for adaptive cardiac remodeling in response to pressure overload stress. Methods: Transverse aortic constriction (TAC) surgery was performed and cardiac function was evaluated by echocardiography and catheterization, followed by mechanical tests and extracellular matrix (ECM) studies. Germ-free mice with cecal microbiota transplantation were used for validation. 16S ribosomal DNA sequencing and PICRUSt2 analysis were applied to predict the key metabolic pathways. ABX mice were supplemented with the derived metabolic products and their efficacy was tested. To elucidate the underlying mechanism, we isolated mouse primary cardiac fibroblasts and treated them with the metabolites. Lastly, G-coupled protein receptor 41 (GPR41) and GPR43 double knockdown cardiac fibroblasts were generated and the anti-fibrogenic effect of metabolites was determined. Results: Cardiac hypertrophy and dysfunction were more severe in ABX-TAC mice compared to the controls. Moreover, TAC-induced fibrosis was more profound in ABX hearts, which was accompanied by disrupted ECM structure making the heart tissues mechanically weaker and more brittle. Reconstruction of healthy gut microbiota in germ-free mice successfully restored cardiac function and prevented excessive fibrosis and ECM disarray under stress. Furthermore, functional prediction identified acetate and propionate as critical mediators in the gut microbiota-modulated cardiac mechanics. Supplementation of acetate and propionate improved heart function, attenuated fibrosis, and reversed ECM disarray after TAC. In addition, treating primary cardiac fibroblasts with acetate and propionate attenuated cell contraction, inhibited myofibroblast formation, and reduced collagen formation after TGF-ß1 stimulus. Finally, knocking down GPR41 and GPR43 receptors in cardiac fibroblasts blunted the inhibitory effects of acetate and propionate. Conclusions: The gut microbiota is a potential therapeutic target for cardiac ECM remodeling and heart structural integrity. By establishing a healthy gut microbiome or replenishing the derived metabolites, we could improve cardiac health under dysbiosis after pressure-overload stress.
Subject(s)
Gastrointestinal Microbiome , Mice , Animals , Propionates/pharmacology , Heart , Fibrosis , AcetatesABSTRACT
We hypothesized that the host microbiome may influence foreign body responses following biomaterial implantation. To test this, we implanted a variety of clinically relevant biomaterials into germ-free or antibiotic-treated mice. Surprisingly, these mice displayed less fibrous tissue deposition, reduced host cell recruitment to the implant site, and differential expression of angiogenic and inflammatory markers. These observations were reversed upon fecal microbiome reconstitution, confirming a causal role of the host microbiome. In a clinically relevant disease model, microbiome-depleted mice cleared hyaluronic acid and bone marrow mononuclear cells from ischemic hind limb tissues more slowly, resulting in an improved therapeutic response. Findings were confirmed in pigs which showed reduced fibrotic responses to a variety of implanted materials. Lastly, we profiled changes in the host microbiome following material implantation, implicating several key bacteria phyla.
Subject(s)
Biocompatible Materials , Gastrointestinal Microbiome , Animals , Anti-Bacterial Agents , Foreign-Body Reaction , Hyaluronic Acid , Mice , SwineABSTRACT
Doxorubicin (DOX) is a potent chemotherapeutic agent known to induce cardiotoxicity. Here we applied one-dimensional scanning multiphoton imaging to investigate the derangement of cardiac dynamics induced by DOX on a zebrafish model. DOX changed the cell morphology and significantly prolonged calcium transient and sarcomere contraction, leading to an arrhythmia-like contractile disorder. The restoration phase of calcium transient dominated the overall prolongation, indicating that DOX perturbed primarily the protein functions responsible for recycling cytosolic calcium ions. This novel finding supplements the existing mechanism of DOX cardiotoxicity. We anticipate that this approach should help mechanistic studies of drug-induced cardiotoxicity or heart diseases.
ABSTRACT
Two-pore channel 2 (TPC2) resides in endolysosomal membranes but also in lysosome-related organelles such as the melanin producing melanosomes. Gain-of-function polymorphisms in hTPC2 are associated with decreased melanin production and blond hair color. Vice versa genetic ablation of TPC2 increases melanin production. We show here an inverse correlation between melanin production and melanoma proliferation, migration, and invasion due to the dual activity of TPC2 in endolysosomes and melanosomes. Our results are supported by both genetic ablation and pharmacological inhibition of TPC2. Mechanistically, our data show that loss/block of TPC2 results in reduced protein levels of MITF, a major regulator of melanoma progression, but an increased activity of the melanin-generating enzyme tyrosinase. TPC2 inhibition thus provides a twofold benefit in melanoma prevention and treatment by increasing, through interference with tyrosinase activity, the synthesis of UV blocking melanin in melanosomes and by decreasing MITF-driven melanoma progression by increased GSK3ß-mediated MITF degradation.
Subject(s)
Calcium Channels/metabolism , Cell Movement/drug effects , Cell Proliferation/drug effects , Endosomes/drug effects , Flavonoids/pharmacology , Melanins/metabolism , Melanoma/drug therapy , Melanosomes/drug effects , Cell Line, Tumor , Endosomes/metabolism , Glycogen Synthase Kinase 3 beta/metabolism , HEK293 Cells , Hair Color/drug effects , Humans , Lysosomes/drug effects , Lysosomes/metabolism , Melanoma/metabolism , Melanosomes/metabolism , Microphthalmia-Associated Transcription Factor/metabolism , Monophenol Monooxygenase/metabolism , Pigmentation/drug effectsABSTRACT
The role of two-pore channel 2 (TPC2), one of the few cation channels localized on endolysosomal membranes, in cancer remains poorly understood. Here, we report that TPC2 knockout reduces proliferation of cancer cells in vitro, affects their energy metabolism, and successfully abrogates tumor growth in vivo. Concurrently, we have developed simplified analogs of the alkaloid tetrandrine as potent TPC2 inhibitors by screening a library of synthesized benzyltetrahydroisoquinoline derivatives. Removal of dispensable substructures of the lead molecule tetrandrine increases antiproliferative properties against cancer cells and impairs proangiogenic signaling of endothelial cells to a greater extent than tetrandrine. Simultaneously, toxic effects on non-cancerous cells are reduced, allowing in vivo administration and revealing a TPC2 inhibitor with antitumor efficacy in mice. Hence, our study unveils TPC2 as valid target for cancer therapy and provides easily accessible tetrandrine analogs as a promising option for effective pharmacological interference.
Subject(s)
Antineoplastic Agents/pharmacology , Calcium Channels/metabolism , Carcinoma, Hepatocellular/drug therapy , Gene Editing , Isoquinolines/pharmacology , Liver Neoplasms/drug therapy , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Apoptosis/drug effects , Calcium Channels/deficiency , Calcium Channels/genetics , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Proliferation/drug effects , Cells, Cultured , Drug Screening Assays, Antitumor , Female , Humans , Isoquinolines/chemical synthesis , Isoquinolines/chemistry , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Mice , Mice, Inbred C57BLABSTRACT
Background: In 1883, Ilya Mechnikov discovered phagocytes and established the concept of phagocytosis by macrophages. In 1908, he was awarded the Nobel Prize in Physiology/Medicine for his findings, which laid the foundations for today's understanding of the innate immune response. Only in the 1960s, Max Cooper and Robert Good significantly advanced our understanding of the immune system by demonstrating that B- and T-cells cooperate to regulate the adaptive immune response. Both, innate and adaptive immune response are essential to effectively protect the individual against infectious agents, such as viruses, bacterial or insect toxins, or allergens. Innate immune responses occur rapidly upon exposure to noxious or infectious agents or organisms, in contrast to the adaptive immune system that needs days rather than hours to develop and acts primarily on the basis of antigen-specific receptors expressed on the surface of B- and T-lymphocytes. In recent years, it has become evident that endosomes and lysosomes are involved in many aspects of immune cell function, such as phagocytosis, antigen presentation and processing by antigen-presenting cells, release of proinflammatory mediators, e.g., by mast cells, or secretion of the pore-forming protein perforin by cytotoxic T lymphocytes. Several lysosomal storage disorders (LSDs) have been associated with defects in immune system function or immune system hyperactivity, such as Gaucher, Fabry, or Niemann-Pick type C1 disease, mucopolysaccharidoses (MPS), gangliosidosis, or juvenile neuronal ceroid lipofuscinosis (JNCL). Beside accumulating evidence on the importance of endolysosomes in immune cell function, recent results suggest direct roles of endolysosomal ion channels, such as the TRPML channels (mucolipins), which are members of the transient receptor potential (TRP) superfamily of non-selective cation channels, for different aspects of immune cell function. The aim of this review is to discuss the current knowledge about the roles of TRPML channels in inflammation and immunity, and to assess their potential as drug targets to influence immune cell functions. Advances: Examples of recently established roles of TRPML channels in immune system function and immune response include the TRPML1-mediated modulation of secretory lysosomes, granzyme B content, and tuning of effector function in NK cells, TRPML1-dependent directional dendritic cell (DC) migration and DC chemotaxis, and the role of TRPML2 in chemokine release from LPS-stimulated macrophages. Outlook: Although our understanding of the functional roles of TRPML channels in inflammation and immunity is still in its infancy, a few interesting findings have been made in the past years, encouraging further and more detailed work on the role of TRPMLs, e.g., in intracellular trafficking and release of chemokines, cytokines, or granzyme B, or in phagocytosis and bacterial toxin and virus trafficking through the endolysosomal machinery.
Subject(s)
Endosomes/metabolism , Inflammation/metabolism , Lysosomal Storage Diseases/metabolism , Lysosomes/metabolism , TRPM Cation Channels/metabolism , Transient Receptor Potential Channels/metabolism , Animals , Humans , Immunity , Protein TransportABSTRACT
Ischemia/reperfusion-induced edema (IRE), one of the most significant causes of mortality after lung transplantation, can be mimicked ex vivo in isolated perfused mouse lungs (IPL). Transient receptor potential vanilloid 4 (TRPV4) is a nonselective cation channel studied in endothelium; however, its role in the lung epithelium remains elusive. Here, we show enhanced IRE in TRPV4-deficient (TRPV4-/-) IPL compared with that of WT controls, indicating a protective role of TRPV4 in maintenance of the alveolar epithelial barrier. By immunohistochemistry, mRNA profiling, and electrophysiological characterization, we detected TRPV4 in bronchial epithelium, alveolar epithelial type I (ATI), and alveolar epithelial type II (ATII) cells. Genetic ablation of TRPV4 resulted in reduced expression of the water-conducting aquaporin-5 (AQP-5) channel in ATI cells. Migration of TRPV4-/- ATI cells was reduced, and cell barrier function was impaired. Analysis of isolated primary TRPV4-/- ATII cells revealed a reduced expression of surfactant protein C, and the TRPV4 activator GSK1016790A induced increases in current densities only in WT ATII cells. Moreover, TRPV4-/- lungs of adult mice developed significantly larger mean chord lengths and altered lung function compared with WT lungs. Therefore, our data illustrate essential functions of TRPV4 channels in alveolar epithelial cells and in protection from edema formation.
Subject(s)
Aquaporin 5/genetics , Edema/genetics , Lung Diseases/genetics , Reperfusion Injury/genetics , TRPV Cation Channels/genetics , Alveolar Epithelial Cells/pathology , Animals , Bronchi/metabolism , Bronchi/pathology , Cell Movement/genetics , Disease Models, Animal , Edema/etiology , Edema/pathology , Humans , Leucine/analogs & derivatives , Leucine/pharmacology , Lung Diseases/etiology , Lung Diseases/pathology , Lung Transplantation/adverse effects , Mice , Mice, Knockout , Reperfusion Injury/complications , Reperfusion Injury/pathology , Sulfonamides/pharmacologyABSTRACT
Ion selectivity is a defining feature of a given ion channel and is considered immutable. Here we show that ion selectivity of the lysosomal ion channel TPC2, which is hotly debated (Calcraft et al., 2009; Guo et al., 2017; Jha et al., 2014; Ruas et al., 2015; Wang et al., 2012), depends on the activating ligand. A high-throughput screen identified two structurally distinct TPC2 agonists. One of these evoked robust Ca2+-signals and non-selective cation currents, the other weaker Ca2+-signals and Na+-selective currents. These properties were mirrored by the Ca2+-mobilizing messenger, NAADP and the phosphoinositide, PI(3,5)P2, respectively. Agonist action was differentially inhibited by mutation of a single TPC2 residue and coupled to opposing changes in lysosomal pH and exocytosis. Our findings resolve conflicting reports on the permeability and gating properties of TPC2 and they establish a new paradigm whereby a single ion channel mediates distinct, functionally-relevant ionic signatures on demand.
Subject(s)
Calcium Channel Agonists/pharmacology , Calcium Channels/metabolism , Macrophages/metabolism , Raloxifene Hydrochloride/pharmacology , Animals , Benzylisoquinolines/pharmacology , Calcium/metabolism , Calcium Channel Agonists/chemistry , Calcium Channels/genetics , Fluphenazine/pharmacology , Gene Expression Regulation/drug effects , HEK293 Cells , HeLa Cells , Humans , Ionomycin/pharmacology , Macrophages/drug effects , Mice , NADP/analogs & derivatives , NADP/metabolism , Phosphatidylinositol Phosphates/pharmacology , Single Molecule Imaging , Sodium/metabolismABSTRACT
To resolve the subcellular distribution of endolysosomal ion channels, we have established a novel experimental approach to selectively patch clamp Rab5 positive early endosomes (EE) versus Rab7/LAMP1-positive late endosomes/lysosomes (LE/LY). To functionally characterize ion channels in endolysosomal membranes with the patch-clamp technique, it is important to develop techniques to selectively enlarge the respective organelles. We found here that two small molecules, wortmannin and latrunculin B, enlarge Rab5-positive EE when combined but not Rab7-, LAMP1-, or Rab11 (RE)-positive vesicles. The two compounds act rapidly, specifically, and are readily applicable in contrast to genetic approaches or previously used compounds such as vacuolin, which enlarges EE, RE, and LE/LY. We apply this approach here to measure currents mediated by TRPML channels, in particular TRPML3, which we found to be functionally active in both EE and LE/LY in overexpressing cells as well as in endogenously expressing CD11b+ lung-tissue macrophages.
Subject(s)
Action Potentials/drug effects , Androstadienes/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Endosomes/metabolism , Thiazolidines/pharmacology , Aminopyridines/pharmacology , CD11b Antigen/metabolism , Endosomes/drug effects , HEK293 Cells , Heterocyclic Compounds, 3-Ring/pharmacology , Humans , Lung/cytology , Lung/metabolism , Lysosomal-Associated Membrane Protein 1/metabolism , Lysosomes/drug effects , Lysosomes/metabolism , Macrophages/cytology , Macrophages/drug effects , Macrophages/metabolism , Macrophages, Peritoneal/cytology , Macrophages, Peritoneal/drug effects , Macrophages, Peritoneal/metabolism , Patch-Clamp Techniques , Transient Receptor Potential Channels/genetics , Transient Receptor Potential Channels/metabolism , Wortmannin , rab GTP-Binding Proteins/metabolism , rab5 GTP-Binding Proteins/metabolism , rab7 GTP-Binding ProteinsABSTRACT
According to proteomics analyses, more than 70 different ion channels and transporters are harbored in membranes of intracellular compartments such as endosomes and lysosomes. Malfunctioning of these channels has been implicated in human diseases such as lysosomal storage disorders, neurodegenerative diseases and metabolic pathologies, as well as in the progression of certain infectious diseases. As a consequence, these channels have engendered very high interest as future drug targets. Detailed electrophysiological characterization of intracellular ion channels is lacking, mainly because standard methods to analyze plasma membrane ion channels, such as the patch-clamp technique, are not readily applicable to intracellular organelles. Here we present a protocol detailing how to implement a manual patch-clamp technique for endolysosomal compartments. In contrast to the alternatively used planar endolysosomal patch-clamp technique, this method is a visually controlled, direct patch-clamp technique similar to conventional patch-clamping. The protocol assumes basic knowledge and experience with patch-clamp methods. Implementation of the method requires up to 1 week, and material preparation takes â¼2-4 d. An individual experiment (i.e., measurement of channel currents across the endolysosomal membrane), including control experiments, can be completed within 1 h. This excludes the time for endolysosome enlargement, which takes between 1 and 48 h, depending on the approach and cell type used. Data analysis requires an additional hour.
Subject(s)
Endosomes/metabolism , Ion Channels/metabolism , Lysosomes/metabolism , Patch-Clamp Techniques/methods , Animals , Cells, Cultured , Humans , MiceABSTRACT
Metastatic invasion is the major cause of cancer-related deaths. In this study, we introduce two-pore channels (TPC), a recently described class of NAADP- and PI(3,5)P2-sensitive Ca2+-permeable cation channels in the endolysosomal system of cells, as candidate targets for the treatment of invasive cancers. Inhibition of the channel abrogated migration of metastatic cancer cells in vitro Silencing or pharmacologic inhibition of the two-pore channel TPC2 reduced lung metastasis of mammary mouse cancer cells. Disrupting TPC function halted trafficking of ß1-integrin, leading to its accumulation in EEA1-positive early endosomes. As a consequence, invasive cancer cells were no longer able to form leading edges, which are required for adequate migration. Our findings link TPC to cancer cell migration and provide a preclinical proof of concept for their candidacy as targets to treat metastatic cancers. Cancer Res; 77(6); 1427-38. ©2017 AACR.