RESUMO
Commensal bacteria serve as an important line of defense against colonisation by opportunisitic pathogens, but the underlying molecular mechanisms remain poorly explored. Here, we show that strains of a commensal bacterium, Haemophilus haemolyticus, make hemophilin, a heme-binding protein that inhibits growth of the opportunistic pathogen, non-typeable Haemophilus influenzae (NTHi) in culture. We purified the NTHi-inhibitory protein from H. haemolyticus and identified the hemophilin gene using proteomics and a gene knockout. An x-ray crystal structure of recombinant hemophilin shows that the protein does not belong to any of the known heme-binding protein folds, suggesting that it evolved independently. Biochemical characterisation shows that heme can be captured in the ferrous or ferric state, and with a variety of small heme-ligands bound, suggesting that hemophilin could function under a range of physiological conditions. Hemophilin knockout bacteria show a limited capacity to utilise free heme for growth. Our data suggest that hemophilin is a hemophore and that inhibition of NTHi occurs by heme starvation, raising the possibility that competition from hemophilin-producing H. haemolyticus could antagonise NTHi colonisation in the respiratory tract.
Assuntos
Haemophilus influenzae/efeitos dos fármacos , Haemophilus/metabolismo , Proteínas Ligantes de Grupo Heme , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/farmacologia , Infecções por Haemophilus/microbiologia , Infecções por Haemophilus/prevenção & controle , Haemophilus influenzae/crescimento & desenvolvimento , Heme/metabolismo , Proteínas Ligantes de Grupo Heme/química , Proteínas Ligantes de Grupo Heme/isolamento & purificação , Proteínas Ligantes de Grupo Heme/farmacologia , HumanosRESUMO
Chromatin remodeling enzymes act to dynamically regulate gene accessibility. In many cases, these enzymes function as large multicomponent complexes that in general comprise a central ATP-dependent Snf2 family helicase that is decorated with a variable number of regulatory subunits. The nucleosome remodeling and deacetylase (NuRD) complex, which is essential for normal development in higher organisms, is one such macromolecular machine. The NuRD complex comprises â¼10 subunits, including the histone deacetylases 1 and 2 (HDAC1 and HDAC2), and is defined by the presence of a CHD family remodeling enzyme, most commonly CHD4 (chromodomain helicase DNA-binding protein 4). The existing paradigm holds that CHD4 acts as the central hub upon which the complex is built. We show here that this paradigm does not, in fact, hold and that CHD4 is a peripheral component of the NuRD complex. A complex lacking CHD4 that has HDAC activity can exist as a stable species. The addition of recombinant CHD4 to this nucleosome deacetylase complex reconstitutes a NuRD complex with nucleosome remodeling activity. These data contribute to our understanding of the architecture of the NuRD complex.
Assuntos
Autoantígenos/metabolismo , DNA Helicases/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Nucleossomos/metabolismo , Animais , Autoantígenos/genética , Linhagem Celular , DNA Helicases/genética , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/genética , Histona Desacetilase 2/metabolismo , Humanos , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Camundongos , Nucleossomos/genéticaRESUMO
There are multiple intrinsic mechanisms for diastolic dysfunction ranging from molecular to structural derangements in ventricular myocardium. The molecular mechanisms regulating the progression from normal diastolic function to severe dysfunction still remain poorly understood. Recent studies suggest a potentially important role of core cardio-enriched transcription factors (TFs) in the control of cardiac diastolic function in health and disease through their ability to regulate the expression of target genes involved in the process of adaptive and maladaptive cardiac remodeling. The current relevant findings on the role of a variety of such TFs (TBX5, GATA-4/6, SRF, MYOCD, NRF2, and PITX2) in cardiac diastolic dysfunction and failure are updated, emphasizing their potential as promising targets for novel treatment strategies. In turn, the new animal models described here will be key tools in determining the underlying molecular mechanisms of disease. Since diastolic dysfunction is regulated by various TFs, which are also involved in cross talk with each other, there is a need for more in-depth research from a biomedical perspective in order to establish efficient therapeutic strategies.
Assuntos
Insuficiência Cardíaca Diastólica/genética , MicroRNAs/genética , Volume Sistólico , Fatores de Transcrição/genética , Animais , Coração/fisiopatologia , Humanos , Camundongos , Miocárdio/metabolismo , Ratos , Transdução de Sinais , Remodelação VentricularRESUMO
New antifungals with unique modes of action are urgently needed to treat the increasing global burden of invasive fungal infections. The fungal inositol polyphosphate kinase (IPK) pathway, comprised of IPKs that convert IP3 to IP8, provides a promising new target due to its impact on multiple, critical cellular functions and, unlike in mammalian cells, its lack of redundancy. Nearly all IPKs in the fungal pathway are essential for virulence, with IP3-4 kinase (IP3-4K) the most critical. The dibenzylaminopurine compound, N2-(m-trifluorobenzylamino)-N6-(p-nitrobenzylamino)purine (TNP), is a commercially available inhibitor of mammalian IPKs. The ability of TNP to be adapted as an inhibitor of fungal IP3-4K has not been investigated. We purified IP3-4K from the human pathogens, Cryptococcus neoformans and Candida albicans, and optimised enzyme and surface plasmon resonance (SPR) assays to determine the half inhibitory concentration (IC50) and binding affinity (KD), respectively, of TNP and 38 analogues. A novel chemical route was developed to efficiently prepare TNP analogues. TNP and its analogues demonstrated inhibition of recombinant IP3-4K from C. neoformans (CnArg1) at low µM IC50s, but not IP3-4K from C. albicans (CaIpk2) and many analogues exhibited selectivity for CnArg1 over the human equivalent, HsIPMK. Our results provide a foundation for improving potency and selectivity of the TNP series for fungal IP3-4K.
Assuntos
Criptococose , Cryptococcus neoformans , Animais , Humanos , Virulência , Antifúngicos/química , Criptococose/tratamento farmacológico , Criptococose/microbiologia , Candida albicans , Inositol/metabolismo , Purinas/metabolismo , MamíferosRESUMO
The finding of a genotype-negative hypertrophic cardiomyopathy (HCM) pedigree with several affected members indicating a familial origin of the disease has driven this study to discover causative gene variants. Genetic testing of the proband and subsequent family screening revealed the presence of a rare variant in the MYBPC3 gene, c.3331-26T>G in intron 30, with evidence supporting cosegregation with the disease in the family. An analysis of potential splice-altering activity using several splicing algorithms consistently yielded low scores. Minigene expression analysis at the mRNA and protein levels revealed that c.3331-26T>G is a spliceogenic variant with major splice-altering activity leading to undetectable levels of properly spliced transcripts or the corresponding protein. Minigene and patient mRNA analyses indicated that this variant induces complete and partial retention of intron 30, which was expected to lead to haploinsufficiency in carrier patients. As most spliceogenic MYBPC3 variants, c.3331-26T>G appears to be non-recurrent, since it was identified in only two additional unrelated probands in our large HCM cohort. In fact, the frequency analysis of 46 known splice-altering MYBPC3 intronic nucleotide substitutions in our HCM cohort revealed 9 recurrent and 16 non-recurrent variants present in a few probands (≤ 4), while 21 were not detected. The identification of non-recurrent elusive MYBPC3 spliceogenic variants that escape detection by in silico algorithms represents a challenge for genetic diagnosis of HCM and contributes to solving a fraction of genotype-negative HCM cases.
Assuntos
Cardiomiopatia Hipertrófica , Proteínas de Transporte , Cardiomiopatia Hipertrófica/diagnóstico , Cardiomiopatia Hipertrófica/genética , Proteínas de Transporte/genética , Proteínas do Citoesqueleto/genética , Haploinsuficiência , Humanos , Mutação , Linhagem , RNA MensageiroRESUMO
Here we report an infant with clinical findings suggestive of Jervell and Lange-Nielsen syndrome (JLNS), including a prolonged QT interval (LQTS) and chronic bilateral sensorineural deafness. NGS analysis revealed one known heterozygous pathogenic missense variant, KCNQ1 p.R259L, previously associated with LQTS but insufficient to explain the cardioauditory disorder. In a screening of proximal intronic regions, we found a heterozygous variant, KCNQ1 c.1686-9 T > C, absent from controls and previously undescribed. Several splicing prediction tools returned low scores for this intronic variant. Driven by the proband's phenotype rather than the neutral predictions, we have characterized this rare intronic variant. Family analysis has shown that the proband inherited the missense and the intronic variants from his mother and father, respectively. A minigene splicing assay revealed that the intronic variant induced an additional transcript, arising from skipping of exon 14, which was translated into a truncated protein in transfected cells. The splice-out of exon 14 creates a frameshift in exon 15 and a stop codon in exon 16, which is the last exon of KCNQ1. This mis-spliced transcript is expected to escape nonsense-mediated decay and predicted to encode a truncated loss-of-function protein, KCNQ1 p.L563Kfs*73. The analysis of endogenous KCNQ1 expression in the blood of the proband's parents detected the aberrant transcript only in the patient's father. Taken together, these analyses confirmed the proband's diagnosis of JLNS1 and indicated that c.1686-9 T > C is a cryptic splice-altering variant, expanding the known genetic spectrum of biallelic KCNQ1 variant combinations leading to JLNS1.
RESUMO
Molecular predisposition of postnatal ventricular myocardium to chamber-dependent (concentric or eccentric) remodeling remains largely elusive. To this end, we compared gene expression in the left (LV) versus right ventricle (RV) in newborn piglets, using a differential display reverse transcription-PCR (DDRT-PCR) technique. Out of more than 5600 DDRT-PCR bands, a total of 153 bands were identified as being differentially displayed. Of these, 96 bands were enriched in the LV, whereas the remaining 57 bands were predominant in the RV. The transcripts, displaying over twofold LV-RV expression differences, were sequenced and identified by BLAST comparison to known mRNA sequences. Among the genes, whose expression was not previously recognized as being chamber-dependent, we identified a small cohort of key regulators of muscle cell growth/proliferation (MAP3K7IP2, MSTN, PHB2, APOBEC3F) and gene expression (PTPLAD1, JMJD1C, CEP290), which may be relevant to the chamber-dependent predisposition of ventricular myocardium to respond differentially to pressure (LV) and volume (RV) overloads after birth. In addition, our data demonstrate chamber-dependent alterations in expression of as yet uncharacterized novel genes, which may also be suitable candidates for association studies in animal models of LV/RV hypertrophy.
Assuntos
Perfilação da Expressão Gênica/métodos , Ventrículos do Coração/metabolismo , Miocárdio/patologia , Remodelação Ventricular/genética , Animais , Animais Recém-Nascidos , Regulação da Expressão Gênica no Desenvolvimento , Coração/anatomia & histologia , Ventrículos do Coração/patologia , Miocárdio/metabolismo , Miostatina/genética , Miostatina/metabolismo , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , SuínosRESUMO
Chromatin remodellers hydrolyse ATP to move nucleosomal DNA against histone octamers. The mechanism, however, is only partially resolved, and it is unclear if it is conserved among the four remodeller families. Here we use single-molecule assays to examine the mechanism of action of CHD4, which is part of the least well understood family. We demonstrate that the binding energy for CHD4-nucleosome complex formation-even in the absence of nucleotide-triggers significant conformational changes in DNA at the entry side, effectively priming the system for remodelling. During remodelling, flanking DNA enters the nucleosome in a continuous, gradual manner but exits in concerted 4-6 base-pair steps. This decoupling of entry- and exit-side translocation suggests that ATP-driven movement of entry-side DNA builds up strain inside the nucleosome that is subsequently released at the exit side by DNA expulsion. Based on our work and previous studies, we propose a mechanism for nucleosome sliding.
Assuntos
Montagem e Desmontagem da Cromatina , Microscopia Intravital , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Nucleossomos/metabolismo , Translocação Genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Histonas/genética , Histonas/metabolismo , Humanos , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Microscopia de Fluorescência , Domínios Proteicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Imagem Individual de MoléculaRESUMO
The coexistence of GLA (Pro259Ser, c.775C>T) and MYBPC3 (c.1351+2T>C) mutations was found in a female patient with hypertrophic cardiomyopathy. Histology documented abundant vacuolisation with osmiophilic lamellar bodies and positive Gb3 immunohistochemistry. In the presence of a hypertrophic cardiomyopathy phenotype, the systematic search for unusual findings is mandatory to rule out a phenocopy.
Assuntos
Cardiomiopatia Hipertrófica/genética , Proteínas de Transporte/genética , DNA/genética , Galactosidases/genética , Predisposição Genética para Doença , Mutação , Miocárdio/metabolismo , Biópsia , Cardiomiopatia Hipertrófica/diagnóstico , Cardiomiopatia Hipertrófica/metabolismo , Análise Mutacional de DNA , Ecocardiografia , Feminino , Galactosidases/metabolismo , Humanos , Pessoa de Meia-Idade , Miocárdio/patologia , Miosinas , Linhagem , FenótipoRESUMO
The nucleosome remodeling and deacetylase (NuRD) complex is essential for metazoan development but has been refractory to biochemical analysis. We present an integrated analysis of the native mammalian NuRD complex, combining quantitative mass spectrometry, cross-linking, protein biochemistry, and electron microscopy to define the architecture of the complex. NuRD is built from a 2:2:4 (MTA, HDAC, and RBBP) deacetylase module and a 1:1:1 (MBD, GATAD2, and Chromodomain-Helicase-DNA-binding [CHD]) remodeling module, and the complex displays considerable structural dynamics. The enigmatic GATAD2 controls the asymmetry of the complex and directly recruits the CHD remodeler. The MTA-MBD interaction acts as a point of functional switching, with the transcriptional regulator PWWP2A competing with MBD for binding to the MTA-HDAC-RBBP subcomplex. Overall, our data address the long-running controversy over NuRD stoichiometry, provide imaging of the mammalian NuRD complex, and establish the biochemical mechanism by which PWWP2A can regulate NuRD composition.
Assuntos
Regulação da Expressão Gênica/genética , Histona Desacetilases/metabolismo , Nucleossomos/metabolismo , Humanos , Modelos MolecularesRESUMO
It has been proposed that the ankyrin repeat domain 1 (ANKRD1) factor (also known as CARP) plays a critical role in transcriptional regulation, myofibrillar assembly and stretch sensing during heart development and cardiac insults. ANKRD1/CARP has also been reported to negatively regulate cardiac gene expression in cell-based promoter-reporter assays. Consequently, rapid up-regulation of the ankrd1 gene in myocardium in response to developmental stimuli or pathological insults has tended to be interpreted in the context of the inhibitory effects of ANKRD1 on cardiomyocyte gene expression. Surprisingly, a total ankrd1 knockout resulted in a complete lack of phenotype, suggesting that ANKRD1/CARP is not crucial for regulation of cardiac gene expression in vivo. In this essay, we summarize (1) the accumulated evidence for the apparent multifunctional properties of this enigmatic protein, (2) the distinct chamber-dependent regulation of ankrd1 expression patterns in the heart, both during development and cardiac injury, and (3) ANKRD1 involvement in networks regulating adaptation of the myocardium to stress. Whenever feasible, we present the results obtained in patients together with those obtained in the relevant animal and cellular models. A close examination of the findings still fails to define ANKRD1 as a negative regulator of cardiac gene expression in vivo, but rather indicates that its augmented expression can represent an adaptive response of the myocardium to stress both during development and various heart insults.
Assuntos
Repetição de Anquirina/genética , Cardiopatias Congênitas/genética , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miocárdio/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Organogênese/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Cardiopatias Congênitas/metabolismo , Humanos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
The most significant mechanism of bacterial resistance to aminoglycosides is the enzymatic inactivation of the drug. Herein, we analyze several key aspects of the aminoglycoside recognition by the resistance enzyme ANT(4') from Staphylococcus aureus, employing NMR complemented with site-directed mutagenesis experiments and measurements of the enzymatic activity on newly synthesized kanamycin derivatives. From a methodological perspective, this analysis provides the first example reported for the use of transferred NOE (trNOE) experiments in the analysis of complex molecular recognition processes, characterized by the existence of simultaneous binding events of the ligand to different regions of a protein receptor. The obtained results show that, in favorable cases, these overlapping binding processes can be isolated employing site-directed mutagenesis and then independently analyzed. From a molecular recognition perspective, this work conclusively shows that the enzyme ANT(4') displays a wide tolerance to conformational variations in the drug. Thus, according to the NMR data, kanamycin-A I/II linkage exhibits an unusual anti-Psi orientation in the ternary complex, which is in qualitative agreement with the previously reported crystallographic complex. In contrast, closely related, kanamycin-B is recognized by the enzyme in the syn-type arrangement for both glycosidic bonds. This observation together with the enzymatic activity displayed by ANT(4') against several synthetic kanamycin derivatives strongly suggests that the spatial distribution of positive charges within the aminoglycoside scaffold is the key feature that governs its preferred binding mode to the protein catalytic region and also the regioselectivity of the adenylation reaction. In contrast, the global shape of the antibiotic does not seem to be a critical factor. This feature represents a qualitative difference between the target A-site RNA and the resistance enzyme ANT(4') as aminoglycoside receptors.
Assuntos
Aminoglicosídeos/análise , Aminoglicosídeos/química , Antibacterianos/química , Farmacorresistência Bacteriana , Compostos de Amônio Quaternário/química , Antibacterianos/farmacologia , Sítios de Ligação , Calorimetria , Catálise , Cátions/química , Farmacorresistência Bacteriana/efeitos dos fármacos , Hidroxilação , Canamicina/análogos & derivados , Canamicina/química , Canamicina/farmacologia , Cinética , Magnésio/química , Modelos Moleculares , Estrutura Molecular , Mutagênese Sítio-Dirigida , Mutação/genética , Ressonância Magnética Nuclear Biomolecular , Staphylococcus aureus/enzimologia , Staphylococcus aureus/genéticaRESUMO
We have developed a set of simple modifications of the green fluorescent protein (GFP)fragment reassembly assay in bacteria that permits: (i)fluorescent microscopy visualization of GFP reassembly only 1-2 h after induction of protein expression, thus approximating the detection of GFP reassembly to the real-time dynamics of protein complex formation in living cells; (ii) spectrofluorometric detection of reassembled GFP fluorescent signals directly in lysates from cell suspension thereby avoiding, in many cases, the need for tag-affinity isolation of protein complexes; and (iii) comparative quantification of signal intensity in numerous cell-sample lysates using a Bio-Rad IQ5 spectrofluorometric detection system (Bio-Rad Laboratories, Madrid, Spain). Collectively, the results demonstrate that the combination of microscopic and spectrofluorometric detection provides a time-saving and sensitive alternative to existing methods of fluorescence complementation analysis.
Assuntos
Teste de Complementação Genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia de Fluorescência/métodos , Fragmentos de Peptídeos/metabolismo , Espectrometria de Fluorescência/métodos , Escherichia coli/citologia , Proteínas de Fluorescência Verde/genética , Ligação Proteica , Mapeamento de Interação de ProteínasRESUMO
There is growing evidence that putative gene regulatory networks including cardio-enriched transcription factors, such as PITX2, TBX5, ZFHX3, and SHOX2, and their effector/target genes along with downstream non-coding RNAs can play a potentially important role in the process of adaptive and maladaptive atrial rhythm remodeling. In turn, expression of atrial fibrillation-associated transcription factors is under the control of upstream regulatory non-coding RNAs. This review broadly explores gene regulatory mechanisms associated with susceptibility to atrial fibrillation-with key examples from both animal models and patients-within the context of both cardiac transcription factors and non-coding RNAs. These two systems appear to have multiple levels of cross-regulation and act coordinately to achieve effective control of atrial rhythm effector gene expression. Perturbations of a dynamic expression balance between transcription factors and corresponding non-coding RNAs can provoke the development or promote the progression of atrial fibrillation. We also outline deficiencies in current models and discuss ongoing studies to clarify remaining mechanistic questions. An understanding of the function of transcription factors and non-coding RNAs in gene regulatory networks associated with atrial fibrillation risk will enable the development of innovative therapeutic strategies.
Assuntos
Fibrilação Atrial/genética , Fibrilação Atrial/metabolismo , Suscetibilidade a Doenças , Regulação da Expressão Gênica , Miocárdio/metabolismo , RNA não Traduzido/genética , Fatores de Transcrição/metabolismo , Animais , Fibrilação Atrial/fisiopatologia , Biomarcadores , Humanos , MicroRNAs/genética , Modelos BiológicosRESUMO
Marfan syndrome (MFS) is an autosomal dominantly inherited connective tissue disorder, mostly caused by mutations in the fibrillin-1 (FBN1) gene. We, by using targeted next-generation sequence analysis, identified a novel intronic FBN1 mutation (the c.2678-15C>A variant) in a MFS patient with aortic dilatation. The computational predictions showed that the heterozygous c.2678-15C>A intronic variant might influence the splicing process by differentially affecting canonical versus cryptic splice site utilization within intron 22 of the FBN1 gene. RT-PCR and Western blot analyses, using FBN1 minigenes transfected into HeLa and COS-7 cells, revealed that the c.2678-15C>A variant disrupts normal splicing of intron 22 leading to aberrant 13-nt intron 22 inclusion, frameshift, and premature termination codon. Collectively, the results strongly suggest that the c.2678-15C>A variant could lead to haploinsufficiency of the FBN1 functional protein and structural connective tissue fragility in MFS complicated by aorta dilation, a finding that further expands on the genetic basis of aortic pathology.
Assuntos
Fibrilina-1/genética , Íntrons/genética , Síndrome de Marfan/genética , Mutação , Adulto , Aorta/patologia , Dilatação Patológica , Heterozigoto , Humanos , Masculino , Proteínas dos MicrofilamentosRESUMO
Chromatin structure and function is regulated by reader proteins recognizing histone modifications and/or histone variants. We recently identified that PWWP2A tightly binds to H2A.Z-containing nucleosomes and is involved in mitotic progression and cranial-facial development. Here, using in vitro assays, we show that distinct domains of PWWP2A mediate binding to free linker DNA as well as H3K36me3 nucleosomes. In vivo, PWWP2A strongly recognizes H2A.Z-containing regulatory regions and weakly binds H3K36me3-containing gene bodies. Further, PWWP2A binds to an MTA1-specific subcomplex of the NuRD complex (M1HR), which consists solely of MTA1, HDAC1, and RBBP4/7, and excludes CHD, GATAD2 and MBD proteins. Depletion of PWWP2A leads to an increase of acetylation levels on H3K27 as well as H2A.Z, presumably by impaired chromatin recruitment of M1HR. Thus, this study identifies PWWP2A as a complex chromatin-binding protein that serves to direct the deacetylase complex M1HR to H2A.Z-containing chromatin, thereby promoting changes in histone acetylation levels.
Assuntos
Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Histona Desacetilases/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Proteínas Repressoras/metabolismo , Acetilação , Animais , Proteínas Cromossômicas não Histona/genética , Células HEK293 , Histona Desacetilases/genética , Histonas/genética , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilação , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Camundongos , Nucleossomos/metabolismo , RNA Interferente Pequeno , Proteínas Repressoras/genética , TransativadoresRESUMO
Despite the importance of MYOC for glaucoma, the protein's normal function(s) and the pathogenic mechanism(s) of MYOC mutations are not clear. Elevated intraocular pressure (IOP) and glaucoma are sometimes induced by corticosteroids, and corticosteroid use can result in substantially increased MYOC expression. It has been suggested, therefore, that steroid-induced MYOC protein levels cause steroid-induced glaucoma and that protein level-increasing mutations in MYOC contribute to glaucoma not associated with steroid use. A causative role of elevated MYOC levels in steroid-induced glaucoma is controversial, however, and it is not clear if elevated MYOC levels can result in IOP elevation. To directly test if increased levels of MYOC can cause IOP elevation and glaucoma, we generated bacterial artificial chromosome transgenic mice that overexpress Myoc at a level similar to that induced by corticosteroid use. These mice do not develop elevated IOP or glaucoma. Our present findings, along with the absence of glaucoma in mice completely lacking MYOC, show that changing the level of MYOC is not pathogenic (from absent to approximately 15 times normal). These findings suggest that noncoding sequence variants are unlikely to influence glaucoma and that disease pathogenesis in primary open-angle glaucoma patients is dependent upon the expression of abnormal mutant proteins. This work does not support a causative role for increased MYOC levels or the MYOC gene in steroid-induced glaucoma.
Assuntos
Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica , Glaucoma de Ângulo Aberto/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Pressão Intraocular/fisiologia , Corticosteroides/efeitos adversos , Corticosteroides/farmacologia , Adulto , Animais , Proteínas do Citoesqueleto , Olho/anatomia & histologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glaucoma de Ângulo Aberto/induzido quimicamente , Glaucoma de Ângulo Aberto/genética , Humanos , Pressão Intraocular/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Nervo Óptico/anatomia & histologia , Nervo Óptico/metabolismo , Regiões Promotoras Genéticas , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/metabolismo , Malha Trabecular/citologia , Malha Trabecular/metabolismoRESUMO
The nucleosome remodelling and deacetylase (NuRD) complex is essential for the development of complex animals. NuRD has roles in regulating gene expression and repairing damaged DNA. The complex comprises at least six proteins with two or more paralogues of each protein routinely identified when the complex is purified from cell extracts. To understand the structure and function of NuRD, a map of direct subunit interactions is needed. Dozens of published studies have attempted to define direct inter-subunit connectivities. We propose that conclusions reported in many such studies are in fact ambiguous for one of several reasons. First, the expression of many NuRD subunits in bacteria is unlikely to lead to folded, active protein. Second, interaction studies carried out in cells that contain endogenous NuRD complex can lead to false positives through bridging of target proteins by endogenous components. Combining existing information on NuRD structure with a protocol designed to minimize false positives, we report a conservative and robust interaction map for the NuRD complex. We also suggest a 3D model of the complex that brings together the existing data on the complex. The issues and strategies discussed herein are also applicable to the analysis of a wide range of multi-subunit complexes. ENZYMES: Micrococcal nuclease (MNase), EC 3.1.31.1; histone deacetylase (HDAC), EC 3.5.1.98.
Assuntos
Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/química , Nucleossomos/química , Mapeamento de Interação de Proteínas/métodos , Animais , Artefatos , Western Blotting , Escherichia coli , Células HEK293 , Células HeLa , Histona Desacetilase 1/química , Humanos , Camundongos , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , Subunidades Proteicas , Coelhos , Proteínas Recombinantes de Fusão/química , ReticulócitosRESUMO
Diastolic heart failure (DHF) was produced in 6-day-old piglets by intravenous administration of Doxorubicin, and ANKRD1 protein and mRNA levels were determined in atrial (A) and ventricular (V) chambers of failing vs control hearts. In controls, ANKRD1 showed a left-right (L-R) asymmetric distribution with protein levels 2-fold higher in the LA as compared to the RA, and 8-fold higher in the LV than the RV. In failing hearts, ANKRD1 levels were augmented about 2-fold in each ventricle but equally reduced in both atria as compared to controls. ANKRD1 downregulation in atria is discussed as a process associated with advanced DHF.
Assuntos
Regulação para Baixo , Insuficiência Cardíaca/metabolismo , Proteínas Musculares/biossíntese , Animais , Antibióticos Antineoplásicos/farmacologia , Antibióticos Antineoplásicos/toxicidade , Diástole , Regulação para Baixo/efeitos dos fármacos , Doxorrubicina/farmacologia , Doxorrubicina/toxicidade , Átrios do Coração/metabolismo , Átrios do Coração/patologia , Insuficiência Cardíaca/induzido quimicamente , Insuficiência Cardíaca/patologia , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Especificidade de Órgãos , SuínosRESUMO
The nucleosome remodeling and deacetylase (NuRD) complex remodels the genome in the context of both gene transcription and DNA damage repair. It is essential for normal development and is distributed across multiple tissues in organisms ranging from mammals to nematode worms. In common with other chromatin-remodeling complexes, however, its molecular mechanism of action is not well understood and only limited structural information is available to show how the complex is assembled. As a step towards understanding the structure of the NuRD complex, we have characterized the interaction between two subunits: the metastasis associated protein MTA1 and the histone-binding protein RBBP4. We show that MTA1 can bind to two molecules of RBBP4 and present negative stain electron microscopy and chemical crosslinking data that allow us to build a low-resolution model of an MTA1-(RBBP4)2 subcomplex. These data build on our understanding of NuRD complex structure and move us closer towards an understanding of the biochemical basis for the activity of this complex.