RESUMO
The sixth family phosphodiesterases (PDE6) are principal effector enzymes of the phototransduction cascade in rods and cones. Maturation of nascent PDE6 protein into a functional enzyme relies on a coordinated action of ubiquitous chaperone HSP90, its specialized cochaperone aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1), and the regulatory Pγ-subunit of PDE6. Deficits in PDE6 maturation and function underlie severe visual disorders and blindness. Here, to elucidate the roles of HSP90, AIPL1, and Pγ in the maturation process, we developed the heterologous expression system of human cone PDE6C in insect cells allowing characterization of the purified enzyme. We demonstrate that in the absence of Pγ, HSP90, and AIPL1 convert the inactive and aggregating PDE6C species into dimeric PDE6C that is predominantly misassembled. Nonetheless, a small fraction of PDE6C is properly assembled and fully functional. From the analysis of mutant mice that lack both rod Pγ and PDE6C, we conclude that, in contrast to the cone enzyme, no maturation of rod PDE6AB occurs in the absence of Pγ. Co-expression of PDE6C with AIPL1 and Pγ in insect cells leads to a fully mature enzyme that is equivalent to retinal PDE6. Lastly, using immature PDE6C and purified chaperone components, we reconstituted the process of the client maturation in vitro. Based on this analysis we propose a scheme for the PDE6 maturation process.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 6 , Células Fotorreceptoras Retinianas Cones , Animais , Humanos , Camundongos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cegueira/genética , Linhagem Celular , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/química , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/deficiência , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Mutação , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/deficiência , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Células Fotorreceptoras Retinianas Cones/química , Células Fotorreceptoras Retinianas Cones/metabolismoRESUMO
Voltage-gated sodium (NaV) channels drive the upstroke of the action potential and are comprised of a pore-forming α-subunit and regulatory ß-subunits. The ß-subunits modulate the gating, trafficking, and pharmacology of the α-subunit. These functions are routinely assessed by ectopic expression in heterologous cells. However, currently available expression systems may not capture the full range of these effects since they contain endogenous ß-subunits. To better reveal ß-subunit functions, we engineered a human cell line devoid of endogenous NaV ß-subunits and their immediate phylogenetic relatives. This new cell line, ß-subunit-eliminated eHAP expression (BeHAPe) cells, were derived from haploid eHAP cells by engineering inactivating mutations in the ß-subunits SCN1B, SCN2B, SCN3B, and SCN4B, and other subfamily members MPZ (myelin protein zero(P0)), MPZL1, MPZL2, MPZL3, and JAML. In diploid BeHAPe cells, the cardiac NaV α-subunit, NaV1.5, was highly sensitive to ß-subunit modulation and revealed that each ß-subunit and even MPZ imparted unique gating properties. Furthermore, combining ß1 and ß2 with NaV1.5 generated a sodium channel with hybrid properties, distinct from the effects of the individual subunits. Thus, this approach revealed an expanded ability of ß-subunits to regulate NaV1.5 activity and can be used to improve the characterization of other α/ß NaV complexes.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.5 , Subunidades Proteicas , Subunidades beta do Canal de Sódio Disparado por Voltagem , Humanos , Potenciais de Ação , Linhagem Celular , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/química , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Fosfoproteínas/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/deficiência , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Subunidades beta do Canal de Sódio Disparado por Voltagem/química , Subunidades beta do Canal de Sódio Disparado por Voltagem/deficiência , Subunidades beta do Canal de Sódio Disparado por Voltagem/genética , Subunidades beta do Canal de Sódio Disparado por Voltagem/metabolismo , MutaçãoRESUMO
Sporadic occurrence of inherited eye disorders has been reported in cattle but so far pathogenic variants were found only for rare forms of cataract but not for retinopathies. The aim of this study was to characterize the phenotype and the genetic aetiology of a recessive form of congenital day-blindness observed in several cases of purebred Original Braunvieh cattle. Electroretinography in an affected calf revealed absent cone-mediated function, whereas the rods continue to function normally. Brain areas involved in vision were morphologically normal. When targeting cones by immunofluorescence, a decrease in cone number and an accumulation of beta subunits of cone cyclic-nucleotide gated channel (CNGB3) in the outer plexiform layer of affected animals was obvious. Achromatopsia is a monogenic Mendelian disease characterized by the loss of cone photoreceptor function resulting in day-blindness, total color-blindness, and decreased central visual acuity. After SNP genotyping and subsequent homozygosity mapping with twelve affected cattle, we performed whole-genome sequencing and variant calling of three cases. We identified a single missense variant in the bovine CNGB3 gene situated in a ~2.5 Mb homozygous genome region on chromosome 14 shared between all cases. All affected cattle were homozygous carriers of the p.Asp251Asn mutation that was predicted to be deleterious, affecting an evolutionary conserved residue. In conclusion, we have evidence for the occurrence of a breed-specific novel CNGB3-related form of recessively inherited achromatopsia in Original Braunvieh cattle which we have designated OH1 showing an allele frequency of the deleterious allele of ~8%. The identification of carriers will enable selection against this inherited disorder. The studied cattle might serve as an animal model to further elucidate the function of CNGB3 in mammals.
Assuntos
Alelos , Defeitos da Visão Cromática/genética , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Mutação de Sentido Incorreto , Subunidades Proteicas/genética , Células Fotorreceptoras Retinianas Cones/metabolismo , Substituição de Aminoácidos , Animais , Asparagina/metabolismo , Ácido Aspártico/metabolismo , Bovinos , Defeitos da Visão Cromática/diagnóstico por imagem , Defeitos da Visão Cromática/metabolismo , Defeitos da Visão Cromática/patologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/deficiência , Eletrorretinografia , Feminino , Expressão Gênica , Frequência do Gene , Homozigoto , Masculino , Fenótipo , Subunidades Proteicas/deficiência , Células Fotorreceptoras Retinianas Cones/patologia , Células Fotorreceptoras Retinianas Bastonetes/citologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Sequenciamento Completo do GenomaRESUMO
Bacteria play an integral role in shaping plant growth and development. However, the genetic factors that facilitate plant-bacteria interactions remain largely unknown. Here, we demonstrated the importance of two bacterial genetic factors that facilitate the interactions between plant-growth-promoting (PGP) bacteria in the genus Caulobacter and the host plant Arabidopsis. Using homologous recombination, we disrupted the cytochrome ubiquinol oxidase (cyo) operon in both C. vibrioides CB13 and C. segnis TK0059 by knocking out the expression of cyoB (critical subunit of the cyo operon) and showed that the mutant strains were unable to enhance the growth of Arabidopsis. In addition, disruption of the cyo operon, metabolomic reconstructions, and pH measurements suggested that both elevated cyoB expression and acid production by strain CB13 contribute to the previously observed inhibition of Arabidopsis seed germination. We also showed that the crescent shape of the PGP bacterial strain C. crescentus CB15 contributes to its ability to enhance plant growth. Thus, we have identified specific genetic factors that explain how select Caulobacter strains interact with Arabidopsis plants.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Caulobacter/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Arabidopsis/anatomia & histologia , Arabidopsis/microbiologia , Proteínas de Bactérias/metabolismo , Caulobacter/classificação , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Expressão Gênica , Germinação , Recombinação Homóloga , Concentração de Íons de Hidrogênio , Filogenia , Subunidades Proteicas/deficiência , Subunidades Proteicas/genética , Espécies Reativas de Oxigênio/metabolismoRESUMO
Ca2+/calmodulin-dependent kinase II (CaMKII) regulates synaptic plasticity in multiple ways, supposedly including the secretion of neuromodulators like brain-derived neurotrophic factor (BDNF). Here, we show that neuromodulator secretion is indeed reduced in mouse α- and ßCaMKII-deficient (αßCaMKII double-knockout [DKO]) hippocampal neurons. However, this was not due to reduced secretion efficiency or neuromodulator vesicle transport but to 40% reduced neuromodulator levels at synapses and 50% reduced delivery of new neuromodulator vesicles to axons. αßCaMKII depletion drastically reduced neuromodulator expression. Blocking BDNF secretion or BDNF scavenging in wild-type neurons produced a similar reduction. Reduced neuromodulator expression in αßCaMKII DKO neurons was restored by active ßCaMKII but not inactive ßCaMKII or αCaMKII, and by CaMKII downstream effectors that promote cAMP-response element binding protein (CREB) phosphorylation. These data indicate that CaMKII regulates neuromodulation in a feedback loop coupling neuromodulator secretion to ßCaMKII- and CREB-dependent neuromodulator expression and axonal targeting, but CaMKIIs are dispensable for the secretion process itself.
Assuntos
Astrócitos/metabolismo , Fator Neurotrófico Derivado do Encéfalo/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Cálcio/metabolismo , Neurônios/metabolismo , Subunidades Proteicas/genética , Animais , Astrócitos/citologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/deficiência , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Retroalimentação Fisiológica , Regulação da Expressão Gênica , Hipocampo/citologia , Hipocampo/metabolismo , Camundongos , Camundongos Transgênicos , Neurônios/citologia , Fosforilação , Cultura Primária de Células , Subunidades Proteicas/deficiência , Sinapses/fisiologia , Transmissão Sináptica , Imagem com Lapso de TempoRESUMO
NMDA receptors (NMDARs) are critical for physiological synaptic plasticity, learning, and memory and for pathological plasticity and neuronal death. The GluN1 subunit is encoded by a single gene, GRIN1, with 8 splice variants, but whether the diversity generated by this splicing has physiological consequences remains enigmatic. Here, we generate mice lacking from the GluN1 exon 5-encoded N1 cassette (GluN1a mice) or compulsorily expressing this exon (GluN1b mice). Despite no differences in basal synaptic transmission, long-term potentiation in the hippocampus is significantly enhanced in GluN1a mice compared with that in GluN1b mice. Furthermore, GluN1a mice learn more quickly and have significantly better spatial memory performance than do GluN1b mice. In addition, in human iPSC-derived neurons in autism spectrum disorder NMDARs show characteristics of N1-lacking GluN1. Our findings indicate that alternative splicing of GluN1 is a mechanism for controlling physiological long-lasting synaptic potentiation, learning, and memory.
Assuntos
Processamento Alternativo , Transtorno do Espectro Autista/genética , Potenciação de Longa Duração/genética , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Animais , Transtorno do Espectro Autista/metabolismo , Transtorno do Espectro Autista/patologia , Morte Celular/genética , Diferenciação Celular , Potenciais Pós-Sinápticos Excitadores/fisiologia , Éxons , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/deficiência , Neurônios/patologia , Cultura Primária de Células , Subunidades Proteicas/deficiência , Subunidades Proteicas/genética , Receptores de N-Metil-D-Aspartato/deficiência , Memória Espacial/fisiologia , Sinapses/metabolismo , Transmissão SinápticaRESUMO
The 26S proteasome is a key player in the degradation of ubiquitinated proteins, comprising a 20S core particle (CP) and a 19S regulatory particle (RP). The RP is further divided into base and lid subcomplexes, which are assembled independently from each other. We have previously demonstrated the assembly pathway of the CP and the base by observing assembly intermediates resulting from knockdowns of each proteasome subunit and the assembly chaperones. In this study, we examine the assembly pathway of the mammalian lid, which remains to be elucidated. We show that the lid assembly pathway is conserved between humans and yeast. The final step is the incorporation of Rpn12 into the assembly intermediate consisting of two modular complexes, Rpn3-7-15 and Rpn5-6-8-9-11, in both humans and yeast. Furthermore, we dissect the assembly pathways of the two modular complexes by the knockdown of each lid subunit.
Assuntos
Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Subunidades Proteicas/deficiência , Subunidades Proteicas/genética , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
AP-2 complex is widely distributed in eukaryotes in the form of heterotetramer that functions in the uptake of membrane proteins during mammalian/plant clathrin-mediated endocytosis. However, its biological function remains mysterious in pathogenic fungi. In this study, the wheat scab fungus, Fusarium graminearum, was used to characterise the biological function of the AP-2 complex. Our study shows that FgAP-2 complex plays a critical role in the maintenance of hyphal polarity. Lack of any subunit (FgAP2α , FgAP2ß , FgAP2σ , and FgAP2mu ) of the FgAP-2 complex significantly affects the fungal vegetative growth, conidial morphology, and germination. Remarkably, FgAP-2 complex is important for the fungal pathogenicity, especially during colonisation and extension after infecting the host. The FgAP-2 complex is expressed ubiquitously at all developmental stages but having more concentrated protein distribution at the subapical collar and septa in young growing hyphae. Although FgAP-2 complex displays similar dynamic behaviour to the actin patch components and accumulates at endocytic sites, it is dispensable for general endocytosis. We further demonstrated that FgAP-2 complex is required for polar localisation of the lipid flippases FgDnfA and FgDnfB, which led to the proposal that FgAP-2 functions as a cargo-specific adaptor that promotes polar growth and colonising ability of F. graminearum.
Assuntos
Complexo 2 de Proteínas Adaptadoras/genética , Proteínas Fúngicas/genética , Fusarium/genética , Fusarium/patogenicidade , Regulação Fúngica da Expressão Gênica , Proteínas de Transferência de Fosfolipídeos/genética , Actinas/genética , Actinas/metabolismo , Complexo 2 de Proteínas Adaptadoras/metabolismo , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Endocitose/genética , Proteínas Fúngicas/metabolismo , Fusarium/crescimento & desenvolvimento , Fusarium/metabolismo , Deleção de Genes , Hifas/genética , Hifas/crescimento & desenvolvimento , Hifas/metabolismo , Hifas/patogenicidade , Isoenzimas/genética , Isoenzimas/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Doenças das Plantas/microbiologia , Subunidades Proteicas/deficiência , Subunidades Proteicas/genética , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/metabolismo , Esporos Fúngicos/patogenicidade , Triticum/microbiologia , Técnicas do Sistema de Duplo-Híbrido , VirulênciaRESUMO
The nucleosome, composed of DNA and a histone core, is the basic structural unit of chromatin. The fission yeast Schizosaccharomyces pombe has two genes of histone H2A, hta1+ and hta2+; these genes encode two protein species of histone H2A (H2Aα and H2Aß, respectively), which differ in three amino acid residues, and only hta2+ is upregulated during meiosis. However, it is unknown whether S. pombe H2Aα and H2Aß have functional differences. Therefore, in this study, we examined the possible functional differences between H2Aα and H2Aß during meiosis in S. pombe. We found that deletion of hta2+, but not hta1+, causes defects in chromosome segregation and spore formation during meiosis. Meiotic defects in hta2+ deletion cells were rescued by expressing additional copies of hta1+ or by expressing hta1+ from the hta2 promoter. This indicated that the defects were caused by insufficient amounts of histone H2A, and not by the amino acid residue differences between H2Aα and H2Aß. Microscopic observation attributed the chromosome segregation defects to anaphase bridge formation in a chromosomal region at the repeats of ribosomal RNA genes (rDNA repeats). These results suggest that histone H2A insufficiency affects the chromatin structures of rDNA repeats, leading to chromosome missegregation in S. pombe.
Assuntos
Segregação de Cromossomos/fisiologia , Cromossomos Fúngicos/metabolismo , DNA Ribossômico/genética , Histonas/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/metabolismo , Anáfase , Cromatina/metabolismo , Histonas/deficiência , Histonas/metabolismo , Regiões Promotoras Genéticas , Subunidades Proteicas/deficiência , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Esporos Fúngicos/metabolismo , Regulação para CimaRESUMO
BACKGROUND: Innate lymphoid cells (ILCs) are a newly discovered family of immune cells that have similar cytokine-secreting profiles as T helper cell subsets. Although ILCs are critical for host defense against infections and tissue homeostasis, their roles in tumor development are not well established. METHODS: We studied the function of ILC3 cells in the liver for the development of hepatocellular carcinoma (HCC) in murine HCC models using flow cytometry, adoptive transfer, and in vitro functional assays. FINDINGS: We found that ILC3 lacking the natural cytotoxicity-triggering receptor (NCR-ILC3) promoted the development of HCC in response to interleukin 23 (IL-23). IL-23 serum level is elevated in HCC patients and its high expression is associated with poor clinical outcomes. We found that IL-23 could promote tumor development in murine HCC tumor models. IL-23 promoted the expansion of NCR-ILC3 and its differentiation from group 1 ILCs (ILC1s). Furthermore, NCR-ILC3 initiated IL-17 production upon IL-23 stimulation and directly inhibited CD8+ T cell immunity by promoting lymphocyte apoptosis and limiting their proliferation. INTERPRETATION: Together, our findings suggest that NCR-ILC3 initiates the IL-17-rich immunosuppressive tumor microenvironment and promotes the development of HCC, thus may serve as a promising target for future cancer immunotherapy. FUND: This work was supported by grants from National Natural Science Foundation of China (81471586, 81571556), the Priority Academic Program Development of Jiangsu Higher Education Institutions, the collaborative Innovation Center of Hematology, start-up grant from National University of Singapore, the Cancer Prevention and Research Institute of Texas CPRIT (RR180017), and the National Cancer Institute's Cancer Center Support (Core) Grant CA016672 (to The University of Texas MD Anderson Cancer Center).
Assuntos
Carcinoma Hepatocelular/patologia , Interleucina-17/metabolismo , Interleucina-23/metabolismo , Neoplasias Hepáticas/patologia , Animais , Apoptose , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Técnicas de Cocultura , Modelos Animais de Doenças , Imunidade Inata , Interleucina-12/metabolismo , Interleucina-17/análise , Interleucina-23/análise , Interleucina-23/genética , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/metabolismo , Linfócitos/citologia , Linfócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Subunidades Proteicas/deficiência , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transplante HomólogoRESUMO
Human DNA polymerase δ is normally present in unstressed, non-dividing cells as a heterotetramer (Pol δ4). Its smallest subunit, p12, is transiently degraded in response to UV damage, as well as during the entry into S-phase, resulting in the conversion of Pol δ4 to a trimer (Pol δ3). In order to further understand the specific cellular roles of these two forms of Pol δ, the gene (POLD4) encoding p12 was disrupted by CRISPR/Cas9 to produce p12 knockout (p12KO) cells. Thus, Pol δ4 is absent in p12KO cells, leaving Pol δ3 as the sole source of Pol δ activity. GFP reporter assays revealed that the p12KO cells exhibited a defect in homologous recombination (HR) repair, indicating that Pol δ4, but not Pol δ3, is required for HR. Expression of Flag-tagged p12 in p12KO cells to restore Pol δ4 alleviated the HR defect. These results establish a specific requirement for Pol δ4 in HR repair. This leads to the prediction that p12KO cells should be more sensitive to chemotherapeutic agents, and should exhibit synthetic lethal killing by PARP inhibitors. These predictions were confirmed by clonogenic cell survival assays of p12KO cells treated with cisplatin and mitomycin C, and with the PARP inhibitors Olaparib, Talazoparib, Rucaparib, and Niraparib. The sensitivity to PARP inhibitors in H1299-p12KO cells was alleviated by expression of Flag-p12. These findings have clinical significance, as the expression levels of p12 could be a predictive biomarker of tumor response to PARP inhibitors. In addition, small cell lung cancers (SCLC) are known to exhibit a defect in p12 expression. Analysis of several SCLC cell lines showed that they exhibit hypersensitivity to PARP inhibitors, providing evidence that loss of p12 expression could represent a novel molecular basis for HR deficiency.
Assuntos
DNA Polimerase III/antagonistas & inibidores , DNA Polimerase III/genética , Técnicas de Inativação de Genes , Recombinação Homóloga/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Subunidades Proteicas/deficiência , Subunidades Proteicas/genética , Linhagem Celular Tumoral , Cisplatino/farmacologia , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA Polimerase III/deficiência , Células HeLa , Recombinação Homóloga/efeitos dos fármacos , Humanos , Mitomicina/farmacologiaRESUMO
Voltage-gated Kv1.1 potassium channel α-subunits, encoded by the Kcna1 gene, have traditionally been regarded as neural-specific with no expression or function in the heart. However, recent data revealed that Kv1.1 subunits are expressed in atria where they may have an overlooked role in controlling repolarization and arrhythmia susceptibility independent of the nervous system. To explore this concept in more detail and to identify functional and molecular effects of Kv1.1 channel impairment in the heart, atrial cardiomyocyte patch-clamp electrophysiology and gene expression analyses were performed using Kcna1 knockout ( Kcna1-/-) mice. Specifically, we hypothesized that Kv1.1 subunits contribute to outward repolarizing K+ currents in mouse atria and that their absence prolongs cardiac action potentials. In voltage-clamp experiments, dendrotoxin-K (DTX-K), a Kv1.1-specific inhibitor, significantly reduced peak outward K+ currents in wild-type (WT) atrial cells but not Kcna1-/- cells, demonstrating an important contribution by Kv1.1-containing channels to mouse atrial repolarizing currents. In current-clamp recordings, Kcna1-/- atrial myocytes exhibited significant action potential prolongation which was exacerbated in right atria, effects that were partially recapitulated in WT cells by application of DTX-K. Quantitative RT-PCR measurements showed mRNA expression remodeling in Kcna1-/- atria for several ion channel genes that contribute to the atrial action potential including the Kcna5, Kcnh2, and Kcnj2 potassium channel genes and the Scn5a sodium channel gene. This study demonstrates a previously undescribed heart-intrinsic role for Kv1.1 subunits in mediating atrial repolarization, thereby adding a new member to the already diverse collection of known K+ channels in the heart.
Assuntos
Potenciais de Ação/fisiologia , Átrios do Coração/metabolismo , Canal de Potássio Kv1.1/antagonistas & inibidores , Canal de Potássio Kv1.1/genética , Miócitos Cardíacos/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Feminino , Átrios do Coração/citologia , Átrios do Coração/efeitos dos fármacos , Canal de Potássio Kv1.1/deficiência , Masculino , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Subunidades Proteicas/deficiência , Subunidades Proteicas/genéticaRESUMO
Polyglutamine (polyQ) expansion in Ataxin-7 (ATXN7) results in spinocerebellar ataxia type 7 (SCA7) and causes visual impairment. SCA7 photoreceptors progressively lose their outer segments (OSs), a structure essential for their visual function. ATXN7 is a subunit of the transcriptional coactivator Spt-Ada-Gcn5 Acetyltransferase complex, implicated in the development of the visual system in flies. To determine the function of ATXN7 in the vertebrate eye, we have inactivated ATXN7 in zebrafish. While ATXN7 depletion in flies led to gross retinal degeneration, in zebrafish, it primarily results in ocular coloboma, a structural malformation responsible for pediatric visual impairment in humans. ATXN7 inactivation leads to elevated Hedgehog signaling in the forebrain, causing an alteration of proximo-distal patterning of the optic vesicle during early eye development and coloboma. At later developmental stages, malformations of photoreceptors due to incomplete formation of their OSs are observed and correlate with altered expression of crx, a key transcription factor involved in the formation of photoreceptor OS. Therefore, we propose that a primary toxic effect of polyQ expansion is the alteration of ATXN7 function in the daily renewal of OS in SCA7. Together, our data indicate that ATXN7 plays an essential role in vertebrate eye morphogenesis and photoreceptor differentiation, and its loss of function may contribute to the development of human coloboma.
Assuntos
Ataxina-7/deficiência , Coloboma/etiologia , Coloboma/metabolismo , Predisposição Genética para Doença , Células Fotorreceptoras/metabolismo , Subunidades Proteicas/deficiência , Transativadores/genética , Animais , Animais Geneticamente Modificados , Biomarcadores , Padronização Corporal/genética , Diferenciação Celular , Coloboma/patologia , Modelos Animais de Doenças , Edição de Genes , Regulação da Expressão Gênica , Histonas/metabolismo , Imuno-Histoquímica , Modelos Biológicos , Nervo Óptico/embriologia , Nervo Óptico/metabolismo , Organogênese/genética , Fenótipo , Células Fotorreceptoras/patologia , Processamento de Proteína Pós-Traducional , Transativadores/química , Transativadores/metabolismo , Peixe-ZebraRESUMO
The α7-nicotinic acetylcholine receptor (α7-nAChR) is a ligand-gated ion channel that is expressed widely in vertebrates and is the principal high-affinity α-bungarotoxin (α-bgtx) binding protein in the mammalian CNS. α7-nAChRs associate with proteins that can modulate its properties. The α7-nAChR interactome is the summation of proteins interacting or associating with α7-nAChRs in a protein complex. To identify an α7-nAChR interactome in neural tissue, we isolated α-bgtx-affinity protein complexes from wild-type and α7-nAChR knockout (α7 KO) mouse whole brain tissue homogenates using α-bgtx-affinity beads. Affinity precipitated proteins were trypsinized and analyzed with an Orbitrap Fusion mass spectrometer. Proteins isolated with the α7-nAChR specific ligand, α-bgtx, were determined to be α7-nAChR associated proteins. The α7-nAChR subunit and 120 additional proteins were identified. Additionally, 369 proteins were identified as binding to α-bgtx in the absence of α7-nAChR expression, thereby identifying nonspecific proteins for α7-nAChR investigations using α-bgtx enrichment. These results expand on our previous investigations of α7-nAChR interacting proteins using α-bgtx-affinity bead isolation by controlling for differences between α7-nAChR and α-bgtx-specific proteins, developing an improved protein isolation methodology, and incorporating the latest technology in mass spectrometry. The α7-nAChR interactome identified in this study includes proteins associated with the expression, localization, function, or modulation of α7-nAChRs, and it provides a foundation for future studies to elucidate how these interactions contribute to human disease.
Assuntos
Encéfalo/metabolismo , Bungarotoxinas/química , Mapeamento de Interação de Proteínas/métodos , Subunidades Proteicas/química , Proteoma/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/química , Animais , Encéfalo/citologia , Bungarotoxinas/metabolismo , Cromatografia de Afinidade , Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/citologia , Neurônios/metabolismo , Peptídeos/análise , Ligação Proteica , Subunidades Proteicas/deficiência , Subunidades Proteicas/genética , Proteólise , Proteoma/genética , Proteômica/métodos , Receptor Nicotínico de Acetilcolina alfa7/deficiência , Receptor Nicotínico de Acetilcolina alfa7/genéticaRESUMO
Synaptic vesicle and active zone proteins are required for synaptogenesis. The molecular mechanisms for coordinated synthesis of these proteins are not understood. Using forward genetic screens, we identified the conserved THO nuclear export complex (THOC) as an important regulator of presynapse development in C. elegans dopaminergic neurons. In THOC mutants, synaptic messenger RNAs are retained in the nucleus, resulting in dramatic decrease of synaptic protein expression, near complete loss of synapses, and compromised dopamine function. CRE binding protein (CREB) interacts with THOC to mark synaptic transcripts for efficient nuclear export. Deletion of Thoc5, a THOC subunit, in mouse dopaminergic neurons causes severe defects in synapse maintenance and subsequent neuronal death in the substantia nigra compacta. These cellular defects lead to abrogated dopamine release, ataxia, and animal death. Together, our results argue that nuclear export mechanisms can select specific mRNAs and be a rate-limiting step for neuronal differentiation and survival.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Neurônios Dopaminérgicos/metabolismo , Proteínas Nucleares/genética , Sinapses/metabolismo , Transporte Ativo do Núcleo Celular , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Sinalização do Cálcio , Núcleo Celular/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutagênese , Mutação de Sentido Incorreto , Proteínas Nucleares/deficiência , Proteínas Nucleares/metabolismo , Subunidades Proteicas/deficiência , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismoRESUMO
Phosphoinositides are bioactive lipids essential in the regulation of cell signaling as well as cytoskeleton and membrane dynamics. Their metabolism is highly active in blood platelets where they play a critical role during activation, at least through two well identified pathways involving phospholipase C and phosphoinositide 3-kinases (PI3K). Here, using a sensitive high-performance liquid chromatography-mass spectrometry method recently developed, we monitored for the first time the profiling of phosphatidylinositol (PI), PIP, PIP2 and PIP3 molecular species (fatty-acyl profiles) in human and mouse platelets during the course of stimulation by thrombin and collagen-related peptide. Furthermore, using class IA PI3K p110α or p110ß deficient mouse platelets and a pharmacological inhibitor, we show the crucial role of p110ß and the more subtle role of p110α in the production of PIP3 molecular species following stimulation. This comprehensive platelet phosphoinositides profiling provides important resources for future studies and reveals new information on phosphoinositides biology, similarities and differences in mouse and human platelets and unexpected dramatic increase in low-abundance molecular species of PIP2 during stimulation, opening new perspectives in phosphoinositide signaling in platelets.
Assuntos
Plaquetas/efeitos dos fármacos , Classe I de Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Animais , Plaquetas/citologia , Plaquetas/metabolismo , Proteínas de Transporte/farmacologia , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Classe I de Fosfatidilinositol 3-Quinases/deficiência , Inibidores Enzimáticos/farmacologia , Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peptídeos/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Cultura Primária de Células , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/deficiência , Subunidades Proteicas/genética , Pirimidinonas/farmacologia , Trombina/farmacologia , ortoaminobenzoatos/farmacologiaRESUMO
Proteomic analyses were carried out on isolated mitochondrial samples of C. albicans from gene-deleted mutants (nuo1Δ, nuo2Δ and goa1Δ) as well as the parental strain in order to better understand the contribution of these three fungal-specific mitochondrial ETC complex I (CI) subunits to cellular activities. Herein, we identify 2333 putative proteins from four strains, in which a total of 663 proteins (28.5%) are putatively located in mitochondria. Comparison of protein abundances between mutants and the parental strain reveal 146 differentially-expressed proteins, of which 78 are decreased and 68 are increased in at least one mutant. The common changes across the three mutants include the down-regulation of nuclear-encoded CI subunit proteins as well as phospholipid, ergosterol and cell wall mannan synthesis, and up-regulated proteins in CIV and the alternative oxidase (AOX2). As for gene-specific functions, we find that NUO1 participates in nucleotide synthesis and ribosomal biogenesis; NUO2 is involved in vesicle trafficking; and GOA1 appears to regulate membrane transporter proteins, ROS removal, and substrates trafficking between peroxisomes and mitochondria. The proteomic view of general as well as mutant-specific proteins further extends our understanding of the functional roles of non-mammalian CI-specific subunit proteins in cell processes. Particularly intriguing is the confirmation of a regulatory role for GOA1 on ETC function, a protein found almost exclusively in Candida species. SIGNIFICANCE: Fungal mitochondria are critical for fungal pathogenesis. The absence of any of the three fungal specific CI subunits in mitochondria causes an avirulence phenotype of C. albicans in a murine model of invasive disease. As model yeast (Saccharomyces cerevisiae) lacks a CI and is rarely a pathogen of humans, C. albicans is a better choice for establishing a link between mitochondrial CI and pathogenesis. Apart from the general effects of CI mutants on respiration, previous phenotyping of these mutants were quite similar to each other or to CI conservative subunit. By comparison to transcriptional data, the proteomic data obtained in this study indicate that biosynthetic events in each mutant such as cell wall and cell membrane phospholipids and ergosterol are generally decreased in both transcriptomal and translational levels. However, in the case of mitochondrial function, glycolysis/gluconeogenesis, and ROS scavengers, often gene changes are opposite that of proteomic data in mutants. We hypothesize that the loss of energy production in mutants is compensated by increases in protein levels of glycolysis, gluconeogenesis, and anti-ROS scavengers that at least extend mutant survival.
Assuntos
Candida albicans/enzimologia , Complexo I de Transporte de Elétrons/deficiência , Proteínas Fúngicas/análise , Mitocôndrias/enzimologia , Proteoma/análise , Deleção de Genes , Subunidades Proteicas/deficiência , Saccharomyces cerevisiaeRESUMO
Chromatin remodelers have emerged as prominent regulators of epigenetic processes and potential drivers of various human pathologies. The multi-subunit chromatin-remodeling SWI/SNF complex determines gene expression programs and, consequently, contributes to the differentiation, maturation and plasticity of neurons. Here, we investigate the elusive biological function of Bcl7a and Bcl7b, two newly identified subunits of the SWI/SNF complex that are highly expressed throughout the brain. We generated ubiquitous and neuron-specific Bcl7a and Bcl7b single and double knockout mice. We provide evidence that Bcl7b is dispensable for animal survival as well as behavioral plasticity. Conversely, ubiquitous Bcl7a knockout results in perinatal lethality, while genetic deletion of Bcl7a in postmitotic neurons elicits motor abnormalities and affects dendritic branching of Purkinje cells, with no obvious synergistic relationship with Bcl7b. Collectively, our findings reveal novel insights into the cellular processes linked to BCL7-containing SWI/SNF complexes and their unrecognized roles in the brain.
Assuntos
Comportamento Animal/fisiologia , Proteínas dos Microfilamentos/metabolismo , Animais , Peso Corporal , Encéfalo/metabolismo , Células Cultivadas , Feminino , Locomoção/fisiologia , Masculino , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/deficiência , Proteínas dos Microfilamentos/genética , Subunidades Proteicas/deficiência , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Células de Purkinje/citologia , Células de Purkinje/metabolismoRESUMO
Salmonella enterica Enteritidis forms biofilms and survives in agricultural environments, infecting poultry and eggs. Bacteria in biofilms are difficult to eradicate compared to planktonic cells, causing serious problems in industry and public health. In this study, we evaluated the role of ihfA and ihfB in biofilm formation by S. enterica Enteritidis by employing different microbiology techniques. Our data indicate that ihf mutant strains are impaired in biofilm formation, showing a reduction in matrix formation and a decrease in viability and metabolic activity. Phenotypic analysis also showed that deletion of ihf causes a deficiency in curli fimbriae expression, cellulose production and pellicle formation. These results show that integration host factor has an important regulatory role in biofilm formation by S. enterica Enteritidis.
Assuntos
Biofilmes/crescimento & desenvolvimento , Fímbrias Bacterianas/genética , Regulação Bacteriana da Expressão Gênica , Fatores Hospedeiros de Integração/genética , Plâncton/genética , Salmonella enteritidis/genética , Celulose/biossíntese , Fímbrias Bacterianas/metabolismo , Deleção de Genes , Aptidão Genética , Fatores Hospedeiros de Integração/deficiência , Plâncton/crescimento & desenvolvimento , Plâncton/metabolismo , Polissacarídeos Bacterianos/biossíntese , Polissacarídeos Bacterianos/deficiência , Subunidades Proteicas/deficiência , Subunidades Proteicas/genética , Salmonella enteritidis/crescimento & desenvolvimento , Salmonella enteritidis/metabolismo , Salmonella enteritidis/patogenicidadeRESUMO
The GluA1 subunit of the AMPA receptor has been implicated in schizophrenia. While GluA1 is important for cognition, it is not clear what the role of GluA1 is in hedonic responses that are relevant to the negative symptoms of disorders such as schizophrenia. Here, we tested mice that lack GluA1 (Gria1 -/- mice) on consumption of sucrose solutions using a licking microstructure analysis. GluA1 deletion drastically reduced palatability (as measured by the mean lick cluster size) across a range of sucrose concentrations. Although initial lick rates were reduced, measures of consumption across long periods of access to sucrose solutions were not affected by GluA1 deletion and Gria1 -/- mice showed normal satiety responses to high sucrose concentrations. GluA1 deletion also failed to impair flavour conditioning, in which increased intake of a flavour occurred as a consequence of prior pairing with a high sucrose concentration. These results demonstrate that GluA1 plays a role in responding on the basis of palatability rather than other properties, such as the automatic and learnt post-ingestive, nutritional consequences of sucrose. Therefore, Gria1 -/- mice provide a potential model of anhedonia, adding converging evidence to the role of glutamatergic dysfunction in various symptoms of schizophrenia and related disorders.