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1.
Nat Commun ; 11(1): 4980, 2020 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-33020477

RESUMO

The functions of the proto-oncoprotein c-Myc and the tumor suppressor p53 in controlling cell survival and proliferation are inextricably linked as "Yin and Yang" partners in normal cells to maintain tissue homeostasis: c-Myc induces the expression of ARF tumor suppressor (p14ARF in human and p19ARF in mouse) that binds to and inhibits mouse double minute 2 homolog (MDM2) leading to p53 activation, whereas p53 suppresses c-Myc through a combination of mechanisms involving transcriptional inactivation and microRNA-mediated repression. Nonetheless, the regulatory interactions between c-Myc and p53 are not retained by cancer cells as is evident from the often-imbalanced expression of c-Myc over wildtype p53. Although p53 repression in cancer cells is frequently associated with the loss of ARF, we disclose here an alternate mechanism whereby c-Myc inactivates p53 through the actions of the c-Myc-Inducible Long noncoding RNA Inactivating P53 (MILIP). MILIP functions to promote p53 polyubiquitination and turnover by reducing p53 SUMOylation through suppressing tripartite-motif family-like 2 (TRIML2). MILIP upregulation is observed amongst diverse cancer types and is shown to support cell survival, division and tumourigenicity. Thus our results uncover an inhibitory axis targeting p53 through a pan-cancer expressed RNA accomplice that links c-Myc to suppression of p53.


Assuntos
Neoplasias/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Longo não Codificante/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Carcinogênese , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/genética , RNA Longo não Codificante/genética , Sumoilação , Proteína Supressora de Tumor p53/genética , Ubiquitinação
2.
Nat Commun ; 11(1): 4859, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32978401

RESUMO

Cell death is intrinsically linked with immunity. Disruption of an immune-activated MAPK cascade, consisting of MEKK1, MKK1/2, and MPK4, triggers cell death and autoimmunity through the nucleotide-binding leucine-rich repeat (NLR) protein SUMM2 and the MAPK kinase kinase MEKK2. In this study, we identify a Catharanthus roseus receptor-like kinase 1-like (CrRLK1L), named LETUM2/MEDOS1 (LET2/MDS1), and the glycosylphosphatidylinositol (GPI)-anchored protein LLG1 as regulators of mekk1-mkk1/2-mpk4 cell death. LET2/MDS1 functions additively with LET1, another CrRLK1L, and acts genetically downstream of MEKK2 in regulating SUMM2 activation. LET2/MDS1 complexes with LET1 and promotes LET1 phosphorylation, revealing an intertwined regulation between different CrRLK1Ls. LLG1 interacts with the ectodomain of LET1/2 and mediates LET1/2 transport to the plasma membrane, corroborating its function as a co-receptor of LET1/2 in the mekk1-mkk1/2-mpk4 cell death pathway. Thus, our data suggest that a trimeric complex consisting of two CrRLK1Ls LET1, LET2/MDS1, and a GPI-anchored protein LLG1 that regulates the activation of NLR SUMM2 for initiating cell death and autoimmunity.


Assuntos
Autoimunidade/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas Ligadas por GPI/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Autoimunidade/fisiologia , Proteínas de Transporte/imunologia , Catharanthus/genética , Catharanthus/metabolismo , Morte Celular/genética , Proteínas Ligadas por GPI/genética , Regulação da Expressão Gênica de Plantas , Glicosilfosfatidilinositóis , MAP Quinase Quinase Quinases/genética , Sistema de Sinalização das MAP Quinases , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fosforilação , Proteínas de Plantas/imunologia , Plantas Geneticamente Modificadas , Interferência de RNA , Transcriptoma
3.
Nature ; 585(7826): 609-613, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32939087

RESUMO

Breaks in DNA strands recruit the protein PARP1 and its paralogue PARP2 to modify histones and other substrates through the addition of mono- and poly(ADP-ribose) (PAR)1-5. In the DNA damage responses, this post-translational modification occurs predominantly on serine residues6-8 and requires HPF1, an accessory factor that switches the amino acid specificity of PARP1 and PARP2 from aspartate or glutamate to serine9,10. Poly(ADP) ribosylation (PARylation) is important for subsequent chromatin decompaction and provides an anchor for the recruitment of downstream signalling and repair factors to the sites of DNA breaks2,11. Here, to understand the molecular mechanism by which PARP enzymes recognize DNA breaks within chromatin, we determined the cryo-electron-microscopic structure of human PARP2-HPF1 bound to a nucleosome. This showed that PARP2-HPF1 bridges two nucleosomes, with the broken DNA aligned in a position suitable for ligation, revealing the initial step in the repair of double-strand DNA breaks. The bridging induces structural changes in PARP2 that signal the recognition of a DNA break to the catalytic domain, which licenses HPF1 binding and PARP2 activation. Our data suggest that active PARP2 cycles through different conformational states to exchange NAD+ and substrate, which may enable PARP enzymes to act processively while bound to chromatin. The processes of PARP activation and the PARP catalytic cycle we describe can explain mechanisms of resistance to PARP inhibitors and will aid the development of better inhibitors as cancer treatments12-16.


Assuntos
Proteínas de Transporte/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Biocatálise , Proteínas de Transporte/química , Proteínas de Transporte/ultraestrutura , Microscopia Crioeletrônica , DNA/metabolismo , Reparo do DNA , Ativação Enzimática , Humanos , Modelos Moleculares , NAD/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/ultraestrutura , Nucleossomos/química , Nucleossomos/ultraestrutura , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/ultraestrutura , Domínios Proteicos
4.
Nat Commun ; 11(1): 4055, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792504

RESUMO

Although metastasis is the most common cause of cancer deaths, metastasis-intrinsic dependencies remain largely uncharacterized. We previously reported that metastatic pancreatic cancers were dependent on the glucose-metabolizing enzyme phosphogluconate dehydrogenase (PGD). Surprisingly, PGD catalysis was constitutively elevated without activating mutations, suggesting a non-genetic basis for enhanced activity. Here we report a metabolic adaptation that stably activates PGD to reprogram metastatic chromatin. High PGD catalysis prevents transcriptional up-regulation of thioredoxin-interacting protein (TXNIP), a gene that negatively regulates glucose import. This allows glucose consumption rates to rise in support of PGD, while simultaneously facilitating epigenetic reprogramming through a glucose-fueled histone hyperacetylation pathway. Restoring TXNIP normalizes glucose consumption, lowers PGD catalysis, reverses hyperacetylation, represses malignant transcripts, and impairs metastatic tumorigenesis. We propose that PGD-driven suppression of TXNIP allows pancreatic cancers to avidly consume glucose. This renders PGD constitutively activated and enables metaboloepigenetic selection of additional traits that increase fitness along glucose-replete metastatic routes.


Assuntos
Cromatina/metabolismo , Glucose/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Animais , Transporte Biológico/genética , Transporte Biológico/fisiologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Reprogramação Celular/genética , Reprogramação Celular/fisiologia , Imunoprecipitação da Cromatina , Epigênese Genética/genética , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/genética , Fosfogluconato Desidrogenase/genética , Fosfogluconato Desidrogenase/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismo
5.
Nat Commun ; 11(1): 4187, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32826901

RESUMO

EHBP1 is an adaptor protein that regulates vesicular trafficking by recruiting Rab8 family members and Eps15-homology domain-containing proteins 1/2 (EHD1/2). It also links endosomes to the actin cytoskeleton. However, the underlying molecular mechanism of activation of EHBP1 actin-binding activity is unclear. Here, we show that both termini of EHBP1 have membrane targeting potential. EHBP1 associates with PI(3)P, PI(5)P, and phosphatidylserine via its N-terminal C2 domain. We show that in the absence of Rab8 family members, the C-terminal bivalent Mical/EHBP Rab binding (bMERB) domain forms an intramolecular complex with its central calponin homology (CH) domain and auto-inhibits actin binding. Rab8 binding to the bMERB domain relieves this inhibition. We have analyzed the CH:bMERB auto-inhibited complex and the active bMERB:Rab8 complex biochemically and structurally. Together with structure-based mutational studies, this explains how binding of Rab8 frees the CH domain and allows it to interact with the actin cytoskeleton, leading to membrane tubulation.


Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/metabolismo , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/genética , Proteínas dos Microfilamentos/genética , Modelos Moleculares , Fosfatos de Fosfatidilinositol/metabolismo , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico/fisiologia , Alinhamento de Sequência , Proteínas de Transporte Vesicular , Proteínas rab de Ligação ao GTP/genética
6.
Life Sci ; 258: 118190, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32777299

RESUMO

AIMS: Glycolysis is an important process for cervical carcinoma development. Previous studies have indicated that stress-induced phosphoprotein 1 (STIP1) is associated with development of multiple tumors. Nevertheless, the role and mechanism of STIP1 in glycolysis of cervical carcinoma remain unclear. MAIN METHODS: The association between STIP1 and survival probability and the correlation between STIP1 expression and pyruvate kinase M2 (PKM2) as well as lactate dehydrogenase isoform A (LDHA) levels in cervical carcinoma were analyzed via The Cancer Genome Atlas (TCGA). The expression of STIP1, PKM2, LDHA, and cytochrome c (Cyt C) was measured via western blot or quantitative reverse transcription polymerase chain reaction. Cell viability and apoptosis were examined via cell counting kit 8 and flow cytometry, respectively. Glycolysis was assessed via detection of glucose consumption and lactate production. The protein involved in the Wnt/ß-catenin pathway was measured via western blot. KEY FINDINGS: STIP1 abundance was elevated in cervical carcinoma cells. High expression of STIP1 indicated poor survival probability. Knockdown of STIP1 inhibited cervical carcinoma cell viability and promoted apoptosis. STIP1 expression was positively correlated with PKM2 and LDHA levels in cervical carcinoma. Silence of STIP1 inhibited glycolysis and decreased PKM2 and LDHA expression. Down-regulation of STIP1 repressed the Wnt/ß-catenin pathway. Overexpression of ß-catenin reversed the effect of STIP1 silence on viability, apoptosis, glycolysis, and levels of PKM2 and LDHA. SIGNIFICANCE: STIP1 knockdown suppressed glycolysis in cervical carcinoma by inhibiting PKM2 and LDHA expression and activation of the Wnt/ß-catenin pathway.


Assuntos
Proteínas de Transporte/metabolismo , Regulação para Baixo/genética , Glicólise , Proteínas de Choque Térmico/metabolismo , Lactato Desidrogenase 5/metabolismo , Proteínas de Membrana/metabolismo , Hormônios Tireóideos/metabolismo , Neoplasias do Colo do Útero/genética , Via de Sinalização Wnt , Apoptose , Linhagem Celular Tumoral , Sobrevivência Celular , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Proteínas de Choque Térmico/genética , Humanos , Modelos Biológicos , Neoplasias do Colo do Útero/patologia , Via de Sinalização Wnt/genética
7.
PLoS One ; 15(8): e0235898, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833999

RESUMO

Myo/Nog cells were discovered in the chick embryo epiblast. Their expression of MyoD reflects a commitment to the skeletal muscle lineage and capacity to differentiate into myofibroblasts. Release of Noggin by Myo/Nog cells is essential for normal morphogenesis. Myo/Nog cells rapidly respond to wounding in the skin and eyes. In this report, we present evidence suggesting that Myo/Nog cells phagocytose tattoo ink in tissue sections of human skin and engulf cell corpses in cultures of anterior human lens tissue and magnetic beads injected into the anterior chamber of mice in vivo. Myo/Nog cells are distinct from macrophages in the skin and eyes indicated by the absence of labeling with an antibody to ionized calcium binding adaptor molecule 1. In addition to their primary roles as regulators of BMP signaling and progenitors of myofibroblasts, Myo/Nog cells behave as nonprofessional phagocytes defined as cells whose primary functions are unrelated to phagocytosis but are capable of engulfment.


Assuntos
Miofibroblastos/citologia , Fagócitos/citologia , Células-Tronco/citologia , Animais , Proteínas de Transporte/metabolismo , Diferenciação Celular , Células Cultivadas , Embrião de Galinha , Feminino , Humanos , Cristalino/citologia , Cristalino/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína MyoD/metabolismo , Miofibroblastos/metabolismo , Fagócitos/metabolismo , Fagocitose , Coelhos , Pele/citologia , Pele/metabolismo , Células-Tronco/metabolismo
8.
Nature ; 585(7824): 251-255, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32848248

RESUMO

Mutation of C9orf72 is the most prevalent defect associated with amyotrophic lateral sclerosis and frontotemporal degeneration1. Together with hexanucleotide-repeat expansion2,3, haploinsufficiency of C9orf72 contributes to neuronal dysfunction4-6. Here we determine the structure of the C9orf72-SMCR8-WDR41 complex by cryo-electron microscopy. C9orf72 and SMCR8 both contain longin and DENN (differentially expressed in normal and neoplastic cells) domains7, and WDR41 is a ß-propeller protein that binds to SMCR8 such that the whole structure resembles an eye slip hook. Contacts between WDR41 and the DENN domain of SMCR8 drive the lysosomal localization of the complex in conditions of amino acid starvation. The structure suggested that C9orf72-SMCR8 is a GTPase-activating protein (GAP), and we found that C9orf72-SMCR8-WDR41 acts as a GAP for the ARF family of small GTPases. These data shed light on the function of C9orf72 in normal physiology, and in amyotrophic lateral sclerosis and frontotemporal degeneration.


Assuntos
Esclerose Amiotrófica Lateral/genética , Proteínas Relacionadas à Autofagia/química , Proteína C9orf72/química , Proteína C9orf72/genética , Proteínas de Transporte/química , Microscopia Crioeletrônica , Demência Frontotemporal/genética , Haploinsuficiência , Complexos Multiproteicos/química , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Esclerose Amiotrófica Lateral/metabolismo , Proteínas Relacionadas à Autofagia/deficiência , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/ultraestrutura , Proteína C9orf72/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte/ultraestrutura , Demência Frontotemporal/metabolismo , Humanos , Lisossomos/metabolismo , Modelos Moleculares , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Domínios Proteicos
9.
PLoS Biol ; 18(7): e3000778, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32678845

RESUMO

The evolution of transformed cancer cells into metastatic tumors is, in part, driven by altered intracellular signaling downstream of receptor tyrosine kinases (RTKs). The surface levels and activity of RTKs are governed mainly through clathrin-mediated endocytosis (CME), endosomal recycling, or degradation. In turn, oncogenic signaling downstream of RTKs can reciprocally regulate endocytic trafficking by creating feedback loops in cells to enhance tumor progression. We previously showed that FCH/F-BAR and Double SH3 Domain-Containing Protein (FCHSD2) has a cancer-cell specific function in regulating CME in non-small-cell lung cancer (NSCLC) cells. Here, we report that FCHSD2 loss impacts recycling of the RTKs, epidermal growth factor receptor (EGFR) and proto-oncogene c-Met (MET), and shunts their trafficking into late endosomes and lysosomal degradation. Notably, FCHSD2 depletion results in the nuclear translocation of active extracellular signal-regulated kinase 1 and 2 (ERK1/2), leading to enhanced transcription and up-regulation of EGFR and MET. The small GTPase, Ras-related protein Rab-7A (Rab7), is essential for the FCHSD2 depletion-induced effects. Correspondingly, FCHSD2 loss correlates to higher tumor grades of NSCLC. Clinically, NSCLC patients expressing high FCHSD2 exhibit elevated survival, whereas patients with high Rab7 expression display decreased survival rates. Our study provides new insight into the molecular nexus for crosstalk between oncogenic signaling and RTK trafficking that controls cancer progression.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Proteínas de Transporte/metabolismo , Endocitose , Neoplasias Pulmonares/patologia , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana/metabolismo , Oncogenes , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Linhagem Celular Tumoral , Progressão da Doença , Endossomos/metabolismo , Receptores ErbB/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/enzimologia , Transporte Proteico , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptores da Transferrina/metabolismo , Regulação para Cima , Proteínas rab de Ligação ao GTP/metabolismo
10.
Proc Natl Acad Sci U S A ; 117(32): 19254-19265, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32719146

RESUMO

The appropriate arrangement of myonuclei within skeletal muscle myofibers is of critical importance for normal muscle function, and improper myonuclear localization has been linked to a variety of skeletal muscle diseases, such as centronuclear myopathy and muscular dystrophies. However, the molecules that govern myonuclear positioning remain elusive. Here, we report that skeletal muscle-specific CIP (sk-CIP) is a regulator of nuclear positioning. Genetic deletion of sk-CIP in mice results in misalignment of myonuclei along the myofibers and at specialized structures such as neuromuscular junctions (NMJs) and myotendinous junctions (MTJs) in vivo, impairing myonuclear positioning after muscle regeneration, leading to severe muscle dystrophy in mdx mice, a mouse model of Duchenne muscular dystrophy. sk-CIP is localized to the centrosome in myoblasts and relocates to the outer nuclear envelope in myotubes upon differentiation. Mechanistically, we found that sk-CIP interacts with the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex and the centriole Microtubule Organizing Center (MTOC) proteins to coordinately modulate myonuclear positioning and alignment. These findings indicate that sk-CIP may function as a muscle-specific anchoring protein to regulate nuclear position in multinucleated muscle cells.


Assuntos
Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Miopatias Congênitas Estruturais/fisiopatologia , Proteínas Nucleares/metabolismo , Animais , Proteínas de Transporte/genética , Núcleo Celular/genética , Humanos , Camundongos , Camundongos Endogâmicos mdx , Camundongos Knockout , Músculo Esquelético/fisiopatologia , Miopatias Congênitas Estruturais/genética , Miopatias Congênitas Estruturais/metabolismo , Proteínas Nucleares/genética , Especificidade de Órgãos
11.
Mol Cell Biol ; 40(17)2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601107

RESUMO

Fodrin and its erythroid cell-specific isoform spectrin are actin-associated fibrous proteins that play crucial roles in the maintenance of structural integrity in mammalian cells, which is necessary for proper cell function. Normal cell morphology is altered in diseases such as various cancers and certain neuronal disorders. Fodrin and spectrin are two-chain (αß) molecules that are encoded by paralogous genes and share many features but also demonstrate certain differences. Fodrin (in humans, typically a heterodimer of the products of the SPTAN1 and SPTBN1 genes) is expressed in nearly all cell types and is especially abundant in neuronal tissues, whereas spectrin (in humans, a heterodimer of the products of the SPTA1 and SPTB1 genes) is expressed almost exclusively in erythrocytes. To fulfill a role in such a variety of different cell types, it was anticipated that fodrin would need to be a more versatile scaffold than spectrin. Indeed, as summarized here, domains unique to fodrin and its regulation by Ca2+, calmodulin, and a variety of posttranslational modifications (PTMs) endow fodrin with additional specific functions. However, how fodrin structural variations and misregulated PTMs may contribute to the etiology of various cancers and neurodegenerative diseases needs to be further investigated.


Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Actinas/metabolismo , Animais , Cálcio/metabolismo , Calmodulina/metabolismo , Células Eritroides/metabolismo , Humanos , Neurônios/metabolismo , Espectrina/metabolismo , Espectrina/fisiologia , Relação Estrutura-Atividade
12.
Ecotoxicol Environ Saf ; 202: 110885, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32650140

RESUMO

Silicon (Si) is a metalloid which is gaining worldwide attention of plant scientists due to its ameliorating impact on plants' growth and development. The beneficial response of Si is observed predominantly under numerous abiotic and biotic stress conditions. However, under favorable conditions, most of the plant can grow without it. Therefore, Si has yet not been fully accepted as essential element rather it is being considered as quasi-essential for plants' growth. Si is also known to enhance resilience in plants by reducing the plant's stress. Besides its second most abundance on the earth crust, most of the soils lack plant available form of Si i.e. silicic acid. In this regard, understanding the role of Si in plant metabolism, its uptake from roots and transport to aerial tissues along with its ionomics and proteomics under different circumstances is of great concern. Plants have evolved a well-optimized Si-transport system including various transporter proteins like Low silicon1 (Lsi1), Low silicon2 (Lsi2), Low silicon3 (Lsi3) and Low silicon6 (Lsi6) at specific sub-cellular locations along with the expression profiling that creates precisely coordinated network among these transporters, which also facilitate uptake and accumulation of Si. Though, an ample amount of information is available pertinent to the solute specificity, active sites, transcriptional and post-transcriptional regulation of these transporter genes. Similarly, the information regarding transporters involved in Si accumulation in different organelles is also available particularly in silica cells occurred in poales. But in this review, we have attempted to compile studies related to plants vis à vis Si, its role in abiotic and biotic stress, its uptake in various parts of plants via different types of Si-transporters, expression pattern, localization and the solute specificity. Besides these, this review will also provide the compiled knowledge about the genetic variation among crop plants vis à vis enhanced Si uptake and related benefits.


Assuntos
Plantas/metabolismo , Silício/metabolismo , Transporte Biológico , Proteínas de Transporte/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Raízes de Plantas/metabolismo , Solo
13.
Nat Commun ; 11(1): 3284, 2020 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-32601292

RESUMO

The inner nuclear membrane (INM) selectively accumulates proteins that are essential for nuclear functions; however, overaccumulation of INM proteins results in a range of rare genetic disorders. So far, little is known about how defective, mislocalized, or abnormally accumulated membrane proteins are actively removed from the INM, especially in plants and animals. Here, via analysis of a proximity-labeling proteomic profile of INM-associated proteins in Arabidopsis, we identify critical components for an INM protein degradation pathway. We show that this pathway relies on the CDC48 complex for INM protein extraction and 26S proteasome for subsequent protein degradation. Moreover, we show that CDC48 at the INM may be regulated by a subgroup of PUX proteins, which determine the substrate specificity or affect the ATPase activity of CDC48. These PUX proteins specifically associate with the nucleoskeleton underneath the INM and physically interact with CDC48 proteins to negatively regulate INM protein degradation in plants.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Membrana/metabolismo , Membrana Nuclear/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Membrana/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Membrana Nuclear/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Plantas Geneticamente Modificadas , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteólise , Proteoma/genética , Coloração e Rotulagem/métodos , Espectrometria de Massas em Tandem/métodos , Proteína com Valosina/genética , Proteína com Valosina/metabolismo
14.
Nat Commun ; 11(1): 3422, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32647223

RESUMO

The Integrator complex processes 3'-ends of spliceosomal small nuclear RNAs (snRNAs). Furthermore, it regulates transcription of protein coding genes by terminating transcription after unstable pausing. The molecular basis for Integrator's functions remains obscure. Here, we show that INTS10, Asunder/INTS13 and INTS14 form a separable, functional Integrator module. The structure of INTS13-INTS14 reveals a strongly entwined complex with a unique chain interlink. Unexpected structural homology to the Ku70-Ku80 DNA repair complex suggests nucleic acid affinity. Indeed, the module displays affinity for DNA and RNA but prefers RNA hairpins. While the module plays an accessory role in snRNA maturation, it has a stronger influence on transcription termination after pausing. Asunder/INTS13 directly binds Integrator's cleavage module via a conserved C-terminal motif that is involved in snRNA processing and required for spermatogenesis. Collectively, our data establish INTS10-INTS13-INTS14 as a nucleic acid-binding module and suggest that it brings cleavage module and target transcripts into proximity.


Assuntos
Proteínas de Transporte/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ácidos Nucleicos/metabolismo , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Autoantígeno Ku/química , Mutação/genética , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , RNA/química , RNA/metabolismo , Processamento Pós-Transcricional do RNA , Homologia Estrutural de Proteína
15.
Nat Commun ; 11(1): 3351, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620897

RESUMO

The sodium-leak channel NALCN forms a subthreshold sodium conductance that controls the resting membrane potentials of neurons. The auxiliary subunits of the channel and their functions in mammals are largely unknown. In this study, we demonstrate that two large proteins UNC80 and UNC79 are subunits of the NALCN complex. UNC80 knockout mice are neonatal lethal. The C-terminus of UNC80 contains a domain that interacts with UNC79 and overcomes a soma-retention signal to achieve dendritic localization. UNC80 lacking this domain, as found in human patients, still supports whole-cell NALCN currents but lacks dendritic localization. Our results establish the subunit composition of the NALCN complex, uncover the inter-subunit interaction domains, reveal the functional significance of regulation of dendritic membrane potential by the sodium-leak channel complex, and provide evidence supporting that genetic variations found in individuals with intellectual disability are the causes for the phenotype observed in patients.


Assuntos
Proteínas de Transporte/genética , Deficiência Intelectual/genética , Canais Iônicos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Animais , Proteínas de Transporte/metabolismo , Criança , Análise Mutacional de DNA , Conjuntos de Dados como Assunto , Dendritos/patologia , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Células HEK293 , Hipocampo/citologia , Hipocampo/patologia , Humanos , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/patologia , Canais Iônicos/genética , Masculino , Camundongos , Camundongos Knockout , Mutação , Proteínas do Tecido Nervoso/metabolismo , Cultura Primária de Células , Domínios Proteicos/genética , Índice de Gravidade de Doença , Sequenciamento Completo do Exoma
16.
Nat Commun ; 11(1): 3711, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709891

RESUMO

The skeletal muscle T-tubule is a specialized membrane domain essential for coordinated muscle contraction. However, in the absence of genetically tractable systems the mechanisms involved in T-tubule formation are unknown. Here, we use the optically transparent and genetically tractable zebrafish system to probe T-tubule development in vivo. By combining live imaging of transgenic markers with three-dimensional electron microscopy, we derive a four-dimensional quantitative model for T-tubule formation. To elucidate the mechanisms involved in T-tubule formation in vivo, we develop a quantitative screen for proteins that associate with and modulate early T-tubule formation, including an overexpression screen of the entire zebrafish Rab protein family. We propose an endocytic capture model involving firstly, formation of dynamic endocytic tubules at transient nucleation sites on the sarcolemma, secondly, stabilization by myofibrils/sarcoplasmic reticulum and finally, delivery of membrane from the recycling endosome and Golgi complex.


Assuntos
Contração Muscular/fisiologia , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestrutura , Sarcolema/fisiologia , Sarcolema/ultraestrutura , Animais , Canais de Cálcio/metabolismo , Canais de Cálcio/ultraestrutura , Canais de Cálcio Tipo L/metabolismo , Proteínas de Transporte/metabolismo , Biologia do Desenvolvimento , Complexo de Golgi/metabolismo , Masculino , Microscopia Eletrônica , Proteínas Musculares/química , Músculo Esquelético/química , Miofibrilas/metabolismo , Sarcolema/química , Retículo Sarcoplasmático/metabolismo , Peixe-Zebra
17.
PLoS One ; 15(7): e0232137, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32614896

RESUMO

In sarcomeres, α-actinin crosslinks thin filaments and anchors them at the Z-disc. Drosophila melanogaster Zasp52 also localizes at Z-discs and interacts with α-actinin via its extended PDZ domain, thereby contributing to myofibril assembly and maintenance, yet the detailed mechanism of Zasp52 function is unknown. Here we show a strong genetic interaction between actin and Zasp52 during indirect flight muscle assembly, indicating that this interaction plays a critical role during myofibril assembly. Our results suggest that Zasp52 contains an actin-binding site, which includes the extended PDZ domain and the ZM region. Zasp52 binds with micromolar affinity to monomeric actin. A co-sedimentation assay indicates that Zasp52 can also bind to F-actin. Finally, we use in vivo rescue assays of myofibril assembly to show that the α-actinin-binding domain of Zasp52 is not sufficient for a full rescue of Zasp52 mutants suggesting additional contributions of Zasp52 actin-binding to myofibril assembly.


Assuntos
Actinas/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Drosophila/metabolismo , Miofibrilas/metabolismo , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Domínios PDZ , Ligação Proteica
18.
Proc Natl Acad Sci U S A ; 117(29): 17429-17437, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32636270

RESUMO

Biogenesis of plant microRNAs (miRNAs) takes place in nuclear dicing bodies (D-bodies), where the ribonulease III-type enzyme Dicer-like 1 (DCL1) processes primary transcripts of miRNAs (pri-miRNAs) into miRNA/miRNA* (*, passenger strand) duplexes from either base-to-loop or loop-to-base directions. Hyponastic Leaves 1 (HYL1), a double-stranded RNA-binding protein, is crucial for efficient and accurate processing. However, whether HYL1 has additional function remains unknown. Here, we report that HYL1 plays a noncanonical role in protecting pri-miRNAs from nuclear exosome attack in addition to ensuring processing. Loss of functions in SOP1 or HEN2, two cofactors of the nucleoplasmic exosome, significantly suppressed the morphological phenotypes of hyl1-2 Remarkably, mature miRNAs generated from loop-to-base processing were partially but preferentially restored in the hyl1 sop1 and hyl1 hen2 double mutants. Accordingly, loop-to-base-processed pri-miRNAs accumulated to higher levels in double mutants. In addition, dysfunction of HEN2, but not of SOP1, in hyl1-2 resulted in overaccumulation of many base-to-loop-processed pri-miRNAs, with most of their respective miRNAs unaffected. In summary, our findings reveal an antagonistic action of exosome in pri-miRNA biogenesis and uncover dual roles of HYL1 in stabilizing and processing of pri-miRNAs.


Assuntos
Núcleo Celular/metabolismo , Exossomos/metabolismo , MicroRNAs/metabolismo , Proteínas de Ligação a RNA/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Regulação da Expressão Gênica de Plantas , MicroRNAs/genética , Plantas Geneticamente Modificadas , Proteínas de Ligação a RNA/genética , Ribonuclease III
19.
Arch Microbiol ; 202(8): 2071-2081, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32488560

RESUMO

Streptococcus intermedius is a causative agent of brain or liver abscesses. S. intermedius produces intermedilysin that plays a pivotal role in pathogenicity. We identified other pathogenic factors and described a fibronectin binding protein (FBP) homolog of S. intermedius (FbpI) that mediated bacterial adhesion to epithelial cells and virulence for mice. The amino acid sequence of FbpI is similar to that of atypical FBPs, which do not possess a conventional secretion signal and an anchoring motif. A full-length recombinant FbpI (rFbpI) bound to immobilized fibronectin in a dose-dependent manner. The fibronectin binding activity of an N-terminal construct of rFbpI comprising the translation initiation methionine of the open reading frame to lysine 265 (rFbpI-N) bound immobilized fibronectin to a much lesser extent compared with rFbpI. A construct comprising the C-terminal domain (alanine 266 to methionine 549; rFbpI-C) bound immobilized fibronectin equivalently to rFbpI. Adherence of the isogenic mutant ΔfbpI to cultured epithelial cells and immobilized fibronectin was significantly lower than that of the wild-type strain. Abscess formation of ΔfbpI reduced in a mouse infection model compared with that in the wild-type. Thus, FbpI may play a role in bacterial adhesion to host cells and represent a critical pathogenic factor of S. intermedius.


Assuntos
Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/patologia , Streptococcus intermedius/genética , Streptococcus intermedius/patogenicidade , Virulência/genética , Animais , Aderência Bacteriana , Bacteriocinas , Proteínas de Transporte/metabolismo , Modelos Animais de Doenças , Fibronectinas/metabolismo , Humanos , Camundongos , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptococcus intermedius/metabolismo
20.
Am J Physiol Gastrointest Liver Physiol ; 319(2): G175-G188, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32538140

RESUMO

Gastrokines (GKNs) are anti-inflammatory proteins secreted by gastric epithelial (surface mucous and pit) cells, with their aberrant loss of expression causally linked to premalignant inflammation and gastric cancer (GC). Transcriptional mechanisms accounting for GKN expression loss have not been elucidated. Using human clinical cohorts, mouse transgenics, bioinformatics, and transfection/reporter assays, we report a novel mechanism of GKN gene transcriptional regulation and its impairment in GC. GKN1/GKN2 loss is highly coordinated, with both genes showing parallel downregulation during human and mouse GC development, suggesting joint transcriptional control. In BAC transgenic studies, we defined a 152-kb genomic region surrounding the human GKN1/GKN2 genes sufficient to direct their tissue- and lineage-restricted expression. A screen of the 152-kb region for candidate regulatory elements identified a DNase I hypersensitive site (CR2) located 4 kb upstream of the GKN1 gene. CR2 showed overlapping enrichment of enhancer-related histone marks (H3K27Ac), a consensus binding site (GRE) for the glucocorticoid receptor (GR), strong GR occupancy in ChIP-seq data sets and, critically, exhibited dexamethasone-sensitive enhancer activity in reporter assays. Strikingly, GR showed progressive expression loss, paralleling that of GKN1/2, in human and mouse GC, suggesting desensitized glucocorticoid signaling as a mechanism underlying GKN loss. Finally, mouse adrenalectomy studies revealed a critical role for endogenous glucocorticoids in sustaining correct expression (and anti-inflammatory restraint) of GKNs in vivo. Together, these data link the coordinate expression of GKNs to a glucocorticoid-responsive and likely shared transcriptional enhancer mechanism, with its compromised activation contributing to dual GKN loss during GC progression.NEW & NOTEWORTHY Gastrokine 2 (GKN2) is an anti-inflammatory protein produced by the gastric epithelium. GKN2 expression is progressively lost during gastric cancer (GC), which is believed to play a casual role in GC development. Here, we use bacterial artificial chromosome transgenic studies to identify a glucocorticoid-responsive enhancer element that likely governs expression of GKN1/GKN2, which, via parallel expression loss of the anti-inflammatory glucocorticoid receptor, reveals a novel mechanism to explain the loss of GKN2 during GC pathogenesis.


Assuntos
Proteínas de Transporte/metabolismo , Glucocorticoides/farmacologia , Hormônios Peptídicos/metabolismo , Neoplasias Gástricas/metabolismo , Células A549 , Animais , Proteínas de Transporte/genética , Cromossomos Artificiais Bacterianos , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Família Multigênica , Hormônios Peptídicos/genética
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