RESUMEN
The transmembrane 6 superfamily member 2 (TM6SF2) loss-of-function variant rs58542926 is a genetic risk factor for nonalcoholic fatty liver disease and progression to fibrosis but is paradoxically associated with lower levels of hepatically derived triglyceride-rich lipoproteins. TM6SF2 is expressed predominantly in liver and small intestine, sites for triglyceride-rich lipoprotein biogenesis and export. In light of this, we hypothesized that TM6SF2 may exhibit analogous effects on both liver and intestine lipid homeostasis. To test this, we genotyped rs58542926 in 983 bariatric surgery patients from the Geisinger Medical Center for Nutrition and Weight Management, Geisinger Health System, in Pennsylvania and from 3,556 study participants enrolled in the Amish Complex Disease Research Program. Although these two cohorts have different metabolic profiles, carriers in both cohorts had improved fasting lipid profiles. Importantly, following a high-fat challenge, carriers in the Amish Complex Disease Research Program cohort exhibited significantly lower postprandial serum triglycerides, suggestive of a role for TM6SF2 in the small intestine. To gain further insight into this putative role, effects of TM6SF2 deficiency were studied in a zebrafish model and in cultured human Caco-2 enterocytes. In both systems TM6SF2 deficiency resulted in defects in small intestine metabolism in response to dietary lipids, including significantly increased lipid accumulation, decreased lipid clearance, and increased endoplasmic reticulum stress. CONCLUSIONS: These data strongly support a role of TM6SF2 in the regulation of postprandial lipemia, potentially through a similar function for TM6SF2 in the lipidation and/or export of both hepatically and intestinally derived triglyceride-rich lipoproteins. (Hepatology 2017;65:1526-1542).
Asunto(s)
Estrés del Retículo Endoplásmico , Intestino Delgado/metabolismo , Metabolismo de los Lípidos/genética , Hígado/metabolismo , Proteínas de la Membrana/genética , Animales , Secuencia de Bases , Células CACO-2 , Enterocitos/metabolismo , Hígado Graso/genética , Femenino , Hepatocitos/metabolismo , Homeostasis , Humanos , Intestino Delgado/ultraestructura , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Persona de Mediana Edad , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple , Periodo Posprandial , Triglicéridos/biosíntesis , Triglicéridos/sangre , Tunicamicina , Pez CebraRESUMEN
Despite significant advances over recent years, the treatment of T cell acute lymphoblastic leukemia (T-ALL) remains challenging. We have recently shown that a subset of T-ALL cases exhibited constitutive activation of the lymphocyte-specific protein tyrosine kinase (LCK) and were consequently responsive to treatments with LCK inhibitors and degraders such as dasatinib and dasatinib-based PROTACs. Here we report the design, synthesis and in vitro/vivo evaluation of SJ45566, a potent and orally bioavailable LCK PROTAC.
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Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/antagonistas & inhibidores , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Humanos , Administración Oral , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Animales , Ratones , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/síntesis química , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Antineoplásicos/síntesis química , Antineoplásicos/farmacocinética , Disponibilidad Biológica , Línea Celular Tumoral , Relación Estructura-ActividadRESUMEN
Molecular-glue degraders are small molecules that induce a specific interaction between an E3 ligase and a target protein, resulting in the target proteolysis. The discovery of molecular glue degraders currently relies mostly on screening approaches. Here, we describe screening of a library of cereblon (CRBN) ligands against a panel of patient-derived cancer cell lines, leading to the discovery of SJ7095, a potent degrader of CK1α, IKZF1 and IKZF3 proteins. Through a structure-informed exploration of structure activity relationship (SAR) around this small molecule we develop SJ3149, a selective and potent degrader of CK1α protein in vitro and in vivo. The structure of SJ3149 co-crystalized in complex with CK1α + CRBN + DDB1 provides a rationale for the improved degradation properties of this compound. In a panel of 115 cancer cell lines SJ3149 displays a broad antiproliferative activity profile, which shows statistically significant correlation with MDM2 inhibitor Nutlin-3a. These findings suggest potential utility of selective CK1α degraders for treatment of hematological cancers and solid tumors.
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Antineoplásicos , Neoplasias , Humanos , Antineoplásicos/farmacología , Antineoplásicos/química , Línea Celular , Neoplasias/tratamiento farmacológico , Proteolisis , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Genome editing using the CRISPR-Cas9 platform creates precise modifications in cells and whole organisms. Although knockout (KO) mutations can occur at high frequencies, determining the editing rates in a pool of cells or selecting clones that contain only KO alleles can be a challenge. User-defined knock-in (KI) modifications are achieved at much lower rates, making the identification of correctly modified clones even more challenging. The high-throughput format of targeted next-generation sequencing (NGS) provides a platform allowing sequence information to be gathered from a one to thousands of samples. However, it also poses a challenge in terms of analyzing the large amount of data that is generated. In this chapter, we present and discuss CRIS.py, a simple and highly versatile Python-based program for analyzing NGS data for genome-editing outcomes. CRIS.py can be used to analyze sequencing results for any kind of modification or multiplex modifications specified by the user. Moreover, CRIS.py runs on all fastq files found in a directory, thereby concurrently analyzing all uniquely indexed samples. CRIS.py results are consolidated into two summary files, which allows users to sort and filter results and quickly identify the clones (or animals) of greatest interest.
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Sistemas CRISPR-Cas , Edición Génica , Animales , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Mutación , Modelos AnimalesRESUMEN
Thalidomide and its analogues are frequently used in PROTAC design. However, they are known to be inherently unstable, undergoing hydrolysis even in commonly utilized cell culture media. We recently reported that phenyl glutarimide (PG)-based PROTACs displayed improved chemical stability and, consequently, improved protein degradation efficacy and cellular potency. Our optimization efforts, aiming to further improve the chemical stability and eliminate the racemization-prone chiral center in PG, led us to the development of phenyl dihydrouracil (PD)-based PROTACs. Here we describe the design and synthesis of LCK-directing PD-PROTACs and compare their physicochemical and pharmacological properties to those of the corresponding IMiD and PG analogues.
RESUMEN
B cell lineage acute lymphoblastic leukemia (B-ALL) is composed of diverse molecular subtypes, and while transcriptional and DNA methylation profiling has been extensively examined, the chromatin landscape is not well characterized for many subtypes. We therefore mapped chromatin accessibility using ATAC-seq in primary B-ALL cells from 156 patients spanning ten molecular subtypes and present this dataset as a resource. Differential chromatin accessibility and transcription factor (TF) footprint profiling were employed and identified B-ALL cell of origin, TF-target gene interactions enriched in B-ALL, and key TFs associated with accessible chromatin sites preferentially active in B-ALL. We further identified over 20% of accessible chromatin sites exhibiting strong subtype enrichment and candidate TFs that maintain subtype-specific chromatin architectures. Over 9,000 genetic variants were uncovered, contributing to variability in chromatin accessibility among patient samples. Our data suggest that distinct chromatin architectures are driven by diverse TFs and inherited genetic variants that promote unique gene-regulatory networks.
RESUMEN
Ezakiella coagulans and Fenollaria massiliensis are two obligate anaerobic bacteria in the family Peptoniphilaceae and are both uncommon members of the human vaginal microbiota. We isolated a strain of each bacterium from the same vaginal swab specimen and here report the first complete genome sequences of the two species.
RESUMEN
The ability of mitochondria to buffer a rapid rise in cytosolic Ca2+ is a hallmark of proper cell homeostasis. Here, we employed m-3M3FBS, a putative phospholipase C (PLC) agonist, to explore the relationships between intracellular Ca2+ imbalance, mitochondrial physiology, and cell death. m-3M3FBS induced a potent dose-dependent Ca2+ release from the endoplasmic reticulum (ER), followed by a rise in intra-mitochondrial Ca2+. When the latter exceeded the organelle buffering capacity, an abrupt mitochondrial inner membrane permeabilization (MIMP) occurred, releasing matrix contents into the cytosol. MIMP was followed by cell death that was independent of Bcl-2 family members and inhibitable by the intracellular Ca2+ chelator BAPTA-AM. Cyclosporin A (CsA), capable of blocking the mitochondrial permeability transition (MPT), completely prevented cell death induced by m-3M3FBS. However, CsA acted upstream of mitochondria by preventing Ca2+ release from ER stores. Therefore, loss of Ca2+ intracellular balance and mitochondrial Ca2+ overload followed by MIMP induced a cell death process that is distinct from Bcl-2 family-regulated mitochondrial outer membrane permeabilization (MOMP). Further, the inhibition of cell death by CsA or its analogues can be independent of effects on the MPT.
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Calcio , Membranas Mitocondriales , Apoptosis , Calcio/metabolismo , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
BACKGROUND: Vaginal bacterial communities dominated by Lactobacillus species are associated with a reduced risk of various adverse health outcomes. However, somewhat unexpectedly, many healthy women have microbiota that are not dominated by lactobacilli. To determine the factors that drive vaginal community composition we characterized the genetic composition and transcriptional activities of vaginal microbiota in healthy women. RESULTS: We demonstrate that the abundance of a species is not always indicative of its transcriptional activity and that impending changes in community composition can be predicted from metatranscriptomic data. Functional comparisons highlight differences in the metabolic activities of these communities, notably in their degradation of host produced mucin but not glycogen. Degradation of mucin by communities not dominated by Lactobacillus may play a role in their association with adverse health outcomes. Finally, we show that the transcriptional activities of L. crispatus, L. iners, and Gardnerella vaginalis vary with the taxonomic composition of the communities in which they reside. Notably, L. iners and G. vaginalis both demonstrate lower expression of their cholesterol-dependent cytolysins when co-resident with Lactobacillus spp. and higher expression when co-resident with other facultative and obligate anaerobes. The pathogenic potential of these species may depend on the communities in which they reside and thus could be modulated by interventional strategies. CONCLUSIONS: Our results provide insight to the functional ecology of the vaginal microbiota, demonstrate the diagnostic potential of metatranscriptomic data, and reveal strategies for the management of these ecosystems.
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Microbiota , Vagina , Bacterias/genética , Femenino , Humanos , Lactobacillus/genética , Microbiota/genética , Mucinas , ARN Ribosómico 16S/genética , Vagina/microbiologíaRESUMEN
Lactobacillus iners is a common member of the human vaginal microbiota, with a genome size smaller than that of other lactobacilli. Here, we report the complete genome sequences of six L. iners strains isolated from different vaginal swab specimens. Three strains were found to harbor â¼100-kbp plasmids, which were not known previously.