RESUMO
Calcium (Ca2+ ) is a physiological key factor, and the precise modulation of free cytosolic Ca2+ levels regulates multiple cellular functions. Store-operated Ca2+ entry (SOCE) is a major mechanism controlling Ca2+ homeostasis, and is mediated by the concerted activity of the Ca2+ sensor STIM1 and the Ca2+ channel ORAI1. Dominant gain-of-function mutations in STIM1 or ORAI1 cause tubular aggregate myopathy (TAM) or Stormorken syndrome, whereas recessive loss-of-function mutations are associated with immunodeficiency. Here, we report the identification and functional characterization of novel ORAI1 mutations in TAM patients. We assess basal activity and SOCE of the mutant ORAI1 channels, and we demonstrate that the G98S and V107M mutations generate constitutively permeable ORAI1 channels, whereas T184M alters the channel permeability only in the presence of STIM1. These data indicate a mutation-dependent pathomechanism and a genotype/phenotype correlation, as the ORAI1 mutations associated with the most severe symptoms induce the strongest functional cellular effect. Examination of the non-muscle features of our patients strongly suggests that TAM and Stormorken syndrome are spectra of the same disease. Overall, our results emphasize the importance of SOCE in skeletal muscle physiology, and provide new insights in the pathomechanisms involving aberrant Ca2+ homeostasis and leading to muscle dysfunction.
Assuntos
Ativação do Canal Iônico/genética , Mutação de Sentido Incorreto , Miopatias Congênitas Estruturais/genética , Proteína ORAI1/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Transtornos Plaquetários/genética , Transtornos Plaquetários/metabolismo , Cálcio/metabolismo , Células Cultivadas , Dislexia/genética , Dislexia/metabolismo , Eritrócitos Anormais/metabolismo , Feminino , Células HEK293 , Humanos , Ictiose/genética , Ictiose/metabolismo , Masculino , Camundongos Knockout , Microscopia de Fluorescência/métodos , Transtornos de Enxaqueca/genética , Transtornos de Enxaqueca/metabolismo , Miose/genética , Miose/metabolismo , Fadiga Muscular/genética , Miopatias Congênitas Estruturais/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Linhagem , Homologia de Sequência de Aminoácidos , Baço/anormalidades , Baço/metabolismo , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismoRESUMO
Tubular aggregates are regular arrays of membrane tubules accumulating in muscle with age. They are found as secondary features in several muscle disorders, including alcohol- and drug-induced myopathies, exercise-induced cramps, and inherited myasthenia, but also exist as a pure genetic form characterized by slowly progressive muscle weakness. We identified dominant STIM1 mutations as a genetic cause of tubular-aggregate myopathy (TAM). Stromal interaction molecule 1 (STIM1) is the main Ca(2+) sensor in the endoplasmic reticulum, and all mutations were found in the highly conserved intraluminal Ca(2+)-binding EF hands. Ca(2+) stores are refilled through a process called store-operated Ca(2+) entry (SOCE). Upon Ca(2+)-store depletion, wild-type STIM1 oligomerizes and thereby triggers extracellular Ca(2+) entry. In contrast, the missense mutations found in our four TAM-affected families induced constitutive STIM1 clustering, indicating that Ca(2+) sensing was impaired. By monitoring the calcium response of TAM myoblasts to SOCE, we found a significantly higher basal Ca(2+) level in TAM cells and a dysregulation of intracellular Ca(2+) homeostasis. Because recessive STIM1 loss-of-function mutations were associated with immunodeficiency, we conclude that the tissue-specific impact of STIM1 loss or constitutive activation is different and that a tight regulation of STIM1-dependent SOCE is fundamental for normal skeletal-muscle structure and function.
Assuntos
Cálcio/metabolismo , Proteínas de Membrana/metabolismo , Miopatias Congênitas Estruturais/patologia , Proteínas de Neoplasias/metabolismo , Adolescente , Adulto , Idoso , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Criança , Feminino , Homeostase , Humanos , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Músculos/patologia , Músculos/ultraestrutura , Mutação/genética , Mioblastos/metabolismo , Mioblastos/patologia , Miopatias Congênitas Estruturais/genética , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Linhagem , Fenótipo , Molécula 1 de Interação Estromal , Adulto JovemRESUMO
Centronuclear myopathies are congenital muscle disorders characterized by type I myofibre predominance and an increased number of muscle fibres with nuclear centralization. The severe neonatal X-linked form is due to mutations in MTM1, autosomal recessive centronuclear myopathy with neonatal or childhood onset results from mutations in BIN1 (amphiphysin 2), and dominant cases were previously associated to mutations in DNM2 (dynamin 2). Our aim was to determine the genetic basis and physiopathology of patients with mild dominant centronuclear myopathy without mutations in DNM2. We hence established and characterized a homogeneous cohort of nine patients from five families with a progressive adult-onset centronuclear myopathy without facial weakness, including three sporadic cases and two families with dominant disease inheritance. All patients had similar histological and ultrastructural features involving type I fibre predominance and hypotrophy, as well as prominent nuclear centralization and clustering. We identified heterozygous BIN1 mutations in all patients and the molecular diagnosis was complemented by functional analyses. Two mutations in the N-terminal amphipathic helix strongly decreased the membrane-deforming properties of amphiphysin 2 and three stop-loss mutations resulted in a stable protein containing 52 supernumerary amino acids. Immunolabelling experiments revealed abnormal central accumulation of dynamin 2, caveolin-3, and the autophagic marker p62, and general membrane alterations of the triad, the sarcolemma, and the basal lamina as potential pathological mechanisms. In conclusion, we identified BIN1 as the second gene for dominant centronuclear myopathy. Our data provide the evidence that specific BIN1 mutations can cause either recessive or dominant centronuclear myopathy and that both disorders involve different pathomechanisms.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Mutação/genética , Miopatias Congênitas Estruturais/genética , Proteínas Nucleares/genética , Proteínas Supressoras de Tumor/genética , Adulto , Idade de Início , Dinamina II/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/metabolismoRESUMO
BACKGROUND: Tubular aggregate myopathies (TAMs) are muscle disorders characterised by abnormal accumulations of densely packed single-walled or double-walled membrane tubules in muscle fibres. Recently, STIM1, encoding a major calcium sensor of the endoplasmic reticulum, was identified as a TAM gene. METHODS: The present study aims to define the clinical, histological and ultrastructural phenotype of tubular aggregate myopathy and to assess the STIM1 mutation spectrum. RESULTS: We describe six new TAM families harbouring one known and four novel STIM1 mutations. All identified mutations are heterozygous missense mutations affecting highly conserved amino acids in the calcium-binding EF-hand domains, demonstrating the presence of a mutation hot spot for TAM. We show that the mutations induce constitutive STIM1 clustering, strongly suggesting that calcium sensing and consequently calcium homoeostasis is impaired. Histological and ultrastructural analyses define a common picture with tubular aggregates labelled with Gomori trichrome and Nicotinamide adenine dinucleotide (NADH) tetrazolium reductase, substantiating their endoplasmic reticulum origin. The aggregates were observed in both fibre types and were often accompanied by nuclear internalisation and fibre size variability. The phenotypical spectrum ranged from childhood onset progressive muscle weakness and elevated creatine kinase levels to adult-onset myalgia without muscle weakness and normal CK levels. CONCLUSIONS: The present study expands the phenotypical spectrum of STIM1-related tubular aggregate myopathy. STIM1 should therefore be considered for patients with tubular aggregate myopathies involving either muscle weakness or myalgia as the first and predominant clinical sign.
Assuntos
Proteínas de Membrana/genética , Músculo Esquelético/patologia , Mutação , Miopatias Congênitas Estruturais/diagnóstico , Miopatias Congênitas Estruturais/genética , Proteínas de Neoplasias/genética , Fenótipo , Adulto , Idoso , Sequência de Aminoácidos , Animais , Biópsia , Cálcio/metabolismo , Linhagem Celular , Análise Mutacional de DNA , Feminino , Humanos , Masculino , Proteínas de Membrana/química , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestrutura , Miopatias Congênitas Estruturais/metabolismo , Proteínas de Neoplasias/química , Linhagem , Conformação Proteica , Alinhamento de Sequência , Molécula 1 de Interação EstromalRESUMO
Retinal amacrine cells are a diverse set of interneurons within the inner nuclear layer. The canonical Wnt pathway is highly active within mature amacrine cells, but its role remains unclear. Leucine-rich repeat containing G-protein receptor 5 (Lgr5) is a newly identified component of the Wnt receptor complex that potentiates beta-catenin signaling. In multiple epithelial organs Lgr5 marks adult tissue stem cells. We investigated the expression of this gene using Lgr5-eGFP-IRES-CreER transgenic reporter mice. In the eye, Lgr5 was exclusively expressed in glycinergic amacrine cells in adult mice. Amacrine cells are post-mitotic and represent the first neuronal and non-stem cell lineage to express Lgr5. We further interrogated the spatiotemporal labeling of individual amacrine cells with controlled fluorophore expression. This "fluorofilling" technique provides a tool to study amacrine morphology and dissect neural networks.
Assuntos
Células Amácrinas/metabolismo , Regulação da Expressão Gênica , Glicinérgicos/farmacologia , Receptores Acoplados a Proteínas G/genética , Retina/metabolismo , Células Amácrinas/citologia , Células Amácrinas/efeitos dos fármacos , Animais , Camundongos , Camundongos Transgênicos , Receptores Acoplados a Proteínas G/biossíntese , Retina/citologia , Transdução de SinaisRESUMO
CAR-T therapy is a promising, novel treatment modality for B-cell malignancies and yet many patients relapse through a variety of means, including loss of CAR-T cells and antigen escape. To investigate leukemia-intrinsic CAR-T resistance mechanisms, we performed genome-wide CRISPR-Cas9 loss-of-function screens in an immunocompetent murine model of B-cell acute lymphoblastic leukemia (B-ALL) utilizing a modular guide RNA library. We identified IFNγR/JAK/STAT signaling and components of antigen processing and presentation pathway as key mediators of resistance to CAR-T therapy in vivo; intriguingly, loss of this pathway yielded the opposite effect in vitro (sensitized leukemia to CAR-T cells). Transcriptional characterization of this model demonstrated upregulation of these pathways in tumors relapsed after CAR-T treatment, and functional studies showed a surprising role for natural killer (NK) cells in engaging this resistance program. Finally, examination of data from B-ALL patients treated with CAR-T revealed an association between poor outcomes and increased expression of JAK/STAT and MHC-I in leukemia cells. Overall, our data identify an unexpected mechanism of resistance to CAR-T therapy in which tumor cell interaction with the in vivo tumor microenvironment, including NK cells, induces expression of an adaptive, therapy-induced, T-cell resistance program in tumor cells.
Assuntos
Linfoma de Burkitt , Leucemia , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Receptores de Antígenos Quiméricos , Humanos , Animais , Camundongos , RNA Guia de Sistemas CRISPR-Cas , Imunoterapia Adotiva , Linfócitos T , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Microambiente TumoralRESUMO
Generating reliable preclinical data in animal models of disease is essential in therapy development. Here, we performed statistical analysis and joint longitudinal-survival modeling of the progressive phenotype observed in Mtm1-/y mice, a reliable model for myotubular myopathy. Analysis of historical data was used to generate a model for phenotype progression, which was then confirmed with phenotypic data from a new colony of mice derived via in vitro fertilization in an independent animal house, highlighting the reproducibility of disease phenotype in Mtm1-/y mice. These combined data were used to refine the phenotypic parameters analyzed in these mice and improve the model generated for expected disease progression. The disease progression model was then used to test the therapeutic efficacy of Dnm2 targeting. Dnm2 reduction by antisense oligonucleotides blocked or postponed disease development, and resulted in a significant dose-dependent improvement outside the expected disease progression in untreated Mtm1-/y mice. This provides an example of optimizing disease analysis and testing therapeutic efficacy in a preclinical model, which can be applied by scientists testing therapeutic approaches using neuromuscular disease models in different laboratories. This article has an associated First Person interview with the joint first authors of the paper.
Assuntos
Miopatias Congênitas Estruturais , Proteínas Tirosina Fosfatases não Receptoras , Animais , Progressão da Doença , Dinamina II/genética , Humanos , Camundongos , Músculo Esquelético , Mutação , Miopatias Congênitas Estruturais/genética , Fenótipo , Proteínas Tirosina Fosfatases não Receptoras/genética , Reprodutibilidade dos TestesRESUMO
The immunostimulatory intracellular domains (ICDs) of chimaeric antigen receptors (CARs) are essential for converting antigen recognition into antitumoural function. Although there are many possible combinations of ICDs, almost all current CARs rely on combinations of CD3ð, CD28 and 4-1BB. Here we show that a barcoded library of 700,000 unique CD19-specific CARs with diverse ICDs cloned into lentiviral vectors and transduced into Jurkat T cells can be screened at high throughput via cell sorting and next-generation sequencing to optimize CAR signalling for antitumoural functions. By using this screening approach, we identified CARs with new ICD combinations that, compared with clinically available CARs, endowed human primary T cells with comparable tumour control in mice and with improved proliferation, persistence, exhaustion and cytotoxicity after tumour rechallenge in vitro. The screening strategy can be adapted to other disease models, cell types and selection conditions, and could be used to improve adoptive cell therapies and to expand their utility to new disease indications.
Assuntos
Neoplasias , Receptores de Antígenos de Linfócitos T/análise , Receptores de Antígenos Quiméricos , Animais , Antígenos CD28/metabolismo , Humanos , Imunoterapia Adotiva , Camundongos , Neoplasias/metabolismo , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos TRESUMO
Myotubular myopathy, also called X-linked centronuclear myopathy (XL-CNM), is a severe congenital disease targeted for therapeutic trials. To date, biomarkers to monitor disease progression and therapy efficacy are lacking. The Mtm1 -/y mouse is a faithful model for XL-CNM, due to myotubularin 1 (MTM1) loss-of-function mutations. Using both an unbiased approach (RNA sequencing [RNA-seq]) and a directed approach (qRT-PCR and protein level), we identified decreased Mstn levels in Mtm1 -/y muscle, leading to low levels of myostatin in muscle and plasma. Myostatin (Mstn or growth differentiation factor 8 [Gdf8]) is a protein released by myocytes and inhibiting muscle growth and differentiation. Decreasing Dnm2 by genetic cross with Dnm2 +/- mice or by antisense oligonucleotides blocked or postponed disease progression and resulted in an increase in circulating myostatin. In addition, plasma myostatin levels inversely correlated with disease severity and with Dnm2 mRNA levels in muscles. Altered Mstn levels were associated with a generalized disruption of the myostatin pathway. Importantly, in two different forms of CNMs we identified reduced circulating myostatin levels in plasma from patients. This provides evidence of a blood-based biomarker that may be used to monitor disease state in XL-CNM mice and patients and supports monitoring circulating myostatin during clinical trials for myotubular myopathy.
RESUMO
Skeletal muscle development and regeneration are tightly regulated processes. How the intracellular organization of muscle fibers is achieved during these steps is unclear. Here, we focus on the cellular and physiological roles of amphiphysin 2 (BIN1), a membrane remodeling protein mutated in both congenital and adult centronuclear myopathies (CNM), that is ubiquitously expressed and has skeletal muscle-specific isoforms. We created and characterized constitutive muscle-specific and inducible Bin1 homozygous and heterozygous knockout mice targeting either ubiquitous or muscle-specific isoforms. Constitutive Bin1-deficient mice died at birth from lack of feeding due to a skeletal muscle defect. T-tubules and other organelles were misplaced and altered, supporting a general early role for BIN1 in intracellular organization, in addition to membrane remodeling. Although restricted deletion of Bin1 in unchallenged adult muscles had no impact, the forced switch from the muscle-specific isoforms to the ubiquitous isoforms through deletion of the in-frame muscle-specific exon delayed muscle regeneration. Thus, ubiquitous BIN1 function is necessary for muscle development and function, whereas its muscle-specific isoforms fine tune muscle regeneration in adulthood, supporting that BIN1 CNM with congenital onset are due to developmental defects, whereas later onset may be due to regeneration defects.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Regeneração/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Animais , Animais Recém-Nascidos , Éxons/genética , Comportamento Alimentar , Homozigoto , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/ultraestrutura , Músculo Esquelético/ultraestrutura , Especificidade de Órgãos , Isoformas de Proteínas/metabolismo , Deleção de Sequência , Análise de SobrevidaRESUMO
OBJECTIVE: Patients with BRAF-mutant colorectal cancer (CRC) have a poor prognosis. Molecular status is not currently used to select which drug to use in combination with radiotherapy. Our aim was to identify drugs that radiosensitise CRC cells with known BRAF status. METHODS: We screened 298 oncological drugs with and without ionising radiation in colorectal cancer cells isogenic for BRAF. Hits from rank product analysis were validated in a 16-cell line panel of human CRC cell lines, using clonogenic survival assays and xenograft models in vivo. RESULTS: Most consistently identified hits were drugs targeting cell growth/proliferation or DNA damage repair. The most effective class of drugs that radiosensitised wild-type and mutant cell lines was PARP inhibitors. In clonogenic survival assays, talazoparib produced a radiation enhancement ratio of 1.9 in DLD1 (BRAF-wildtype) cells and 1.8 in RKO (BRAF V600E) cells. In DLD1 xenografts, talazoparib significantly increased the inhibitory effect of radiation on tumour growth (P ≤ 0.01). CONCLUSIONS: Our method for screening large drug libraries for radiosensitisation has identified PARP inhibitors as promising radiosensitisers of colorectal cancer cells with wild-type and mutant BRAF backgrounds.
RESUMO
Autosomal dominant centronuclear myopathy (CNM) caused by mutations in the gene coding for amphiphysin-2 (BIN1) typically presents in adulthood with progressive muscle weakness. We report a Dutch family with AD CNM due to a novel BIN1 mutation (c.53T>A (p.Val18Glu)), strongly impairing the membrane tubulation activity of amphiphysin-2. The main features were mild proximal weakness with pronounced myalgia, exercise intolerance and large muscle mass, with a childhood onset in the youngest generation and mild cognitive features. This suggests BIN1 mutations should be considered in patients with isolated exercise intolerance and myalgia, even in childhood.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Mutação , Miopatias Congênitas Estruturais/genética , Proteínas Nucleares/genética , Proteínas Supressoras de Tumor/genética , Idade de Início , Idoso , Criança , Família , Feminino , Genes Dominantes , Humanos , Masculino , Pessoa de Meia-Idade , Miopatias Congênitas Estruturais/epidemiologia , Miopatias Congênitas Estruturais/patologia , FenótipoRESUMO
Cisplatin and its platinum analogs, carboplatin and oxaliplatin, are some of the most widely used cancer chemotherapeutics. Although cisplatin and carboplatin are used primarily in germ cell, breast and lung malignancies, oxaliplatin is instead used almost exclusively to treat colorectal and other gastrointestinal cancers. Here we utilize a unique, multi-platform genetic approach to study the mechanism of action of these clinically established platinum anti-cancer agents, as well as more recently developed cisplatin analogs. We show that oxaliplatin, unlike cisplatin and carboplatin, does not kill cells through the DNA-damage response. Rather, oxaliplatin kills cells by inducing ribosome biogenesis stress. This difference in drug mechanism explains the distinct clinical implementation of oxaliplatin relative to cisplatin, and it might enable mechanistically informed selection of distinct platinum drugs for distinct malignancies. These data highlight the functional diversity of core components of front-line cancer therapy and the potential benefits of applying a mechanism-based rationale to the use of our current arsenal of anti-cancer drugs.
Assuntos
Antineoplásicos/farmacologia , Carboplatina/farmacologia , Cisplatino/farmacologia , Neoplasias , Biogênese de Organelas , Compostos Organoplatínicos/farmacologia , Ribossomos/efeitos dos fármacos , Animais , Western Blotting , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Humanos , Camundongos , Oxaliplatina , Fenantridinas/farmacologia , Compostos de Platina/farmacologia , Análise de Componente Principal , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estresse Fisiológico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We present the clinical, morphological and molecular data of an Italian family with centronuclear myopathy, carrying a novel pathogenic mutation of BIN1 gene in heterozygous state, consistent with autosomal dominant inheritance. The proband, a 56-years-old man suffered of lower limbs myalgia and slight CK elevation. Clinical examination revealed no muscle weakness, short stature, mild symmetric eyelid ptosis, scapular winging, ankle retraction and well-developed muscles. Muscle biopsy showed nuclear centralization and clustering, deep sarcolemmal invaginations and type 1 fibers hypotrophy. Muscle MRI revealed fatty infiltration of posterior legs compartments, lumbar paraspinal and serratus muscles. By sequencing BIN1, we identified a heterozygous pathogenic mutation [c.107C>A (p.A36E)], and we demonstrate that the mutation strongly impairs the membrane tubulation property of the protein. One affected sister with similar phenotype carried the same mutation. Our findings expand the clinical, morphological and genetic spectrum of the autosomal dominant CNM associated with BIN1 mutations.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Creatina Quinase/metabolismo , Mialgia/fisiopatologia , Miopatias Congênitas Estruturais , Proteínas Nucleares/genética , Proteínas Supressoras de Tumor/genética , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mialgia/etiologia , Miopatias Congênitas Estruturais/genética , Miopatias Congênitas Estruturais/metabolismo , Miopatias Congênitas Estruturais/patologia , Miopatias Congênitas Estruturais/fisiopatologia , LinhagemRESUMO
The objective of this study was to determine and compare the in vitro responses of equine large colon arterial and venous rings to vasodilatory neuropeptides; calcitonin gene-related peptide (CGRP); substance P (SP); vasoactive intestinal polypeptide (VIP); and acetylcholine (ACh), a standard nonpeptide endothelium-dependent vasodilator. Responses of vessel rings to graded concentrations (10(-11) M to 10(-5) M) of each drug were determined in endothelium-intact, denuded, and Nomega-nitro-L-arginine methyl ester (L-NAME, 10(-5) M)-treated rings that were pre-contracted with norepinephrine. Percentage maximal relaxation (PMR), defined as the % decrease from the contracted state, was determined. Because all rings did not relax at least 50%, EC50 values could not be consistently calculated. Arterial rings with intact endothelium were more sensitive to CGRP, compared with VIP and SP, and venous rings of all conditions were more sensitive to VIP than CGRP or SP. Overall, arteries had a greater PMR for ACh compared with SP and VIP. Intact and L-NAME treated arteries had a greater PMR than denuded arteries; there were no differences in PMR of intact and L-NAME treated arteries. Veins had a greater PMR for VIP than CGRP, SP, or ACh. Calcitonin gene-related peptide caused greater relaxation in intact arteries, whereas VIP causes greater relaxation in veins. Arterial relaxation was dependent upon the presence of intact endothelium. The response of veins to VIP among the conditions tested was not different, suggesting VIP has direct actions on venous smooth muscle. These neuropeptides modulate vasomotor tone via vasorelaxation in colonic arteries and veins.
Assuntos
Colo/irrigação sanguínea , Endotélio Vascular/fisiologia , Neuropeptídeos/farmacologia , Óxido Nítrico/fisiologia , Vasodilatadores/farmacologia , Acetilcolina/farmacologia , Animais , Artérias/efeitos dos fármacos , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Colo/efeitos dos fármacos , Colo/metabolismo , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Cavalos , Técnicas In Vitro , Contração Muscular , Óxido Nítrico/metabolismo , Norepinefrina/farmacologia , Substância P/farmacologia , Peptídeo Intestinal Vasoativo/farmacologia , Veias/efeitos dos fármacosRESUMO
Nucleus positioning is key for intracellular organization, cell differentiation, and organ development and is affected in many diseases, including myopathies due to alteration in amphiphysin-2 (BIN1). The actin and microtubule cytoskeletons are essential for nucleus positioning, but their crosstalk in this process is sparsely characterized. Here, we report that impairment of amphiphysin/BIN1 in Caenorhabditis elegans, mammalian cells, or muscles from patients with centronuclear myopathy alters nuclear position and shape. We show that AMPH-1/BIN1 binds to nesprin and actin, as well as to the microtubule-binding protein CLIP170 in both species. Expression of the microtubule-anchoring CAP-GLY domain of CLIP170 fused to the nuclear-envelope-anchoring KASH domain of nesprin rescues nuclear positioning defects of amph-1 mutants. Amphiphysins thus play a central role in linking the nuclear envelope with the actin and microtubule cytoskeletons. We propose that BIN1 has a direct and evolutionarily conserved role in nuclear positioning, altered in myopathies.
Assuntos
Núcleo Celular/genética , Proteínas dos Microfilamentos/genética , Proteínas Associadas aos Microtúbulos/genética , Miopatias Congênitas Estruturais/genética , Proteínas de Neoplasias/genética , Proteínas do Tecido Nervoso/genética , Membrana Nuclear/genética , Proteínas Nucleares/genética , Actinas/genética , Animais , Células COS , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Forma Celular/genética , Chlorocebus aethiops , Citoplasma/metabolismo , Citoesqueleto/genética , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Células HEK293 , Humanos , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/genética , Microtúbulos/metabolismo , Complexos Multiproteicos , Músculo Esquelético/metabolismo , Miopatias Congênitas Estruturais/metabolismo , Miopatias Congênitas Estruturais/patologia , Proteínas de Neoplasias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismoRESUMO
OBJECTIVE: To establish an in vivo method for matrix metalloproteinase (MMP)-2 and MMP-9 induction in horses via IV administration of lipopolysaccharide (LPS) and to evaluate the ability of doxycycline, oxytetracycline, flunixin meglumine, and pentoxifylline to inhibit equine MMP-2 and MMP-9 production. ANIMALS: 29 adult horses of various ages and breeds and either sex. PROCEDURES: In part 1, horses received an IV administration of LPS (n = 5) or saline (0.9% NaCl) solution (5). Venous blood samples were collected before and at specified times for 24 hours after infusion. Plasma was harvested and analyzed for MMP-2 and MMP-9 activities via zymography. In part 2, horses received doxycycline (n = 5), oxytetracycline (5), flunixin meglumine (5), or pentoxifylline (4) before and for up to 12 hours after administration of LPS. Plasma was obtained and analyzed, and results were compared with results from the LPS-infused horses of part 1. RESULTS: Administration of LPS significantly increased MMP-2 and MMP-9 activities in the venous circulation of horses. All MMP inhibitors significantly decreased LPS-induced increases in MMP activities but to differing degrees. Pentoxifylline and oxytetracycline appeared to be the most effective MMP-2 and MMP-9 inhibitors, whereas doxycycline and flunixin meglumine were more effective at inhibiting MMP-2 activity than MMP-9 activity. CONCLUSIONS AND CLINICAL RELEVANCE: IV administration of LPS to horses caused increased venous plasma activities of MMP-2 and MMP-9. These MMP activities were reduced by pentoxifylline and oxytetracycline, suggesting that further evaluation of these medications for treatment and prevention of MMP-associated diseases in horses is indicated.
Assuntos
Endotoxemia/veterinária , Inibidores Enzimáticos/farmacologia , Cavalos/sangue , Lipopolissacarídeos/farmacologia , Metaloproteinase 2 da Matriz/sangue , Metaloproteinase 9 da Matriz/sangue , Animais , Temperatura Corporal/efeitos dos fármacos , Clonixina/análogos & derivados , Clonixina/farmacologia , Doxiciclina/farmacologia , Endotoxemia/enzimologia , Indução Enzimática/efeitos dos fármacos , Feminino , Frequência Cardíaca/efeitos dos fármacos , Masculino , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/biossíntese , Oxitetraciclina/farmacologia , Pentoxifilina/farmacologia , Distribuição Aleatória , Taxa Respiratória/efeitos dos fármacosRESUMO
There is increasing evidence that oncogenic transformation modifies the metabolic program of cells. A common alteration is the upregulation of glycolysis, and efforts to target glycolytic enzymes for anticancer therapy are under way. Here, we performed a genome-wide haploid genetic screen to identify resistance mechanisms to 3-bromopyruvate (3-BrPA), a drug candidate that inhibits glycolysis in a poorly understood fashion. We identified the SLC16A1 gene product, MCT1, as the main determinant of 3-BrPA sensitivity. MCT1 is necessary and sufficient for 3-BrPA uptake by cancer cells. Additionally, SLC16A1 mRNA levels are the best predictor of 3-BrPA sensitivity and are most elevated in glycolytic cancer cells. Furthermore, forced MCT1 expression in 3-BrPA-resistant cancer cells sensitizes tumor xenografts to 3-BrPA treatment in vivo. Our results identify a potential biomarker for 3-BrPA sensitivity and provide proof of concept that the selectivity of cancer-expressed transporters can be exploited for delivering toxic molecules to tumors.
Assuntos
Transportadores de Ácidos Monocarboxílicos/genética , Piruvatos/metabolismo , Simportadores/genética , Animais , Transporte Biológico/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Glicólise/genética , Humanos , Camundongos , Camundongos Nus , Transportadores de Ácidos Monocarboxílicos/metabolismo , Piruvatos/farmacologia , Simportadores/metabolismoRESUMO
Heterozygous mutations in dynamin 2 (DNM2) have been linked to dominant Charcot-Marie-Tooth neuropathy and centronuclear myopathy. We report the first homozygous mutation in the DNM2 protein p.Phe379Val, in three consanguineous patients with a lethal congenital syndrome associating akinesia, joint contractures, hypotonia, skeletal abnormalities, and brain and retinal hemorrhages. In vitro membrane tubulation, trafficking and GTPase assays are consistent with an impact of the DNM2p.Phe379Val mutation on endocytosis. Although DNM2 has been previously implicated in axonal and muscle maintenance, the clinical manifestation in our patients taken together with our expression analysis profile during mouse embryogenesis and knockdown approaches in zebrafish resulting in defects in muscle organization and angiogenesis support a pleiotropic role for DNM2 during fetal development in vertebrates and humans.