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1.
Nat Commun ; 12(1): 714, 2021 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-33514705

RESUMO

Polycomb repressive complex 2 (PRC2) is a histone methyltransferase critical for maintaining gene silencing during eukaryotic development. In mammals, PRC2 activity is regulated in part by the selective incorporation of one of two paralogs of the catalytic subunit, EZH1 or EZH2. Each of these enzymes has specialized biological functions that may be partially explained by differences in the multivalent interactions they mediate with chromatin. Here, we present two cryo-EM structures of PRC2:EZH1, one as a monomer and a second one as a dimer bound to a nucleosome. When bound to nucleosome substrate, the PRC2:EZH1 dimer undergoes a dramatic conformational change. We demonstrate that mutation of a divergent EZH1/2 loop abrogates the nucleosome-binding and methyltransferase activities of PRC2:EZH1. Finally, we show that PRC2:EZH1 dimers are more effective than monomers at promoting chromatin compaction, and the divergent EZH1/2 loop is essential for this function, thereby tying together the methyltransferase, nucleosome-binding, and chromatin-compaction activities of PRC2:EZH1. We speculate that the conformational flexibility and the ability to dimerize enable PRC2 to act on the varied chromatin substrates it encounters in the cell.


Assuntos
Cromatina/metabolismo , Inativação Gênica , Complexo Repressor Polycomb 2/ultraestrutura , Animais , Linhagem Celular , Histonas/genética , Histonas/metabolismo , Modelos Moleculares , Mutação , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Multimerização Proteica , Células Sf9 , Spodoptera , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo
2.
Proc Natl Acad Sci U S A ; 117(35): 21740-21746, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817533

RESUMO

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) anion channel is essential for epithelial salt-water balance. CFTR mutations cause cystic fibrosis, a lethal incurable disease. In cells CFTR is activated through the cAMP signaling pathway, overstimulation of which during cholera leads to CFTR-mediated intestinal salt-water loss. Channel activation is achieved by phosphorylation of its regulatory (R) domain by cAMP-dependent protein kinase catalytic subunit (PKA). Here we show using two independent approaches--an ATP analog that can drive CFTR channel gating but is unsuitable for phosphotransfer by PKA, and CFTR mutants lacking phosphorylatable serines--that PKA efficiently opens CFTR channels through simple binding, under conditions that preclude phosphorylation. Unlike when phosphorylation happens, CFTR activation by PKA binding is completely reversible. Thus, PKA binding promotes release of the unphosphorylated R domain from its inhibitory position, causing full channel activation, whereas phosphorylation serves only to maintain channel activity beyond termination of the PKA signal. The results suggest two levels of CFTR regulation in cells: irreversible through phosphorylation, and reversible through R-domain binding to PKA--and possibly also to other members of a large network of proteins known to interact with the channel.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Canais de Ânion Dependentes de Voltagem/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Ânions/metabolismo , Fenômenos Biofísicos , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Ativação do Canal Iônico/fisiologia , Mutagênese Sítio-Dirigida , Nucleotídeos/metabolismo , Oócitos/metabolismo , Técnicas de Patch-Clamp/métodos , Fosforilação , Ligação Proteica/fisiologia , Serina/metabolismo , Canais de Ânion Dependentes de Voltagem/fisiologia , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo
3.
Nucleic Acids Res ; 48(17): 9538-9549, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32766790

RESUMO

Posttranslational modifications (PTMs) of histones represent a crucial regulatory mechanism of nucleosome and chromatin dynamics in various of DNA-based cellular processes, such as replication, transcription and DNA damage repair. Lysine succinylation (Ksucc) is a newly identified histone PTM, but its regulation and function in chromatin remain poorly understood. Here, we utilized an expressed protein ligation (EPL) strategy to synthesize histone H4 with site-specific succinylation at K77 residue (H4K77succ), an evolutionarily conserved succinylation site at the nucleosomal DNA-histone interface. We then assembled mononucleosomes with the semisynthetic H4K77succ in vitro. We demonstrated that this succinylation impacts nucleosome dynamics and promotes DNA unwrapping from the histone surface, which allows proteins such as transcription factors to rapidly access buried regions of the nucleosomal DNA. In budding yeast, a lysine-to-glutamic acid mutation, which mimics Ksucc, at the H4K77 site reduced nucleosome stability and led to defects in DNA damage repair and telomere silencing in vivo. Our findings revealed this uncharacterized histone modification has important roles in nucleosome and chromatin dynamics.


Assuntos
DNA/metabolismo , Histonas/síntese química , Histonas/metabolismo , Lisina/metabolismo , Nucleossomos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cromatina/química , Cromatina/metabolismo , Transferência Ressonante de Energia de Fluorescência , Histonas/genética , Lisina/química , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/síntese química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo
4.
Proc Natl Acad Sci U S A ; 117(28): 16154-16159, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601228

RESUMO

The metaphase spindle is a dynamic structure orchestrating chromosome segregation during cell division. Recently, soft matter approaches have shown that the spindle behaves as an active liquid crystal. Still, it remains unclear how active force generation contributes to its characteristic spindle-like shape. Here we combine theory and experiments to show that molecular motor-driven forces shape the structure through a barreling-type instability. We test our physical model by titrating dynein activity in Xenopus egg extract spindles and quantifying the shape and microtubule orientation. We conclude that spindles are shaped by the interplay between surface tension, nematic elasticity, and motor-driven active forces. Our study reveals how motor proteins can mold liquid crystalline droplets and has implications for the design of active soft materials.


Assuntos
Metáfase/fisiologia , Fuso Acromático/fisiologia , Animais , Fenômenos Biomecânicos , Dineínas/antagonistas & inibidores , Dineínas/metabolismo , Elasticidade , Cristais Líquidos , Metáfase/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Microtúbulos/fisiologia , Mitose , Fuso Acromático/química , Fuso Acromático/efeitos dos fármacos , Tensão Superficial , Proteínas de Xenopus/antagonistas & inibidores , Proteínas de Xenopus/metabolismo , Xenopus laevis
5.
Arch Biochem Biophys ; 689: 108436, 2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32492375

RESUMO

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels belong to the superfamily of voltage-gated potassium (Kv) and cyclic nucleotide-gated (CNG) channels. HCN channels contain the glycine-tyrosine-glycine (GYG) sequence that forms part of the selectivity filter, a similar structure than some potassium channels; however, they permeate both sodium and potassium, giving rise to an inward current. Yet a second amino acid sequence, leucine-cysteine-isoleucine (LCI), next to GYG, is well-preserved in all HCNs but not in the selective potassium channels. In this study we used site-directed mutagenesis and electrophysiology in frog oocytes to determine whether the LCI sequence affects the kinetics of HCN2 currents. Permeability and voltage dependence were evaluated, and we found a role of LCI in the gating mechanism combined with changes in ion permeability. The I residue resulted critical to this function.


Assuntos
Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/química , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Ativação do Canal Iônico , Potenciais da Membrana , Mutagênese Sítio-Dirigida , Oócitos/metabolismo , Permeabilidade , Potássio/metabolismo , Sódio/metabolismo , Xenopus/genética , Proteínas de Xenopus/química , Proteínas de Xenopus/genética
6.
Am J Physiol Cell Physiol ; 319(2): C371-C380, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32579473

RESUMO

Cation-coupled chloride cotransporters (CCC) play a role in modulating intracellular chloride concentration ([Cl-]i) and cell volume. Cell shrinkage and cell swelling are accompanied by an increase or decrease in [Cl-]i, respectively. Cell shrinkage and a decrease in [Cl-]i increase the activity of NKCCs (Na-K-Cl cotransporters: NKCC1, NKCC2, and Na-Cl) and inhibit the activity of KCCs (K-Cl cotransporters: KCC1 to KCC4), wheras cell swelling and an increase in [Cl-]i activate KCCs and inhibit NKCCs; thus, it is unlikely that the same kinase is responsible for both effects. WNK1 and WNK4 are chloride-sensitive kinases that modulate the activity of CCC in response to changes in [Cl-]i. Here, we showed that WNK3, another member of the serine-threonine kinase WNK family with known effects on CCC, is not sensitive to [Cl-]i but can be regulated by changes in extracellular tonicity. In contrast, WNK4 is highly sensitive to [Cl-]i but is not regulated by changes in cell volume. The activity of WNK3 toward NaCl cotransporter is not affected by eliminating the chloride-binding site of WNK3, further confirming that the kinase is not sensitive to chloride. Chimeric WNK3/WNK4 proteins were produced, and analysis of the chimeras suggests that sequences within the WNK's carboxy-terminal end may modulate the chloride affinity. We propose that WNK3 is a cell volume-sensitive kinase that translates changes in cell volume into phosphorylation of CCC.


Assuntos
Tamanho Celular , Proteínas Serina-Treonina Quinases/genética , Simportadores de Cloreto de Sódio/metabolismo , Proteínas de Xenopus/genética , Animais , Cloretos/química , Cloretos/metabolismo , Citoplasma/química , Citoplasma/metabolismo , Humanos , Oócitos/química , Oócitos/metabolismo , Fosforilação/genética , Proteínas Serina-Treonina Quinases/metabolismo , Simportadores de Cloreto de Sódio/química , Xenopus/genética , Xenopus/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismo
7.
BMC Mol Cell Biol ; 21(1): 39, 2020 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-32466750

RESUMO

BACKGROUND: Members of the T-box family of DNA-binding proteins play a prominent role in the differentiation of the three primary germ layers. VegT, Brachyury, and Eomesodermin function as transcriptional activators and, in addition to directly activating the transcription of endoderm- and mesoderm-specific genes, serve as regulators of growth factor signaling during induction of these germ layers. In contrast, the T-box gene, tbx2, is expressed in the embryonic ectoderm, where Tbx2 functions as a transcriptional repressor and inhibits mesendodermal differentiation by the TGFß ligand Activin. Tbx2 misexpression also promotes dorsal ectodermal fate via inhibition of the BMP branch of the TGFß signaling network. RESULTS: Here, we report a physical association between Tbx2 and both Smad1 and Smad2, mediators of BMP and Activin/Nodal signaling, respectively. We perform structure/function analysis of Tbx2 to elucidate the roles of both Tbx2-Smad interaction and Tbx2 DNA-binding in germ layer suppression. CONCLUSION: Our studies demonstrate that Tbx2 associates with intracellular mediators of the Activin/Nodal and BMP/GDF pathways. We identify a novel repressor domain within Tbx2, and have determined that Tbx2 DNA-binding activity is required for repression of TGFß signaling. Finally, our data also point to overlapping yet distinct mechanisms for Tbx2-mediated repression of Activin/Nodal and BMP/GDF signaling.


Assuntos
Ativinas/farmacologia , Ectoderma/metabolismo , Camadas Germinativas/metabolismo , Proteína Smad1/metabolismo , Proteína Smad2/metabolismo , Proteínas com Domínio T/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Padronização Corporal , Proteínas Morfogenéticas Ósseas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Fatores de Diferenciação de Crescimento/metabolismo , Fosforilação , Domínios Proteicos/genética , Transdução de Sinais/genética , Proteína Smad1/genética , Proteína Smad2/genética , Proteínas com Domínio T/química , Proteínas com Domínio T/genética , Fator de Crescimento Transformador beta/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis
8.
J Mol Biol ; 432(10): 3149-3158, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32277988

RESUMO

Ligand-independent activation of receptor tyrosine kinases (RTKs) allows for dissecting out the receptor-specific signaling outcomes from the pleiotropic effects of the ligands. In this regard, RTK intracellular domains (ICD) are of interest due to their ability to recapitulate signaling activity in a ligand-independent manner when fused to chemical or optical dimerizing domains. A common strategy for synthetic activation of RTKs involves membrane tethering of dimerizer-RTK ICD fusions. Depending on the intrinsic signaling capacity, however, this approach could entail undesirable baseline signaling activity in the absence of stimulus, thereby diminishing the system's sensitivity. Here, we observed toxicity in early Xenopus laevis embryos when using such a conventional optogenetic design for the fibroblast growth factor receptor (FGFR). To surpass this challenge, we developed a cytoplasm-to-membrane translocation approach, where FGFR ICD is recruited from the cytoplasm to the plasma membrane by light, followed by its subsequent activation via homo-association. This strategy results in the optical activation of FGFR with low background activity and high sensitivity, which allows for the light-mediated formation of ectopic tail-like structures in developing X. laevis embryos. We further generalized this strategy by developing optogenetic platforms to control three neurotrophic tropomyosin receptor kinases, TrkA, TrkB, and TrkC. We envision that these ligand-independent optogenetic RTKs will provide useful toolsets for the delineation of signaling sub-circuits in developing vertebrate embryos.


Assuntos
Optogenética/métodos , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Xenopus laevis/crescimento & desenvolvimento , Animais , Membrana Celular/metabolismo , Citoplasma/metabolismo , Desenvolvimento Embrionário , Células HEK293 , Humanos , Transporte Proteico , Receptores Proteína Tirosina Quinases/química , Transdução de Sinais , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética
9.
Nat Commun ; 11(1): 1345, 2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-32165637

RESUMO

In several metazoans, the number of active replication origins in embryonic nuclei is higher than in somatic ones, ensuring rapid genome duplication during synchronous embryonic cell divisions. High replication origin density can be restored by somatic nuclear reprogramming. However, mechanisms underlying high replication origin density formation coupled to rapid cell cycles are poorly understood. Here, using Xenopus laevis, we show that SSRP1 stimulates replication origin assembly on somatic chromatin by promoting eviction of histone H1 through its N-terminal domain. Histone H1 removal derepresses ORC and MCM chromatin binding, allowing efficient replication origin assembly. SSRP1 protein decays at mid-blastula transition (MBT) when asynchronous somatic cell cycles start. Increasing levels of SSRP1 delay MBT and, surprisingly, accelerate post-MBT cell cycle speed and embryo development. These findings identify a major epigenetic mechanism regulating DNA replication and directly linking replication origin assembly, cell cycle duration and embryo development in vertebrates.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismo , Animais , Blástula/embriologia , Blástula/metabolismo , Cromatina/genética , Cromatina/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Grupo de Alta Mobilidade , Histonas/química , Histonas/genética , Domínios Proteicos , Origem de Replicação , Proteínas de Xenopus/genética , Xenopus laevis/embriologia
10.
Biochim Biophys Acta Biomembr ; 1862(6): 183240, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32119862

RESUMO

The IP3 receptor binding protein released with inositol 1,4,5-trisphosphate (IRBIT) plays important roles in the regulation of intracellular Ca2+ signaling and intracellular pH. The mammals express two IRBIT paralogs, i.e., IRBIT1 (encoded by AHCYL1) and IRBIT2 (encoded by AHCYL2). The clawed frog Xenopus laevis oocyte is widely used for biophysical studies on ion channels and transporters. It remains unknown whether endogenous IRBIT is expressed in Xenopus oocytes. Here, we cloned from frog oocyte irbit2.L and irbit2.S, orthologs of mammalian IRBIT2. When over-expressed, the frog IRBITs powerfully stimulate the electrogenic Na+/HCO3- cotransporter NBCe1-B as mouse IRBIT2-V2 does. Expression of an isolated Nt fragment of NBCe1-B containing the IRBIT-binding domain greatly decreases NBCe1-B activity in oocytes, suggesting that the basal activity of NBCe1-B contains a large component derived from the stimulation by endogenous frog IRBIT. The frog IRBITs are highly homologous to the mammalian ones in the carboxyl-terminal region, but varies greatly in the amino-terminal (Nt) appendage. Interestingly, truncation study showed that the Nt appendage of IRBIT1 and the long Nt appendage of IRBIT2-V2 modestly enhance, whereas the short Nt appendage of IRBIT2-V4 greatly inhibits the functional interaction between IRBIT and NBCe1-B. Finally, Ala-substitution of Ser68, a key phosphorylation site in the PEST domain of IRBIT, causes distinct functional consequences depending on the structural context of the Nt appendage in different IRBIT isoforms. We conclude that the Nt appendage of IRBITs is not necessary for, but plays an important regulatory role in the functional interaction between IRBIT and NBCe1-B.


Assuntos
Inositol 1,4,5-Trifosfato/fisiologia , Simportadores de Sódio-Bicarbonato/metabolismo , Animais , Inositol 1,4,5-Trifosfato/metabolismo , Camundongos , Fragmentos de Peptídeos/farmacologia , Ligação Proteica/efeitos dos fármacos , Simportadores de Sódio-Bicarbonato/efeitos dos fármacos , Proteínas de Xenopus/metabolismo , Xenopus laevis
11.
Nature ; 579(7800): 603-608, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32132710

RESUMO

Acetaldehyde is a highly reactive, DNA-damaging metabolite that is produced upon alcohol consumption1. Impaired detoxification of acetaldehyde is common in the Asian population, and is associated with alcohol-related cancers1,2. Cells are protected against acetaldehyde-induced damage by DNA crosslink repair, which when impaired causes Fanconi anaemia (FA), a disease resulting in failure to produce blood cells and a predisposition to cancer3,4. The combined inactivation of acetaldehyde detoxification and the FA pathway induces mutation, accelerates malignancies and causes the rapid attrition of blood stem cells5-7. However, the nature of the DNA damage induced by acetaldehyde and how this is repaired remains a key question. Here we generate acetaldehyde-induced DNA interstrand crosslinks and determine their repair mechanism in Xenopus egg extracts. We find that two replication-coupled pathways repair these lesions. The first is the FA pathway, which operates using excision-analogous to the mechanism used to repair the interstrand crosslinks caused by the chemotherapeutic agent cisplatin. However, the repair of acetaldehyde-induced crosslinks results in increased mutation frequency and an altered mutational spectrum compared with the repair of cisplatin-induced crosslinks. The second repair mechanism requires replication fork convergence, but does not involve DNA incisions-instead the acetaldehyde crosslink itself is broken. The Y-family DNA polymerase REV1 completes repair of the crosslink, culminating in a distinct mutational spectrum. These results define the repair pathways of DNA interstrand crosslinks caused by an endogenous and alcohol-derived metabolite, and identify an excision-independent mechanism.


Assuntos
Acetaldeído/química , Reagentes para Ligações Cruzadas/química , Dano ao DNA , Reparo do DNA , Replicação do DNA/fisiologia , DNA/química , Etanol/química , Anemia de Fanconi/metabolismo , Animais , Cisplatino/química , Cisplatino/farmacologia , Dano ao DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/metabolismo , Etanol/farmacologia , Mutagênese/efeitos dos fármacos , Nucleotidiltransferases/metabolismo , Mutação Puntual/efeitos dos fármacos , Mutação Puntual/genética , Xenopus , Proteínas de Xenopus/metabolismo
12.
PLoS Genet ; 16(2): e1008590, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32053595

RESUMO

The 1.6 Mbp deletion on chromosome 3q29 is associated with a range of neurodevelopmental disorders, including schizophrenia, autism, microcephaly, and intellectual disability. Despite its importance towards neurodevelopment, the role of individual genes, genetic interactions, and disrupted biological mechanisms underlying the deletion have not been thoroughly characterized. Here, we used quantitative methods to assay Drosophila melanogaster and Xenopus laevis models with tissue-specific individual and pairwise knockdown of 14 homologs of genes within the 3q29 region. We identified developmental, cellular, and neuronal phenotypes for multiple homologs of 3q29 genes, potentially due to altered apoptosis and cell cycle mechanisms during development. Using the fly eye, we screened for 314 pairwise knockdowns of homologs of 3q29 genes and identified 44 interactions between pairs of homologs and 34 interactions with other neurodevelopmental genes. Interestingly, NCBP2 homologs in Drosophila (Cbp20) and X. laevis (ncbp2) enhanced the phenotypes of homologs of the other 3q29 genes, leading to significant increases in apoptosis that disrupted cellular organization and brain morphology. These cellular and neuronal defects were rescued with overexpression of the apoptosis inhibitors Diap1 and xiap in both models, suggesting that apoptosis is one of several potential biological mechanisms disrupted by the deletion. NCBP2 was also highly connected to other 3q29 genes in a human brain-specific interaction network, providing support for the relevance of our results towards the human deletion. Overall, our study suggests that NCBP2-mediated genetic interactions within the 3q29 region disrupt apoptosis and cell cycle mechanisms during development.


Assuntos
Encéfalo/embriologia , Cromossomos Humanos Par 3/genética , Proteínas de Drosophila/genética , Desenvolvimento Embrionário/genética , Deficiência Intelectual/genética , Complexo Proteico Nuclear de Ligação ao Cap/genética , Proteínas de Xenopus/genética , Animais , Apoptose/genética , Encéfalo/patologia , Ciclo Celular/genética , Deleção Cromossômica , Deficiências do Desenvolvimento/genética , Deficiências do Desenvolvimento/patologia , Modelos Animais de Doenças , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Embrião não Mamífero , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Redes Reguladoras de Genes , Humanos , Deficiência Intelectual/patologia , Complexo Proteico Nuclear de Ligação ao Cap/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis
13.
J Clin Invest ; 130(2): 813-826, 2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-31904590

RESUMO

Multipass membrane proteins have a myriad of functions, including transduction of cell-cell signals, ion transport, and photoreception. Insertion of these proteins into the membrane depends on the endoplasmic reticulum (ER) membrane protein complex (EMC). Recently, birth defects have been observed in patients with variants in the gene encoding a member of this complex, EMC1. Patient phenotypes include congenital heart disease, craniofacial malformations, and neurodevelopmental disease. However, a molecular connection between EMC1 and these birth defects is lacking. Using Xenopus, we identified defects in neural crest cells (NCCs) upon emc1 depletion. We then used unbiased proteomics and discovered a critical role for emc1 in WNT signaling. Consistent with this, readouts of WNT signaling and Frizzled (Fzd) levels were reduced in emc1-depleted embryos, while NCC defects could be rescued with ß-catenin. Interestingly, other transmembrane proteins were mislocalized upon emc1 depletion, providing insight into additional patient phenotypes. To translate our findings back to humans, we found that EMC1 was necessary for human NCC development in vitro. Finally, we tested patient variants in our Xenopus model and found the majority to be loss-of-function alleles. Our findings define molecular mechanisms whereby EMC1 dysfunction causes disease phenotypes through dysfunctional multipass membrane protein topogenesis.


Assuntos
Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Complexos Multiproteicos/metabolismo , Crista Neural/embriologia , Transtornos do Neurodesenvolvimento/metabolismo , Via de Sinalização Wnt , Proteínas de Xenopus/metabolismo , Animais , Modelos Animais de Doenças , Retículo Endoplasmático/genética , Retículo Endoplasmático/patologia , Membranas Intracelulares/patologia , Complexos Multiproteicos/genética , Crista Neural/patologia , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/patologia , Xenopus , Proteínas de Xenopus/genética
14.
Int J Mol Sci ; 21(3)2020 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-31979419

RESUMO

The TFF peptides xP1 and xP4 from Xenopus laevis are orthologs of TFF1 and TFF2, respectively. xP1 is secreted as a monomer from gastric surface mucous cells and is generally not associated with mucins, whereas xP4 is a typical secretory peptide from esophageal goblet cells, and gastric mucous neck and antral gland cells tightly associated as a lectin with the ortholog of mucin MUC6. Both TFF peptides have diverse protective functions, xP1 as a scavenger for reactive oxygen species preventing oxidative damage and xP4 as a constituent of the water-insoluble adherent inner mucus barrier. Here, we present localization studies using immunofluorescence and immunoelectron microscopy. xP1 is concentrated in dense cores of secretory granules of surface mucous cells, whereas xP4 mixes with MUC6 in esophageal goblet cells. Of note, we observe two different types of goblet cells, which differ in their xP4 synthesis, and this is even visible morphologically at the electron microscopic level. xP4-negative granules are recognized by their halo, which is probably the result of shrinkage during the processing of samples for electron microscopy. Probably, the tight lectin binding of xP4 and MUC6 creates a crosslinked mucous network forming a stabile granule matrix, which prevents shrinkage.


Assuntos
Mucosa Esofágica/metabolismo , Mucosa Gástrica/metabolismo , Células Caliciformes/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Vesículas Secretórias/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Secreções Corporais/metabolismo , Mucosa Esofágica/ultraestrutura , Esôfago/metabolismo , Esôfago/ultraestrutura , Imunofluorescência , Mucosa Gástrica/ultraestrutura , Células Caliciformes/citologia , Células Caliciformes/ultraestrutura , Lectinas/metabolismo , Microscopia Eletrônica , Mucina-6/metabolismo , Mucinas/metabolismo , Proteínas de Xenopus/ultraestrutura , Xenopus laevis
15.
J Biol Chem ; 295(9): 2724-2735, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31996376

RESUMO

Embryonic cell fate specification and axis patterning requires integration of several signaling pathways that orchestrate region-specific gene expression. The transcription factor signal transducer and activator of transcription 3 (Stat3) plays important roles during early development, but it is unclear how Stat3 is activated. Here, using Xenopus as a model, we analyzed the post-translational regulation and functional consequences of Stat3 activation in dorsoventral axis patterning. We show that Stat3 phosphorylation, lysine methylation, and transcriptional activity increase before gastrulation and induce ventral mesoderm formation. Down syndrome critical region gene 6 (DSCR6), a RIPPLY family member that induces dorsal mesoderm by releasing repressive polycomb group proteins from chromatin, bound to the Stat3 C-terminal region and antagonized its transcriptional and ventralizing activities by interfering with its lysine methylation. Enhancer of zeste 2 polycomb-repressive complex 2 subunit (Ezh2) also bound to this region; however, its methyltransferase activity was required for Stat3 methylation and activation. Loss of Ezh2 resulted in dorsalization of ventral mesoderm and formation of a secondary axis. Furthermore, interference with Ezh2 phosphorylation also prevented Stat3 lysine methylation and transcriptional activity. Thus, inhibition of either Ezh2 phosphorylation or Stat3 lysine methylation compensated for the absence of DSCR6 function. These results reveal that DSCR6 and Ezh2 critically and post-translationally regulate Stat3 transcriptional activity. Ezh2 promotes Stat3 activation in ventral mesoderm formation independently of epigenetic regulation, whereas DSCR6 specifies dorsal fate by counteracting this ventralizing activity. This antagonism helps pattern the mesoderm along the dorsoventral axis, representing a critical facet of cell identity regulation during development.


Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/fisiologia , Proteínas Repressoras/fisiologia , Fator de Transcrição STAT3/metabolismo , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/fisiologia , Xenopus/crescimento & desenvolvimento , Animais , Padronização Corporal , Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/citologia , Processamento de Proteína Pós-Traducional , Fatores de Transcrição
16.
Genesis ; 58(3-4): e23354, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31909537

RESUMO

To investigate microRNA (miR) functions in early eye development, we asked whether eye field transcription factors (EFTFs) are targets of miR-dependent regulation in Xenopus embryos. Argonaute (AGO) ribonucleoprotein complexes, including miRs and targeted mRNAs, were coimmunoprecipitated from transgenic embryos expressing myc-tagged AGO under the control of the rax1 promoter; mRNAs for all EFTFs coimmunoprecipitated with Ago in late neurulae. Computational predictions of miR binding sites within EFTF 3'UTRs identified miR-199a-3p ("miR-199") as a candidate regulator of EFTFs, and miR-199 was shown to regulate rax1 in vivo. Targeted overexpression of miR-199 led to small eyes, a reduction in EFTF expression, and reduced cell proliferation. Inhibition of interactions between mir-199 and the rax1 3'UTR reversed the small eye phenotype. Although targeted knockdown of miR-199 left the eye field intact, it reduced optic cup outgrowth and disrupted eye formation. Computational identification of candidate miR-199 targets within the Xenopus transcriptome led to the identification of ptk7 as a candidate regulator. Targeted overexpression of ptk7 resulted in abnormal optic cup formation and a reduction or loss of eye development, recapitulating the range of eye phenotypes seen following miR-199 knockdown. Our results indicate that miR-199 plays both positive and negative regulatory roles in eye development.


Assuntos
Olho/embriologia , Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/genética , Interferência de RNA , Xenopus laevis/embriologia , Xenopus laevis/genética , Animais , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Estudos de Associação Genética , Mutação com Perda de Função , Organogênese/genética , Fenótipo , Ligação Proteica , Receptores Proteína Tirosina Quinases/genética , Reprodutibilidade dos Testes , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo
17.
Nat Commun ; 11(1): 270, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31937751

RESUMO

Phase separation of substrates and effectors is proposed to enhance biological reaction rates and efficiency. Targeting protein for Xklp2 (TPX2) is an effector of branching microtubule nucleation in spindles and functions with the substrate tubulin by an unknown mechanism. Here we show that TPX2 phase separates into a co-condensate with tubulin, which mediates microtubule nucleation in vitro and in isolated cytosol. TPX2-tubulin co-condensation preferentially occurs on pre-existing microtubules, the site of branching microtubule nucleation, at the endogenous and physiologically relevant concentration of TPX2. Truncation and chimera versions of TPX2 suggest that TPX2-tubulin co-condensation enhances the efficiency of TPX2-mediated branching microtubule nucleation. Finally, the known inhibitor of TPX2, the importin-α/ß heterodimer, regulates TPX2 condensation in vitro and, consequently, branching microtubule nucleation activity in isolated cytosol. Our study demonstrates how regulated phase separation can simultaneously enhance reaction efficiency and spatially coordinate microtubule nucleation, which may facilitate rapid and accurate spindle formation.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Proteínas de Ciclo Celular/química , Citosol/metabolismo , Carioferinas/metabolismo , Meiose , Proteínas Associadas aos Microtúbulos/química , Centro Organizador dos Microtúbulos/metabolismo , Óvulo/citologia , Óvulo/metabolismo , Fuso Acromático/metabolismo , Proteínas de Xenopus/química , Xenopus laevis
18.
Methods Mol Biol ; 2041: 87-106, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646482

RESUMO

Xenopus embryos are one of the most used animal models in developmental biology and are well suited for apprehending functions of signaling pathways during embryogenesis. To do so, it is necessary to be able to detect expression pattern of the key genes of these signaling pathways. Here we describe the whole-mount in situ hybridization technique to investigate the expression pattern of ectonucleotidases and purinergic receptors during embryonic development.


Assuntos
5'-Nucleotidase/metabolismo , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ/métodos , Receptores Purinérgicos/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , 5'-Nucleotidase/genética , Animais , Embrião não Mamífero/citologia , Desenvolvimento Embrionário , Feminino , Sondas RNA , Receptores Purinérgicos/genética , Transdução de Sinais , Proteínas de Xenopus/genética , Xenopus laevis/embriologia
19.
Biochim Biophys Acta Gen Subj ; 1864(3): 129482, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31734465

RESUMO

BACKGROUND: Asymmetric arginine dimethylation of histone H4R3 to H4R3me2a by protein arginine methyltransferase 1 (PRMT1) has been implicated to play a key role in gene activation throughout vertebrates. PRMT1 knockout in mouse leads to embryonic lethality. This and the uterus-enclosed nature of the mouse embryo make it difficult to determine the development role of PRMT1 in mammals. METHODS: We took advantage of the external development of the diploid anuran Xenopus tropicalis and adapted the TALEN genome editing technology to knock out PRMT1 in order to investigate how PRMT1 participates in vertebrate development. RESULTS: We observed that PRMT1 knockout had no apparent effect on embryogenesis because normally feeding tadpoles were formed, despite the reduced asymmetric H4R3 di-methylation (H4R3me2a) due to the knockout. However, PRMT1 knockout tadpoles had severely reduced growth even with normal growth hormone gene expression. These tadpoles were also stalled in development shortly after feeding began at stages 44/45 and died within 2 weeks, well before the onset of metamorphosis. In situ analyses revealed broad cessation or drastic reduction in cell proliferation in diverse organs including the eye, brain, spinal cord, liver, and intestine. CONCLUSIONS: Our findings suggest that PRMT1 is not required for embryogenesis but is a key regulator for normal progression of vertebrate development and growth. GENERAL SIGNIFICANCE: The similarities and differences between PRMT1 knockout Xenopus tropicalis and mouse suggest that two distinct phases of vertebrate development: early embryogenesis and subsequent growth/organ maturation, have different but evolutionally conserved requirement for epigenetic modifications.


Assuntos
Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Animais , Arginina/genética , Arginina/metabolismo , Proliferação de Células , Epigênese Genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Técnicas de Inativação de Genes/métodos , Histona Metiltransferases/metabolismo , Histonas/metabolismo , Larva/metabolismo , Masculino , Metamorfose Biológica/genética , Metamorfose Biológica/fisiologia , Metilação , Metiltransferases/metabolismo , Processamento de Proteína Pós-Traducional , Ativação Transcricional , Xenopus/genética , Xenopus/crescimento & desenvolvimento , Xenopus/metabolismo , Proteínas de Xenopus/metabolismo
20.
Mol Cell ; 77(5): 1080-1091.e8, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-31862156

RESUMO

Enzymatic processing of DNA underlies all DNA repair, yet inappropriate DNA processing must be avoided. In vertebrates, double-strand breaks are repaired predominantly by non-homologous end joining (NHEJ), which directly ligates DNA ends. NHEJ has the potential to be highly mutagenic because it uses DNA polymerases, nucleases, and other enzymes that modify incompatible DNA ends to allow their ligation. Using frog egg extracts that recapitulate NHEJ, we show that end processing requires the formation of a "short-range synaptic complex" in which DNA ends are closely aligned in a ligation-competent state. Furthermore, single-molecule imaging directly demonstrates that processing occurs within the short-range complex. This confinement of end processing to a ligation-competent complex ensures that DNA ends undergo ligation as soon as they become compatible, thereby minimizing mutagenesis. Our results illustrate how the coordination of enzymatic catalysis with higher-order structural organization of substrate maximizes the fidelity of DNA repair.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Instabilidade Genômica , Animais , DNA Ligases/genética , DNA Ligases/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteína Quinase Ativada por DNA/genética , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo , Modelos Genéticos , Complexos Multiproteicos , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Imagem Individual de Molécula , Fatores de Tempo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis
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