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1.
Artigo em Inglês | MEDLINE | ID: mdl-34816323

RESUMO

Natural killer (NK) cells are cytotoxic lymphocytes that play a major role in the innate immune system. NK cells exhibit potent cytotoxic activity against cancer cells and virally infected cells without antigen priming. These unique cytotoxic properties make NK cells a promising therapeutic against cancer. Limitations of NK cell therapy include deficiencies in high clinical efficacy often due to a need for a high NK cell to target cell ratio to achieve effective killing. In order to address the suboptimal efficacy of current adoptive NK cell therapy, a high throughput screen (HTS) was designed and performed to identify drug-like compounds that increase NK cytotoxic activity against tumor cells without affecting the normal cells. This screen was performed in a 384-well plate format utilizing an expanded primary NK cell product and ovarian cancer cells as a target cell (TC) line. Of the 8000 diverse small molecules screened, 16 hits were identified (0.2% hit rate) based on both a robust Z (RZ) score < -3 and a greater than 10% increase in NK cell killing. A validation screen had a confirmation rate of 70%. Select compounds were further validated and characterized by additional cytotoxicity assays including activity against multiple blood cancer and solid tumor cell lines, with no effect on primary human T cells. This work demonstrates that high-throughput screening can be reliably used to identify compounds that increase NK tumoricidal activity in vitro that can be further investigated and translated for potential clinical application. Précis: Our work led to the identification of promising compound that potently increases NK cell-mediated killing of a variety of different cancer cells, but no impact on the killing of normal cells. This compound demonstrates the utility of this assay.

2.
Chem Sci ; 12(30): 10388-10394, 2021 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-34377425

RESUMO

The spiroindimicins are a unique class of chlorinated indole alkaloids characterized by three heteroaromatic rings structured around a congested spirocyclic stereocenter. Here, we report the first total synthesis of (+)-spiroindimicin A, which bears a challenging C-3'/C-5''-linked spiroindolenine. We detail our initial efforts to effect a biomimetic oxidative spirocyclization from its proposed natural precursor, lynamicin D, and describe how these studies shaped our final abiotic 9-step solution to this complex alkaloid built around a key Pd-catalyzed asymmetric spirocyclization. Scalable access to spiroindimicins A, H, and their congeners has enabled discovery of their activity against several parasites relevant to human health, providing potential starting points for new therapeutics for the neglected tropical diseases leishmaniasis and African sleeping sickness.

3.
J Med Chem ; 64(15): 10951-10966, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34260245

RESUMO

Influenza viruses cause approximately half a million deaths every year worldwide. Vaccines are available but partially effective, and the number of antiviral medications is limited. Thus, it is crucial to develop therapeutic strategies to counteract this major pathogen. Influenza viruses enter the host cell via their hemagglutinin (HA) proteins. The HA subtypes of influenza A virus are phylogenetically classified into groups 1 and 2. Here, we identified an inhibitor of the HA protein, a tertiary aryl sulfonamide, that prevents influenza virus entry and replication. This compound shows potent antiviral activity against diverse H1N1, H5N1, and H3N2 influenza viruses encoding HA proteins from both groups 1 and 2. Synthesis of derivatives of this aryl sulfonamide identified moieties important for antiviral activity. This compound may be considered as a lead for drug development with the intent to be used alone or in combination with other influenza A virus antivirals to enhance pan-subtype efficacy.


Assuntos
Antivirais/farmacologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A/efeitos dos fármacos , Sulfonamidas/farmacologia , Antivirais/síntese química , Antivirais/química , Relação Dose-Resposta a Droga , Estrutura Molecular , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/química , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
4.
J Med Chem ; 64(5): 2739-2761, 2021 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-33620219

RESUMO

Malaria control programs continue to be threatened by drug resistance. To identify new antimalarials, we conducted a phenotypic screen and identified a novel tetrazole-based series that shows fast-kill kinetics and a relatively low propensity to develop high-level resistance. Preliminary structure-activity relationships were established including identification of a subseries of related amides with antiplasmodial activity. Assaying parasites with resistance to antimalarials led us to test whether the series had a similar mechanism of action to chloroquine (CQ). Treatment of synchronized Plasmodium falciparum parasites with active analogues revealed a pattern of intracellular inhibition of hemozoin (Hz) formation reminiscent of CQ's action. Drug selections yielded only modest resistance that was associated with amplification of the multidrug resistance gene 1 (pfmdr1). Thus, we have identified a novel chemical series that targets the historically druggable heme polymerization pathway and that can form the basis of future optimization efforts to develop a new malaria treatment.


Assuntos
Amidas/farmacologia , Antimaláricos/farmacologia , Hemoglobinas/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Tetrazóis/farmacologia , Amidas/síntese química , Amidas/farmacocinética , Antimaláricos/síntese química , Antimaláricos/farmacocinética , Resistência Microbiana a Medicamentos/efeitos dos fármacos , Hemeproteínas/antagonistas & inibidores , Células Hep G2 , Humanos , Estrutura Molecular , Testes de Sensibilidade Parasitária , Plasmodium falciparum/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/farmacocinética , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade , Tetrazóis/síntese química , Tetrazóis/farmacocinética
5.
Sci Rep ; 10(1): 20662, 2020 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-33244070

RESUMO

PDA is a major cause of US cancer-related deaths. Oncogenic Kras presents in 90% of human PDAs. Kras mutations occur early in pre-neoplastic lesions but are insufficient to cause PDA. Other contributing factors early in disease progression include chronic pancreatitis, alterations in epigenetic regulators, and tumor suppressor gene mutation. GPCRs activate heterotrimeric G-proteins that stimulate intracellular calcium and oncogenic Kras signaling, thereby promoting pancreatitis and progression to PDA. By contrast, Rgs proteins inhibit Gi/q-coupled GPCRs to negatively regulate PDA progression. Rgs16::GFP is expressed in response to caerulein-induced acinar cell dedifferentiation, early neoplasia, and throughout PDA progression. In genetically engineered mouse models of PDA, Rgs16::GFP is useful for pre-clinical rapid in vivo validation of novel chemotherapeutics targeting early lesions in patients following successful resection or at high risk for progressing to PDA. Cultured primary PDA cells express Rgs16::GFP in response to cytotoxic drugs. A histone deacetylase inhibitor, TSA, stimulated Rgs16::GFP expression in PDA primary cells, potentiated gemcitabine and JQ1 cytotoxicity in cell culture, and Gem + TSA + JQ1 inhibited tumor initiation and progression in vivo. Here we establish the use of Rgs16::GFP expression for testing drug combinations in cell culture and validation of best candidates in our rapid in vivo screen.


Assuntos
Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Antineoplásicos/farmacologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/patologia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Células Acinares/efeitos dos fármacos , Células Acinares/metabolismo , Células Acinares/patologia , Adenocarcinoma/metabolismo , Animais , Cálcio/metabolismo , Carcinogênese/efeitos dos fármacos , Carcinogênese/metabolismo , Carcinogênese/patologia , Carcinoma Ductal Pancreático/metabolismo , Desdiferenciação Celular/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Ceruletídeo/metabolismo , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Progressão da Doença , Proteínas de Ligação ao GTP/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Camundongos , Ductos Pancreáticos/efeitos dos fármacos , Ductos Pancreáticos/metabolismo , Neoplasias Pancreáticas/metabolismo , Pancreatite/tratamento farmacológico , Pancreatite/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas RGS/metabolismo , Transdução de Sinais/efeitos dos fármacos
6.
ACS Infect Dis ; 6(8): 2057-2072, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32686409

RESUMO

The few frontline antileishmanial drugs are poorly effective and toxic. To search for new drugs for this neglected tropical disease, we tested the activity of compounds in the Medicines for Malaria Venture (MMV) "Pathogen Box" against Leishmania amazonensis axenic amastigotes. Screening yielded six discovery antileishmanial compounds with EC50 values from 50 to 480 nM. Concentration-response assays demonstrated that the best hit, MMV676477, had mid-nanomolar cytocidal potency against intracellular Leishmania amastigotes, Trypanosoma brucei, and Plasmodium falciparum, suggesting broad antiparasitic activity. We explored structure-activity relationships (SAR) within a small group of MMV676477 analogs and observed a wide potency range (20-5000 nM) against axenic Leishmania amastigotes. Compared to MMV676477, our most potent analog, SW41, had ∼5-fold improved antileishmanial potency. Multiple lines of evidence suggest that MMV676477 selectively disrupts Leishmania tubulin dynamics. Morphological studies indicated that MMV676477 and analogs affected L. amazonensis during cell division. Differential centrifugation showed that MMV676477 promoted partitioning of cellular tubulin toward the polymeric form in parasites. Turbidity assays with purified Leishmania and porcine tubulin demonstrated that MMV676477 promoted leishmanial tubulin polymerization in a concentration-dependent manner. Analogs' antiparasitic activity correlated with their ability to facilitate purified Leishmania tubulin polymerization. Chemical cross-linking demonstrated binding of the MMV676477 scaffold to purified Leishmania tubulin, and competition studies established a correlation between binding and antileishmanial activity. Our studies demonstrate that MMV676477 is a potent antiparasitic compound that preferentially promotes Leishmania microtubule polymerization. Due to its selectivity for and broad-spectrum activity against multiple parasites, this scaffold shows promise for antiparasitic drug development.


Assuntos
Leishmania , Malária , Animais , Antiparasitários/farmacologia , Polimerização , Suínos , Tubulina (Proteína)
7.
Biofabrication ; 12(3): 035019, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32408287

RESUMO

High-throughput screening (HTS) is a well-established approach for tumor-specific drug development because of its high efficiency and customizable selection of antineoplastic drugs. However, there is still a lack of an appropriate cell-based HTS specific for migratory cancer cells. In the study presented here, we created a novel assay (mHTS): a single-cell-level screening method targeting migratory cancer cells and can be applied in a high-throughput manner. This mHTS platform is based on microchannel devices (providing physical confinement during cell migration and limit migrating cells' proliferation rate) assembled 96-well plate (fitting to HTS manner). To determine the feasibility of this assay, we quantified the anti-migratory and anti-viability effects of several molecules (Cytochalasin D, Doxorubicin and AZD-6244) on migrating (creeping inside microchannel) glioblastoma multiforme (GBM) cells. After analyzing migration screening data that was collected on a single-cell-level, we were able to compare those drug's effects on cancer cells' migration velocity and uncovered the migration inhibiting potential of AZD (500 nM and 1000 nM). Viability data based on single-cell-level screening also allowed us to further understand the same drug's different lethality toward migrating and normal 2D cultured cancer cells. The Pre-classification of subpopulations enables us to study the heterogeneity of cancer and ensures our method's feasibility for a high-throughput manner. All these results proved our mHTS platform is suitable for single-cell-level anti-migration drug screening and has potential feasibility in promoting the development of anti-migratory-cancer-drug in a high-throughput manner.


Assuntos
Movimento Celular , Ensaios de Triagem em Larga Escala , Neoplasias/patologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Estudos de Viabilidade , Humanos , Camundongos
8.
Cell Chem Biol ; 27(5): 622-634.e6, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32330442

RESUMO

Destabilizing domains (DDs), such as a mutated form of Escherichia coli dihydrofolate reductase (ecDHFR), confer instability and promote protein degradation. However, when combined with small-molecule stabilizers (e.g., the antibiotic trimethoprim), DDs allow positive regulation of fusion protein abundance. Using a combinatorial screening approach, we identified and validated 17 unique 2,4-diaminopyrimidine/triazine-based ecDHFR DD stabilizers, at least 15 of which were ineffective antibiotics against E. coli and S. aureus. Identified stabilizers functioned in vivo to control an ecDHFR DD-firefly luciferase in the mouse eye and/or the liver. Next, stabilizers were leveraged to perform synergistic dual functions in vitro (HeLa cell death sensitization) and in vivo (repression of ocular inflammation) by stabilizing a user-defined ecDHFR DD while also controlling endogenous signaling pathways. Thus, these newly identified pharmacological chaperones allow for simultaneous control of compound-specific endogenous and user-defined genetic pathways, the combination of which may provide synergistic effects in complex biological scenarios.


Assuntos
Antibacterianos/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Antagonistas do Ácido Fólico/farmacologia , Pirimidinas/farmacologia , Tetra-Hidrofolato Desidrogenase/metabolismo , Animais , Antibacterianos/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Feminino , Antagonistas do Ácido Fólico/química , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pirimidinas/química , Tetra-Hidrofolato Desidrogenase/química , Triazinas/química , Triazinas/farmacologia , Trimetoprima/análogos & derivados , Trimetoprima/farmacologia
9.
PLoS Pathog ; 16(4): e1008407, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32240278

RESUMO

Influenza A viruses are human pathogens with limited therapeutic options. Therefore, it is crucial to devise strategies for the identification of new classes of antiviral medications. The influenza A virus genome is constituted of 8 RNA segments. Two of these viral RNAs are transcribed into mRNAs that are alternatively spliced. The M1 mRNA encodes the M1 protein but is also alternatively spliced to yield the M2 mRNA during infection. M1 to M2 mRNA splicing occurs at nuclear speckles, and M1 and M2 mRNAs are exported to the cytoplasm for translation. M1 and M2 proteins are critical for viral trafficking, assembly, and budding. Here we show that gene knockout of the cellular protein NS1-BP, a constituent of the M mRNA speckle-export pathway and a binding partner of the virulence factor NS1 protein, inhibits M mRNA nuclear export without altering bulk cellular mRNA export, providing an avenue to preferentially target influenza virus. We performed a high-content, image-based chemical screen using single-molecule RNA-FISH to label viral M mRNAs followed by multistep quantitative approaches to assess cellular mRNA and cell toxicity. We identified inhibitors of viral mRNA biogenesis and nuclear export that exhibited no significant activity towards bulk cellular mRNA at non-cytotoxic concentrations. Among the hits is a small molecule that preferentially inhibits nuclear export of a subset of viral and cellular mRNAs without altering bulk cellular mRNA export. These findings underscore specific nuclear export requirements for viral mRNAs and phenocopy down-regulation of the mRNA export factor UAP56. This RNA export inhibitor impaired replication of diverse influenza A virus strains at non-toxic concentrations. Thus, this screening strategy yielded compounds that alone or in combination may serve as leads to new ways of treating influenza virus infection and are novel tools for studying viral RNA trafficking in the nucleus.


Assuntos
Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Antivirais/farmacologia , Núcleo Celular/virologia , Vírus da Influenza A/metabolismo , Influenza Humana/virologia , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Avaliação Pré-Clínica de Medicamentos , Humanos , Vírus da Influenza A/genética , RNA Mensageiro/genética , RNA Viral/genética , Replicação Viral/efeitos dos fármacos
10.
Nat Commun ; 9(1): 2050, 2018 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-29784984

RESUMO

The originally published version of this Article contained an error in the spelling of the author Nathaniel W. Oswald, which was incorrectly given as Nathaniel W. Olswald. This has now been corrected in both the PDF and HTML versions of the Article.

11.
Nat Commun ; 8(1): 2270, 2017 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-29273768

RESUMO

Drugs that mirror the cellular effects of starvation mimics are considered promising therapeutics for common metabolic disorders, such as obesity, liver steatosis, and for ageing. Starvation, or caloric restriction, is known to activate the transcription factor EB (TFEB), a master regulator of lipid metabolism and lysosomal biogenesis and function. Here, we report a nanotechnology-enabled high-throughput screen to identify small-molecule agonists of TFEB and discover three novel compounds that promote autophagolysosomal activity. The three lead compounds include the clinically approved drug, digoxin; the marine-derived natural product, ikarugamycin; and the synthetic compound, alexidine dihydrochloride, which is known to act on a mitochondrial target. Mode of action studies reveal that these compounds activate TFEB via three distinct Ca2+-dependent mechanisms. Formulation of these compounds in liver-tropic biodegradable, biocompatible nanoparticles confers hepatoprotection against diet-induced steatosis in murine models and extends lifespan of Caenorhabditis elegans. These results support the therapeutic potential of small-molecule TFEB activators for the treatment of metabolic and age-related disorders.


Assuntos
Autofagia/efeitos dos fármacos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/agonistas , Biguanidas/farmacologia , Caenorhabditis elegans/efeitos dos fármacos , Digoxina/farmacologia , Inibidores Enzimáticos/farmacologia , Lactamas/farmacologia , Longevidade/efeitos dos fármacos , Síndrome Metabólica/metabolismo , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Caenorhabditis elegans/metabolismo , Cálcio/metabolismo , Restrição Calórica , Dieta Hiperlipídica , Fígado Gorduroso/metabolismo , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Inanição
12.
Genes Dev ; 31(17): 1770-1783, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28982760

RESUMO

Direct reprogramming of fibroblasts to cardiomyocytes represents a potential means of restoring cardiac function following myocardial injury. AKT1 in the presence of four cardiogenic transcription factors, GATA4, HAND2, MEF2C, and TBX5 (AGHMT), efficiently induces the cardiac gene program in mouse embryonic fibroblasts but not adult fibroblasts. To identify additional regulators of adult cardiac reprogramming, we performed an unbiased screen of transcription factors and cytokines for those that might enhance or suppress the cardiogenic activity of AGHMT in adult mouse fibroblasts. Among a collection of inducers and repressors of cardiac reprogramming, we discovered that the zinc finger transcription factor 281 (ZNF281) potently stimulates cardiac reprogramming by genome-wide association with GATA4 on cardiac enhancers. Concomitantly, ZNF281 suppresses expression of genes associated with inflammatory signaling, suggesting the antagonistic convergence of cardiac and inflammatory transcriptional programs. Consistent with an inhibitory influence of inflammatory pathways on cardiac reprogramming, blockade of these pathways with anti-inflammatory drugs or components of the nucleosome remodeling deacetylase (NuRD) complex, which associate with ZNF281, stimulates cardiac gene expression. We conclude that ZNF281 acts at a nexus of cardiac and inflammatory gene programs, which exert opposing influences on fibroblast to cardiac reprogramming.


Assuntos
Reprogramação Celular/genética , Regulação da Expressão Gênica/genética , Fatores de Transcrição/metabolismo , Anti-Inflamatórios/farmacologia , Reprogramação Celular/efeitos dos fármacos , Fibroblastos/fisiologia , Fator de Transcrição GATA4/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Estudo de Associação Genômica Ampla , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Proteínas Repressoras , Transcriptoma
13.
Mol Biol Cell ; 20(24): 5290-305, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19846667

RESUMO

Molecular mechanisms for cell migration, especially how signaling and cytoskeletal systems are integrated, are not understood well. Here, we examined the role of CARMIL (capping protein, Arp2/3, and Myosin-I linker) family proteins in migrating cells. Vertebrates express three conserved genes for CARMIL, and we examined the functions of the two CARMIL genes expressed in migrating human cultured cells. Both isoforms, CARMIL1 and 2, were necessary for cell migration, but for different reasons. CARMIL1 localized to lamellipodia and macropinosomes, and loss of its function caused loss of lamellipodial actin, along with defects in protrusion, ruffling, and macropinocytosis. CARMIL1-knockdown cells showed loss of activation of Rac1, and CARMIL1 was biochemically associated with the GEF Trio. CARMIL2, in contrast, colocalized with vimentin intermediate filaments, and loss of its function caused a distinctive multipolar phenotype. Loss of CARMIL2 also caused decreased levels of myosin-IIB, which may contribute to the polarity phenotype. Expression of one CARMIL isoform was not able to rescue the knockdown phenotypes of the other. Thus, the two isoforms are both important for cell migration, but they have distinct functions.


Assuntos
Proteínas de Transporte/metabolismo , Movimento Celular , Actinas/metabolismo , Sequência de Aminoácidos , Proteínas de Transporte/química , Polaridade Celular , Clonagem Molecular , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Proteínas dos Microfilamentos , Microtúbulos/metabolismo , Dados de Sequência Molecular , Miosina Tipo II/metabolismo , Filogenia , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Transporte Proteico , Pseudópodes/metabolismo , Homologia de Sequência de Aminoácidos , Vimentina/metabolismo , Cicatrização , Proteínas rac1 de Ligação ao GTP/metabolismo
14.
J Biol Chem ; 281(47): 36347-59, 2006 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-16987810

RESUMO

CKIP-1 is a pleckstrin homology domain-containing protein that induces alterations of the actin cytoskeleton and cell morphology when expressed in human osteosarcoma cells. CKIP-1 interacts with the heterodimeric actin-capping protein in cells, so we postulated that this interaction was responsible for the observed cytoskeletal and morphological effects of CKIP-1. To test this postulate, we used peptide "walking arrays" and alignments of CKIP-1 with CARMIL, another CP-binding protein, to identify Arg-155 and Arg-157 of CKIP-1 as residues potentially required for its interactions with CP. CKIP-1 mutants harboring Arg-155 and Arg-157 substitutions exhibited greatly decreased CP binding, while retaining wild-type localization, the ability to interact with protein kinase CK2, and self-association. To examine the phenotype associated with expression of these mutants, we generated tetracycline-inducible human osteosarcoma cells lines expressing R155E,R157E mutants of CKIP-1. Examination of these cell lines reveals that CKIP-1 R155E,R157E did not induce the distinct changes in cell morphology and the actin cytoskeleton that are characteristic of wild-type CKIP-1 demonstrating that the interaction between CKIP-1 and CP is required for these cellular effects.


Assuntos
Actinas/química , Proteínas de Transporte/fisiologia , Sequência de Aminoácidos , Animais , Arginina/química , Proteínas de Transporte/metabolismo , Caseína Quinase II/metabolismo , Linhagem Celular Tumoral , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Dados de Sequência Molecular , Mutação , Osteossarcoma/metabolismo , Peptídeos/química , Faloidina/farmacologia , Ligação Proteica , Homologia de Sequência de Aminoácidos
15.
J Biol Chem ; 281(28): 19196-203, 2006 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-16707503

RESUMO

CD2-associated protein (CD2AP) is a scaffold molecule that plays a critical role in the maintenance of the kidney filtration barrier. Little, however, is understood about its mechanism of function. We used mass spectrometry to identify CD2AP-interacting proteins. Many of the proteins that we identified suggest a role for CD2AP in endocytosis and actin regulation. To address the role of CD2AP in regulation of the actin cytoskeleton, we focused on characterizing the interaction of CD2AP with actin-capping protein CP. We identified a novel binding motif LXHXTXXRPK(X)6P present in CD2AP that is also found in its homolog Cin85 and other capping protein-associated proteins such as CARMIL and CKIP-1. CD2AP inhibits the function of capping protein in vitro. Therefore, our results support a role of CD2AP in the regulation of the actin cytoskeleton.


Assuntos
Actinas/química , Proteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Galinhas , Proteínas do Citoesqueleto , Citoesqueleto/metabolismo , Endocitose , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas dos Microfilamentos/química , Dados de Sequência Molecular , Ligação Proteica , Homologia de Sequência de Aminoácidos
16.
Curr Biol ; 15(19): 1755-61, 2005 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-16213823

RESUMO

Regulation of microtubule dynamics and organization in mitosis by a number of microtubule-associated proteins (MAPs) is required for proper bipolar spindle assembly, yet the precise mechanisms by which many MAPs function are poorly understood. One interesting class of MAPs is known to localize to the nucleus during interphase yet fulfill important spindle functions during mitosis. We have identified Xenopus nuclear factor 7 (Xnf7), a developmental regulator of dorsal-ventral patterning, as a microtubule-binding protein that also associates with the nuclear import receptor importin alpha/beta. Xnf7 localized to interphase nuclei and metaphase spindles both in Xenopus egg extracts and cultured cells. Xnf7-depleted spindles were hypersensitive to microtubule-depolymerizing agents. Functional characterization of Xnf7 revealed that it binds directly to microtubules, exhibits RING-finger-dependent E3-ubiquitin-ligase activity, and has C-terminal-dependent microtubule-bundling activity. The minimal microtubule-bundling domain of Xnf7 was sufficient to rescue the spindle-hypersensitivity phenotype. Thus, we have identified Xnf7 as a nuclear MAP whose microtubule-bundling activity, but not E3-ligase activity, contributes to microtubule organization and spindle integrity. Characterization of the multiple activities of Xnf7 may have implications for understanding human diseases caused by mutations in related proteins.


Assuntos
Microtúbulos/metabolismo , Mitose/fisiologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fuso Acromático/fisiologia , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus/metabolismo , Animais , Western Blotting , Primers do DNA , Proteínas de Ligação a DNA , Proteínas do Ovo , Imuno-Histoquímica , Carioferinas/metabolismo , Microscopia Eletrônica , Microscopia de Fluorescência , Microtúbulos/fisiologia , Microtúbulos/ultraestrutura , Mutagênese Sítio-Dirigida , Ligação Proteica , Fuso Acromático/ultraestrutura , Xenopus/genética
17.
Mol Cell ; 17(2): 277-90, 2005 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-15664196

RESUMO

How kinetochore proteins form a dynamic interface with microtubules is largely unknown. In budding yeast, the 10-protein Dam1 complex is an Aurora kinase target that plays essential roles maintaining the integrity of the mitotic spindle and regulating interactions with the kinetochore. Here, we investigated the biochemical properties of purified Dam1 complex. The complex oligomerized into rings around microtubules. Ring formation was facilitated by microtubules but could occur in their absence. Mutant alleles led to partially assembled complexes or reduced microtubule binding. The interaction between rings and microtubules is mediated by the C termini of both Dam1 and alphabeta-tubulin. Ring formation promotes microtubule assembly, stabilizes against disassembly, and promotes bundling. A GTP-tubulin lattice is the preferred binding partner for the complex, and Dam1 rings can exhibit lateral mobility on microtubules. These observations suggest a mechanism by which the kinetochore can recognize and stay attached to the plus ends of microtubules.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Animais , Bovinos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/isolamento & purificação , Proteínas do Citoesqueleto , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/metabolismo , Hidrazinas/metabolismo , Substâncias Macromoleculares , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/isolamento & purificação , Microtúbulos/ultraestrutura , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo
18.
Curr Opin Struct Biol ; 13(2): 256-61, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12727521

RESUMO

Tubulin is known to exist in at least two main conformations: straight when bound to GTP or buried within the microtubule lattice, and curved when bound to GDP. The latter is most obvious during microtubule depolymerization, when protofilaments bend and peel off from microtubule ends. The curved, low-energy subunits form tantalizing ring structures in the presence of stabilizing divalent cations. Interestingly, cellular factors and antimitotic agents that act by depolymerizing microtubules can induce the formation of rings. In these rings, tubulin dimers generally appear kinked at the monomer-monomer interface, either to the same or to a lesser extent than at the dimer-dimer interface, with each agent giving rise to particular subtleties in the structures of the rings and the tubulin dimer itself that may reflect their distinctive mechanisms of action. How these kinks relate to what happens when the stored energy of GTP hydrolysis is released, freeing GDP*tubulin into an unconstrained state, remains an open question.


Assuntos
Proteínas dos Microtúbulos , Microtúbulos/química , Microtúbulos/ultraestrutura , Modelos Moleculares , Tubulina (Proteína)/química , Tubulina (Proteína)/ultraestrutura , Sítios de Ligação , Depsipeptídeos , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Microtúbulos/metabolismo , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Ligação Proteica , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Estatmina , Relação Estrutura-Atividade , Tubulina (Proteína)/metabolismo
19.
J Mol Biol ; 316(3): 817-28, 2002 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-11866534

RESUMO

The stability of microtubules during the cell-cycle is regulated by a number of cellular factors, some of which stabilize microtubules and others that promote breakdown. XKCM1 is a kinesin-like protein that induces microtubule depolymerization and is required for mitotic spindle assembly. We have examined the binding and depolymerization effects of XKCM1 on different tubulin polymers in order to learn about its mechanism of action. Zinc-induced tubulin polymers, characterized by an anti-parallel protofilament arrangement, are depolymerized by XKCM1, indicating that this enzyme acts on a single protofilament. GDP-tubulin rings, which correspond to the low-energy state of tubulin, are stable only under conditions that inhibit XKCM1 depolymerizing activity, but can be stabilized by XKCM1 bound to AMPPNP. Tubulin polymers made of subtilisin-treated tubulin (lacking the tubulin C-terminal tail) are resistant to XKCM1-induced depolymerization, suggesting that the interaction of the acidic tail of tubulin with basic residues in XKCM1 unique to Kin I proteins is required for depolymerization.


Assuntos
Cinesina/química , Cinesina/metabolismo , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Proteínas de Xenopus , Sequência de Aminoácidos , Animais , Biopolímeros/química , Biopolímeros/metabolismo , Domínio Catalítico , Bovinos , Guanosina Difosfato/metabolismo , Cinesina/ultraestrutura , Microscopia Eletrônica , Microtúbulos/química , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Quaternária de Proteína/efeitos dos fármacos , Alinhamento de Sequência , Subtilisina/metabolismo , Suínos , Tubulina (Proteína)/ultraestrutura , Zinco/farmacologia
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