RESUMEN
Effective interplay between the uterus and the embryo is essential for pregnancy establishment, however, convenient methods to screen embryo implantation success and maternal uterine response in experimental mouse models are currently lacking. Here we report 3DMOUSEneST, a groundbreaking method for analyzing mouse implantation sites based on label-free higher harmonic generation microscopy, providing unprecedented insights into the embryo-uterine dynamics during early pregnancy. The 3DMOUSEneST method incorporates second-harmonic generation microscopy to image the three-dimensional structure formed by decidual fibrillar collagen, named 'decidual nest', and third-harmonic generation microscopy to evaluate early conceptus (defined as the embryo and extraembryonic tissues) growth. We demonstrate that decidual nest volume is a measurable indicator of decidualization efficacy and correlates with the probability of early pregnancy progression based on a logistic regression analysis using Smad1/5 and Smad2/3 conditional knockout mice with known implantation defects. 3DMOUSEneST has great potential to become a principal method for studying decidual fibrillar collagen and characterizing mouse models associated with early embryonic lethality and fertility issues.
RESUMEN
A pharmacologic approach to male contraception remains a longstanding challenge in medicine. Toward this objective, we explored the spermatogenic effects of a selective small-molecule inhibitor (JQ1) of the bromodomain and extraterminal (BET) subfamily of epigenetic reader proteins. Here, we report potent inhibition of the testis-specific member BRDT, which is essential for chromatin remodeling during spermatogenesis. Biochemical and crystallographic studies confirm that occupancy of the BRDT acetyl-lysine binding pocket by JQ1 prevents recognition of acetylated histone H4. Treatment of mice with JQ1 reduced seminiferous tubule area, testis size, and spermatozoa number and motility without affecting hormone levels. Although JQ1-treated males mate normally, inhibitory effects of JQ1 evident at the spermatocyte and round spermatid stages cause a complete and reversible contraceptive effect. These data establish a new contraceptive that can cross the blood:testis boundary and inhibit bromodomain activity during spermatogenesis, providing a lead compound targeting the male germ cell for contraception.
Asunto(s)
Azepinas/farmacología , Anticonceptivos Masculinos/farmacología , Proteínas Nucleares/antagonistas & inhibidores , Triazoles/farmacología , Animales , Azepinas/química , Barrera Hematotesticular , Anticonceptivos Masculinos/química , Femenino , Humanos , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Modelos Moleculares , Proteínas Nucleares/química , Estructura Terciaria de Proteína , Recuento de Espermatozoides , Motilidad Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Testículo/citología , Testículo/efectos de los fármacos , Triazoles/químicaRESUMEN
EPH receptors (EPHs), the largest family of tyrosine kinases, phosphorylate downstream substrates upon binding of ephrin cell surface-associated ligands. In a large cohort of endometriotic lesions from individuals with endometriosis, we found that EPHA2 and EPHA4 expressions are increased in endometriotic lesions relative to normal eutopic endometrium. Because signaling through EPHs is associated with increased cell migration and invasion, we hypothesized that chemical inhibition of EPHA2/4 could have therapeutic value. We screened DNA-encoded chemical libraries (DECL) to rapidly identify EPHA2/4 kinase inhibitors. Hit compound, CDD-2693, exhibited picomolar/nanomolar kinase activity against EPHA2 (Ki: 4.0 nM) and EPHA4 (Ki: 0.81 nM). Kinome profiling revealed that CDD-2693 bound to most EPH family and SRC family kinases. Using NanoBRET target engagement assays, CDD-2693 had nanomolar activity versus EPHA2 (IC50: 461 nM) and EPHA4 (IC50: 40 nM) but was a micromolar inhibitor of SRC, YES, and FGR. Chemical optimization produced CDD-3167, having picomolar biochemical activity toward EPHA2 (Ki: 0.13 nM) and EPHA4 (Ki: 0.38 nM) with excellent cell-based potency EPHA2 (IC50: 8.0 nM) and EPHA4 (IC50: 2.3 nM). Moreover, CDD-3167 maintained superior off-target cellular selectivity. In 12Z endometriotic epithelial cells, CDD-2693 and CDD-3167 significantly decreased EFNA5 (ligand) induced phosphorylation of EPHA2/4, decreased 12Z cell viability, and decreased IL-1ß-mediated expression of prostaglandin synthase 2 (PTGS2). CDD-2693 and CDD-3167 decreased expansion of primary endometrial epithelial organoids from patients with endometriosis and decreased Ewing's sarcoma viability. Thus, using DECL, we identified potent pan-EPH inhibitors that show specificity and activity in cellular models of endometriosis and cancer.
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Inhibidores de Proteínas Quinasas , Humanos , Femenino , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Endometriosis/tratamiento farmacológico , Endometriosis/metabolismo , Endometriosis/patología , ADN/metabolismo , Receptores de la Familia Eph/metabolismo , Receptores de la Familia Eph/antagonistas & inhibidores , Receptor EphA2/metabolismo , Receptor EphA2/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/química , Movimiento Celular/efectos de los fármacosRESUMEN
Spermatozoa have a streamlined shape to swim through the oviduct to fertilize oocytes. To become svelte spermatozoa, spermatid cytoplasm must be eliminated in several steps including sperm release, which is part of spermiation. Although this process has been well observed, the molecular mechanisms that underlie it remain unclear. In male germ cells, there are membraneless organelles called nuage, which are observed by electron microscopy in various forms of dense material. Reticulated body (RB) and chromatoid body remnant (CR) are two types of nuage in spermatids, but the functions of both are unknown. Using CRISPR/Cas9 technology, we deleted the entire coding sequence of testis-specific serine kinase substrate (TSKS) in mice and demonstrate that TSKS is essential for male fertility through the formation of both RB and CR, prominent sites of TSKS localization. Due to the lack of TSKS-derived nuage (TDN), the cytoplasmic contents cannot be eliminated from spermatid cytoplasm in Tsks knockout mice, resulting in excess residual cytoplasm with an abundance of cytoplasmic materials and inducing an apoptotic response. In addition, ectopic expression of TSKS in cells results in formation of amorphous nuage-like structures; dephosphorylation of TSKS helps to induce nuage, while phosphorylation of TSKS blocks the formation. Our results indicate that TSKS and TDN are essential for spermiation and male fertility by eliminating cytoplasmic contents from the spermatid cytoplasm.
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Proteínas del Citoesqueleto , Gránulos de Ribonucleoproteína de Células Germinales , Fosfoproteínas , Espermátides , Animales , Masculino , Ratones , Citoplasma , Citosol , Ratones Noqueados , Semen , Proteínas del Citoesqueleto/genética , Fosfoproteínas/genéticaRESUMEN
The cochlea's ability to discriminate sound frequencies is facilitated by a special topography along its longitudinal axis known as tonotopy. Auditory hair cells located at the base of the cochlea respond to high-frequency sounds, whereas hair cells at the apex respond to lower frequencies. Gradual changes in morphological and physiological features along the length of the cochlea determine each region's frequency selectivity, but it remains unclear how tonotopy is established during cochlear development. Recently, sonic hedgehog (SHH) was proposed to initiate the establishment of tonotopy by conferring regional identity to the primordial cochlea. Here, using mouse genetics, we provide in vivo evidence that regional identity in the embryonic cochlea acts as a framework upon which tonotopy-specific properties essential for frequency selectivity in the mature cochlea develop. We found that follistatin (FST) is required for the maintenance of apical cochlear identity, but dispensable for its initial induction. In a fate-mapping analysis, we found that FST promotes expansion of apical cochlear cells, contributing to the formation of the apical cochlear domain. SHH, in contrast, is required both for the induction and maintenance of apical identity. In the absence of FST or SHH, mice produce a short cochlea lacking its apical domain. This results in the loss of apex-specific anatomical and molecular properties and low-frequency-specific hearing loss.
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Folistatina , Proteínas Hedgehog , Animales , Ratones , Folistatina/genética , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Cóclea/fisiología , Audición/fisiología , Mamíferos/metabolismoRESUMEN
BRDT, BRD2, BRD3, and BRD4 comprise the bromodomain and extraterminal (BET) subfamily which contain two similar tandem bromodomains (BD1 and BD2). Selective BD1 inhibition phenocopies effects of tandem BET BD inhibition both in cancer models and, as we and others have reported of BRDT, in the testes. To find novel BET BD1 binders, we screened >4.5 billion molecules from our DNA-encoded chemical libraries with BRDT-BD1 or BRDT-BD2 proteins in parallel. A compound series enriched only by BRDT-BD1 was resynthesized off-DNA, uncovering a potent chiral compound, CDD-724, with >2,000-fold selectivity for inhibiting BRDT-BD1 over BRDT-BD2. CDD-724 stereoisomers exhibited remarkable differences in inhibiting BRDT-BD1, with the R-enantiomer (CDD-787) being 50-fold more potent than the S-enantiomer (CDD-786). From structureactivity relationship studies, we produced CDD-956, which maintained picomolar BET BD1 binding potency and high selectivity over BET BD2 proteins and had improved stability in human liver microsomes over CDD-787. BROMOscan profiling confirmed the excellent pan-BET BD1 affinity and selectivity of CDD-787 and CDD-956 on BD1 versus BD2 and all other BD-containing proteins. A cocrystal structure of BRDT-BD1 bound with CDD-956 was determined at 1.82 Å and revealed BRDT-BD1specific contacts with the αZ and αC helices that explain the high affinity and selectivity for BET BD1 versus BD2. CDD-787 and CDD-956 maintain cellular BD1-selectivity in NanoBRET assays and show potent antileukemic activity in acute myeloid leukemia cell lines. These BET BD1-specific and highly potent compounds are structurally unique and provide insight into the importance of chirality to achieve BET specificity.
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Antiinflamatorios no Esteroideos , Antineoplásicos , Anticonceptivos Masculinos , Descubrimiento de Drogas , Proteínas Nucleares , Bibliotecas de Moléculas Pequeñas , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/aislamiento & purificación , Antiinflamatorios no Esteroideos/farmacología , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Antineoplásicos/farmacología , Anticonceptivos Masculinos/química , Anticonceptivos Masculinos/aislamiento & purificación , Anticonceptivos Masculinos/farmacología , ADN/genética , Humanos , Masculino , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/química , Dominios Proteicos , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/aislamiento & purificación , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-ActividadRESUMEN
Imaging mass spectrometry (IMS) is a powerful tool for mapping the spatial distribution of unlabeled drugs and metabolites that may find application in assessing drug delivery, explaining drug efficacy, and identifying potential toxicity. This study focuses on determining the spatial distribution of the antidepressant duloxetine, which is widely prescribed despite common adverse effects (liver injury, constant headaches) whose mechanisms are not fully understood. We used high-resolution IMS with matrix-assisted laser desorption/ionization to examine the distribution of duloxetine and its major metabolites in four mouse organs where it may contribute to efficacy or toxicity: brain, liver, kidney, and spleen. In none of these tissues is duloxetine or its metabolites homogeneously distributed, which has implications for both efficacy and toxicity. We found duloxetine to be similarly distributed in spleen red pulp and white pulp but differentially distributed in different anatomic regions of the liver, kidney, and brain, with dose-dependent patterns. Comparison with hematoxylin and eosin staining of tissue sections reveals that the ion images of endogenous lipids help delineate anatomic regions in the brain and kidney, while heme ion images assist in differentiating regions within the spleen. These endogenous metabolites may serve as a valuable resource for examining the spatial distribution of other drugs in tissues when staining images are not available. These findings may facilitate future mechanistic studies of the therapeutic and adverse effects of duloxetine. In the current work, we did not perform absolute quantification of duloxetine, which will be reported in due course. SIGNIFICANCE STATEMENT: The study utilized imaging mass spectrometry to examine the spatial distribution of duloxetine and its primary metabolites in mouse brain, liver, kidney, and spleen. These results may pave the way for future investigations into the mechanisms behind duloxetine's therapeutic and adverse effects. Furthermore, the mass spectrometry images of specific endogenous metabolites such as heme could be valuable in analyzing the spatial distribution of other drugs within tissues in scenarios where histological staining images are unavailable.
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Antidepresivos , Encéfalo , Clorhidrato de Duloxetina , Riñón , Hígado , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Bazo , Animales , Clorhidrato de Duloxetina/metabolismo , Ratones , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Bazo/metabolismo , Bazo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/diagnóstico por imagen , Riñón/metabolismo , Riñón/efectos de los fármacos , Hígado/metabolismo , Hígado/efectos de los fármacos , Antidepresivos/metabolismo , Distribución Tisular , Masculino , Ratones Endogámicos C57BLRESUMEN
The mammalian sperm midpiece has a unique double-helical structure called the mitochondrial sheath that wraps tightly around the axoneme. Despite the remarkable organization of the mitochondrial sheath, the molecular mechanisms involved in mitochondrial sheath formation are unclear. In the process of screening testis-enriched genes for functions in mice, we identified armadillo repeat-containing 12 (ARMC12) as an essential protein for mitochondrial sheath formation. Here, we engineered Armc12-null mice, FLAG-tagged Armc12 knock-in mice, and TBC1 domain family member 21 (Tbc1d21)-null mice to define the functions of ARMC12 in mitochondrial sheath formation in vivo. We discovered that absence of ARMC12 causes abnormal mitochondrial coiling along the flagellum, resulting in reduced sperm motility and male sterility. During spermiogenesis, sperm mitochondria in Armc12-null mice cannot elongate properly at the mitochondrial interlocking step which disrupts abnormal mitochondrial coiling. ARMC12 is a mitochondrial peripheral membrane protein and functions as an adherence factor between mitochondria in cultured cells. ARMC12 in testicular germ cells interacts with mitochondrial proteins MIC60, VDAC2, and VDAC3 as well as TBC1D21 and GK2, which are required for mitochondrial sheath formation. We also observed that TBC1D21 is essential for the interaction between ARMC12 and VDAC proteins in vivo. These results indicate that ARMC12 uses integral mitochondrial membrane proteins VDAC2 and VDAC3 as scaffolds to link mitochondria and works cooperatively with TBC1D21. Thus, our studies have revealed that ARMC12 regulates spatiotemporal mitochondrial dynamics to form the mitochondrial sheath through cooperative interactions with several proteins on the sperm mitochondrial surface.
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Proteínas del Dominio Armadillo/genética , Proteínas Activadoras de GTPasa/genética , Infertilidad Masculina/genética , Proteínas de Microfilamentos/genética , Dinámicas Mitocondriales/genética , Animales , Axonema/genética , Humanos , Infertilidad Masculina/patología , Masculino , Ratones , Ratones Noqueados , Proteínas de Transporte de Membrana Mitocondrial/genética , Motilidad Espermática/genética , Cola del Espermatozoide/patología , Cola del Espermatozoide/ultraestructura , Espermátides/metabolismo , Espermatogénesis/genética , Espermatozoides/patología , Espermatozoides/ultraestructura , Testículo/metabolismo , Canal Aniónico 2 Dependiente del Voltaje/genética , Canales Aniónicos Dependientes del Voltaje/genéticaRESUMEN
The biological mechanisms underpinning learning are unclear. Mounting evidence has suggested that adult hippocampal neurogenesis is involved although a causal relationship has not been well defined. Here, using high-resolution genetic mapping of adult neurogenesis, combined with sequencing information, we identify follistatin (Fst) and demonstrate its involvement in learning and adult neurogenesis. We confirmed that brain-specific Fst knockout (KO) mice exhibited decreased hippocampal neurogenesis and demonstrated that FST is critical for learning. Fst KO mice exhibit deficits in spatial learning, working memory, and long-term potentiation (LTP). In contrast, hippocampal overexpression of Fst in KO mice reversed these impairments. By utilizing RNA sequencing and chromatin immunoprecipitation, we identified Asic4 as a target gene regulated by FST and show that Asic4 plays a critical role in learning deficits caused by Fst deletion. Long-term overexpression of hippocampal Fst in C57BL/6 wild-type mice alleviates age-related decline in cognition, neurogenesis, and LTP. Collectively, our study reveals the functions for FST in adult neurogenesis and learning behaviors.
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Canales Iónicos Sensibles al Ácido/metabolismo , Folistatina/fisiología , Hipocampo/metabolismo , Neurogénesis , Plasticidad Neuronal , Aprendizaje Espacial/fisiología , Canales Iónicos Sensibles al Ácido/genética , Animales , Cognición , Femenino , Potenciación a Largo Plazo , Masculino , Memoria , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Sinapsis/fisiologíaRESUMEN
Skeletal muscle and bone homeostasis are regulated by members of the myostatin/GDF-11/activin branch of the transforming growth factor-ß superfamily, which share many regulatory components, including inhibitory extracellular binding proteins and receptors that mediate signaling. Here, we present the results of genetic studies demonstrating a critical role for the binding protein follistatin (FST) in regulating both skeletal muscle and bone. Using an allelic series corresponding to varying expression levels of endogenous Fst, we show that FST acts in an exquisitely dose-dependent manner to regulate both muscle mass and bone density. Moreover, by employing a genetic strategy to target Fst expression only in the posterior (caudal) region of the animal, we show that the effects of Fst loss are mostly restricted to the posterior region, implying that locally produced FST plays a much more important role than circulating FST with respect to regulation of muscle and bone. Finally, we show that targeting receptors for these ligands specifically in osteoblasts leads to dramatic increases in bone mass, with trabecular bone volume fraction being increased by 12- to 13-fold and bone mineral density being increased by 8- to 9-fold in humeri, femurs, and lumbar vertebrae. These findings demonstrate that bone, like muscle, has an enormous inherent capacity for growth that is normally kept in check by this signaling system and suggest that the extent to which this regulatory mechanism may be used throughout the body to regulate tissue mass may be more significant than previously appreciated.
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Desarrollo Óseo/fisiología , Folistatina/metabolismo , Músculo Esquelético/crecimiento & desarrollo , Factor de Crecimiento Transformador beta/metabolismo , Alelos , Animales , Densidad Ósea , Folistatina/genética , Regulación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/fisiología , Heterocigoto , Homeostasis , Ratones , Familia de Multigenes , Transducción de Señal , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Bromodomain testis (BRDT), a member of the bromodomain and extraterminal (BET) subfamily that includes the cancer targets BRD2, BRD3, and BRD4, is a validated contraceptive target. All BET subfamily members have two tandem bromodomains (BD1 and BD2). Knockout mice lacking BRDT-BD1 or both bromodomains are infertile. Treatment of mice with JQ1, a BET BD1/BD2 nonselective inhibitor with the highest affinity for BRD4, disrupts spermatogenesis and reduces sperm number and motility. To assess the contribution of each BRDT bromodomain, we screened our collection of DNA-encoded chemical libraries for BRDT-BD1 and BRDT-BD2 binders. High-enrichment hits were identified and resynthesized off-DNA and examined for their ability to compete with JQ1 in BRDT and BRD4 bromodomain AlphaScreen assays. These studies identified CDD-1102 as a selective BRDT-BD2 inhibitor with low nanomolar potency and >1,000-fold selectivity over BRDT-BD1. Structure-activity relationship studies of CDD-1102 produced a series of additional BRDT-BD2/BRD4-BD2 selective inhibitors, including CDD-1302, a truncated analog of CDD-1102 with similar activity, and CDD-1349, an analog with sixfold selectivity for BRDT-BD2 versus BRD4-BD2. BROMOscan bromodomain profiling confirmed the great affinity and selectivity of CDD-1102 and CDD-1302 on all BET BD2 versus BD1 with the highest affinity for BRDT-BD2. Cocrystals of BRDT-BD2 with CDD-1102 and CDD-1302 were determined at 2.27 and 1.90 Å resolution, respectively, and revealed BRDT-BD2 specific contacts that explain the high affinity and selectivity of these compounds. These BD2-specific compounds and their binding to BRDT-BD2 are unique compared with recent reports and enable further evaluation of their nonhormonal contraceptive potential in vitro and in vivo.
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Azepinas/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Anticonceptivos Masculinos/farmacología , Proteínas Nucleares/antagonistas & inhibidores , Factores de Transcripción/antagonistas & inhibidores , Triazoles/farmacología , Animales , Azepinas/química , Sitios de Unión , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Clonación Molecular , Anticonceptivos Masculinos/química , Cristalografía por Rayos X , Descubrimiento de Drogas , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Ligandos , Masculino , Ratones , Simulación del Acoplamiento Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Relación Estructura-Actividad Cuantitativa , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Testículo/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Triazoles/químicaRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed more than 4 million humans globally, but there is no bona fide Food and Drug Administration-approved drug-like molecule to impede the COVID-19 pandemic. The sluggish pace of traditional therapeutic discovery is poorly suited to producing targeted treatments against rapidly evolving viruses. Here, we used an affinity-based screen of 4 billion DNA-encoded molecules en masse to identify a potent class of virus-specific inhibitors of the SARS-CoV-2 main protease (Mpro) without extensive and time-consuming medicinal chemistry. CDD-1714, the initial three-building-block screening hit (molecular weight [MW] = 542.5 g/mol), was a potent inhibitor (inhibition constant [Ki] = 20 nM). CDD-1713, a smaller two-building-block analog (MW = 353.3 g/mol) of CDD-1714, is a reversible covalent inhibitor of Mpro (Ki = 45 nM) that binds in the protease pocket, has specificity over human proteases, and shows in vitro efficacy in a SARS-CoV-2 infectivity model. Subsequently, key regions of CDD-1713 that were necessary for inhibitory activity were identified and a potent (Ki = 37 nM), smaller (MW = 323.4 g/mol), and metabolically more stable analog (CDD-1976) was generated. Thus, screening of DNA-encoded chemical libraries can accelerate the discovery of efficacious drug-like inhibitors of emerging viral disease targets.
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Proteasas 3C de Coronavirus/antagonistas & inhibidores , Proteasas 3C de Coronavirus/genética , Descubrimiento de Drogas/métodos , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/genética , Animales , COVID-19/virología , Células Cultivadas , Proteasas 3C de Coronavirus/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática , Ingeniería Genética , Humanos , Modelos Moleculares , Conformación Molecular , Estructura Molecular , SARS-CoV-2/metabolismo , Relación Estructura-Actividad , Replicación Viral , Tratamiento Farmacológico de COVID-19RESUMEN
Ovarian cancer (OC) is one of the deadliest cancers affecting the female reproductive system. It may present little or no symptoms at the early stages and typically unspecific symptoms at later stages. High-grade serous ovarian cancer (HGSC) is the subtype responsible for most ovarian cancer deaths. However, very little is known about the metabolic course of this disease, particularly in its early stages. In this longitudinal study, we examined the temporal course of serum lipidome changes using a robust HGSC mouse model and machine learning data analysis. Early progression of HGSC was marked by increased levels of phosphatidylcholines and phosphatidylethanolamines. In contrast, later stages featured more diverse lipid alterations, including fatty acids and their derivatives, triglycerides, ceramides, hexosylceramides, sphingomyelins, lysophosphatidylcholines, and phosphatidylinositols. These alterations underscored unique perturbations in cell membrane stability, proliferation, and survival during cancer development and progression, offering potential targets for early detection and prognosis of human ovarian cancer.
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Cistadenocarcinoma Seroso , Neoplasias Ováricas , Ratones , Animales , Femenino , Humanos , Lipidómica , Estudios Longitudinales , Neoplasias Ováricas/metabolismo , Esfingomielinas/metabolismo , Cistadenocarcinoma Seroso/metabolismoRESUMEN
Infertility afflicts up to 15% of couples globally each year with men a contributing factor in 50% of these cases. Globozoospermia is a rare condition found in infertile men, which is characterized by defective acrosome biogenesis leading to the production of round-headed sperm. Here, we report that family with sequence similarity 209 (Fam209) is required for acrosome biogenesis in mouse sperm. FAM209 is a small transmembrane protein conserved among mammals. Loss of Fam209 results in fertility defects that are secondary to abnormalities in acrosome biogenesis during spermiogenesis, reminiscent of globozoospermia. Analysis of the FAM209 proteome identified DPY19L2, whose human orthologue is involved in the majority of globozoospermia cases. Although mutations in human and mouse Dpy19l2 have been shown to cause globozoospermia, no in vivo interacting partners of DPY19L2 have been identified until now. FAM209 colocalizes with DPY19L2 at the inner nuclear membrane to maintain the developing acrosome. Here, we identified FAM209 as the first interacting partner of DPY19L2, and the second protein that is essential for acrosome biogenesis that localizes to the inner nuclear membrane.
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Acrosoma , Infertilidad Masculina , Animales , Fertilidad/genética , Infertilidad Masculina/genética , Masculino , Ratones , Espermatogénesis/genética , EspermatozoidesRESUMEN
Meiosis has a principal role in sexual reproduction to generate haploid gametes in both sexes. During meiosis, the cell nucleus hosts a dynamic environment where some genes are transcriptionally activated, and some are inactivated at the same time. This becomes possible through subnuclear compartmentalization. The sex body, sequestering X and Y chromosomes during male meiosis and creating an environment for the meiotic sex chromosome inactivation (MSCI) is one of the best known and studied subnuclear compartments. Herein, we show that MRNIP forms droplet-like accumulations that fuse together to create a distinct subnuclear compartment that partially overlaps with the sex body chromatin during diplotene. We demonstrate that Mrnip-/- spermatocytes have impaired DNA double-strand break (DSB) repair, they display reduced sex body formation and defective MSCI. We show that Mrnip-/- undergoes critical meiocyte loss at the diplotene stage. Furthermore, we determine that DNA DSBs (induced by SPO11) and synapsis initiation (facilitated by SYCP1) precede Mrnip expression in testes. Altogether, our findings indicate that in addition to an emerging role in DNA DSB repair, MRNIP has an essential function in spermatogenesis during meiosis I by forming drop-like accumulations interacting with the sex body.
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Espermatocitos , Espermatogénesis , Animales , Cromatina/genética , Cromatina/metabolismo , Femenino , Fertilidad , Masculino , Meiosis , Ratones , Espermatocitos/metabolismo , Espermatogénesis/genética , Cromosoma Y/genéticaRESUMEN
Sperm-oocyte membrane fusion is one of the most important events for fertilization. So far, IZUMO1 and Fertilization Influencing Membrane Protein (FIMP) on the sperm membrane and CD9 and JUNO (IZUMO1R/FOLR4) on the oocyte membrane have been identified as fusion-required proteins. However, the molecular mechanisms for sperm-oocyte fusion are still unclear. Here, we show that testis-enriched genes, sperm-oocyte fusion required 1 (Sof1/Llcfc1/1700034O15Rik), transmembrane protein 95 (Tmem95), and sperm acrosome associated 6 (Spaca6), encode sperm proteins required for sperm-oocyte fusion in mice. These knockout (KO) spermatozoa carry IZUMO1 but cannot fuse with the oocyte plasma membrane, leading to male sterility. Transgenic mice which expressed mouse Sof1, Tmem95, and Spaca6 rescued the sterility of Sof1, Tmem95, and Spaca6 KO males, respectively. SOF1 and SPACA6 remain in acrosome-reacted spermatozoa, and SPACA6 translocates to the equatorial segment of these spermatozoa. The coexpression of SOF1, TMEM95, and SPACA6 in IZUMO1-expressing cultured cells did not enhance their ability to adhere to the oocyte membrane or allow them to fuse with oocytes. SOF1, TMEM95, and SPACA6 may function cooperatively with IZUMO1 and/or unknown fusogens in sperm-oocyte fusion.
Asunto(s)
Reacción Acrosómica , Proteínas de la Membrana , Proteínas de Plasma Seminal , Espermatozoides/fisiología , Reacción Acrosómica/genética , Reacción Acrosómica/fisiología , Animales , Femenino , Infertilidad Masculina/genética , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Proteínas de Plasma Seminal/genética , Proteínas de Plasma Seminal/metabolismoRESUMEN
DNA-encoded chemical libraries are collections of compounds individually coupled to unique DNA tags serving as amplifiable identification barcodes. By bridging split-and-pool combinatorial synthesis with the ligation of unique encoding DNA oligomers, million- to billion-member libraries can be synthesized for use in hundreds of healthcare target screens. Although structural diversity and desirable molecular property ranges generally guide DNA-encoded chemical library design, recent reports have highlighted the utility of focused DNA-encoded chemical libraries that are structurally biased for a class of protein targets. Herein, a protease-focused DNA-encoded chemical library was designed that utilizes chemotypes known to engage conserved catalytic protease residues. The three-cycle library features functional moieties such as guanidine, which interacts strongly with aspartate of the protease catalytic triad, as well as mild electrophiles such as sulfonamide, urea, and carbamate. We developed a DNA-compatible method for guanidinylation of amines and reduction of nitriles. Employing these optimized reactions, we constructed a 9.8-million-membered DNA-encoded chemical library. Affinity selection of the library with thrombin, a common protease, revealed a number of enriched features which ultimately led to the discovery of a 1 nM inhibitor of thrombin. Thus, structurally focused DNA-encoded chemical libraries have tremendous potential to find clinically useful high-affinity hits for the rapid discovery of drugs for targets (e.g., proteases) with essential functions in infectious diseases (e.g., severe acute respiratory syndrome coronavirus 2) and relevant healthcare conditions (e.g., male contraception).
Asunto(s)
ADN/química , ADN/metabolismo , Descubrimiento de Drogas , Biblioteca de Genes , Inhibidores de Proteasas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Trombina/antagonistas & inhibidores , Técnicas Químicas Combinatorias , Humanos , Inhibidores de Proteasas/química , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
Sperm-oocyte fusion is a critical event in mammalian fertilization, categorized by three indispensable proteins. Sperm membrane protein IZUMO1 and its counterpart oocyte membrane protein JUNO make a protein complex allowing sperm to interact with the oocyte, and subsequent sperm-oocyte fusion. Oocyte tetraspanin protein CD9 also contributes to sperm-oocyte fusion. However, the fusion process cannot be explained solely by these three essential factors. In this study, we focused on analyzing a testis-specific gene 4930451I11Rik and generated mutant mice using the CRISPR/Cas9 system. Although IZUMO1 remained in 4930451I11Rik knockout (KO) spermatozoa, the KO spermatozoa were unable to fuse with oocytes and the KO males were severely subfertile. 4930451I11Rik encodes two isoforms: a transmembrane (TM) form and a secreted form. Both CRISPR/Cas9-mediated TM deletion and transgenic (Tg) rescue with the TM form revealed that only the TM form plays a critical role in sperm-oocyte fusion. Thus, we renamed this TM form Fertilization Influencing Membrane Protein (FIMP). The mCherry-tagged FIMP TM form was localized to the sperm equatorial segment where the sperm-oocyte fusion event occurs. Thus, FIMP is a sperm-specific transmembrane protein that is necessary for the sperm-oocyte fusion process.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/metabolismo , Espermatozoides/metabolismo , Testículo/metabolismo , Secuencia de Aminoácidos , Animales , Fertilización In Vitro , Humanos , Inmunoglobulinas/genética , Inmunoglobulinas/metabolismo , Infertilidad Masculina/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Oocitos/fisiología , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Interacciones Espermatozoide-Óvulo/fisiologíaRESUMEN
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.
Asunto(s)
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
BACKGROUND: Ubiquitination is a post-translational modification required for a number of physiological functions regulating protein homeostasis, such as protein degradation. The endoplasmic reticulum (ER) quality control system recognizes and degrades proteins no longer needed in the ER through the ubiquitin-proteasome pathway. E2 and E3 enzymes containing a transmembrane domain have been shown to function in ER quality control. The ER transmembrane protein UBE2J1 is a E2 ubiquitin-conjugating enzyme reported to be essential for spermiogenesis at the elongating spermatid stage. Spermatids from Ube2j1 KO male mice are believed to have defects in the dislocation step of ER quality control. However, associated E3 ubiquitin-protein ligases that function during spermatogenesis remain unknown. RESULTS: We identified four evolutionarily conserved testis-specific E3 ubiquitin-protein ligases [RING finger protein 133 (Rnf133); RING finger protein 148 (Rnf148); RING finger protein 151 (Rnf151); and Zinc finger SWIM-type containing 2 (Zswim2)]. Using the CRISPR/Cas9 system, we generated and analyzed the fertility of mutant mice with null alleles for each of these E3-encoding genes, as well as double and triple knockout (KO) mice. Male fertility, male reproductive organ, and sperm-associated parameters were analyzed in detail. Fecundity remained largely unaffected in Rnf148, Rnf151, and Zswim2 KO males; however, Rnf133 KO males displayed severe subfertility. Additionally, Rnf133 KO sperm exhibited abnormal morphology and reduced motility. Ultrastructural analysis demonstrated that cytoplasmic droplets were retained in Rnf133 KO spermatozoa. Although Rnf133 and Rnf148 encode paralogous genes that are chromosomally linked and encode putative ER transmembrane E3 ubiquitin-protein ligases based on their protein structures, there was limited functional redundancy of these proteins. In addition, we identified UBE2J1 as an E2 ubiquitin-conjugating protein that interacts with RNF133. CONCLUSIONS: Our studies reveal that RNF133 is a testis-expressed E3 ubiquitin-protein ligase that plays a critical role for sperm function during spermiogenesis. Based on the presence of a transmembrane domain in RNF133 and its interaction with the ER containing E2 protein UBE2J1, we hypothesize that these ubiquitin-regulatory proteins function together in ER quality control during spermatogenesis.