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1.
Nat Commun ; 10(1): 2641, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201325

RESUMO

Epsilon toxin (Etx), a potent pore forming toxin (PFT) produced by Clostridium perfringens, is responsible for the pathogenesis of enterotoxaemia of ruminants and has been suggested to play a role in multiple sclerosis in humans. Etx is a member of the aerolysin family of ß-PFTs (aß-PFTs). While the Etx soluble monomer structure was solved in 2004, Etx pore structure has remained elusive due to the difficulty of isolating the pore complex. Here we show the cryo-electron microscopy structure of Etx pore assembled on the membrane of susceptible cells. The pore structure explains important mutant phenotypes and suggests that the double ß-barrel, a common feature of the aß-PFTs, may be an important structural element in driving efficient pore formation. These insights provide the framework for the development of novel therapeutics to prevent human and animal infections, and are relevant for nano-biotechnology applications.


Assuntos
Toxinas Bacterianas/química , Clostridium perfringens/ultraestrutura , Animais , Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Toxinas Bacterianas/metabolismo , Biotecnologia/métodos , Linhagem Celular , Infecções por Clostridium/microbiologia , Infecções por Clostridium/prevenção & controle , Clostridium perfringens/genética , Clostridium perfringens/metabolismo , Clostridium perfringens/patogenicidade , Microscopia Crioeletrônica , Cães , Enterotoxemia/microbiologia , Enterotoxemia/prevenção & controle , Modelos Moleculares , Mutagênese Sítio-Dirigida , Nanotecnologia/métodos , Conformação Proteica em Folha beta/genética , Multimerização Proteica/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
2.
Nat Cell Biol ; 21(6): 768-777, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31061466

RESUMO

Controlling cellular processes with light can help elucidate their underlying mechanisms. Here we present zapalog, a small-molecule dimerizer that undergoes photolysis when exposed to blue light. Zapalog dimerizes any two proteins tagged with the FKBP and DHFR domains until exposure to light causes its photolysis. Dimerization can be repeatedly restored with uncleaved zapalog. We implement this method to investigate mitochondrial motility and positioning in cultured neurons. Using zapalog, we tether mitochondria to constitutively active kinesin motors, forcing them down the axon towards microtubule (+) ends until their instantaneous release via blue light, which results in full restoration of their endogenous motility. We find that one-third of stationary mitochondria cannot be pulled away from their position and that these firmly anchored mitochondria preferentially localize to VGLUT1-positive presynapses. Furthermore, inhibition of actin polymerization with latrunculin A reduces this firmly anchored pool. On release from exogenous motors, mitochondria are preferentially recaptured at presynapses.


Assuntos
Axônios/metabolismo , Mitocôndrias/genética , Fotólise , Multimerização Proteica/efeitos da radiação , Actinas/antagonistas & inibidores , Animais , Axônios/química , Axônios/efeitos da radiação , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Células COS , Cercopithecus aethiops , Cinesina/química , Luz , Microtúbulos/genética , Microtúbulos/efeitos da radiação , Mitocôndrias/química , Mitocôndrias/efeitos da radiação , Neurônios/química , Neurônios/efeitos da radiação , Polimerização/efeitos dos fármacos , Domínios Proteicos/genética , Domínios Proteicos/efeitos da radiação , Multimerização Proteica/genética , Sinapses/química , Sinapses/genética , Sinapses/efeitos da radiação , Proteínas de Ligação a Tacrolimo/química , Proteínas de Ligação a Tacrolimo/genética , Tiazolidinas/farmacologia , Proteína Vesicular 1 de Transporte de Glutamato/genética
3.
Phys Chem Chem Phys ; 21(22): 12036-12043, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31135803

RESUMO

The experimental finding that α-synuclein (αS) occurs physiologically as a helically folded tetramer begs the question: why are helical tetramers the most populated multimers? While the helical tetramer is known to resist aggregation, the assembly mechanism of αS peptides remains largely unknown. By rationally designing a series of helical multimers from dimer to octamer, we characterized the free energy landscape of wild-type and mutated multimers using molecular dynamics computer simulations. Competition between supramolecular packing and solvation results in well-hydrated dimers and trimers, and more screened pentamers to octamers, with the helical tetramer possessing the most balanced structure with the lowest activation energy. Our data suggest that familial mutants are very sensitive to alterations in monomer packing that would in turn raise the energy barriers for multimerization. Finally, the hypothesis that the αS tetramer forms a soluble, benign "dead end" to circumvent aggregation is supported by its computed very weak association with negatively charged cell membranes.


Assuntos
Agregados Proteicos , alfa-Sinucleína/química , Humanos , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Mutação , Fosfatidilserinas/química , Agregados Proteicos/genética , Conformação Proteica , Multimerização Proteica/genética , Termodinâmica , Água/química , alfa-Sinucleína/genética
4.
Nat Commun ; 10(1): 1068, 2019 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-30842409

RESUMO

The small heat-shock protein HSP27 is a redox-sensitive molecular chaperone that is expressed throughout the human body. Here, we describe redox-induced changes to the structure, dynamics, and function of HSP27 and its conserved α-crystallin domain (ACD). While HSP27 assembles into oligomers, we show that the monomers formed upon reduction are highly active chaperones in vitro, but are susceptible to self-aggregation. By using relaxation dispersion and high-pressure nuclear magnetic resonance (NMR) spectroscopy, we observe that the pair of ß-strands that mediate dimerisation partially unfold in the monomer. We note that numerous HSP27 mutations associated with inherited neuropathies cluster to this dynamic region. High levels of sequence conservation in ACDs from mammalian sHSPs suggest that the exposed, disordered interface present in free monomers or oligomeric subunits may be a general, functional feature of sHSPs.


Assuntos
Proteínas de Choque Térmico HSP27/metabolismo , Doenças do Sistema Nervoso Periférico/genética , Agregação Patológica de Proteínas/genética , Multimerização Proteica/genética , Desdobramento de Proteína , Proteínas de Choque Térmico HSP27/química , Proteínas de Choque Térmico HSP27/genética , Mutação , Ressonância Magnética Nuclear Biomolecular , Oxirredução , Conformação Proteica em Folha beta/genética , Estrutura Quaternária de Proteína/fisiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
5.
Proc Natl Acad Sci U S A ; 116(9): 3562-3571, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30808748

RESUMO

The N-terminal region of the huntingtin protein, encoded by exon-1, comprises an amphiphilic domain (httNT), a polyglutamine (Q n ) tract, and a proline-rich sequence. Polyglutamine expansion results in an aggregation-prone protein responsible for Huntington's disease. Here, we study the earliest events involved in oligomerization of a minimalistic construct, httNTQ7, which remains largely monomeric over a sufficiently long period of time to permit detailed quantitative NMR analysis of the kinetics and structure of sparsely populated [Formula: see text] oligomeric states, yet still eventually forms fibrils. Global fitting of concentration-dependent relaxation dispersion, transverse relaxation in the rotating frame, and exchange-induced chemical shift data reveals a bifurcated assembly mechanism in which the NMR observable monomeric species either self-associates to form a productive dimer (τex ∼ 30 µs, K diss ∼ 0.1 M) that goes on to form a tetramer ([Formula: see text] µs; K diss ∼ 22 µM), or exchanges with a "nonproductive" dimer that does not oligomerize further (τex ∼ 400 µs; K diss ∼ 0.3 M). The excited state backbone chemical shifts are indicative of a contiguous helix (residues 3-17) in the productive dimer/tetramer, with only partial helical character in the nonproductive dimer. A structural model of the productive dimer/tetramer was obtained by simulated annealing driven by intermolecular paramagnetic relaxation enhancement data. The tetramer comprises a D 2 symmetric dimer of dimers with largely hydrophobic packing between the helical subunits. The structural model, validated by EPR distance measurements, illuminates the role of the httNT domain in the earliest stages of prenucleation and oligomerization, before fibril formation.


Assuntos
Amiloide/genética , Proteína Huntingtina/genética , Doença de Huntington/genética , Amiloide/química , Amiloide/ultraestrutura , Cristalografia por Raios X , Citoesqueleto/química , Citoesqueleto/genética , Éxons/genética , Proteína Huntingtina/química , Proteína Huntingtina/ultraestrutura , Doença de Huntington/patologia , Cinética , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Peptídeos/química , Peptídeos/genética , Polímeros/química , Domínios Proteicos/genética , Multimerização Proteica/genética , Relação Estrutura-Atividade
6.
Nucleic Acids Res ; 47(7): 3550-3567, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30698745

RESUMO

Activation of the checkpoint protein Tel1 requires the Mre11-Rad50-Xrs2 (MRX) complex, which recruits Tel1 at DNA double-strand breaks (DSBs) through direct interaction between Tel1 and Xrs2. However, in vitro Tel1 activation by MRX requires ATP binding to Rad50, suggesting a role also for the MR subcomplex in Tel1 activation. Here we describe two separation-of-functions alleles, mre11-S499P and rad50-A78T, which we show to specifically affect Tel1 activation without impairing MRX functions in DSB repair. Both Mre11-S499P and Rad50-A78T reduce Tel1-MRX interaction leading to poor Tel1 association at DSBs and consequent loss of Tel1 activation. The Mre11-S499P variant reduces Mre11-Rad50 interaction, suggesting an important role for MR complex formation in Tel1 activation. Molecular dynamics simulations show that the wild type MR subcomplex bound to ATP lingers in a tightly 'closed' conformation, while ADP presence leads to the destabilization of Rad50 dimer and of Mre11-Rad50 association, both events being required for MR conformational transition to an open state. By contrast, MRA78T undertakes complex opening even if Rad50 is bound to ATP, indicating that defective Tel1 activation caused by MRA78T results from destabilization of the ATP-bound conformational state.


Assuntos
Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas de Saccharomyces cerevisiae/genética , Ativação Transcricional/genética , Trifosfato de Adenosina/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Dano ao DNA/genética , Reparo do DNA/genética , DNA Fúngico/genética , Conformação Molecular , Complexos Multiproteicos/genética , Ligação Proteica/genética , Multimerização Proteica/genética , Saccharomyces cerevisiae/genética , Transdução de Sinais/genética
7.
Yakugaku Zasshi ; 139(1): 53-60, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-30606929

RESUMO

Adiponectin, the most abundant adipose tissue-derived adipocytokine, improves insulin sensitivity and has anti-inflammatory properties. Disulfide-bond A oxidoreductase-like protein (DsbA-L) is a key molecule in the multimerization of adiponectin (i.e., activation of adiponectin). In mice, liver-specific knockout of the Dsba-L gene impaired the mitochondrial function in hepatocytes and exacerbated the high-fat-diet-induced fatty liver. In addition, the DsbA-L mRNA level is negatively correlated with body mass index (BMI) in humans. We recently investigated the clinical impact of the DsbA-L gene on lifestyle-related diseases in Japanese subjects. We confirmed the influence of the common DsbA-L rs1917760 polymorphism on the multimerization of adiponectin, as well as the association of the polymorphism with the risk of obesity and non-alcoholic fatty liver disease, using prediction models based on a non-linear mixed effect model and/or structural equation models among elderly participants in a health screening program. We also observed a decreasing effect of DsbA-L polymorphism on the DsbA-L mRNA level in peripheral blood mononuclear cells, and an increasing effect of the polymorphism on the prevalence of excessive weight among schizophrenia patients at a high risk for obesity. These findings suggest that DsbA-L may be a key molecule associated with the development and progression of obesity and its related diseases. Therefore, genotyping the DsbA-L polymorphism and identifying patients at a high risk of developing obesity may help prevent obesity and its complications by facilitating targeted prevention and treatment programs for high-risk individuals.


Assuntos
Adiponectina/metabolismo , Estudos de Associação Genética , Glutationa Transferase/genética , Obesidade/genética , Farmacogenética , Multimerização Proteica/genética , Adiponectina/fisiologia , Animais , Índice de Massa Corporal , Humanos , Camundongos , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Hepatopatia Gordurosa não Alcoólica/terapia , Obesidade/prevenção & controle , Obesidade/terapia , Polimorfismo Genético , RNA Mensageiro , Risco
8.
Mutat Res ; 814: 1-6, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30648609

RESUMO

HNF4α is a culprit gene product for a monogenic and dominantly-inherited form of diabetes, referred to as MODY1 (Maturity Onset Diabetes of the Young type 1). Reduced HNF4α activities have been linked to impaired insulin secretion and ß-cell function. Numerous mutations have been identified from the patients and they have been instructive as to the individual residue's role in protein structure-function and dysfunction. As a member of the nuclear receptor (NR) superfamily, HNF4α is made of characteristic modular domains and it functions exclusively as a homodimer despite its sequence homology to RXR, a common heterodimer partner of non-steroidal NRs. Transcription factors commonly dimerize to enhance their molecular functions mainly by facilitating the recognition of double helix target DNAs that display an intrinsic pseudo-2-fold symmetry and the recruitment of the remainder of the main transcriptional machinery. HNF4α is no exception and its dimerization is maintained by the ligand binding domain (LBD) mainly through the leucine-zipper-like interactions at the stalk of two interacting helices. Although many MODY1 mutations have been previously characterized, including DNA binding disruptors, ligand binding disruptors, coactivator binding disruptors, and protein stability disruptors, protein dimerization disruptors have not been formally reported. In this report, we present a set of data for the two MODY1 mutations found right at the dimerization interface (L332 P and L328del mutations) which clearly exhibit the disruptive effects of directly affecting dimerization, protein stability, and transcriptional activities. These data reinforced the fact that MODY mutations are loss-of-function mutations and HNF4α dimerization is essential for its optimal function and normal physiology.


Assuntos
Diabetes Mellitus Tipo 2/genética , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Mutação , Domínios e Motivos de Interação entre Proteínas/genética , Multimerização Proteica , Dimerização , Células HeLa , Fator 4 Nuclear de Hepatócito/química , Humanos , Mutação com Perda de Função/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica , Multimerização Proteica/genética , Estabilidade Proteica , Estrutura Quaternária de Proteína/genética , Ativação Transcricional/genética
9.
Nat Commun ; 10(1): 251, 2019 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-30651569

RESUMO

Gene regulatory mechanisms rely on a complex network of RNA processing factors to prevent untimely gene expression. In fission yeast, the highly conserved ortholog of human ERH, called Erh1, interacts with the YTH family RNA binding protein Mmi1 to form the Erh1-Mmi1 complex (EMC) implicated in gametogenic gene silencing. However, the structural basis of EMC assembly and its functions are poorly understood. Here, we present the co-crystal structure of the EMC that consists of Erh1 homodimers interacting with Mmi1 in a 2:2 stoichiometry via a conserved molecular interface. Structure-guided mutation of the Mmi1Trp112 residue, which is required for Erh1 binding, causes defects in facultative heterochromatin assembly and gene silencing while leaving Mmi1-mediated transcription termination intact. Indeed, EMC targets masked in mmi1∆ due to termination defects are revealed in mmi1W112A. Our study delineates EMC requirements in gene silencing and identifies an ERH interface required for interaction with an RNA binding protein.


Assuntos
Proteínas de Transporte/metabolismo , Inativação Gênica , Ligação Proteica/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/genética , Montagem e Desmontagem da Cromatina/genética , Regulação Fúngica da Expressão Gênica , Meiose/genética , Multimerização Proteica/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Terminação da Transcrição Genética , Fatores de Poliadenilação e Clivagem de mRNA/química , Fatores de Poliadenilação e Clivagem de mRNA/genética
10.
Biochemistry ; 58(6): 742-754, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30562452

RESUMO

More than 50 different intronic and exonic autosomal dominant mutations in the tau gene have been linked to the neurodegenerative disorder frontotemporal dementia with Parkinsonism linked to chromosome-17 (FTDP-17). Although the pathological and clinical presentation of this disorder is heterogeneous among patients, the deposition of tau as pathological aggregates is a common feature. Collectively, FTDP-17 mutations have been shown to alter tau's ability to stabilize microtubules, enhance its aggregation, alter mRNA splicing, or induce its hyperphosphorylation, among other effects. Previous in vitro studies from our lab revealed that these mutations differ markedly from each other in the longest 2N4R isoform of tau. However, it is not entirely known whether the effect of a single mutation varies when compared between different isoforms of tau. Differences in the isoelectric points of the N-terminal region of tau isoforms lead to changes in their biochemical properties, raising the possibility that isoforms could also be disproportionately affected by disease-related mechanisms such as mutations. We therefore performed a comparative study of three FTDP-17 mutations present in different regions of tau (R5L, P301L, and R406W) in the three 4R isoforms of tau. We observed significant differences in the effect these mutations exert on the total amount and kinetics of aggregation, aggregate length distributions, and microtubule stabilizing propensity of 4R tau isoforms for all three selected mutants. These results demonstrate that different combinations of FTDP-17 mutations and tau isoforms are functionally distinct and could have important implications for our understanding of disease and animal models of tauopathies.


Assuntos
Microtúbulos/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismo , Escherichia coli/genética , Humanos , Cinética , Mutação , Polimerização , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica/genética , Tubulina (Proteína)/metabolismo
11.
Int J Mol Sci ; 19(7)2018 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-29958424

RESUMO

Biosynthesis of heme represents a complex process that involves multiple stages controlled by different enzymes. The first of these proteins is a pyridoxal 5′-phosphate (PLP)-dependent homodimeric enzyme, 5-aminolevulinate synthase (ALAS), that catalyzes the rate-limiting step in heme biosynthesis, the condensation of glycine with succinyl-CoA. Genetic mutations in human erythroid-specific ALAS (ALAS2) are associated with two inherited blood disorders, X-linked sideroblastic anemia (XLSA) and X-linked protoporphyria (XLPP). XLSA is caused by diminished ALAS2 activity leading to decreased ALA and heme syntheses and ultimately ineffective erythropoiesis, whereas XLPP results from “gain-of-function” ALAS2 mutations and consequent overproduction of protoporphyrin IX and increase in Zn2+-protoporphyrin levels. All XLPP-linked mutations affect the intrinsically disordered C-terminal tail of ALAS2. Our earlier molecular dynamics (MD) simulation-based analysis showed that the activity of ALAS2 could be regulated by the conformational flexibility of the active site loop whose structural features and dynamics could be changed due to mutations. We also revealed that the dynamic behavior of the two protomers of the ALAS2 dimer differed. However, how the structural dynamics of ALAS2 active site loop and C-terminal tail dynamics are related to each other and contribute to the homodimer asymmetry remained unanswered questions. In this study, we used bioinformatics and computational biology tools to evaluate the role(s) of the C-terminal tail dynamics in the structure and conformational dynamics of the murine ALAS2 homodimer active site loop. To assess the structural correlation between these two regions, we analyzed their structural displacements and determined their degree of correlation. Here, we report that the dynamics of ALAS2 active site loop is anti-correlated with the dynamics of the C-terminal tail and that this anti-correlation can represent a molecular basis for the functional and dynamic asymmetry of the ALAS2 homodimer.


Assuntos
5-Aminolevulinato Sintetase/química , Anemia Sideroblástica/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Heme/química , 5-Aminolevulinato Sintetase/genética , Anemia Sideroblástica/patologia , Animais , Domínio Catalítico , Biologia Computacional , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Heme/biossíntese , Heme/genética , Humanos , Camundongos , Simulação de Dinâmica Molecular , Mutação/genética , Multimerização Proteica/genética
12.
J Virol ; 92(19)2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30021903

RESUMO

Inflammasomes play a key role in host innate immune responses to viral infection by caspase-1 (Casp-1) activation to facilitate interleukin-1ß (IL-1ß) secretion, which contributes to the host antiviral defense. The NLRP3 inflammasome consists of the cytoplasmic sensor molecule NLRP3, adaptor protein ASC, and effector protein pro-caspase-1 (pro-Casp-1). NLRP3 and ASC promote pro-Casp-1 cleavage, leading to IL-1ß maturation and secretion. However, as a countermeasure, viral pathogens have evolved virulence factors to antagonize inflammasome pathways. Here we report that V gene knockout Sendai virus [SeV V(-)] induced markedly greater amounts of IL-1ß than wild-type SeV in infected THP1 macrophages. Deficiency of NLRP3 in cells inhibited SeV V(-)-induced IL-1ß secretion, indicating an essential role for NLRP3 in SeV V(-)-induced IL-1ß activation. Moreover, SeV V protein inhibited the assembly of NLRP3 inflammasomes, including NLRP3-dependent ASC oligomerization, NLRP3-ASC association, NLRP3 self-oligomerization, and intermolecular interactions between NLRP3 molecules. Furthermore, a high correlation between the NLRP3-binding capacity of V protein and the ability to block inflammasome complex assembly was observed. Therefore, SeV V protein likely inhibits NLRP3 self-oligomerization by interacting with NLRP3 and inhibiting subsequent recruitment of ASC to block NLRP3-dependent ASC oligomerization, in turn blocking full activation of the NLRP3 inflammasome and thus blocking IL-1ß secretion. Notably, the inhibitory action of SeV V protein on NLRP3 inflammasome activation is shared by other paramyxovirus V proteins, such as Nipah virus and human parainfluenza virus type 2. We thus reveal a mechanism by which paramyxovirus inhibits inflammatory responses by inhibiting NLRP3 inflammasome complex assembly and IL-1ß activation.IMPORTANCE The present study demonstrates that the V protein of SeV, Nipah virus, and human parainfluenza virus type 2 interacts with NLRP3 to inhibit NLRP3 inflammasome activation, potentially suggesting a novel strategy by which viruses evade the host innate immune response. As all members of the Paramyxovirinae subfamily carry similar V genes, this new finding may also lead to identification of novel therapeutic targets for paramyxovirus infection and related diseases.


Assuntos
Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Infecções por Respirovirus/metabolismo , Vírus Sendai/metabolismo , Proteínas Virais/metabolismo , Caspase 1/genética , Caspase 1/metabolismo , Células HEK293 , Humanos , Inflamassomos/genética , Interleucina-1beta/genética , Macrófagos/patologia , Macrófagos/virologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Multimerização Proteica/genética , Infecções por Respirovirus/genética , Infecções por Respirovirus/patologia , Vírus Sendai/genética , Células THP-1 , Proteínas Virais/genética
13.
Oncol Rep ; 40(1): 488-494, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29750295

RESUMO

Mutations in the p53 gene are associated with more than half of all human cancers. These mutations often cause a disruption of the tumor-suppressor function of p53 and induce genomic instabilities. Wild­type p53 requires tetramerization to function as an initiator of cell cycle arrest and apoptosis. Although alterations in p53 tetramerization caused by mutation have been well studied, there are few cell lines containing an endogenous mutation in the tetramerization domain of p53. Here, we report the discovery of a canine mammary gland tumor cell line CTB­m2, which contains the Leu332Gln (L332Q) mutation corresponding to Leu344 in the tetramerization domain of human p53. Although CTB­m2 cells are genetically heterozygous for the Leu332Gln mutation, the mutant mRNA was almost exclusively expressed. CTB­m2 cells showed enhanced cell proliferation compared to wild­type p53-expressing CTB­m cells of the same lineage. A p53 tetramerization reporter assay showed that the ability of the p53 mutant to form tetramers was significantly lower than that of wild­type p53. An immunoblot analysis of cross-linked p53 oligomerized forms demonstrated that the L332Q mutant lacked the ability to form tetramers but retained the ability to form dimers. These data suggest that the p53 mutant cell line CTB­m2 could be a useful tool for analyzing the precise tetramerization mechanisms of p53 and verifying the effects of therapeutic agents against tumors expressing p53 mutants that lack the ability to tetramerize.


Assuntos
Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Animais/genética , Proteína Supressora de Tumor p53/genética , Substituição de Aminoácidos/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Cães , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias Mamárias Animais/patologia , Mutação , Multimerização Proteica/genética , Proteína Supressora de Tumor p53/química
15.
Biochim Biophys Acta Mol Basis Dis ; 1864(7): 2448-2457, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29660491

RESUMO

Interallelic interactions of membrane proteins are not taken into account while evaluating the pathogenicity of sequence variants in autosomal recessive disorders. Podocin, a membrane-anchored component of the slit diaphragm, is encoded by NPHS2, the major gene mutated in hereditary podocytopathies. We formerly showed that its R229Q variant is only pathogenic when trans-associated to specific 3' mutations and suggested the causal role of an abnormal C-terminal dimerization. Here we show by FRET analysis and size exclusion chromatography that podocin oligomerization occurs exclusively through the C-terminal tail (residues 283-382): principally through the first C-terminal helical region (H1, 283-313), which forms a coiled coil as shown by circular dichroism spectroscopy, and through the 332-348 region. We show the principal role of the oligomerization sites in mediating interallelic interactions: while the monomer-forming R286Tfs*17 podocin remains membranous irrespective of the coexpressed podocin variant identity, podocin variants with an intact H1 significantly influence each other's localization (r2 = 0.68, P = 9.2 × 10-32). The dominant negative effect resulting in intracellular retention of the pathogenic F344Lfs*4-R229Q heterooligomer occurs in parallel with a reduction in the FRET efficiency, suggesting the causal role of a conformational rearrangement. On the other hand, oligomerization can also promote the membrane localization: it can prevent the endocytosis of F344Lfs*4 or F344* podocin mutants induced by C-terminal truncation. In conclusion, C-terminal oligomerization of podocin can mediate both a dominant negative effect and interallelic complementation. Interallelic interactions of NPHS2 are not restricted to the R229Q variant and have to be considered in compound heterozygous individuals.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular , Nefropatias , Proteínas de Membrana , Mutação de Sentido Incorreto , Podócitos/metabolismo , Multimerização Proteica/genética , Substituição de Aminoácidos , Linhagem Celular Transformada , Transferência Ressonante de Energia de Fluorescência , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Nefropatias/genética , Nefropatias/metabolismo , Nefropatias/patologia , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Podócitos/patologia , Domínios Proteicos
16.
J Dermatol Sci ; 91(1): 35-42, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29631773

RESUMO

BACKGROUND: Piebaldism is a pigmentary disorder characterized by a white forelock and depigmented patches. Although the loss-of-function mutations in the KIT gene underlie the disease, the intracellular dynamics of the mutant KIT are largely unknown. We herein report a Japanese family with piebaldism in which the affected members showed a mild phenotype. OBJECTIVE: The objective of this study is to investigate the functions and intracellular dynamics of the mutant KIT protein. METHODS: We performed genetic analyses of the KIT gene using peripheral blood cells. We analyzed the intracellular localization of the mutant KIT protein in HEK293T cells transfected with wild-type (Wt) and/or mutant KIT genes. Immunoprecipitation analyses, immunoblotting and immunofluorescence studies were performed using antibodies against KIT and downstream signaling proteins. Glycosidase digestion analysis was performed to clarify the intracellular localization of KIT protein. RESULTS: A genetic analysis revealed a novel heterozygous mutation c.645_650delTGTGTC which results in the in-frame deletion of Val216 and Ser217 in the extracellular domain of KIT. Immunoprecipitation analyses confirmed that the wild and mutant KIT formed a heterodimer after treatment with stem cell factor (SCF); however, the phosphorylation of the downstream signaling factors was decreased. In an immunofluorescence study, the mutant KIT accumulated predominantly in the endoplasmic reticulum (ER) and was sparsely expressed on the cell surface. A glycosidase digestion study revealed that the mutant KIT is predominantly localized in the ER. CONCLUSION: These data reveal an aberrant function and intracellular localization of mutant KIT protein in piebaldism.


Assuntos
Piebaldismo/genética , Proteínas Proto-Oncogênicas c-kit/genética , Transdução de Sinais/genética , Fator de Células-Tronco/metabolismo , Adulto , Sequência de Bases/genética , Pré-Escolar , Análise Mutacional de DNA , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Feminino , Células HEK293 , Heterozigoto , Humanos , Fenótipo , Fosforilação , Piebaldismo/sangue , Ligação Proteica , Dobramento de Proteína , Multimerização Proteica/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Deleção de Sequência
17.
J Biosci Bioeng ; 126(1): 1-8, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29506820

RESUMO

Lunasin is a novel promising health-beneficial peptide derived from soybean. However, the application of lunasin is limited by its high cost. In this study, we developed a successful protocol for expression of a dimer formation protein containing 4 tandem repeated lunasin analogs (lunasin-4) in Pichia pastoris. The expression level at the optimal condition (initial pH 7.0, 1.0% final methanol concentration and induction for 72 h at 26 °C) was 0.24 mg/mL cell-free broth. Lunasin analog, obtained from purified lunasin-4 protein through enterokinase digestion and ultrafiltration, significantly decreased (p < 0.05) the release of nitric oxide (NO), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages in a dose-dependent manner. In addition, intracellular signaling array analysis demonstrated down-regulated levels of phosphorylated Akt, mechanistic target of rapamycin (mTOR) and p70 s6 kinase (p70s6k) and an up-regulated level of glycogen synthase kinase-3ß (GSK-3ß) after lunasin analog treatment. These results suggest that lunasin analog exerted anti-inflammatory activities in LPS-stimulated RAW264.7 cells partly via inhibiting the activation of Akt/mTOR/p70s6k signaling pathway. In conclusion, this study provides a potential strategy for recombinant production of bioactive lunasin in industry.


Assuntos
Anti-Inflamatórios , Macrófagos/metabolismo , Pichia/genética , Proteínas de Soja/genética , Sequências de Repetição em Tandem/genética , Animais , Anti-Inflamatórios/metabolismo , Células Cultivadas , Clonagem Molecular/métodos , Regulação Fúngica da Expressão Gênica , Lipopolissacarídeos , Macrófagos/efeitos dos fármacos , Camundongos , Organismos Geneticamente Modificados , Pichia/metabolismo , Multimerização Proteica/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Soja/metabolismo
18.
J Virol ; 92(11)2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29540592

RESUMO

During hepatitis B virus (HBV) infections, subviral particles (SVP) consisting only of viral envelope proteins and lipids are secreted. Heterologous expression of the small envelope protein S in mammalian cells is sufficient for SVP generation. S is synthesized as a transmembrane protein with N-terminal (TM1), central (TM2), and hydrophobic C-terminal (HCR) transmembrane domains. The loops between TM1 and TM2 (the cytosolic loop [CL]) and between TM2 and the HCR (the luminal loop [LL]) are located in the cytosol and the endoplasmic reticulum (ER) lumen, respectively. To define the domains of S mediating oligomerization during SVP morphogenesis, S mutants were characterized by expression in transiently transfected cells. Mutation of 12 out of 15 amino acids of TM1 to alanines, as well as the deletion of HCR, still allowed SVP formation, demonstrating that these two domains are not essential for contacts between S proteins. Furthermore, the oligomerization of S was measured with a fluorescence-activated cell sorter (FACS)-based Förster resonance energy transfer (FRET) assay. This approach demonstrated that the CL, TM2, and the LL independently contributed to S oligomerization, while TM1 and the HCR played minor roles. Apparently, intermolecular homo-oligomerization of the CL, TM2, and the LL drives S protein aggregation. Detailed analyses revealed that the point mutation C65S in the CL, the mutation of 13 out of 19 amino acids of TM2 to alanine residues, and the simultaneous replacement of all 8 cysteine residues in the LL by serine residues blocked the abilities of these domains to support S protein interactions. Altogether, specific domains and residues in the HBV S protein that are required for oligomerization and SVP generation were defined.IMPORTANCE The small hepatitis B virus envelope protein S has the intrinsic ability to direct the morphogenesis of spherical 20-nm subviral lipoprotein particles. Such particles expressed in yeast or mammalian cells represent the antigenic component of current hepatitis B vaccines. Our knowledge about the steps leading from the initial, monomeric, transmembrane translation product of S to SVP is very limited, as is our information on the structure of the complex main epitope of SVP that induces the formation of protective antibodies after vaccination. This study contributes to our understanding of the oligomerization process of S chains during SVP formation and shows that the cytoplasmic loop, one membrane-embedded domain, and the luminal loop of S independently drive S-S oligomerization.


Assuntos
Antígenos de Superfície da Hepatite B/metabolismo , Domínios Proteicos/genética , Multimerização Proteica/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Hepatite B/patologia , Hepatite B/virologia , Vírus da Hepatite B/metabolismo , Humanos , Deleção de Sequência/genética , Montagem de Vírus/genética
19.
Genes Dev ; 32(5-6): 430-447, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29549180

RESUMO

The p53 tumor suppressor protein is the most well studied as a regulator of transcription in the nucleus, where it exists primarily as a tetramer. However, there are other oligomeric states of p53 that are relevant to its regulation and activities. In unstressed cells, p53 is normally held in check by MDM2 that targets p53 for transcriptional repression, proteasomal degradation, and cytoplasmic localization. Here we discovered a hydrophobic region within the MDM2 N-terminal domain that binds exclusively to the dimeric form of the p53 C-terminal domain in vitro. In cell-based assays, MDM2 exhibits superior binding to, hyperdegradation of, and increased nuclear exclusion of dimeric p53 when compared with tetrameric wild-type p53. Correspondingly, impairing the hydrophobicity of the newly identified N-terminal MDM2 region leads to p53 stabilization. Interestingly, we found that dimeric mutant p53 is partially unfolded and is a target for ubiquitin-independent degradation by the 20S proteasome. Finally, forcing certain tumor-derived mutant forms of p53 into dimer configuration results in hyperdegradation of mutant p53 and inhibition of p53-mediated cancer cell migration. Gaining insight into different oligomeric forms of p53 may provide novel approaches to cancer therapy.


Assuntos
Neoplasias/fisiopatologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Citoplasma/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Mutação , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Domínios Proteicos , Multimerização Proteica/genética , Proteólise , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genética
20.
Biochim Biophys Acta Biomembr ; 1860(5): 1105-1113, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29391167

RESUMO

The ß-secretase (BACE1) features a unique sulfur rich motif (M462xxxC466xxxM470xxxC474xxxC478) in its transmembrane helix (BACE1-TM) which is characteristic for proteins involved in copper ion storage and transport. While this motif has been shown to promote BACE1-TM trimerization and binding of copper ions in vitro, the structural basis for the interaction of copper ions with the BACE1-TM is still not well understood. Using molecular dynamics (MD) simulations, we show that membrane embedded BACE1-TMs adopt a flexible trimeric structure that binds and conducts copper ions through variable coordination. In coarse-grained (CG) MD simulations, the spontaneous assembly of BACE1-TMs trimers results in a right-handed helix packing arrangement. In subsequent atomistic MD simulations the sulfur rich motif defines characteristic copper ion coordination sites along a constricted partially solvated axial pore. Sliding and tilting of BACE1-TMs along smooth A459xxxA463/464xxA467 surfaces, facilitated by a central P472 induced kink, enables copper ions to alternate between different coordination sites, including the prominent C466 and M470. We shed light into the structural arrangement of BACE1-TM trimers and propose a mechanism for copper ion conduction that might also apply to other proteins involved in metal ion transport.


Assuntos
Secretases da Proteína Precursora do Amiloide/química , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/metabolismo , Cobre/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Ácido Aspártico Endopeptidases/genética , Sequências Hélice-Alça-Hélice/genética , Humanos , Transporte de Íons/genética , Íons/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica/genética , Domínios e Motivos de Interação entre Proteínas/genética , Multimerização Proteica/genética
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