RESUMEN
Upon detecting pathogens or cell stress, several NOD-like receptors (NLRs) form inflammasome complexes with the adapter ASC and caspase-1, inducing gasdermin D (GSDMD)-dependent cell death and maturation and release of IL-1ß and IL-18. The triggers and activation mechanisms of several inflammasome-forming sensors are not well understood. Here we show that mitochondrial damage activates the NLRP10 inflammasome, leading to ASC speck formation and caspase-1-dependent cytokine release. While the AIM2 inflammasome can also sense mitochondrial demise by detecting mitochondrial DNA (mtDNA) in the cytosol, NLRP10 monitors mitochondrial integrity in an mtDNA-independent manner, suggesting the recognition of distinct molecular entities displayed by the damaged organelles. NLRP10 is highly expressed in differentiated human keratinocytes, in which it can also assemble an inflammasome. Our study shows that this inflammasome surveils mitochondrial integrity. These findings might also lead to a better understanding of mitochondria-linked inflammatory diseases.
Asunto(s)
Citocinas , Inflamasomas , Humanos , Inflamasomas/metabolismo , Caspasa 1/metabolismo , Citocinas/metabolismo , Muerte Celular , ADN Mitocondrial/genética , Interleucina-1beta/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismoRESUMEN
Primary cilia project from the surface of most vertebrate cells and are key in sensing extracellular signals and locally transducing this information into a cellular response. Recent findings show that primary cilia are not merely static organelles with a distinct lipid and protein composition. Instead, the function of primary cilia relies on the dynamic composition of molecules within the cilium, the context-dependent sensing and processing of extracellular stimuli, and cycles of assembly and disassembly in a cell- and tissue-specific manner. Thereby, primary cilia dynamically integrate different cellular inputs and control cell fate and function during tissue development. Here, we review the recently emerging concept of primary cilia dynamics in tissue development, organization, remodeling, and function.
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Cilios , Orgánulos , Cilios/metabolismo , Diferenciación CelularRESUMEN
Primary cilia are hair-like protrusions of the plasma membrane that function as cellular antennae and are present on most cells in the human body. Primary cilia dysfunction leads to severe diseases, commonly termed 'ciliopathies'. A significant symptom of certain ciliopathies is obesity, and current research aims to identify contributing mechanisms of obesity development in these patients. Western lifestyle-associated factors can trigger chronic inflammation, or metaflammation, which can also attribute to obesity-associated metabolic disorders. However, obese individuals can also be 'metabolically healthy', as discussed for a subset of patients with obesity and ciliopathy. Here, we propose that primary cilia signaling might modulate specific immune cell phenotypes, behaviors, and functions, which might impact inflammatory responses in the context of ciliopathies and beyond.
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Cilios , Ciliopatías , Cilios/metabolismo , Ciliopatías/genética , Ciliopatías/metabolismo , Humanos , Obesidad , Transducción de SeñalRESUMEN
Navigation of sperm in fluid flow, called rheotaxis, provides long-range guidance in the mammalian oviduct. The rotation of sperm around their longitudinal axis (rolling) promotes rheotaxis. Whether sperm rolling and rheotaxis require calcium (Ca2+ ) influx via the sperm-specific Ca2+ channel CatSper, or rather represent passive biomechanical and hydrodynamic processes, has remained controversial. Here, we study the swimming behavior of sperm from healthy donors and from infertile patients that lack functional CatSper channels, using dark-field microscopy, optical tweezers, and microfluidics. We demonstrate that rolling and rheotaxis persist in CatSper-deficient human sperm. Furthermore, human sperm undergo rolling and rheotaxis even when Ca2+ influx is prevented. Finally, we show that rolling and rheotaxis also persist in mouse sperm deficient in both CatSper and flagellar Ca2+ -signaling domains. Our results strongly support the concept that passive biomechanical and hydrodynamic processes enable sperm rolling and rheotaxis, rather than calcium signaling mediated by CatSper or other mechanisms controlling transmembrane Ca2+ flux.
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Hidrodinámica , Motilidad Espermática , Espermatozoides/fisiología , Animales , Fenómenos Biomecánicos , Calcio/metabolismo , Canales de Calcio/genética , Canales de Calcio/metabolismo , Señalización del Calcio , Humanos , Masculino , Ratones , Proteínas de Plasma Seminal/genética , Proteínas de Plasma Seminal/metabolismoRESUMEN
The primary cilium constitutes an organelle that orchestrates signal transduction independently from the cell body. Dysregulation of this intricate molecular architecture leads to severe human diseases, commonly referred to as ciliopathies. However, the molecular underpinnings how ciliary signaling orchestrates a specific cellular output remain elusive. By combining spatially resolved optogenetics with RNA sequencing and imaging, we reveal a novel cAMP signalosome that is functionally distinct from the cytoplasm. We identify the genes and pathways targeted by the ciliary cAMP signalosome and shed light on the underlying mechanisms and downstream signaling. We reveal that chronic stimulation of the ciliary cAMP signalosome transforms kidney epithelia from tubules into cysts. Counteracting this chronic cAMP elevation in the cilium by small molecules targeting activation of phosphodiesterase-4 long isoforms inhibits cyst growth. Thereby, we identify a novel concept of how the primary cilium controls cellular functions and maintains tissue integrity in a specific and spatially distinct manner and reveal novel molecular components that might be involved in the development of one of the most common genetic diseases, polycystic kidney disease.
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Quistes , Enfermedades Renales Poliquísticas , Cilios/metabolismo , Quistes/metabolismo , Expresión Génica , Humanos , Riñón , Enfermedades Renales Poliquísticas/genética , Enfermedades Renales Poliquísticas/metabolismoRESUMEN
Cell survival, tissue integrity and organismal health depend on the ability to maintain functional protein networks even under conditions that threaten protein integrity. Protection against such stress conditions involves the adaptation of folding and degradation machineries, which help to preserve the protein network by facilitating the refolding or disposal of damaged proteins. In multicellular organisms, cells are permanently exposed to stress resulting from mechanical forces. Yet, for long time mechanical stress was not recognized as a primary stressor that perturbs protein structure and threatens proteome integrity. The identification and characterization of protein folding and degradation systems, which handle force-unfolded proteins, marks a turning point in this regard. It has become apparent that mechanical stress protection operates during cell differentiation, adhesion and migration and is essential for maintaining tissues such as skeletal muscle, heart and kidney as well as the immune system. Here, we provide an overview of recent advances in our understanding of mechanical stress protection.
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Pliegue de Proteína , Proteostasis , Supervivencia Celular , Proteoma/metabolismo , Estrés MecánicoRESUMEN
The flagellum is essential for sperm motility and fertilization in vivo. The axoneme is the main component of the flagella, extending through its entire length. An axoneme is comprised of two central microtubules surrounded by nine doublets, the nexin-dynein regulatory complex, radial spokes, and dynein arms. Failure to properly assemble components of the axoneme in a sperm flagellum, leads to fertility alterations. To understand this process in detail, we have defined the function of an uncharacterized gene, Cfap97 domain containing 1 (Cfap97d1). This gene is evolutionarily conserved in mammals and multiple other species, including Chlamydomonas. We have used two independently generated Cfap97d1 knockout mouse models to study the gene function in vivo. Cfap97d1 is exclusively expressed in testes starting from post-natal day 20 and continuing throughout adulthood. Deletion of the Cfap97d1 gene in both mouse models leads to sperm motility defects (asthenozoospermia) and male subfertility. In vitro fertilization (IVF) of cumulus-intact oocytes with Cfap97d1 deficient sperm yielded few embryos whereas IVF with zona pellucida-free oocytes resulted in embryo numbers comparable to that of the control. Knockout spermatozoa showed abnormal motility characterized by frequent stalling in the anti-hook position. Uniquely, Cfap97d1 loss caused a phenotype associated with axonemal doublet heterogeneity linked with frequent loss of the fourth doublet in the sperm stored in the epididymis. This study demonstrates that Cfap97d1 is required for sperm flagellum ultra-structure maintenance, thereby playing a critical role in sperm function and male fertility in mice.
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Axonema/genética , Proteínas del Citoesqueleto/genética , Dineínas/genética , Infertilidad Masculina/genética , Animales , Chlamydomonas/genética , Cilios/genética , Cilios/patología , Fertilización In Vitro , Humanos , Infertilidad Masculina/patología , Masculino , Ratones , Ratones Noqueados , Motilidad Espermática/genética , Cola del Espermatozoide/metabolismo , Cola del Espermatozoide/patología , Espermatozoides/crecimiento & desarrollo , Espermatozoides/patología , Testículo/crecimiento & desarrollo , Testículo/patologíaRESUMEN
CRELD1 (Cysteine-Rich with EGF-Like Domains 1) is a risk gene for non-syndromic atrioventricular septal defects in human patients. In a mouse model, Creld1 has been shown to be essential for heart development, particularly in septum and valve formation. However, due to the embryonic lethality of global Creld1 knockout (KO) mice, its cell type-specific function during peri- and postnatal stages remains unknown. Here, we generated conditional Creld1 KO mice lacking Creld1 either in the endocardium (KOTie2) or the myocardium (KOMyHC). Using a combination of cardiac phenotyping, histology, immunohistochemistry, RNA-sequencing, and flow cytometry, we demonstrate that Creld1 function in the endocardium is dispensable for heart development. Lack of myocardial Creld1 causes extracellular matrix remodeling and trabeculation defects by modulation of the Notch1 signaling pathway. Hence, KOMyHC mice die early postnatally due to myocardial hypoplasia. Our results reveal that Creld1 not only controls the formation of septa and valves at an early stage during heart development, but also cardiac maturation and function at a later stage. These findings underline the central role of Creld1 in mammalian heart development and function.
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Moléculas de Adhesión Celular/genética , Proteínas de la Matriz Extracelular/genética , Regulación del Desarrollo de la Expresión Génica , Corazón/embriología , Corazón/fisiología , Miocardio/metabolismo , Organogénesis/genética , Animales , Biomarcadores , Moléculas de Adhesión Celular/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Ratones Noqueados , Análisis de la Célula IndividualRESUMEN
Soluble adenylyl cyclase (sAC: ADCY10) has been genetically confirmed to be essential for male fertility in mice and humans. In mice, ex vivo studies of dormant, caudal epididymal sperm demonstrated that sAC is required for initiating capacitation and activating motility. We now use an improved sAC inhibitor, TDI-10229, for a comprehensive analysis of sAC function in mouse and human sperm. In contrast to caudal epididymal mouse sperm, human sperm are collected post-ejaculation, after sAC activity has already been stimulated. In addition to preventing the capacitation-induced stimulation of sAC and protein kinase A activities, tyrosine phosphorylation, alkalinization, beat frequency and acrosome reaction in dormant mouse sperm, sAC inhibitors interrupt each of these capacitation-induced changes in ejaculated human sperm. Furthermore, we show for the first time that sAC is required during acrosomal exocytosis in mouse and human sperm. These data define sAC inhibitors as candidates for non-hormonal, on-demand contraceptives suitable for delivery via intravaginal devices in women.
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Inhibidores de Adenilato Ciclasa/farmacología , Fertilización/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Adenilil Ciclasas/genética , Adenilil Ciclasas/fisiología , Animales , Células Cultivadas , Femenino , Fertilización/genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Noqueados , Embarazo , Espermatozoides/fisiologíaRESUMEN
Cilia are hair-like membrane protrusions that emanate from the surface of most vertebrate cells and are classified into motile and primary cilia. Motile cilia move fluid flow or propel cells, while also fulfill sensory functions. Primary cilia are immotile and act as a cellular antenna, translating environmental cues into cellular responses. Ciliary dysfunction leads to severe diseases, commonly termed ciliopathies. The molecular details underlying ciliopathies and ciliary function are, however, not well understood. Since cilia are small subcellular compartments, imaging-based approaches have been used to study them. However, tools to comprehensively analyze images are lacking. Automatic analysis approaches require commercial software and are limited to 2D analysis and only a few parameters. The widely used manual analysis approaches are time consuming, user-biased, and difficult to compare. Here, we present CiliaQ, a package of open-source, freely available, and easy-to-use ImageJ plugins. CiliaQ allows high-throughput analysis of 2D and 3D, static or time-lapse images from fluorescence microscopy of cilia in cell culture or tissues, and outputs a comprehensive list of parameters for ciliary morphology, length, bending, orientation, and fluorescence intensity, making it broadly applicable. We envision CiliaQ as a resource and platform for reproducible and comprehensive analysis of ciliary function in health and disease.
Asunto(s)
Cilios/metabolismo , Imagen Óptica/métodos , Proteínas/química , Animales , Línea Celular , Membrana Celular/ultraestructura , Cilios/ultraestructura , Humanos , Ratones , Microscopía Fluorescente , Programas InformáticosRESUMEN
The nonlysosomal glucosylceramidase ß2 (GBA2) catalyzes the hydrolysis of glucosylceramide to glucose and ceramide. Mutations in the human GBA2 gene have been associated with hereditary spastic paraplegia (HSP), autosomal-recessive cerebellar ataxia (ARCA), and the Marinesco-Sjögren-like syndrome. However, the underlying molecular mechanisms are ill-defined. Here, using biochemistry, immunohistochemistry, structural modeling, and mouse genetics, we demonstrate that all but one of the spastic gait locus #46 (SPG46)-connected mutations cause a loss of GBA2 activity. We demonstrate that GBA2 proteins form oligomeric complexes and that protein-protein interactions are perturbed by some of these mutations. To study the pathogenesis of GBA2-related HSP and ARCA in vivo, we investigated GBA2-KO mice as a mammalian model system. However, these mice exhibited a high phenotypic variance and did not fully resemble the human phenotype, suggesting that mouse and human GBA2 differ in function. Whereas some GBA2-KO mice displayed a strong locomotor defect, others displayed only mild alterations of the gait pattern and no signs of cerebellar defects. On a cellular level, inhibition of GBA2 activity in isolated cerebellar neurons dramatically affected F-actin dynamics and reduced neurite outgrowth, which has been associated with the development of neurological disorders. Our results shed light on the molecular mechanism underlying the pathogenesis of GBA2-related HSP and ARCA and reveal species-specific differences in GBA2 function in vivo.
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Ataxia Cerebelosa/metabolismo , Locomoción/genética , Mutación con Pérdida de Función , Paraplejía Espástica Hereditaria/metabolismo , beta-Glucosidasa/metabolismo , Animales , Biocatálisis , Ataxia Cerebelosa/genética , Glucosilceramidasa , Humanos , Ratones , Ratones Noqueados , Paraplejía Espástica Hereditaria/genética , Especificidad de la Especie , beta-Glucosidasa/antagonistas & inhibidores , beta-Glucosidasa/deficiencia , beta-Glucosidasa/genéticaRESUMEN
The second messenger 3',5'-cyclic nucleoside adenosine monophosphate (cAMP) plays a key role in signal transduction across prokaryotes and eukaryotes. Cyclic AMP signaling is compartmentalized into microdomains to fulfil specific functions. To define the function of cAMP within these microdomains, signaling needs to be analyzed with spatio-temporal precision. To this end, optogenetic approaches and genetically encoded fluorescent biosensors are particularly well suited. Synthesis and hydrolysis of cAMP can be directly manipulated by photoactivated adenylyl cyclases (PACs) and light-regulated phosphodiesterases (PDEs), respectively. In addition, many biosensors have been designed to spatially and temporarily resolve cAMP dynamics in the cell. This review provides an overview about optogenetic tools and biosensors to shed light on the subcellular organization of cAMP signaling.
Asunto(s)
Técnicas Biosensibles , AMP Cíclico/metabolismo , Optogenética , Transducción de Señal , Fracciones Subcelulares/metabolismo , Adenilil Ciclasas/metabolismo , Fluorescencia , Transferencia Resonante de Energía de Fluorescencia , Hidrolasas Diéster Fosfóricas/metabolismoRESUMEN
The shortening of sarcomeres that co-ordinates the pump function of the heart is stimulated by electrically-mediated increases in [Ca2+]. This process of excitation-contraction coupling (ECC) is subject to modulation by neurohormonal mediators that tune the output of the heart to meet the needs of the organism. Endothelin-1 (ET-1) is a potent modulator of cardiac function with effects on contraction amplitude, chronotropy and automaticity. The actions of ET-1 are evident during normal adaptive physiological responses and increased under pathophysiological conditions, such as following myocardial infarction and during heart failure, where ET-1 levels are elevated. In myocytes, ET-1 acts through ETA- or ETB-G protein-coupled receptors (GPCRs). Although well studied in atrial myocytes, the influence and mechanisms of action of ET-1 upon ECC in ventricular myocytes are not fully resolved. We show in rat ventricular myocytes that ET-1 elicits a biphasic effect on fractional shortening (initial transient negative and sustained positive inotropy) and increases the peak amplitude of systolic Ca2+ transients in adult rat ventricular myocytes. The negative inotropic phase was ETB receptor-dependent, whereas the positive inotropic response and increase in peak amplitude of systolic Ca2+ transients required ETA receptor engagement. Both effects of ET-1 required phospholipase C (PLC)-activity, although distinct signalling pathways downstream of PLC elicited the effects of each ET receptor. The negative inotropic response involved inositol 1,4,5-trisphosphate (InsP3) signalling and protein kinase C epsilon (PKCε). The positive inotropic action and the enhancement in Ca2+ transient amplitude induced by ET-1 were independent of InsP3 signalling, but suppressed by PKCε. Serine 302 in cardiac myosin binding protein-C was identified as a PKCε substrate that when phosphorylated contributed to the suppression of contraction and Ca2+ transients by PKCε following ET-1 stimulation. Thus, our data provide a new role and mechanism of action for InsP3 and PKCε in mediating the negative inotropic response and in restraining the positive inotropy and enhancement in Ca2+ transients following ET-1 stimulation.
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Proteínas Portadoras/metabolismo , Endotelina-1/farmacología , Ventrículos Cardíacos/citología , Contracción Miocárdica , Miocitos Cardíacos/metabolismo , Proteína Quinasa C-epsilon/metabolismo , Animales , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Cardiotónicos/farmacología , Citosol/metabolismo , Acoplamiento Excitación-Contracción/efectos de los fármacos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Fosforilación/efectos de los fármacos , Proteína Quinasa C-epsilon/antagonistas & inhibidores , Ratas Wistar , Receptores de Endotelina/metabolismo , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/metabolismo , Fosfolipasas de Tipo C/metabolismoRESUMEN
The lysosomal acid ß-glucosidase GBA1 and the non-lysosomal ß-glucosidase GBA2 degrade glucosylceramide (GlcCer) to glucose and ceramide in different cellular compartments. Loss of GBA2 activity and the resulting accumulation of GlcCer results in male infertility, whereas mutations in the GBA1 gene and loss of GBA1 activity cause the lipid-storage disorder Gaucher disease. However, the role of GBA2 in Gaucher disease pathology and its relationship to GBA1 is not well understood. Here, we report a GBA1-dependent down-regulation of GBA2 activity in patients with Gaucher disease. Using an experimental approach combining cell biology, biochemistry, and mass spectrometry, we show that sphingosine, the cytotoxic metabolite accumulating in Gaucher cells through the action of GBA2, directly binds to GBA2 and inhibits its activity. We propose a negative feedback loop, in which sphingosine inhibits GBA2 activity in Gaucher cells, preventing further sphingosine accumulation and, thereby, cytotoxicity. Our findings add a new chapter to the understanding of the complex molecular mechanism underlying Gaucher disease and the regulation of ß-glucosidase activity in general.
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Regulación hacia Abajo , Enfermedad de Gaucher/enzimología , Regulación Enzimológica de la Expresión Génica , Modelos Biológicos , Esfingosina/metabolismo , beta-Glucosidasa/biosíntesis , Animales , Línea Celular , Enfermedad de Gaucher/genética , Glucosilceramidasa , Glucosilceramidas/genética , Glucosilceramidas/metabolismo , Humanos , Masculino , Ratones , Esfingosina/genética , beta-Glucosidasa/genéticaRESUMEN
Glycosphingolipids are key elements of cellular membranes, thereby, controlling a variety of cellular functions. Accumulation of the simple glycosphingolipid glucosylceramide results in life-threatening lipid storage-diseases or in male infertility. How glucosylceramide regulates cellular processes is ill defined. Here, we reveal that glucosylceramide accumulation in GBA2 knockout-mice alters cytoskeletal dynamics due to a more ordered lipid organization in the plasma membrane. In dermal fibroblasts, accumulation of glucosylceramide augments actin polymerization and promotes microtubules persistence, resulting in a higher number of filopodia and lamellipodia and longer microtubules. Similar cytoskeletal defects were observed in male germ and Sertoli cells from GBA2 knockout-mice. In particular, the organization of F-actin structures in the ectoplasmic specialization and microtubules in the sperm manchette is affected. Thus, glucosylceramide regulates cytoskeletal dynamics, providing mechanistic insights into how glucosylceramide controls signaling pathways not only during sperm development, but also in other cell types.
Asunto(s)
Actinas/metabolismo , Citoesqueleto/genética , Glucosilceramidas/genética , Metabolismo de los Lípidos/genética , beta-Glucosidasa/genética , Actinas/química , Animales , Membrana Celular/metabolismo , Membrana Celular/patología , Citoesqueleto/metabolismo , Citoesqueleto/patología , Fibroblastos/metabolismo , Glucosilceramidas/química , Glucosilceramidas/metabolismo , Humanos , Masculino , Ratones , Ratones Noqueados , Microtúbulos/genética , Microtúbulos/metabolismo , Microtúbulos/patología , Seudópodos/genética , Seudópodos/metabolismo , Seudópodos/patología , Células de Sertoli/metabolismo , Células de Sertoli/patología , beta-Glucosidasa/metabolismoRESUMEN
Mammals synthesize, cell-type specifically, the diastereomeric hexosylceramides, ß-galactosylceramide (GalCer) and ß-glucosylceramide (GlcCer), which are involved in several diseases, such as sphingolipidosis, diabetes, chronic kidney diseases, or cancer. In contrast, Bacteroides fragilis, a member of the human gut microbiome, and the marine sponge, Agelas mauritianus, produce α-GalCer, one of the most potent stimulators for invariant natural killer T cells. To dissect the contribution of these individual stereoisomers to pathologies, we established a novel hydrophilic interaction chromatography-based LC-MS2 method and separated (R > 1.5) corresponding diastereomers from each other, independent of their lipid anchors. Testing various bacterial and mammalian samples, we could separate, identify (including the lipid anchor composition), and quantify endogenous ß-GlcCer, ß-GalCer, and α-GalCer isomers without additional derivatization steps. Thereby, we show a selective decrease of ß-GlcCers versus ß-GalCers in cell-specific models of GlcCer synthase-deficiency and an increase of specific ß-GlcCers due to loss of ß-glucoceramidase 2 activity. Vice versa, ß-GalCer increased specifically when cerebroside sulfotransferase (Gal3st1) was deleted. We further confirm ß-GalCer as substrate of globotriaosylceramide synthase for galabiaosylceramide synthesis and identify additional members of the human gut microbiome to contain immunogenic α-GalCers. Finally, this method is shown to separate corresponding hexosylsphingosine standards, promoting its applicability in further investigations.
Asunto(s)
Bacterias/metabolismo , Ceramidas/química , Ceramidas/metabolismo , Animales , Microbioma Gastrointestinal , Humanos , Ratones , EstereoisomerismoRESUMEN
KEY POINTS: In human sperm, proton flux across the membrane is controlled by the voltage-gated proton channel Hv1. We show that sperm harbour both Hv1 and an N-terminally cleaved isoform termed Hv1Sper. The pH-control of Hv1Sper and Hv1 is distinctively different. Hv1Sper and Hv1 can form heterodimers that combine features of both constituents. Cleavage and heterodimerization of Hv1 might represent an adaptation to the specific requirements of pH control in sperm. ABSTRACT: In human sperm, the voltage-gated proton channel Hv1 controls the flux of protons across the flagellar membrane. Here, we show that sperm harbour Hv1 and a shorter isoform, termed Hv1Sper. Hv1Sper is generated from Hv1 by removal of 68 amino acids from the N-terminus by post-translational proteolytic cleavage. The pH-dependent gating of the channel isoforms is distinctly different. In both Hv1 and Hv1Sper, the conductance-voltage relationship is determined by the pH difference across the membrane (∆pH). However, simultaneous changes in intracellular and extracellular pH that leave ΔpH constant strongly shift the activation curve of Hv1Sper but not that of Hv1, demonstrating that cleavage of the N-terminus tunes pH sensing in Hv1. Moreover, we show that Hv1 and Hv1Sper assemble as heterodimers that combine features of both constituents. We suggest that cleavage and heterodimerization of Hv1 represents an adaptation to the specific requirements of pH control in sperm.
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Activación del Canal Iónico/fisiología , Canales Iónicos/fisiología , Espermatozoides/fisiología , Animales , Línea Celular , Células HEK293 , Humanos , Concentración de Iones de Hidrógeno , Canales Iónicos/metabolismo , Masculino , Ratones Endogámicos C57BL , Oocitos/fisiología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Mucosa Respiratoria , Serina Proteasas/metabolismo , Inhibidores de Serina Proteinasa/farmacología , Espermatozoides/efectos de los fármacos , Espermatozoides/metabolismo , Sulfonas/farmacología , Xenopus laevisRESUMEN
STUDY QUESTION: How and where is pro-ovastacin activated and how does active ovastacin regulate zona pellucida hardening (ZPH) and successful fertilization? STUDY FINDING: Ovastacin is partially active before exocytosis and pre-hardens the zona pellucida (ZP) before fertilization. WHAT IS KNOWN ALREADY: The metalloproteinase ovastacin is stored in cortical granules, it cleaves zona pellucida protein 2 (ZP2) upon fertilization and thereby destroys the ZP sperm ligand and triggers ZPH. Female mice deficient in the extracellular circulating ovastacin-inhibitor fetuin-B are infertile due to pre-mature ZPH. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: We isolated oocytes from wild-type and ovastacin-deficient (Astlnull) FVB mice before and after fertilization (in vitro and in vivo) and quantified ovastacin activity and cleavage of ZP2 by immunoblot. We assessed ZPH by measuring ZP digestion time using α-chymotrypsin and by determining ZP2 cleavage. We determined cellular distribution of ovastacin by immunofluorescence using domain-specific ovastacin antibodies. Experiments were performed at least in triplicate with a minimum of 20 oocytes. Data were pre-analyzed using Shapiro-Wilk test. In case of normal distribution, significance was determined via two-sided Student's t-test, whereas in case of non-normal distribution via Mann-Whitney U-test. MAIN RESULTS AND THE ROLE OF CHANCE: Metaphase II (MII) oocytes contained both inactive pro-ovastacin and activated ovastacin. Immunoblot and ZP digestion assays revealed a partial cleavage of ZP2 even before fertilization in wild-type mice. Partial cleavage coincided with germinal-vesicle breakdown and MII, despite the presence of fetuin-B protein, an endogenous ovastacin inhibitor, in the follicular and oviductal fluid. Upon exocytosis, part of the C-terminal domain of ovastacin remained attached to the plasmalemma, while the N-terminal active ovastacin domain was secreted. This finding may resolve previously conflicting data showing that ovastacin acts both as an oolemmal receptor termed SAS1B (sperm acrosomal SLLP1 binding protein; SLLP, sperm lysozyme like protein) and a secreted protease mediating ZP2 cleavage. LIMITATIONS, REASONS FOR CAUTION: For this study, only oocytes isolated from wild-type and ovastacin-deficient FVB mice were investigated. Some experiments involved oocyte activation by the Ca2+ ionophore A23187 to trigger ZPH. WIDER IMPLICATIONS OF THE FINDINGS: This study provides a detailed spatial and temporal view of pre-mature cleavage of ZP2 by ovastacin, which is known to adversely affect IVF rate in mice and humans. LARGE SCALE DATA: None. STUDY FUNDING AND COMPETING INTEREST(S): This work was supported by the Center of Natural Sciences and Medicine and by a start-up grant of the Johannes Gutenberg University Mainz to W.S., and by a grant from Deutsche Forschungsgemeinschaft and by the START program of the Medical Faculty of RWTH Aachen University to J.F. and W.J.D. There are no competing interests to declare.
Asunto(s)
Fetuína-B/genética , Metaloproteasas/genética , Oocitos/metabolismo , Glicoproteínas de la Zona Pelúcida/genética , Zona Pelúcida/metabolismo , Animales , Quimotripsina/química , Exocitosis , Femenino , Fertilización In Vitro , Fetuína-B/metabolismo , Regulación del Desarrollo de la Expresión Génica , Masculino , Metaloproteasas/metabolismo , Metafase , Ratones , Oocitos/citología , Oocitos/crecimiento & desarrollo , Cultivo Primario de Células , Proteolisis , Transducción de Señal , Espermatozoides/citología , Espermatozoides/fisiología , Glicoproteínas de la Zona Pelúcida/metabolismoRESUMEN
Synthetic endocrine disrupting chemicals (EDCs), omnipresent in food, household, and personal care products, have been implicated in adverse trends in human reproduction, including infertility and increasing demand for assisted reproduction. Here, we study the action of 96 ubiquitous EDCs on human sperm. We show that structurally diverse EDCs activate the sperm-specific CatSper channel and, thereby, evoke an intracellular Ca(2+) increase, a motility response, and acrosomal exocytosis. Moreover, EDCs desensitize sperm for physiological CatSper ligands and cooperate in low-dose mixtures to elevate Ca(2+) levels in sperm. We conclude that EDCs interfere with various sperm functions and, thereby, might impair human fertilization.
Asunto(s)
Disruptores Endocrinos/farmacología , Espermatozoides/efectos de los fármacos , Espermatozoides/fisiología , Acrosoma/metabolismo , Potenciales de Acción/efectos de los fármacos , Unión Competitiva , Calcio/metabolismo , Canales de Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Disruptores Endocrinos/química , Exocitosis/efectos de los fármacos , Humanos , Ligandos , Masculino , Unión Proteica , Motilidad Espermática/efectos de los fármacosRESUMEN
The second messengers cAMP and cGMP activate their target proteins by binding to a conserved cyclic nucleotide-binding domain (CNBD). Here, we identify and characterize an entirely novel CNBD-containing protein called CRIS (cyclic nucleotide receptor involved in sperm function) that is unrelated to any of the other members of this protein family. CRIS is exclusively expressed in sperm precursor cells. Cris-deficient male mice are either infertile due to a lack of sperm resulting from spermatogenic arrest, or subfertile due to impaired sperm motility. The motility defect is caused by altered Ca(2+) regulation of flagellar beat asymmetry, leading to a beating pattern that is reminiscent of sperm hyperactivation. Our results suggest that CRIS interacts during spermiogenesis with Ca(2+)-regulated proteins that--in mature sperm--are involved in flagellar bending.