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1.
Nat Rev Neurosci ; 21(4): 183-196, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32152523

RESUMO

Wallerian degeneration is a widespread mechanism of programmed axon degeneration. In the three decades since the discovery of the Wallerian degeneration slow (WldS) mouse, research has generated extensive knowledge of the molecular mechanisms underlying Wallerian degeneration, demonstrated its involvement in non-injury disorders and found multiple ways to block it. Recent developments have included: the detection of NMNAT2 mutations that implicate Wallerian degeneration in rare human diseases; the capacity for lifelong rescue of a lethal condition related to Wallerian degeneration in mice; the discovery of 'druggable' enzymes, including SARM1 and MYCBP2 (also known as PHR1), in Wallerian pathways; and the elucidation of protein structures to drive further understanding of the underlying mechanisms and drug development. Additionally, new data have indicated the potential of these advances to alleviate a number of common disorders, including chemotherapy-induced and diabetic peripheral neuropathies, traumatic brain injury, and amyotrophic lateral sclerosis.


Assuntos
Degeneração Walleriana/metabolismo , Animais , Proteínas do Domínio Armadillo/metabolismo , Proteínas do Citoesqueleto/metabolismo , Modelos Animais de Doenças , Drosophila melanogaster , Humanos , Camundongos , Camundongos Transgênicos , NAD/metabolismo , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Transdução de Sinais , Pesquisa Médica Translacional
2.
PLoS One ; 14(12): e0226061, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31856229

RESUMO

Collective migration plays critical roles in animal development, physiological events, and cancer metastasis. However, the molecular mechanisms of collective cell migration are not well understood. Drosophila border cells represent an excellent in vivo genetic model to study collective cell migration and identify novel regulatory genes for cell migration. Using the Mosaic Analysis with a Repressible Cell Marker (MARCM) system, we screened 240 P-element insertion lines to identify essential genes for border cell migration. Two genes were uncovered, including dlg5 (discs large 5) and CG31689. Further analysis showed that Dlg5 regulates the apical-basal polarity and cluster integrity in border cell clusters. Dlg5 is enriched in lateral surfaces between border cells and central polar cells but also shows punctate localization between border cells. We found that the distribution of Dlg5 in border cell clusters is regulated by Armadillo. Structure-function analysis revealed that the N-terminal Coiled-coil domain and the C-terminal PDZ3-PDZ4-SH3-GUK domains but not the PDZ1-PDZ2 domains of Dlg5 are required for BC migration. The Coiled-coil domain and the PDZ4-SH3-GUK domains are critical for Dlg5's cell surface localization in border cell clusters.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Guanilato Quinases/metabolismo , Oogênese , Animais , Proteínas do Domínio Armadillo/metabolismo , Membrana Celular/metabolismo , Movimento Celular , Polaridade Celular , Drosophila/crescimento & desenvolvimento , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/química , Genes Reporter , Guanilato Quinases/antagonistas & inibidores , Guanilato Quinases/química , Óvulo/crescimento & desenvolvimento , Óvulo/metabolismo , Domínios Proteicos , Proteína Quinase C/metabolismo , Interferência de RNA , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo
3.
Exp Neurol ; 321: 113040, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31445042

RESUMO

Traumatic brain injury (TBI) often damages axons in white matter tracts and causes corpus callosum (CC) atrophy in chronic TBI patients. Injured axons encounter irreversible damage if transected, or alternatively may maintain continuity and subsequently either recover or degenerate. Secondary mechanisms can cause further axon damage, myelin pathology, and neuroinflammation. Molecular mechanisms regulating the progression of white matter pathology indicate potential therapeutic targets. SARM1 is essential for execution of the conserved axon death pathway. We examined white matter pathology following mild TBI with CC traumatic axonal injury in mice with Sarm1 gene deletion (Sarm1-/-). High resolution ultrastructural analysis at 3 days post-TBI revealed dramatically reduced axon damage in Sarm1-/- mice, as compared to Sarm1+/+ wild-type controls. Sarm1 deletion produced larger axons with thinner myelin, and attenuated TBI induced demyelination, i.e. myelin loss along apparently intact axons. At 6 weeks post-TBI, Sarm1-/- mice had less demyelination and thinner myelin than Sarm1+/+ mice, but axonal protection was no longer observed. We next used Thy1-YFP crosses to assess Sarm1 involvement in white matter neurodegeneration and neuroinflammation at 8 weeks post-TBI, when significant CC atrophy indicates chronic pathology. Thy1-YFP expression demonstrated continued CC axon damage yet absence of overt cortical pathology. Importantly, significant CC atrophy in Thy1-YFP/Sarm1+/+ mice was associated with reduced neurofilament immunolabeling of axons. Both effects were attenuated in Thy1-YFP/Sarm1-/- mice. Surprisingly, Thy1-YFP/Sarm1-/- mice had increased CC astrogliosis. This study demonstrates that Sarm1 inactivation reduces demyelination, and white matter atrophy after TBI, while the post-injury stage impacts when axon protection is effective.


Assuntos
Proteínas do Domínio Armadillo/deficiência , Lesões Encefálicas Traumáticas/patologia , Proteínas do Citoesqueleto/deficiência , Doenças Desmielinizantes/patologia , Substância Branca/patologia , Animais , Atrofia/metabolismo , Atrofia/patologia , Axônios/metabolismo , Axônios/patologia , Lesões Encefálicas Traumáticas/metabolismo , Doenças Desmielinizantes/metabolismo , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Substância Branca/metabolismo
4.
Science ; 365(6455): 793-799, 2019 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-31439792

RESUMO

SARM1 (sterile alpha and TIR motif containing 1) is responsible for depletion of nicotinamide adenine dinucleotide in its oxidized form (NAD+) during Wallerian degeneration associated with neuropathies. Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors recognize pathogen effector proteins and trigger localized cell death to restrict pathogen infection. Both processes depend on closely related Toll/interleukin-1 receptor (TIR) domains in these proteins, which, as we show, feature self-association-dependent NAD+ cleavage activity associated with cell death signaling. We further show that SARM1 SAM (sterile alpha motif) domains form an octamer essential for axon degeneration that contributes to TIR domain enzymatic activity. The crystal structures of ribose and NADP+ (the oxidized form of nicotinamide adenine dinucleotide phosphate) complexes of SARM1 and plant NLR RUN1 TIR domains, respectively, reveal a conserved substrate binding site. NAD+ cleavage by TIR domains is therefore a conserved feature of animal and plant cell death signaling pathways.


Assuntos
Proteínas do Domínio Armadillo/química , Proteínas do Citoesqueleto/química , NAD+ Nucleosidase/química , NAD/metabolismo , Proteínas de Plantas/química , Domínios Proteicos , Receptores Imunológicos/química , Animais , Proteínas do Domínio Armadillo/metabolismo , Axônios/enzimologia , Axônios/patologia , Sítios de Ligação , Morte Celular , Sequência Conservada , Cristalografia por Raios X , Proteínas do Citoesqueleto/metabolismo , Células HEK293 , Humanos , Camundongos , NAD+ Nucleosidase/metabolismo , NADP/metabolismo , Neurônios/enzimologia , Proteínas de Plantas/metabolismo , Multimerização Proteica , Receptores Imunológicos/metabolismo , Degeneração Walleriana/enzimologia , Degeneração Walleriana/patologia
5.
Science ; 365(6455): 799-803, 2019 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-31439793

RESUMO

Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors activate cell death and confer disease resistance by unknown mechanisms. We demonstrate that plant Toll/interleukin-1 receptor (TIR) domains of NLRs are enzymes capable of degrading nicotinamide adenine dinucleotide in its oxidized form (NAD+). Both cell death induction and NAD+ cleavage activity of plant TIR domains require known self-association interfaces and a putative catalytic glutamic acid that is conserved in both bacterial TIR NAD+-cleaving enzymes (NADases) and the mammalian SARM1 (sterile alpha and TIR motif containing 1) NADase. We identify a variant of cyclic adenosine diphosphate ribose as a biomarker of TIR enzymatic activity. TIR enzymatic activity is induced by pathogen recognition and functions upstream of the genes enhanced disease susceptibility 1 (EDS1) and N requirement gene 1 (NRG1), which encode regulators required for TIR immune function. Thus, plant TIR-NLR receptors require NADase function to transduce recognition of pathogens into a cell death response.


Assuntos
Arabidopsis/enzimologia , Arabidopsis/imunologia , Domínio Catalítico , NAD+ Nucleosidase/química , NAD/metabolismo , Receptores Imunológicos/química , Substituição de Aminoácidos , Arabidopsis/microbiologia , Proteínas de Arabidopsis/metabolismo , Proteínas do Domínio Armadillo/química , Biomarcadores/análise , Biomarcadores/metabolismo , Morte Celular , Sequência Conservada , ADP-Ribose Cíclica/análise , ADP-Ribose Cíclica/metabolismo , Proteínas do Citoesqueleto/química , Proteínas de Ligação a DNA/metabolismo , Ácido Glutâmico/química , Ácido Glutâmico/genética , Interações Hospedeiro-Patógeno
6.
Nat Commun ; 10(1): 3084, 2019 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-31300652

RESUMO

Resistance to inhibitors of cholinesterase 8A (Ric8A) is an essential regulator of G protein α-subunits (Gα), acting as a guanine nucleotide exchange factor and a chaperone. We report two crystal structures of Ric8A, one in the apo form and the other in complex with a tagged C-terminal fragment of Gα. These structures reveal two principal domains of Ric8A: an armadillo-fold core and a flexible C-terminal tail. Additionally, they show that the Gα C-terminus binds to a highly-conserved patch on the concave surface of the Ric8A armadillo-domain, with selectivity determinants residing in the Gα sequence. Biochemical analysis shows that the Ric8A C-terminal tail is critical for its stability and function. A model of the Ric8A/Gα complex derived from crosslinking mass spectrometry and molecular dynamics simulations suggests that the Ric8A C-terminal tail helps organize the GTP-binding site of Gα. This study lays the groundwork for understanding Ric8A function at the molecular level.


Assuntos
Proteínas do Domínio Armadillo/ultraestrutura , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/ultraestrutura , Chaperonas Moleculares/ultraestrutura , Animais , Proteínas do Domínio Armadillo/genética , Proteínas do Domínio Armadillo/metabolismo , Bovinos , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Espalhamento a Baixo Ângulo , Difração de Raios X
7.
Immunity ; 50(6): 1412-1424.e6, 2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31076360

RESUMO

Assembly of inflammasomes after infection or injury leads to the release of interleukin-1ß (IL-1ß) and to pyroptosis. After inflammasome activation, cells either pyroptose or enter a hyperactivated state defined by IL-1ß secretion without cell death, but what controls these different outcomes is unknown. Here, we show that removal of the Toll-IL-1R protein SARM from macrophages uncouples inflammasome-dependent cytokine release and pyroptosis, whereby cells displayed increased IL-1ß production but reduced pyroptosis. Correspondingly, increasing SARM in cells caused less IL-1ß release and more pyroptosis. SARM suppressed IL-1ß by directly restraining the NLRP3 inflammasome and, hence, caspase-1 activation. Consistent with a role for SARM in pyroptosis, Sarm1-/- mice were protected from lipopolysaccharide (LPS)-stimulated sepsis. Pyroptosis-inducing, but not hyperactivating, NLRP3 stimulants caused SARM-dependent mitochondrial depolarization. Thus, SARM-dependent mitochondrial depolarization distinguishes NLRP3 activators that cause pyroptosis from those that do not, and SARM modulation represents a cell-intrinsic mechanism to regulate cell fate after inflammasome activation.


Assuntos
Proteínas do Domínio Armadillo/metabolismo , Citocinas/metabolismo , Proteínas do Citoesqueleto/metabolismo , Inflamassomos/metabolismo , Animais , Proteínas do Domínio Armadillo/genética , Biomarcadores , Sobrevivência Celular , Proteínas do Citoesqueleto/genética , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Ligação Proteica , Piroptose , Transdução de Sinais
8.
Mol Biol Cell ; 30(12): 1555-1574, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30969890

RESUMO

Eukaryotic cell survival requires maintenance of plasma membrane (PM) homeostasis in response to environmental insults and changes in lipid metabolism. In yeast, a key regulator of PM homeostasis is target of rapamycin (TOR) complex 2 (TORC2), a multiprotein complex containing the evolutionarily conserved TOR protein kinase isoform Tor2. PM localization is essential for TORC2 function. One core TORC2 subunit (Avo1) and two TORC2--associated regulators (Slm1 and Slm2) contain pleckstrin homology (PH) domains that exhibit specificity for binding phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P2). To investigate the roles of PtdIns4,5P2 and constituent subunits of TORC2, we used auxin-inducible degradation to systematically eliminate these factors and then examined localization, association, and function of the remaining TORC2 components. We found that PtdIns4,5P2 depletion significantly reduced TORC2 activity, yet did not prevent PM localization or disassembly of TORC2. Moreover, truncated Avo1 (lacking its C-terminal PH domain) was still recruited to the PM and supported growth. Even when all three PH-containing proteins were absent, the remaining TORC2 subunits were PM-bound. Revealingly, Avo3 localized to the PM independent of both Avo1 and Tor2, whereas both Tor2 and Avo1 required Avo3 for their PM anchoring. Our findings provide new mechanistic information about TORC2 and pinpoint Avo3 as pivotal for TORC2 PM localization and assembly in vivo.


Assuntos
Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas do Domínio Armadillo/química , Proteínas do Domínio Armadillo/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Domínios Proteicos , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo
9.
J Steroid Biochem Mol Biol ; 191: 105316, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31014964

RESUMO

Primary bilateral macronodular adrenocortical hyperplasia (PBMAH) is a rare cause of ACTH-independent Cushing syndrome (CS), which has been associated with ectopic G-protein coupled receptors (GPCRs) in the adrenal cortex. We recently studied a 51-year-old male with PBMAH who presented with severe CS and hyperestronemia, manifesting clinically with a Cushingoid appearance, gynecomastia, and telangiectasias. Analysis of adrenal tissues following bilateral adrenalectomy showed high expression of P450 aromatase (CYP19A1). The patient carried a germline non-sense pathogenic variant in ARMC5 (p.R173*), with two independent somatic pathogenic variants identified in the right (p.S571*) and left (p.Q235*) adrenal tissues, respectively. The expression of ARMC5 was drastically decreased in the hyperplastic regions when compared to either the adjacent non-hyperplastic regions and samples from PBMAH without pathogenic variants in ARMC5. We found expression of CYP19A1 in other cases of PBMAH, although there were no differences in aromatase expression between ARMC5-mutant and ARMC5-non-mutant cases. We conclude that in select cases, PBMAH can be associated with aromatase expression resulting in elevated estrogens, irrespective of sex. Additionally, CYP19A1 expression does not appear to depend on the ARMC5 variant status.


Assuntos
Aromatase/genética , Síndrome de Cushing/genética , Proteínas Supressoras de Tumor/genética , Glândulas Suprarrenais/metabolismo , Glândulas Suprarrenais/patologia , Proteínas do Domínio Armadillo , Aromatase/análise , Síndrome de Cushing/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Mutação Puntual , Proteínas Supressoras de Tumor/análise , Regulação para Cima
10.
Int J Mol Sci ; 20(5)2019 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-30832344

RESUMO

Hypertension is the leading cause of cardiovascular disease in the United States, affecting up to one-third of adults. When compared to other ethnic or racial groups in the United States, African Americans and other people of African descent show a higher incidence of hypertension and its related comorbidities; however, the genetics of hypertension in these populations has not been studied adequately. Several genes have been identified to play a role in the genetics of hypertension. They include genes regulating the renin-aldosterone-angiotensin system (RAAS), such as Sodium Channel Epithelial 1 Beta Subunit (SCNN1B), Armadillo Repeat Containing 5 (ARMC5), G Protein-Coupled Receptor Kinase 4 (GRK4), and Calcium Voltage-Gated Channel Subunit Alpha1 D (CACNA1D). In this review, we focus on recent genetic findings available in the public domain for potential differences between African Americans and other populations. We also cover some recent and relevant discoveries in the field of low-renin hypertension from our laboratory at the National Institutes of Health. Understanding the different genetics of hypertension among various groups is essential for effective precision-guided medical therapy of high blood pressure.


Assuntos
Afro-Americanos/genética , Hipertensão/genética , Proteínas do Domínio Armadillo , Canais de Cálcio Tipo L/genética , Canais Epiteliais de Sódio/genética , Quinase 4 de Receptor Acoplado a Proteína G/genética , Humanos , Hipertensão/etnologia , Proteínas Supressoras de Tumor/genética
11.
Cell Physiol Biochem ; 52(3): 606-616, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30907988

RESUMO

BACKGROUND/AIMS: Aberrant expression of miR-106b is a specific symptom of many solid carcinomas. Overexpression of miR-106b has been observed in gastric cancer. The effect of miR-106b on gastric cancer has been investigated in different cell culture models. However, the effect of miR-106b on metastasis of early gastric cancer (EGC) remains unknown. METHODS: In the study, qRT-PCR, FISH, western blot, luciferase reporter assay, migration and invasion assays, flow cytometry and TUNEL staining were used to investigate the effect of miR-106b on metastasis of EGC. RESULTS: To explore the function of miR-106b in EGC, we investigated the downstream signaling of miR-106b and found that ALEX1 was a direct target of miR-106 in gastric cancer cells. Up-regulation of ALEX1 effectively rescued the cell apoptosis induced by miR-106b inhibitor and promoted the expression levels of phosphorylation of JAK1 and STAT3. Moreover, overexpression of JAK1 reduced the cell apoptosis induced by miR-106b inhibitor and decreased the expression levels of the apoptotic proteins in gastric cancer cells. Furthermore, down-regulation of miR-106b promoted apoptosis of gastric cancer cells via inhibiting JAK1/STAT3 signaling pathway in vitro and in vivo. In addition, GLPG0643, a JAK1 inhibitor, enhanced the inhibitory effect of miR-106b inhibitor on gastric cancer growth in vivo. CONCLUSION: These findings provided a potential therapeutic manner for the treatment of metastasis of EGC in clinic.


Assuntos
Proteínas do Domínio Armadillo/metabolismo , MicroRNAs/metabolismo , Proteínas Oncogênicas/metabolismo , Neoplasias Gástricas/patologia , Animais , Antagomirs/metabolismo , Apoptose , Proteínas do Domínio Armadillo/química , Proteínas do Domínio Armadillo/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo , Humanos , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 1/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Metástase Neoplásica , Proteínas Oncogênicas/química , Proteínas Oncogênicas/genética , Inibidores de Proteínas Quinases/farmacologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias Gástricas/metabolismo , Regulação para Cima
12.
Nat Commun ; 10(1): 104, 2019 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-30631047

RESUMO

AMP-activated protein kinase (AMPK) is a key regulator of cellular energy homeostasis. Although AMPK has been studied extensively in cellular processes, understanding of its substrates and downstream functional network, and their contributions to cell fate and disease development, remains incomplete. To elucidate the AMPK-dependent signaling pathways, we performed global quantitative phosphoproteomic analysis using wild-type and AMPKα1/α2-double knockout cells and discovered 160 AMPK-dependent phosphorylation sites. Further analysis using an AMPK consensus phosphorylation motif indicated that 32 of these sites are likely direct AMPK phosphorylation sites. We validated one uncharacterized protein, ARMC10, and demonstrated that the S45 site of ARMC10 can be phosphorylated by AMPK both in vitro and in vivo. Moreover, ARMC10 overexpression was sufficient to promote mitochondrial fission, whereas ARMC10 knockout prevented AMPK-mediated mitochondrial fission. These results demonstrate that ARMC10 is an effector of AMPK that participates in dynamic regulation of mitochondrial fission and fusion.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas do Domínio Armadillo/metabolismo , Fosfoproteínas/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Proteínas do Domínio Armadillo/genética , Linhagem Celular Tumoral , Células HEK293 , Humanos , Dinâmica Mitocondrial , Fosfoproteínas/genética , Fosforilação , Proteoma/genética , Homologia de Sequência de Aminoácidos , Serina/genética , Serina/metabolismo , Especificidade por Substrato
13.
Biochem Pharmacol ; 161: 52-62, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30633870

RESUMO

SARM is the fifth and most conserved member of the Toll/Il-1 Receptor (TIR) adaptor family. However, unlike the other TIR adaptors, MyD88, Mal, TRIF and TRAM, SARM does not participate in transducing signals downstream of TLRs. By contrast SARM inhibits TLR signalling by interacting with the adaptors TRIF and MyD88. In addition, SARM also has positive roles in innate immunity by activating specific transcriptional programs following immune challenge. SARM has a pivotal role in activating different forms of cell death following cellular stress and viral infection. Many of these functions of mammalian SARM are also reflected in SARM orthologues in lower organisms such as C. elegans and Drosophila. SARM expression is particularly enriched in neurons of the CNS and SARM has a critical role in neuronal death and in axon degeneration. Recent fascinating molecular insights have been revealed as to the molecular mechanism of SARM mediated axon degeneration. SARM has been shown to deplete NAD+ by possessing intrinsic NADase activity in the TIR domain of the protein. This activity can be activated experimentally by forced dimerization of the TIR domain. It is thought that this activity of SARM is normally switched off by the axo-protective activities of NMNAT2 which maintain low levels of the NAD+ precursor NMN. Therefore, there is now great excitement in the field of SARM research as targeting this enzymatic activity of SARM may lead to the development of new therapies for neurodegenerative diseases such as multiple sclerosis and motor neuron disease.


Assuntos
Proteínas do Domínio Armadillo/imunologia , Proteínas do Domínio Armadillo/metabolismo , Morte Celular/fisiologia , Proteínas do Citoesqueleto/imunologia , Proteínas do Citoesqueleto/metabolismo , Imunidade Inata/fisiologia , Animais , Humanos
14.
Cell Rep ; 26(2): 322-329.e3, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30625314

RESUMO

Calcium homeostasis in the lumen of the endoplasmic reticulum is required for correct processing and trafficking of transmembrane proteins, and defects in protein trafficking can impinge on cell signaling pathways. We show here that mutations in the endoplasmic reticulum calcium pump SERCA disrupt Wingless signaling by sequestering Armadillo/ß-catenin away from the signaling pool. Armadillo remains bound to E-cadherin, which is retained in the endoplasmic reticulum when calcium levels there are reduced. Using hypomorphic and null SERCA alleles in combination with the loss of the plasma membrane calcium channel Orai allowed us to define three distinct thresholds of endoplasmic reticulum calcium. Wingless signaling is sensitive to even a small reduction, while Notch and Hippo signaling are disrupted at intermediate levels, and elimination of SERCA function results in apoptosis. These differential and opposing effects on three oncogenic signaling pathways may complicate the use of SERCA inhibitors as cancer therapeutics.


Assuntos
Caderinas/metabolismo , Retículo Endoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Via de Sinalização Wnt , Animais , Apoptose , Proteínas do Domínio Armadillo/metabolismo , Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Sinalização do Cálcio , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Notch/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Fatores de Transcrição/metabolismo
15.
J Exp Med ; 216(2): 294-303, 2019 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-30642945

RESUMO

Axonal degeneration (AxD) following nerve injury, chemotherapy, and in several neurological disorders is an active process driven by SARM1, an injury-activated NADase. Axons of SARM1-null mice exhibit greatly delayed AxD after transection and in models of neurological disease, suggesting that inhibiting SARM1 is a promising strategy to reduce pathological AxD. Unfortunately, no drugs exist to target SARM1. We, therefore, developed SARM1 dominant-negatives that potently block AxD in cellular models of axotomy and neuropathy. To assess efficacy in vivo, we used adeno-associated virus-mediated expression of the most potent SARM1 dominant-negative and nerve transection as a model of severe AxD. While axons of vehicle-treated mice degenerate rapidly, axons of mice expressing SARM1 dominant-negative can remain intact for >10 d after transection, similar to the protection observed in SARM1-null mice. We thus developed a novel in vivo gene therapeutic to block pathological axon degeneration by inhibiting SARM1, an approach that may be applied clinically to treat manifold neurodegenerative diseases characterized by axon loss.


Assuntos
Proteínas do Domínio Armadillo , Axônios/metabolismo , Proteínas do Citoesqueleto , Dependovirus , Marcação de Genes , Terapia Genética , Degeneração Neural , Animais , Proteínas do Domínio Armadillo/antagonistas & inibidores , Proteínas do Domínio Armadillo/genética , Proteínas do Domínio Armadillo/metabolismo , Axônios/patologia , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Modelos Animais de Doenças , Células HEK293 , Humanos , Camundongos , Camundongos Transgênicos , Degeneração Neural/genética , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Degeneração Neural/terapia , Transdução Genética
16.
Oncogene ; 38(20): 3871-3885, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30683884

RESUMO

Metastasis begins with a subset of local tumor cells acquiring the potential to invade into surrounding tissues, and remains to be a major obstacle for cancer treatments. More than 90% of cancer patients died from tumor metastasis, instead of primary tumor growth. The canonical Wnt/ß-catenin pathway plays essential roles in promoting tumor formation, yet its function in regulating tumor metastasis and the underlying mechanisms remain controversial. Here we employed well-established Drosophila tumor models to investigate the regulating mechanism of Wingless (Wg) pathway in tumor invasion. Our results showed that Wg signaling is necessary and sufficient for cell polarity disruption-induced cell migration and molecular changes reminiscent of epithelial-mesenchymal transition (EMT). Moreover, reducing Wg signaling suppressed lgl-/-/RasV12-induced tumor invasion, and cooperation between Arm and RasV12 is sufficient to induce tumor invasion. Mechanistically, we found that cell polarity disruption activates JNK signaling, which in turn upregulate wg expression through transcription factor activator protein-1 (AP-1). We identified a consensus AP-1 binding site located in the 2nd intron of wg, and confirmed that it is essential for AP-1 induced wg transcription both in vitro and in vivo. Lastly, we confirmed that the transcriptional activation of WNT by AP-1 is conserved in human cancer cells. These evidences reveal a positive role of Wnt/ß-catenin pathway in tumor invasion, and provide a conserved mechanism that connects JNK and Wnt signaling in regulating tumor progression.


Assuntos
Proteínas de Drosophila/metabolismo , Neoplasias/patologia , Fator de Transcrição AP-1/metabolismo , Proteína Wnt1/metabolismo , Células A549 , Animais , Animais Geneticamente Modificados , Proteínas do Domínio Armadillo/genética , Proteínas do Domínio Armadillo/metabolismo , Sítios de Ligação , Movimento Celular/genética , Polaridade Celular , Drosophila/citologia , Drosophila/genética , Proteínas de Drosophila/genética , Células HeLa , Humanos , IMP Desidrogenase/genética , IMP Desidrogenase/metabolismo , Íntrons , Sistema de Sinalização das MAP Quinases , Células MCF-7 , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fator de Transcrição AP-1/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/fisiologia , Proteína Wnt1/genética
17.
Hum Mol Genet ; 28(10): 1620-1628, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30608580

RESUMO

Missense mutations in the gene, MAP3K1, are a common cause of 46,XY gonadal dysgenesis, accounting for 15-20% of cases [Ostrer, 2014, Disorders of sex development (DSDs): an update. J. Clin. Endocrinol. Metab., 99, 1503-1509]. Functional studies demonstrated that all of these mutations cause a protein gain-of-function that alters co-factor binding and increases phosphorylation of the downstream MAP kinase pathway targets, MAPK11, MAP3K and MAPK1. This dysregulation of the MAP kinase pathway results in increased CTNNB1, increased expression of WNT4 and FOXL2 and decreased expression of SRY and SOX9. Unique and recurrent pathogenic mutations cluster in three semi-contiguous domains outside the kinase region of the protein, a newly identified N-terminal domain that shares homology with the Guanine Exchange Factor (residues Met164 to Glu231), a Plant HomeoDomain (residues Met442 to Trp495) and an ARMadillo repeat domain (residues Met566 to Glu862). Despite the presence of the mutation clusters and clinical data, there exists a dearth of mechanistic insights behind the development imbalance. In this paper, we use structural modeling and functional data of these mutations to understand alterations of the MAP3K1 protein and the effects on protein folding, binding and downstream target phosphorylation. We show that these mutations have differential effects on protein binding depending on the domains in which they occur. These mutations increase the binding of the RHOA, MAP3K4 and FRAT1 proteins and generally decrease the binding of RAC1. Thus, pathologies in MAP3K1 disrupt the balance between the pro-kinase activities of the RHOA and MAP3K4 binding partners and the inhibitory activity of RAC1.


Assuntos
Transtornos do Desenvolvimento Sexual/genética , MAP Quinase Quinase Quinase 1/genética , MAP Quinase Quinase Quinase 4/genética , Proteínas rac1 de Ligação ao GTP/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas do Domínio Armadillo/genética , Transtorno 46,XY do Desenvolvimento Sexual , Transtornos do Desenvolvimento Sexual/patologia , Feminino , Proteína Forkhead Box L2/genética , Regulação da Expressão Gênica/genética , Disgenesia Gonadal 46 XY/genética , Disgenesia Gonadal 46 XY/patologia , Humanos , MAP Quinase Quinase Quinase 1/química , MAP Quinase Quinase Quinase 4/química , Sistema de Sinalização das MAP Quinases/genética , Masculino , Mutação de Sentido Incorreto/genética , Ligação Proteica/genética , Proteínas Proto-Oncogênicas/genética , Proteína da Região Y Determinante do Sexo/genética , Proteínas rac1 de Ligação ao GTP/química , Proteína rhoA de Ligação ao GTP/química , Proteína rhoA de Ligação ao GTP/genética
18.
Biol Chem ; 400(3): 395-404, 2019 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-30517075

RESUMO

Designed armadillo repeat proteins (dArmRPs) are modular peptide binders composed of N- and C-terminal capping repeats Y and A and a variable number of internal modules M that each specifically recognize two amino acids of the target peptide. Complementary fragments of dArmRPs obtained by splitting the protein between helices H1 and H2 of an internal module show conditional and specific assembly only in the presence of a target peptide (Michel, E., Plückthun, A., and Zerbe, O. (2018). Peptide-guided assembly of repeat protein fragments. Angew. Chem. Int. Ed. 57, 4576-4579). Here, we investigate dArmRP fragments that already spontaneously assemble with high affinity, e.g. those obtained from splits between entire modules or between helices H2 and H3. We find that the interaction of the peptide with the assembled fragments induces distal conformational rearrangements that suggest an induced fit on a global protein level. A population analysis of an equimolar mixture of an N-terminal and three C-terminal fragments with various affinities for the target peptide revealed predominant assembly of the weakest peptide binder. However, adding a target peptide to this mixture altered the population of the protein complexes such that the combination with the highest affinity for the peptide increased and becomes predominant when adding excess of peptide, highlighting the feasibility of peptide-induced enrichment of best binders from inter-modular fragment mixtures.


Assuntos
Proteínas do Domínio Armadillo/química , Peptídeos/química , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica
19.
Ocul Immunol Inflamm ; 27(5): 699-705, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30395750

RESUMO

Purpose: To investigate whether variants in the ARMC9 gene encoding KU-MEL-1 are associated with Vogt-Koyanagi-Harada (VKH) disease in a Japanese population. Methods: We recruited 380 Japanese patients with VKH disease and 744 Japanese healthy controls to genotype seven single-nucleotide polymorphisms (SNPs) in ARMC9. We also performed imputation analysis of the ARMC9 region and 195 imputed SNPs were included in the statistical analysis. Results: We observed an increased frequency of the A allele of rs28690417 in patients compared with controls (P = 0.0097, odds ratio (OR) = 1.46). The A allele had a dominant effect on VKH disease risk (P = 0.011, OR = 1.51). However, these significant differences disappeared after Bonferroni correction (corrected P > 0.05). The remaining 201 SNPs did not show any significant association with disease risk. Conclusions: Our study suggests that ARMC9 variants do not play a critical role in the development of VKH disease.


Assuntos
Proteínas do Domínio Armadillo/genética , Predisposição Genética para Doença , Síndrome Uveomeningoencefálica/genética , Adulto , Grupo com Ancestrais do Continente Asiático/genética , Estudos de Casos e Controles , Feminino , Frequência do Gene , Genótipo , Humanos , Japão , Masculino , Pessoa de Meia-Idade , Razão de Chances , Polimorfismo de Nucleotídeo Único
20.
Mol Biol Cell ; 30(3): 411-426, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30540525

RESUMO

Actomyosin contractility can influence the canonical Wnt signaling pathway in processes like mesoderm differentiation and tissue stiffness during tumorigenesis. We identified that increased nonmuscle myosin II activation and cellular contraction inhibited Wnt target gene transcription in developing Drosophila imaginal disks. Genetic interactions studies were used to show that this effect was due to myosin-induced accumulation of cortical F-actin resulting in clustering and accumulation of E-cadherin to the adherens junctions. This results in E-cadherin titrating any available ß-catenin, the Wnt pathway transcriptional coactivator, to the adherens junctions in order to maintain cell-cell adhesion under contraction. We show that decreased levels of cytoplasmic ß-catenin result in insufficient nuclear translocation for full Wnt target gene transcription. Previous studies have identified some of these interactions, but we present a thorough analysis using the wing disk epithelium to show the consequences of modulating myosin phosphatase. Our work elucidates a mechanism in which the dynamic promotion of actomyosin contractility refines patterning of Wnt transcription during development and maintenance of epithelial tissue in organisms.


Assuntos
Actomiosina/metabolismo , Junções Aderentes/metabolismo , Drosophila melanogaster/metabolismo , Via de Sinalização Wnt , Actinas/metabolismo , Animais , Proteínas do Domínio Armadillo/metabolismo , Caderinas/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Proteínas de Drosophila/metabolismo , Homeostase , Humanos , Modelos Biológicos , Miosina Tipo II/metabolismo , Estabilidade Proteica , Fatores de Transcrição/metabolismo
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