RESUMO
Oocytes are among the longest-lived cells in the body and need to preserve their cytoplasm to support proper embryonic development. Protein aggregation is a major threat for intracellular homeostasis in long-lived cells. How oocytes cope with protein aggregation during their extended life is unknown. Here, we find that mouse oocytes accumulate protein aggregates in specialized compartments that we named endolysosomal vesicular assemblies (ELVAs). Combining live-cell imaging, electron microscopy, and proteomics, we found that ELVAs are non-membrane-bound compartments composed of endolysosomes, autophagosomes, and proteasomes held together by a protein matrix formed by RUFY1. Functional assays revealed that in immature oocytes, ELVAs sequester aggregated proteins, including TDP-43, and degrade them upon oocyte maturation. Inhibiting degradative activity in ELVAs leads to the accumulation of protein aggregates in the embryo and is detrimental for embryo survival. Thus, ELVAs represent a strategy to safeguard protein homeostasis in long-lived cells.
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Vesículas Citoplasmáticas , Oócitos , Agregados Proteicos , Animais , Feminino , Camundongos , Autofagossomos , Vesículas Citoplasmáticas/metabolismo , Lisossomos/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Complexo de Endopeptidases do Proteassoma , ProteóliseRESUMO
Lysosomes require an acidic lumen between pH 4.5 and 5.0 for effective digestion of macromolecules. This pH optimum is maintained by proton influx produced by the V-ATPase and efflux through an unidentified "H+ leak" pathway. Here we show that TMEM175, a genetic risk factor for Parkinson's disease (PD), mediates the lysosomal H+ leak by acting as a proton-activated, proton-selective channel on the lysosomal membrane (LyPAP). Acidification beyond the normal range potently activated LyPAP to terminate further acidification of lysosomes. An endogenous polyunsaturated fatty acid and synthetic agonists also activated TMEM175 to trigger lysosomal proton release. TMEM175 deficiency caused lysosomal over-acidification, impaired proteolytic activity, and facilitated α-synuclein aggregation in vivo. Mutational and pH normalization analyses indicated that the channel's H+ conductance is essential for normal lysosome function. Thus, modulation of LyPAP by cellular cues may dynamically tune the pH optima of endosomes and lysosomes to regulate lysosomal degradation and PD pathology.
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Doença de Parkinson , Endossomos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Membranas Intracelulares/metabolismo , Lisossomos/metabolismo , Doença de Parkinson/metabolismo , Canais de Potássio/metabolismo , PrótonsRESUMO
ß-Coronaviruses are a family of positive-strand enveloped RNA viruses that includes the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Much is known regarding their cellular entry and replication pathways, but their mode of egress remains uncertain. Using imaging methodologies and virus-specific reporters, we demonstrate that ß-coronaviruses utilize lysosomal trafficking for egress rather than the biosynthetic secretory pathway more commonly used by other enveloped viruses. This unconventional egress is regulated by the Arf-like small GTPase Arl8b and can be blocked by the Rab7 GTPase competitive inhibitor CID1067700. Such non-lytic release of ß-coronaviruses results in lysosome deacidification, inactivation of lysosomal degradation enzymes, and disruption of antigen presentation pathways. ß-Coronavirus-induced exploitation of lysosomal organelles for egress provides insights into the cellular and immunological abnormalities observed in patients and suggests new therapeutic modalities.
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COVID-19/metabolismo , SARS-CoV-2/metabolismo , Via Secretória , Liberação de Vírus , Fatores de Ribosilação do ADP/metabolismo , Animais , COVID-19/patologia , Feminino , Células HeLa , Compostos Heterocíclicos com 2 Anéis/farmacologia , Humanos , Lisossomos , Camundongos , Tioureia/análogos & derivados , Tioureia/farmacologia , Proteínas rab de Ligação ao GTP/antagonistas & inibidores , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7 , Tratamento Farmacológico da COVID-19RESUMO
Maintaining a highly acidic lysosomal pH is central to cellular physiology. Here, we use functional proteomics, single-particle cryo-EM, electrophysiology, and in vivo imaging to unravel a key biological function of human lysosome-associated membrane proteins (LAMP-1 and LAMP-2) in regulating lysosomal pH homeostasis. Despite being widely used as a lysosomal marker, the physiological functions of the LAMP proteins have long been overlooked. We show that LAMP-1 and LAMP-2 directly interact with and inhibit the activity of the lysosomal cation channel TMEM175, a key player in lysosomal pH homeostasis implicated in Parkinson's disease. This LAMP inhibition mitigates the proton conduction of TMEM175 and facilitates lysosomal acidification to a lower pH environment crucial for optimal hydrolase activity. Disrupting the LAMP-TMEM175 interaction alkalinizes the lysosomal pH and compromises the lysosomal hydrolytic function. In light of the ever-increasing importance of lysosomes to cellular physiology and diseases, our data have widespread implications for lysosomal biology.
Assuntos
Doença de Parkinson , Humanos , Concentração de Íons de Hidrogênio , Proteínas de Membrana Lisossomal/genética , Proteínas de Membrana Lisossomal/metabolismo , Lisossomos/metabolismo , Doença de Parkinson/metabolismo , Canais de Potássio/metabolismoRESUMO
Vacuolar acidification is essential for vacuoles in diverse physiological functions. However, its role in plant defense, and whether and how pathogens affect vacuolar acidification to promote infection remain unknown. Here, we show that Barley stripe mosaic virus (BSMV) replicase γa, but not its mutant γaR569A , directly blocks acidification of vacuolar lumen and suppresses autophagic degradation to promote viral infection in plants. These were achieved via molecular interaction between γa and V-ATPase catalytic subunit B2 (VHA-B2), leading to disruption of the interaction between VHA-B2 and V-ATPase catalytic subunit E (VHA-E), which impairs the membrane localization of VHA-B2 and suppresses V-ATPase activity. Furthermore, a mutant virus BSMVR569A with the R569A point mutation possesses less viral pathogenicity. Interestingly, multiple viral infections block vacuolar acidification. These findings reveal that functional vacuolar acidification is required for plant antiviral defense and disruption of vacuolar acidification could be a general viral counter-defense strategy employed by multiple viruses.
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Nicotiana/virologia , Vírus de Plantas/patogenicidade , Vacúolos/metabolismo , Proteínas do Complexo da Replicase Viral/metabolismo , Proteínas de Plantas/metabolismo , Vírus de Plantas/fisiologia , Ligação Proteica , ATPases Vacuolares Próton-Translocadoras/metabolismo , Vacúolos/virologia , Proteínas do Complexo da Replicase Viral/química , Replicação ViralRESUMO
Endosomal-lysosomal trafficking is accompanied by the acidification of endosomal compartments by the H+-V-ATPase to reach low lysosomal pH. Disruption of the correct pH impairs lysosomal function and the balance of protein synthesis and degradation (proteostasis). Here, we treated mammalian cells with the small dipeptide LLOMe, which is known to permeabilize lysosomal membranes, and find that LLOMe also impacts late endosomes (LEs) by neutralizing their pH without causing membrane permeabilization. We show that LLOMe leads to hyperactivation of Rab7 (herein referring to Rab7a), and disruption of tubulation and mannose-6-phosphate receptor (CI-M6PR; also known as IGF2R) recycling on pH-neutralized LEs. pH neutralization (NH4Cl) and expression of Rab7 hyperactive mutants alone can both phenocopy the alterations in tubulation and CI-M6PR trafficking. Mechanistically, pH neutralization increases the assembly of the V1G1 subunit (encoded by ATP6V1G1) of the V-ATPase on endosomal membranes, which stabilizes GTP-bound Rab7 via RILP, a known interactor of Rab7 and V1G1. We propose a novel pathway by which V-ATPase and RILP modulate LE pH and Rab7 activation in concert. This pathway might broadly contribute to pH control during physiologic endosomal maturation or starvation and during pathologic pH neutralization, which occurs via lysosomotropic compounds and in disease states.
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Proteínas Adaptadoras de Transdução de Sinal , Endossomos , ATPases Vacuolares Próton-Translocadoras , proteínas de unión al GTP Rab7 , Animais , Humanos , Endossomos/metabolismo , Células HeLa , Concentração de Íons de Hidrogênio , Lisossomos/metabolismo , Transporte Proteico , Receptor IGF Tipo 2/metabolismo , Receptor IGF Tipo 2/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo , ATPases Vacuolares Próton-Translocadoras/genéticaRESUMO
Together with its ß-subunit OSTM1, ClC-7 performs 2Cl-/H+ exchange across lysosomal membranes. Pathogenic variants in either gene cause lysosome-related pathologies, including osteopetrosis and lysosomal storage. CLCN7 variants can cause recessive or dominant disease. Different variants entail different sets of symptoms. Loss of ClC-7 causes osteopetrosis and mostly neuronal lysosomal storage. A recently reported de novo CLCN7 mutation (p.Tyr715Cys) causes widespread severe lysosome pathology (hypopigmentation, organomegaly, and delayed myelination and development, "HOD syndrome"), but no osteopetrosis. We now describe two additional HOD individuals with the previously described p.Tyr715Cys and a novel p.Lys285Thr mutation, respectively. Both mutations decreased ClC-7 inhibition by PI(3,5)P2 and affected residues lining its binding pocket, and shifted voltage-dependent gating to less positive potentials, an effect partially conferred to WT subunits in WT/mutant heteromers. This shift predicts augmented pH gradient-driven Cl- uptake into vesicles. Overexpressing either mutant induced large lysosome-related vacuoles. This effect depended on Cl-/H+-exchange, as shown using mutants carrying uncoupling mutations. Fibroblasts from the p.Y715C patient also displayed giant vacuoles. This was not observed with p.K285T fibroblasts probably due to residual PI(3,5)P2 sensitivity. The gain of function caused by the shifted voltage-dependence of either mutant likely is the main pathogenic factor. Loss of PI(3,5)P2 inhibition will further increase current amplitudes, but may not be a general feature of HOD. Overactivity of ClC-7 induces pathologically enlarged vacuoles in many tissues, which is distinct from lysosomal storage observed with the loss of ClC-7 function. Osteopetrosis results from a loss of ClC-7, but osteoclasts remain resilient to increased ClC-7 activity.
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Canais de Cloreto , Doenças por Armazenamento dos Lisossomos , Lisossomos , Humanos , Masculino , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Mutação com Ganho de Função , Células HEK293 , Doenças por Armazenamento dos Lisossomos/genética , Doenças por Armazenamento dos Lisossomos/metabolismo , Doenças por Armazenamento dos Lisossomos/patologia , Lisossomos/metabolismo , Lisossomos/genética , Proteínas de Membrana , Mutação de Sentido Incorreto , Fosfatos de Fosfatidilinositol/metabolismo , Ubiquitina-Proteína Ligases , Vacúolos/metabolismo , Vacúolos/genética , Vacúolos/patologiaRESUMO
Lysosomes are key organelles maintaining cellular homeostasis in health and disease. Here, we report the identification of N-deacetylase and N-sulfotransferase 3 (NDST3) as a potent regulator of lysosomal functions through an unbiased genetic screen. NDST3 constitutes a new member of the histone deacetylase (HDAC) family and catalyzes the deacetylation of α-tubulin. Loss of NDST3 promotes assembly of the V-ATPase holoenzyme on the lysosomal membrane and thereby increases the acidification of the organelle. NDST3 is downregulated in tissues and cells from patients carrying the C9orf72 hexanucleotide repeat expansion linked to the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Deficiency in C9orf72 decreases the level of NDST3, and downregulation of NDST3 exacerbates the proteotoxicity of poly-dipeptides generated from the C9orf72 hexanucleotide repeats. These results demonstrate a previously unknown regulatory mechanism through which microtubule acetylation regulates lysosomal activities and suggest that NDST3 could be targeted to modulate microtubule and lysosomal functions in relevant diseases.
Assuntos
Lisossomos/metabolismo , Sulfotransferases/metabolismo , Tubulina (Proteína)/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Acetilação , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Imunofluorescência , Expressão Gênica , Técnicas de Silenciamento de Genes , Biblioteca Gênica , Genes Reporter , Humanos , Concentração de Íons de Hidrogênio , Macrolídeos/farmacologia , Macrolídeos/toxicidade , Camundongos , Microtúbulos/metabolismo , Modelos Biológicos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Testes Farmacogenômicos/métodos , Variantes Farmacogenômicos , Ligação Proteica , Sulfotransferases/genética , ATPases Vacuolares Próton-Translocadoras/genéticaRESUMO
Coccolithophores are major producers of ocean biogenic calcite, but this process is predicted to be negatively affected by future ocean acidification scenarios. Since coccolithophores calcify intracellularly, the mechanisms through which changes in seawater carbonate chemistry affect calcification remain unclear. Here we show that voltage-gated H+ channels in the plasma membrane of Coccolithus braarudii serve to regulate pH and maintain calcification under normal conditions but have greatly reduced activity in cells acclimated to low pH. This disrupts intracellular pH homeostasis and impairs the ability of C. braarudii to remove H+ generated by the calcification process, leading to specific coccolith malformations. These coccolith malformations can be reproduced by pharmacological inhibition of H+ channels. Heavily calcified coccolithophore species such as C. braarudii, which make the major contribution to carbonate export to the deep ocean, have a large intracellular H+ load and are likely to be most vulnerable to future decreases in ocean pH.
Assuntos
Fitoplâncton , Água do Mar , Calcificação Fisiológica , Carbonatos , Homeostase , Concentração de Íons de Hidrogênio , Oceanos e MaresRESUMO
Ocean warming and acidification driven by anthropogenic carbon emissions pose an existential threat to marine calcifying communities. A similar perturbation to global carbon cycling and ocean chemistry occurred â¼56 Ma during the Paleocene-Eocene thermal maximum (PETM), but microfossil records of the marine biotic response are distorted by sediment mixing. Here, we use the carbon isotope excursion marking the PETM to distinguish planktic foraminifer shells calcified during the PETM from those calcified prior to the event and then isotopically filter anachronous specimens from the PETM microfossil assemblages. We find that nearly one-half of foraminifer shells in a deep-sea PETM record from the central Pacific (Ocean Drilling Program Site 865) are reworked contaminants. Contrary to previous interpretations, corrected assemblages reveal a transient but significant decrease in tropical planktic foraminifer diversity at this open-ocean site during the PETM. The decrease in local diversity was caused by extirpation of shallow- and deep-dwelling taxa as they underwent extratropical migrations in response to heat stress, with one prominent lineage showing signs of impaired calcification possibly due to ocean acidification. An absence of subbotinids in the corrected assemblages suggests that ocean deoxygenation may have rendered thermocline depths uninhabitable for some deeper-dwelling taxa. Latitudinal range shifts provided a rapid-response survival mechanism for tropical planktic foraminifers during the PETM, but the rapidity of ocean warming and acidification projected for the coming centuries will likely strain the adaptability of these resilient calcifiers.
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Ácidos/química , Aquecimento Global , Plâncton , Planeta Terra , Fósseis , IsótoposRESUMO
A better understanding of how environmental change will affect species interactions would significantly aid efforts to scale up predictions of near-future responses to global change from individuals to ecosystems. To address this need, we used meta-analysis to quantify the individual and combined effects of ocean acidification (OA) and warming on consumption rates of predators and herbivores in marine ecosystems. Although the primary studies demonstrated that these environmental variables can have direct effects on consumers, our analyses highlight high variability in consumption rates in response to OA and warming. This variability likely reflects differences in local adaptation among species, as well as important methodological differences. For example, our results suggest that exposure of consumers to OA reduces consumption rates on average, yet consumption rates actually increase when both consumers and their resource(s) are concurrently exposed to the same conditions. We hypothesize that this disparity is due to increased vulnerability of prey or resource(s) in conditions of OA that offset declines in consumption. This hypothesis is supported by an analysis demonstrating clear declines in prey survival in studies that exposed only prey to future OA conditions. Our results illustrate how simultaneous OA and warming produce complex outcomes when species interact. Researchers should further explore other potential sources of variation in response, as well as the prey-driven component of any changes in consumption and the potential for interactive effects of OA and warming.
Assuntos
Ecossistema , Fenômenos Fisiológicos , Mudança Climática , Aquecimento Global , Concentração de Íons de Hidrogênio , Oceanos e Mares , Água do MarRESUMO
Many calcifying organisms utilize metabolic CO2 to generate CaCO3 minerals to harden their shells and skeletons. Carbonic anhydrases are evolutionary ancient enzymes that have been proposed to play a key role in the calcification process, with the underlying mechanisms being little understood. Here, we used the calcifying primary mesenchyme cells (PMCs) of sea urchin larva to study the role of cytosolic (iCAs) and extracellular carbonic anhydrases (eCAs) in the cellular carbon concentration mechanism (CCM). Molecular analyses identified iCAs and eCAs in PMCs and highlight the prominent expression of a glycosylphosphatidylinositol-anchored membrane-bound CA (Cara7). Intracellular pH recordings in combination with CO2 pulse experiments demonstrated iCA activity in PMCs. iCA activity measurements, together with pharmacological approaches, revealed an opposing contribution of iCAs and eCAs on the CCM. H+-selective electrodes were used to demonstrate eCA-catalyzed CO2 hydration rates at the cell surface. Knockdown of Cara7 reduced extracellular CO2 hydration rates accompanied by impaired formation of specific skeletal segments. Finally, reduced pHi regulatory capacities during inhibition and knockdown of Cara7 underscore a role of this eCA in cellular HCO3- uptake. This work reveals the function of CAs in the cellular CCM of a marine calcifying animal. Extracellular hydration of metabolic CO2 by Cara7 coupled to HCO3- uptake mechanisms mitigates the loss of carbon and reduces the cellular proton load during the mineralization process. The findings of this work provide insights into the cellular mechanisms of an ancient biological process that is capable of utilizing CO2 to generate a versatile construction material.
Assuntos
Calcificação Fisiológica , Dióxido de Carbono , Carbono , Inibidores da Anidrase Carbônica , Anidrases Carbônicas , Ouriços-do-Mar , Animais , Bicarbonatos/metabolismo , Carbono/metabolismo , Dióxido de Carbono/metabolismo , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/genética , Anidrases Carbônicas/metabolismo , Glicosilfosfatidilinositóis , Concentração de Íons de Hidrogênio , Prótons , Ouriços-do-Mar/enzimologiaRESUMO
BACKGROUND: The VPS50 protein functions in synaptic and dense core vesicle acidification, and perturbations of VPS50 function produce behavioral changes in Caenorhabditis elegans. Patients with mutations in VPS50 show severe developmental delay and intellectual disability, characteristics that have been associated with autism spectrum disorders (ASDs). The mechanisms that link VPS50 mutations to ASD are unknown. RESULTS: To examine the role of VPS50 in mammalian brain function and behavior, we used the CRISPR/Cas9 system to generate knockouts of VPS50 in both cultured murine cortical neurons and living mice. In cultured neurons, KO of VPS50 did not affect the number of synaptic vesicles but did cause mislocalization of the V-ATPase V1 domain pump and impaired synaptic activity, likely as a consequence of defects in vesicle acidification and vesicle content. In mice, mosaic KO of VPS50 in the hippocampus altered synaptic transmission and plasticity and generated robust cognitive impairments. CONCLUSIONS: We propose that VPS50 functions as an accessory protein to aid the recruitment of the V-ATPase V1 domain to synaptic vesicles and in that way plays a crucial role in controlling synaptic vesicle acidification. Understanding the mechanisms controlling behaviors and synaptic function in ASD-associated mutations is pivotal for the development of targeted interventions, which may open new avenues for therapeutic strategies aimed at ASD and related conditions.
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Camundongos Knockout , Vesículas Sinápticas , Animais , Camundongos , Comportamento Animal/fisiologia , Encéfalo/metabolismo , Neurônios/metabolismo , Neurônios/fisiologia , Sinapses/metabolismo , Sinapses/fisiologia , Transmissão Sináptica , Vesículas Sinápticas/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
BACKGROUND: Endolysosomal compartments are acidic and contain low pH-dependent proteases, and these conditions are exploited by respiratory viruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza virus, for escaping into the cytosol. Moreover, endolysosomes contain various pattern recognition receptors (PRRs), which respond to virus-derived pathogen-associated molecular patterns (PAMPs) by production of proinflammatory cytokines/chemokines. However, excessive proinflammatory responses can lead to a potentially lethal cytokine storm. OBJECTIVES: Here we investigated the endosomal PRR expression profile in primary human small airway epithelial cells (HSAECs), and whether blockade of endolysosomal acidification affects their cytokine/chemokine production after challenge with virus-derived stimulants. METHODS: HSAECs were exposed to stimulants mimicking virus-derived PAMPs, either in the absence or presence of compounds causing blockade of endolysosomal acidification, followed by measurement of cytokine expression and release. RESULTS: We show that Toll-like receptor 3 (TLR3) is the major endosomal PRR expressed by HSAECs, and that TLR3 expression is strongly induced by TLR3 agonists, but not by a range of other PRR agonists. We also demonstrate that TLR3 engagement with its agonists elicits a robust proinflammatory cytokine/chemokine response, which is profoundly suppressed through blockade of endolysosomal acidification, by bafilomycin A1, monensin, or niclosamide. Using TLR3 reporter cells, it was confirmed that TLR3 signaling is strongly induced by Poly(I:C) and that blockade of endolysosomal acidification efficiently blocked TLR3 signaling. Finally, we show that blockade of endolysosomal acidification causes a reduction in the levels of TLR3 mRNA and protein. CONCLUSIONS: These findings show that blockade of endolysosomal acidification suppresses TLR3-dependent cytokine and chemokine production in HSAECs.
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Immune checkpoint inhibitors and adoptive T cell therapies have been valuable additions to the toolbox in the fight against cancer. These treatments have profoundly increased the number of patients with a realistic perspective toward a return to a cancer-free life. Yet, in a number of patients and tumor entities, cancer immunotherapies have been ineffective so far. In solid tumors, immune exclusion and the immunosuppressive tumor microenvironment represent substantial roadblocks to successful therapeutic outcomes. A major contributing factor to the depressed anti-tumor activity of immune cells in tumors is the harsh metabolic environment. Hypoxia, nutrient competition with tumor and stromal cells, and accumulating noxious waste products, including lactic acid, pose massive constraints to anti-tumor immune cells. Numerous strategies are being developed to exploit the metabolic vulnerabilities of tumor cells in the hope that these would also alleviate metabolism-inflicted immune suppression. While promising in principle, especially in combination with immunotherapies, these strategies need to be scrutinized for their effect on tumor-fighting immune cells, which share some of their key metabolic properties with tumor cells. Here, we provide an overview of strategies that seek to tackle lactate metabolism in tumor or immune cells to unleash anti-tumor immune responses, thereby opening therapeutic options for patients whose tumors are currently not treatable.
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Imunoterapia , Neoplasias , Humanos , Neoplasias/metabolismo , Lactatos , Microambiente TumoralRESUMO
Marine fishes excrete excess H+ using basolateral Na+/K+-ATPase (NKA) and apical Na+/H+-exchanger 3 (NHE3) in gill ionocytes. However, the mechanisms that regulate H+ excretion during exposure to environmentally relevant hypercapnia (ERH) remain poorly understood. Here, we explored transcriptomic, proteomic, and cellular responses in gills of juvenile splitnose rockfish (Sebastes diploproa) exposed to three days of ERH conditions (pH ~7.5; ~1,600 µatm pCO2). Blood pH was fully regulated at ~7.75 despite a lack of significant changes in gill (1) mRNAs coding for proteins involved in blood acid-base regulation, (2) total NKA and NHE3 protein abundance, and (3) ionocyte density. However, ERH-exposed rockfish demonstrated increased NKA and NHE3 abundance on the ionocyte plasma membrane coupled with wider apical membranes and greater extension of apical microvilli. The observed gill ionocyte remodeling is consistent with enhanced H+ excretion that maintains blood pH homeostasis during exposure to ERH and does not necessitate changes at the expression or translation levels. These mechanisms of phenotypic plasticity may allow fishes to regulate blood pH during environmentally relevant acid-base challenges, and thus have important implications for both understanding how organisms respond to climate change and for selecting appropriate metrics to evaluate its impact on marine ecosystems.
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The Dja2 knockout (Dja2-/- ) mice had respiratory distress, and >60% died within 2 days after birth. The surviving adult Dja2-/- mice were infertile and the lungs of Dja2-/- mice showed several abnormalities, including the processing defect of prosurfactant protein C in the alveolar epithelial type II cells and the accumulation of glycolipids in enlarged alveolar macrophages. The luminal pH of acidic organelles in Dja2-/- cells was shifted to pH 5.37-5.45. This deviated pH was immediately restored to control levels (pH 4.56-4.65) by the addition of a diuretic, ethyl isopropyl amiloride (EIPA). Although the role of DJA2 in maintaining the pH homeostasis of lysosome-related organelles is currently obscure, this rapid and remarkable pH resilience is best explained by an EIPA-sensitive proton efflux machinery that is disorganized and overactivated due to the loss of Dja2.
Assuntos
Lisossomos , Prótons , Animais , Camundongos , Transporte Biológico , Concentração de Íons de Hidrogênio , Lisossomos/metabolismo , Macrófagos Alveolares , Camundongos Endogâmicos C57BLRESUMO
Intercalated cells (ICs) in the kidney collecting duct have a versatile role in acid-base and electrolyte regulation along with the host immune defense. Located in the terminal kidney tubule segment, ICs are among the first kidney cells to encounter bacteria when bacteria ascend from the bladder into the kidney. ICs have developed several mechanisms to combat bacterial infections of the kidneys. For example, ICs produce antimicrobial peptides (AMPs), which have direct bactericidal activity, and in many cases are upregulated in response to infections. Some AMP genes with IC-specific kidney expression are multiallelic, and having more copies of the gene confers increased resistance to bacterial infections of the kidney and urinary tract. Similarly, studies in human children demonstrate that those with history of UTIs are more likely to have single-nucleotide polymorphisms in IC-expressed AMP genes that impair the AMP's bactericidal activity. In murine models, depleted or impaired ICs result in decreased clearance of bacterial load following transurethral challenge with uropathogenic E. coli. A 2021 study demonstrated that ICs even act as phagocytes and acidify bacteria within phagolysosomes. Several immune signaling pathways have been identified in ICs which may represent future therapeutic targets in managing kidney infections or inflammation. This review's objective is to highlight IC structure and function with an emphasis on current knowledge of IC's diverse innate immune capabilities.
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Infecções Bacterianas , Túbulos Renais Coletores , Infecções Urinárias , Criança , Camundongos , Humanos , Animais , Escherichia coli , Rim/metabolismo , Infecções Urinárias/metabolismo , Infecções Urinárias/microbiologia , Túbulos Renais Coletores/metabolismo , Imunidade Inata , Infecções Bacterianas/metabolismoRESUMO
Cell culture-based screening of a chemical library identified diphenoxylate as an antiviral agent against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The observed 50% effective concentrations ranged between 1.4 and 4.9 µM against the original wild-type strain and its variants. Time-of-addition experiments indicated that diphenoxylate is an entry blocker targeting a host factor involved in viral infection. Fluorescence microscopic analysis visualized that diphenoxylate prevented SARS-CoV-2 particles from penetrating the cell membrane and also impaired endo-lysosomal acidification. Diphenoxylate exhibited a synergistic inhibitory effect on SARS-CoV-2 infection in human lung epithelial Calu-3 cells when combined with a transmembrane serine protease 2 (TMPRSS2) inhibitor, nafamostat. This synergy suggested that efficient antiviral activity is achieved by blocking both TMPRSS2-mediated early and endosome-mediated late SARS-CoV-2 entry pathways. The antiviral efficacy of diphenoxylate against SARS-CoV-2 was reproducible in a human tonsil organoids system. In a transgenic mouse model expressing the obligate SARS-CoV-2 receptor, human angiotensin-converting enzyme 2, intranasal administration of diphenoxylate (10 mg/kg/day) significantly reduced the viral RNA copy number in the lungs by 70% on day 3. This study underscores that diphenoxylate represents a promising core scaffold, warranting further exploration for chemical modifications aimed at developing a new class of clinically effective antiviral drugs against SARS-CoV-2.
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The genetic causes of global developmental delay (GDD) and intellectual disability (ID) are diverse and include variants in numerous ion channels and transporters. Loss-of-function variants in all five endosomal/lysosomal members of the CLC family of Cl- channels and Cl-/H+ exchangers lead to pathology in mice, humans, or both. We have identified nine variants in CLCN3, the gene encoding CIC-3, in 11 individuals with GDD/ID and neurodevelopmental disorders of varying severity. In addition to a homozygous frameshift variant in two siblings, we identified eight different heterozygous de novo missense variants. All have GDD/ID, mood or behavioral disorders, and dysmorphic features; 9/11 have structural brain abnormalities; and 6/11 have seizures. The homozygous variants are predicted to cause loss of ClC-3 function, resulting in severe neurological disease similar to the phenotype observed in Clcn3-/- mice. Their MRIs show possible neurodegeneration with thin corpora callosa and decreased white matter volumes. Individuals with heterozygous variants had a range of neurodevelopmental anomalies including agenesis of the corpus callosum, pons hypoplasia, and increased gyral folding. To characterize the altered function of the exchanger, electrophysiological analyses were performed in Xenopus oocytes and mammalian cells. Two variants, p.Ile607Thr and p.Thr570Ile, had increased currents at negative cytoplasmic voltages and loss of inhibition by luminal acidic pH. In contrast, two other variants showed no significant difference in the current properties. Overall, our work establishes a role for CLCN3 in human neurodevelopment and shows that both homozygous loss of ClC-3 and heterozygous variants can lead to GDD/ID and neuroanatomical abnormalities.