RESUMEN
Autism spectrum disorder (ASD) is a neurodevelopmental disorder (NDD) that affects approximately 4% of males and 1% of females in the United States. While causes of ASD are multi-factorial, single rare genetic variants contribute to around 20% of cases. Here we report a case series of seven unrelated probands (6 males, 1 female) with ASD or another variable NDD phenotype attributed to de novo heterozygous loss of function or missense variants in the gene LARP1. LARP1 encodes an RNA binding protein that post-transcriptionally regulates the stability and translation of thousands of mRNAs, including those regulating cellular metabolism and metabolic plasticity. Using lymphocytes collected and immortalized from an index proband who carries a truncating variant in one allele of LARP1, we demonstrated lower cellular levels of LARP1 protein causing reduced rates of aerobic respiration and glycolysis. As expression of LARP1 increases during neurodevelopment, with higher levels in neurons and astrocytes, we propose that LARP1 haploinsufficiency contributes to ASD or related NDDs through attenuated metabolic activity in the developing fetal brain.
RESUMEN
PURPOSE: Nucleoside analogues form the backbone of many therapeutic regimens in oncology and require the presence of intracellular enzymes for their activation. A ProTide is comprised of a nucleoside fused to a protective phosphoramidate cap. ProTides are easily incorporated into cells whereupon the cap is cleaved and a preactivated nucleoside released. 3'-Deoxyadenosine (3'-dA) is a naturally occurring adenosine analogue with established anticancer activity in vitro but limited bioavailability due to its rapid in vivo deamination by the circulating enzyme adenosine deaminase, poor uptake into cells, and reliance on adenosine kinase for its activation. In order to overcome these limitations, 3'-dA was chemically modified to create the novel ProTide NUC-7738. EXPERIMENTAL DESIGN: We describe the synthesis of NUC-7738. We determine the IC50 of NUC-7738 using pharmacokinetics (PK) and conduct genome-wide analyses to identify its mechanism of action using different cancer model systems. We validate these findings in patients with cancer. RESULTS: We show that NUC-7738 overcomes the cancer resistance mechanisms that limit the activity of 3'-dA and that its activation is dependent on ProTide cleavage by the enzyme histidine triad nucleotide-binding protein 1. PK and tumor samples obtained from the ongoing first-in-human phase I clinical trial of NUC-7738 further validate our in vitro findings and show NUC-7738 is an effective proapoptotic agent in cancer cells with effects on the NF-κB pathway. CONCLUSIONS: Our study provides proof that NUC-7738 overcomes cellular resistance mechanisms and supports its further clinical evaluation as a novel cancer treatment within the growing pantheon of anticancer ProTides.
Asunto(s)
Neoplasias , Nucleósidos , Estudio de Asociación del Genoma Completo , Humanos , Neoplasias/tratamiento farmacológicoRESUMEN
LARP1 is an oncogenic RNA-binding protein required for ribosome biogenesis and cancer cell survival. From published in vitro studies, there is disparity over which of two different LARP1 protein isoforms (termed the long LI-LARP1 and short SI-LARP1) is the canonical. Here, after conducting a series of biochemical and cellular assays, we conclude that LI-LARP1 (NM_033551.3 > NP_056130.2) is the dominantly expressed form. We observe that SI-LARP1 (NM_015315.5> NP_056130.2) is epigenetically repressed and that this repression is evolutionarily conserved in all but a small subclade of mammalian species. As with other LARP family members, there are multiple potential LARP1 mRNA isoforms that appear to be censored within the nucleus. The capacity of the cell to modulate splicing and expression of these apparently 'redundant' mRNAs hints at contextually specific mechanisms of LARP1 expression.
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Autoantígenos/genética , Regulación Neoplásica de la Expresión Génica , Ribonucleoproteínas/genética , Empalme Alternativo , Secuencia de Aminoácidos , Autoantígenos/química , Autoantígenos/metabolismo , Línea Celular Tumoral , Metilación de ADN , Silenciador del Gen , Humanos , Familia de Multigenes , Especificidad de Órganos , Regiones Promotoras Genéticas , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN/genética , ARN/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Antígeno SS-BRESUMEN
Cancer-related inflammation impacts significantly on cancer development and progression. From early stages, neutrophils and macrophages are drawn to pre-neoplastic cells in the epidermis, but before directly interacting, they must first breach the underlying extracellular matrix barrier layer that includes the basement membrane. Using several different skin cancer models and a collagen I-GFP transgenic zebrafish line, we have undertaken correlative light and electron microscopy (CLEM) to capture the moments when immune cells traverse the basement membrane. We show evidence both for active proteolytic burrowing and for the opportunistic use of pre-existing weak spots in the matrix layer. We show that these small holes, as well as much larger, cancer cell-generated or wound-triggered gaps in the matrix barrier, provide portals for immune cells to access cancer cells in the epidermis and thus are rate limiting in cancer progression.
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Membrana Basal/enzimología , Carcinogénesis/inmunología , Matriz Extracelular/metabolismo , Células Caliciformes/citología , Macrófagos/citología , Neutrófilos/citología , Neoplasias Cutáneas/inmunología , Animales , Animales Modificados Genéticamente , Membrana Basal/citología , Membrana Basal/metabolismo , Membrana Basal/ultraestructura , Carcinogénesis/genética , Carcinogénesis/ultraestructura , Proliferación Celular , Colágeno/metabolismo , Modelos Animales de Enfermedad , Epidermis/crecimiento & desarrollo , Epidermis/inmunología , Epidermis/patología , Matriz Extracelular/enzimología , Células Caliciformes/metabolismo , Células Caliciformes/ultraestructura , Macrófagos/enzimología , Macrófagos/inmunología , Macrófagos/ultraestructura , Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/metabolismo , Microscopía Electrónica de Transmisión , Neutrófilos/enzimología , Neutrófilos/inmunología , Neutrófilos/ultraestructura , Proteolisis/efectos de los fármacos , Neoplasias Cutáneas/enzimología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/ultraestructura , Pez CebraRESUMEN
Fibrillar collagen is a major component of many tissues but has been difficult to image in vivo using transgenic approaches because of problems associated with establishing cells and organisms that generate GFP-fusion collagens that can polymerise into functional fibrils. Here we have developed and characterised GFP and mCherry collagen-I fusion zebrafish lines with basal epidermal-specific expression. We use these lines to reveal the dynamic nature of collagen-I fibril deposition beneath the developing embryonic epidermis, as well as the repair of this collagen meshwork following wounding. Transmission electron microscope studies show that these transgenic lines faithfully reproduce the collagen ultrastructure present in wild type larval skin. During skin development we show that collagen I is deposited by basal epidermal cells initially in fine filaments that are largely randomly orientated but are subsequently aligned into a cross-hatch, orthogonal sub-epithelial network by embryonic day 4. Following skin wounding, we see that sub-epidermal collagen is re-established in the denuded domain, initially as randomly orientated wisps that subsequently become bonded to the undamaged collagen and aligned in a way that recapitulates developmental deposition of sub-epidermal collagen. Crossing our GFP-collagen line against one with tdTomato marking basal epidermal cell membranes reveals how much more rapidly wound re-epithelialisation occurs compared to the re-deposition of collagen beneath the healed epidermis. By use of other tissue specific drivers it will be possible to establish zebrafish lines to enable live imaging of collagen deposition and its remodelling in various other organs in health and disease.
Asunto(s)
Animales Modificados Genéticamente , Colágeno Tipo I , Proteínas Fluorescentes Verdes , Imagen Óptica/métodos , Proteínas Recombinantes de Fusión , Piel , Pez Cebra , Animales , Animales Modificados Genéticamente/embriología , Animales Modificados Genéticamente/genética , Colágeno Tipo I/biosíntesis , Colágeno Tipo I/genética , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Piel/citología , Piel/embriología , Pez Cebra/embriología , Pez Cebra/genéticaRESUMEN
A genetically encoded precursor to an aryl nitrene, para-azidophenylalanine, was introduced site specifically into proteins to deduce if distinct environments were capable of caging a reactive organic intermediate. Following photolysis of mutant T4 lysozyme or green fluorescent proteins, EPR spectra showed, respectively, the presence of a triplet nitrene and an anilino radical.
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Azidas/química , Fenilalanina/análogos & derivados , Bacteriófago T4/enzimología , Bacteriófago T4/genética , Espectroscopía de Resonancia por Spin del Electrón , Proteínas Fluorescentes Verdes/química , Iminas/química , Estructura Molecular , Fenilalanina/química , Fotoquímica , Fotólisis , Rayos Ultravioleta , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
There are many approaches to introduce non-native functionality into proteins either translationally or post-translationally. When a noncanonical amino acid (NAA) is incorporated translationally, the host organism's existing translational machinery is relied upon to insert the amino acid by the same well-established mechanisms used by the host to achieve high fidelity insertion of its canonical amino acids. Research into the in vivo incorporation of NAAs has typically concentrated on evolving or engineering aminoacyl tRNA synthetases (aaRSs); however, new studies have increasingly focused on other members of the translational apparatus, for example entire ribosomes, in attempts to increase the fidelity and efficiency of incorporation of ever more structurally diverse NAAs. As the biochemical methods of NAA systems increase in complexity, it is informative to ask whether the 'rules' for canonical translation (i.e. aaRSs, tRNA, ribosomes, elongation factors, amino acid uptake, and metabolism) hold for NAA systems, or whether new rules are warranted. Here, recent advances in introducing novel chemical functionality into proteins are highlighted.
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Aminoácidos/metabolismo , Ingeniería de Proteínas/métodos , Proteínas/química , Proteínas/genética , Aminoácidos/genética , Animales , Codón/genética , Humanos , Proteínas/metabolismo , Biología SintéticaRESUMEN
Coupling the activities of normally disparate proteins into one functional unit has significant potential in terms of constructing novel switching components for synthetic biology or as biosensors. It also provides a means of investigating the basis behind transmission of conformation events between remote sites that is integral to many biological processes, including allostery. Here we describe how the structures and functions of two normally unlinked proteins, namely, the heme binding capability of cytochrome b(562) and the antibiotic degrading beta-lactamase activity of TEM, have been coupled using a directed evolution domain insertion approach. The important small biomolecule heme directly modulates in vivo and in vitro the beta-lactamase activity of selected integral fusion proteins. The presence of heme decreased the concentration of ampicillin tolerated by Escherichia coli and the level of in vitro hydrolysis of nitrocefin by up to 2 orders of magnitude. Variants with the largest switching magnitudes contained insertions at second-shell sites that abut key catalytic residues. Spectrophotometry confirmed that heme bound to the integral fusion proteins in a manner similar to that of cytochrome b(562). Circular dichroism suggested that only subtle structural changes rather than gross folding-unfolding events were responsible for modulating beta-lactamase activity, and size exclusion chromatography confirmed that the integral fusion proteins remained monomeric in both the apo and holo forms. Thus, by sampling a variety of insertion positions and linker sequences, we are able to couple the functions of two unrelated proteins by domain insertion.