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1.
J Struct Biol X ; 10: 100109, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-39188530

RESUMO

Nsp14 is an RNA methyltransferase (MTase) encoded by all coronaviruses. In fact, many viral families, including DNA viruses, encode MTases that catalyze the methylation of the RNA precap structure, resulting in fully capped viral RNA. This capping is crucial for efficient viral RNA translation, stability, and immune evasion. Our previous research identified nsp14 inhibitors based on the chemical scaffold of its methyl donor - the S-adenosyl methionine (SAM) - featuring a modified adenine base and a substituted arylsulfonamide. However, the binding mode of these inhibitors was based only on docking experiments. To uncover atomic details of nsp14 inhibition we solved the crystal structure of nsp14 bound to STM957. The structure revealed the atomic details of nsp14 inhibition such that the 7-deaza-adenine moiety of STM957 forms specific interactions with Tyr368, Ala353, and Phe367, while the arylsulfonamide moiety engages with Asn388 and Phe506. The large aromatic substituent at the 7-deaza position displaces a network of water molecules near the adenine base. Surprisingly, this was recently observed in the case of an unrelated monkeypox MTase VP39, where the 7-deaza modified SAH analogs also displaced water molecules from the vicinity of the active site.

2.
Structure ; 32(4): 433-439.e4, 2024 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-38325369

RESUMO

The cGAS-STING pathway is a crucial part of innate immunity; it serves to detect DNA in the cytoplasm and to defend against certain cancers, viruses, and bacteria. We designed and synthesized fluorinated carbocyclic cGAMP analogs, MD1203 and MD1202D (MDs), to enhance their stability and their affinity for STING. These compounds demonstrated exceptional activity against STING. Despite their distinct chemical modifications relative to the canonical cyclic dinucleotides (CDNs), crystallographic analysis revealed a binding mode with STING that was consistent with the canonical CDNs. Importantly, MDs were resistant to cleavage by viral poxin nucleases and MDs-bound poxin adopted an unliganded-like conformation. Moreover, MDs complexed with poxin showed a conformation distinct from cGAMP bound to poxin, closely resembling their conformation when bound to STING. In conclusion, the development of MD1203 and MD1202D showcases their potential as potent STING activators with remarkable stability against poxin-mediated degradation-a crucial characteristic for future development of antivirals.


Assuntos
Neoplasias , Nucleotídeos Cíclicos , Humanos , Nucleotídeos Cíclicos/química , Nucleotídeos Cíclicos/metabolismo , Nucleotidiltransferases/química , Imunidade Inata
3.
bioRxiv ; 2024 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-37873443

RESUMO

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has led to significant global morbidity and mortality. A crucial viral protein, the non-structural protein 14 (nsp14), catalyzes the methylation of viral RNA and plays a critical role in viral genome replication and transcription. Due to the low mutation rate in the nsp region among various SARS-CoV-2 variants, nsp14 has emerged as a promising therapeutic target. However, discovering potential inhibitors remains a challenge. In this work, we introduce a computational pipeline for the rapid and efficient identification of potential nsp14 inhibitors by leveraging virtual screening and the NCI open compound collection, which contains 250,000 freely available molecules for researchers worldwide. The introduced pipeline provides a cost-effective and efficient approach for early-stage drug discovery by allowing researchers to evaluate promising molecules without incurring synthesis expenses. Our pipeline successfully identified seven promising candidates after experimentally validating only 40 compounds. Notably, we discovered NSC620333, a compound that exhibits a strong binding affinity to nsp14 with a dissociation constant of 427 ± 84 nM. In addition, we gained new insights into the structure and function of this protein through molecular dynamics simulations. We identified new conformational states of the protein and determined that residues Phe367, Tyr368, and Gln354 within the binding pocket serve as stabilizing residues for novel ligand interactions. We also found that metal coordination complexes are crucial for the overall function of the binding pocket. Lastly, we present the solved crystal structure of the nsp14-MTase complexed with SS148 (PDB:8BWU), a potent inhibitor of methyltransferase activity at the nanomolar level (IC50 value of 70 ± 6 nM). Our computational pipeline accurately predicted the binding pose of SS148, demonstrating its effectiveness and potential in accelerating drug discovery efforts against SARS-CoV-2 and other emerging viruses.

4.
Eur J Med Chem ; 259: 115685, 2023 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-37567057

RESUMO

Cyclic dinucleotides (CDNs) trigger the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway, which plays a key role in cytosolic DNA sensing and thus in immunomodulation against infections, cell damage and cancer. However, cancer immunotherapy trials with CDNs have shown immune activation, but not complete tumor regression. Nevertheless, we designed a novel class of CDNs containing vinylphosphonate based on a STING-affinity screening assay. In vitro, acyloxymethyl phosphate/phosphonate prodrugs of these vinylphosphonate CDNs were up to 1000-fold more potent than the clinical candidate ADU-S100. In vivo, the lead prodrug induced tumor-specific T cell priming and facilitated tumor regression in the 4T1 syngeneic mouse model of breast cancer. Moreover, we solved the crystal structure of this ligand bound to the STING protein. Therefore, our findings not only validate the therapeutic potential of vinylphosphonate CDNs but also open up opportunities for drug development in cancer immunotherapy bridging innate and adaptive immunity.


Assuntos
Neoplasias , Nucleotídeos Cíclicos , Animais , Camundongos , Nucleotídeos Cíclicos/farmacologia , Nucleotídeos Cíclicos/metabolismo , DNA , Neoplasias/tratamento farmacológico , Imunoterapia , Imunidade Inata
5.
Bioorg Med Chem Lett ; 76: 129010, 2022 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-36184029

RESUMO

Novel 4-aminoquinazoline-6-carboxamide derivatives bearing differently substituted aryl or heteroaryl groups at position 7 in the core were rationally designed, synthesized and evaluated for biological activity in vitro as phosphatidylinositol 4-kinase IIα (PI4K2A) inhibitors. The straightforward approach described here enabled the sequential, modular synthesis and broad functionalization of the scaffold in a mere six steps. The SAR investigation reported here is based on detailed structural analysis of the conserved binding mode of ATP and other adenine derivatives to the catalytic site of type II PI4Ks, combined with extensive docking studies. Several compounds exhibited significant activity against PI4K2A. Moreover, we solved a crystal structure of PI4K2B in complex with one of our lead ligand candidates, which validated the ligand binding site and pose predicted by our docking-based ligand model. These discoveries suggest that our structure-based approach may be further developed and employed to synthesize new inhibitors with optimized potency and selectivity for this class of PI4Ks.


Assuntos
1-Fosfatidilinositol 4-Quinase , Trifosfato de Adenosina , 1-Fosfatidilinositol 4-Quinase/química , 1-Fosfatidilinositol 4-Quinase/metabolismo , Ligantes , Trifosfato de Adenosina/metabolismo , Adenina , Relação Estrutura-Atividade , Desenho de Fármacos , Simulação de Acoplamento Molecular
6.
Protein Sci ; 31(9): e4395, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36040262

RESUMO

SARS-CoV-2 nsp10-nsp16 complex is a 2'-O-methyltransferase (MTase) involved in viral RNA capping, enabling the virus to evade the immune system in humans. It has been considered a valuable target in the discovery of antiviral therapeutics, as the RNA cap formation is crucial for viral propagation. Through cross-screening of the inhibitors that we previously reported for SARS-CoV-2 nsp14 MTase activity against nsp10-nsp16 complex, we identified two compounds (SS148 and WZ16) that also inhibited nsp16 MTase activity. To further enable the chemical optimization of these two compounds towards more potent and selective dual nsp14/nsp16 MTase inhibitors, we determined the crystal structure of nsp10-nsp16 in complex with each of SS148 and WZ16. As expected, the structures revealed the binding of both compounds to S-adenosyl-L-methionine (SAM) binding pocket of nsp16. However, our structural data along with the biochemical mechanism of action determination revealed an RNA-dependent SAM-competitive pattern of inhibition for WZ16, clearly suggesting that binding of the RNA first may help the binding of some SAM competitive inhibitors. Both compounds also showed some degree of selectivity against human protein MTases, an indication of great potential for chemical optimization towards more potent and selective inhibitors of coronavirus MTases.


Assuntos
Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , Humanos , Metiltransferases/química , RNA Viral/metabolismo , Proteínas não Estruturais Virais/química
7.
Structure ; 30(8): 1146-1156.e11, 2022 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-35690061

RESUMO

Stimulator of interferon genes (STING) is an adaptor protein of the cGAS-STING signaling pathway involved in the sensing of cytosolic DNA. It functions as a receptor for cyclic dinucleotides (CDNs) and, upon their binding, mediates cytokine expression and host immunity. Besides naturally occurring CDNs, various synthetic CDNs, such as ADU-S100, have been reported to effectively activate STING and are being evaluated in clinical trials for the treatment of cancer. Here, we describe the preparation of a unique new class of STING agonists: isonucleotidic cyclic dinucleotides and the synthesis of their prodrugs. The presented CDNs stimulate STING with comparable efficiency to ADU-S100, whereas their prodrugs demonstrate activity up to four orders of magnitude better due to the improved cellular uptake. The compounds are very potent inducers of inflammatory cytokines by peripheral blood mononuclear cells (PBMCs). We also report the X-ray crystal structure of the lead inhibitor bound to the wild-type (WT) STING.


Assuntos
Nucleotídeos Cíclicos , Pró-Fármacos , Citosol/metabolismo , Leucócitos Mononucleares/metabolismo , Proteínas de Membrana/química , Nucleotídeos Cíclicos/metabolismo , Nucleotídeos Cíclicos/farmacologia
8.
FEBS J ; 289(18): 5571-5598, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35338694

RESUMO

Mycobacteria express enzymes from both the de novo and purine-salvage pathways. However, the regulation of these processes and the roles of individual metabolic enzymes have not been sufficiently detailed. Both Mycobacterium tuberculosis (Mtb) and Mycobacterium smegmatis (Msm) possess three guaB genes, but information is only available on guaB2, which encodes an essential inosine 5'-monophosphate dehydrogenase (IMPDH) involved in de novo purine biosynthesis. This study shows that guaB1, annotated in databases as a putative IMPDH, encodes a guanosine 5'-monophosphate reductase (GMPR), which recycles guanosine monophosphate to inosine monophosphate within the purine-salvage pathway and contains a cystathionine-ß-synthase domain (CBS), which is essential for enzyme activity. GMPR activity is allosterically regulated by the ATP/GTP ratio in a pH-dependent manner. Bioinformatic analysis has indicated the presence of GMPRs containing CBS domains across the entire Actinobacteria phylum.


Assuntos
Cistationina , Mycobacterium tuberculosis , Trifosfato de Adenosina , Cistationina beta-Sintase/genética , GMP Redutase/genética , GMP Redutase/metabolismo , Guanosina Monofosfato/metabolismo , Guanosina Trifosfato , IMP Desidrogenase/genética , IMP Desidrogenase/metabolismo , Inosina , Inosina Monofosfato/metabolismo , Mycobacterium tuberculosis/metabolismo
9.
J Chem Theory Comput ; 17(12): 7397-7405, 2021 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-34797064

RESUMO

Expansion of water vapor through a small orifice to a vacuum produces liquid or frozen clusters which in the experiment serve as model particles for atmospheric aerosols. Yet, there are controversies about the shape of these clusters, suggesting that the nucleation process is not fully understood. Such questions can be answered by molecular dynamics simulations; however, they require microsecond-scale runs with thousands of molecules and accurate energy conservation. The available highly parallel codes typically utilize domain decomposition and are inefficient for heterogeneous systems as clusters in a dilute gas. In this work, we present an implementation of molecular dynamics on graphics processing units based on the Verlet list and apply it to several systems for which experimental data are available. We reproduce sufficiently sized clusters but not the experimentally observed clusters of irregular shape.

10.
J Struct Biol ; 208(2): 92-98, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31415898

RESUMO

RNA-dependent RNA polymerase 3Dpol is a key enzyme for the replication of picornaviruses. The viral genome is translated into a single polyprotein that is subsequently proteolytically processed into matured products. The 3Dpol enzyme arises from a stable 3CD precursor that has high proteolytic activity but no polymerase activity. Upon cleavage of the precursor the newly established N-terminus of 3Dpol is liberated and inserts itself into a pocket on the surface of the 3Dpol enzyme. The essential residue for this mechanism is the very first glycine that is conserved among almost all picornaviruses. However, kobuviruses and siciniviruses have a serine residue instead. Intrigued by this anomaly we sought to solve the crystal structure of these 3Dpol enzymes. The structures revealed a unique fold of the 3Dpol N-termini but the very first serine residues were inserted into a charged pocket in a similar manner as the glycine residue in other picornaviruses. These structures revealed a common underlying mechanism of 3Dpol activation that lies in activation of the α10 helix containing a key catalytical residue Asp238 that forms a hydrogen bond with the 2' hydroxyl group of the incoming NTP nucleotide.


Assuntos
Kobuvirus/enzimologia , Picornaviridae/enzimologia , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/metabolismo , Cristalografia por Raios X , Citometria de Fluxo , Células HeLa , Humanos , Ligação de Hidrogênio , Mutagênese Sítio-Dirigida , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , Proteínas Virais/química , Proteínas Virais/genética
11.
J Chem Theory Comput ; 14(5): 2332-2340, 2018 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-29566335

RESUMO

We develop a methodology for direct molecular-level simulation of adiabatic expansion of gas through a small orifice to a vacuum. The gas attains supersonic speeds, cools, and nucleates. The proposed approach combines equations of frictionless fluid dynamics with molecular dynamics simulation in an expanding periodic box. There are two key components of the proposed algorithm: (i) a time-reversible integrator tailored to an expanding system, and (ii) an iterative procedure employed to satisfy the condition of steady flow. For a conical nozzle (opening angle of 60°), the simulations with argon and water vapor predict cluster sizes in agreement with the experiment. Clusters of irregular shapes observed in the experiment [J. Lengyel et al. Phys. Rev. Lett. 2014, 112, 113401] are not reproduced. The role of friction, turbulence, and sonic boom originating at the sharp nozzle edge is discussed.

12.
Sci Rep ; 7(1): 17309, 2017 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-29230036

RESUMO

Most single stranded plus RNA viruses hijack phosphatidylinositol 4-kinases (PI4Ks) to generate membranes highly enriched in phosphatidylinositol 4-phosphate (PI4P). These membranous compartments known as webs, replication factories or replication organelles are essential for viral replication because they provide protection from the innate intracellular immune response while serving as platforms for viral replication. Using purified recombinant proteins and biomimetic model membranes we show that the nonstructural viral 3A protein is sufficient to promote membrane hyper-phosphorylation given the proper intracellular cofactors (PI4KB and ACBD3). However, our bio-mimetic in vitro reconstitution assay revealed that rather than the presence of PI4P specifically, negative charge alone is sufficient for the recruitment of 3Dpol enzymes to the surface of the lipid bilayer. Additionally, we show that membrane tethered viral 3B protein (also known as Vpg) works in combination with the negative charge to increase the efficiency of membrane recruitment of 3Dpol.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Membrana Celular/metabolismo , Kobuvirus/enzimologia , Proteínas de Membrana/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Infecções por Picornaviridae/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Humanos , Proteínas de Membrana/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Infecções por Picornaviridae/virologia , Proteínas não Estruturais Virais/genética
13.
Sci Rep ; 6: 23641, 2016 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-27009356

RESUMO

Phosphatidylinositol 4-kinase beta (PI4KB) is one of four human PI4K enzymes that generate phosphatidylinositol 4-phosphate (PI4P), a minor but essential regulatory lipid found in all eukaryotic cells. To convert their lipid substrates, PI4Ks must be recruited to the correct membrane compartment. PI4KB is critical for the maintenance of the Golgi and trans Golgi network (TGN) PI4P pools, however, the actual targeting mechanism of PI4KB to the Golgi and TGN membranes is unknown. Here, we present an NMR structure of the complex of PI4KB and its interacting partner, Golgi adaptor protein acyl-coenzyme A binding domain containing protein 3 (ACBD3). We show that ACBD3 is capable of recruiting PI4KB to membranes both in vitro and in vivo, and that membrane recruitment of PI4KB by ACBD3 increases its enzymatic activity and that the ACBD3:PI4KB complex formation is essential for proper function of the Golgi.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Membrana Celular/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Animais , Sítios de Ligação , Células COS , Chlorocebus aethiops , Complexo de Golgi/metabolismo , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Fosfatos de Fosfatidilinositol/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína
14.
Acta Crystallogr D Biol Crystallogr ; 71(Pt 7): 1555-63, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26143926

RESUMO

Phosphatidylinositol 4-phosphate (PI4P) is the most abundant monophosphoinositide in eukaryotic cells. Humans have four phosphatidylinositol 4-kinases (PI4Ks) that synthesize PI4P, among which are PI4K IIß and PI4K IIα. In this study, two crystal structures are presented: the structure of human PI4K IIß and the structure of PI4K IIα containing a nucleoside analogue. The former, a complex with ATP, is the first high-resolution (1.9 Å) structure of a PI4K. These structures reveal new details such as high conformational heterogeneity of the lateral hydrophobic pocket of the C-lobe and together provide a structural basis for isoform-specific inhibitor design.


Assuntos
1-Fosfatidilinositol 4-Quinase/química , Desenho de Fármacos , Nucleosídeos/química , Inibidores de Proteínas Quinases/química , 1-Fosfatidilinositol 4-Quinase/antagonistas & inibidores , 1-Fosfatidilinositol 4-Quinase/metabolismo , Trifosfato de Adenosina/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Humanos , Interações Hidrofóbicas e Hidrofílicas , Nucleosídeos/farmacologia , Conformação Proteica , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Inibidores de Proteínas Quinases/farmacologia
15.
FEBS J ; 280(14): 3436-50, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23678861

RESUMO

Tumour necrosis factor (TNF) related apoptosis inducing ligand (TRAIL), a membrane-bound ligand from the TNF family, has attracted significant attention due to its rather specific and effective ability to induce apoptotic death in various types of cancer cells via binding to and activating its pro-apoptotic death receptors. However, a significant number of primary cancer cells often develop resistance to TRAIL treatment, and the signalling platform behind this phenomenon is not fully understood. Upon blocking endosomal acidification by the vacuolar ATPase (V-ATPase) inhibitors bafilomycin A1 (BafA1) or concanamycin A, we observed a significantly reduced initial sensitivity of several, mainly colorectal, tumour cell lines to TRAIL-induced apoptosis. In cells pretreated with these inhibitors, the TRAIL-induced processing of caspase-8 and the aggregation and trafficking of the TRAIL receptor complexes were temporarily attenuated. Nuclear factor κB or mitogen activated protein/stress kinase signalling from the activated TRAIL receptors remained unchanged, and neither possible lysosomal permeabilization nor acid sphingomyelinase was involved in this process. The cell surface expression of TRAIL receptors and their TRAIL-induced internalization were not affected by V-ATPase inhibitors. The inhibitory effect of BafA1, however, was blunted by knockdown of the caspase-8 inhibitor cFLIP. Altogether, the data obtained provide the first evidence that endosomal acidification could represent an important regulatory node in the proximal part of TRAIL-induced pro-apoptotic signalling.


Assuntos
Antineoplásicos/farmacologia , Caspase 8/metabolismo , Endossomos/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , Apoptose , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Linhagem Celular Tumoral , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Regulação para Baixo , Ativação Enzimática , Humanos , Concentração de Íons de Hidrogênio , Macrolídeos/farmacologia , Transporte Proteico , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esfingolipídeos/fisiologia , Esfingomielina Fosfodiesterase/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo
16.
Oncotarget ; 4(4): 584-99, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23603840

RESUMO

Oncogene addiction describes how cancer cells exhibit dependence on single oncogenes to escape apoptosis and senescence. While oncogene addiction constitutes the basis for new cancer treatment strategies targeting individual kinases and pathways activated by oncogenic mutations, the biochemical basis for this addiction is largely unknown. Here we provide evidence for a metabolic rationale behind the addiction to (V600E)BRAF in two malignant melanoma cell lines. Both cell lines display a striking addiction to glycolysis due to underlying dysfunction of oxidative phosphorylation (OXPHOS). Notably, even minor reductions in glycolytic activity lead to increased OXPHOS activity (reversed Warburg effect), however the mitochondria are unable to sustain ATP production. We show that (V600E)BRAF upholds the activity of glycolysis and therefore the addiction to glycolysis de facto becomes an addiction to (V600E)BRAF. Finally, the senescence response associated with inhibition of (V600E)BRAF is rescued by overexpression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), providing direct evidence that oncogene addiction rests on a metabolic foundation.


Assuntos
Glicólise/genética , Melanoma/genética , Melanoma/metabolismo , Fosforilação Oxidativa , Proteínas Proto-Oncogênicas B-raf/genética , Apoptose/genética , Western Blotting , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Oncogenes , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/fisiologia , Transfecção
17.
Mol Immunol ; 48(12-13): 1439-47, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21501873

RESUMO

Death receptor-6 (DR6) apparently participates in the regulation of T-cell activation and/or activity as its genetic disruption results in enhanced CD4+ T-cell expansion, the production of Th2 cytokines, and interestingly also the compromised migration of CD4+ T cells to sites of inflammation. However, the mechanism of regulation of DR6 expression in cells of the immune system is not fully understood. In this communication we show that DR6 is not expressed in resting T cells from human peripheral blood or murine lymph nodes but that its expression is significantly upregulated in CD3 crosslinking- or PMA/ionomycin-activated T lymphocytes. DR6 expression is transiently increased in both activated human CD4+ and CD8+ T cells and it is apparently dependent on the activation of NF-κB and NF-AT signaling pathways. In contrast to primary peripheral blood T cells, the widely used model lymphoblastic leukemia T-cell line Jurkat is DR6-positive and unexpectedly, TCR-mediated stimulation of Jurkat cells strongly downregulates DR6 expression via suppression of its transcription.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Ativação Linfocitária , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/metabolismo , Receptores do Fator de Necrose Tumoral/genética , Animais , Complexo CD3/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Movimento Celular , Humanos , Interleucinas/biossíntese , Células Jurkat , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Células Th2/imunologia , Células Th2/metabolismo , Regulação para Cima
18.
Biochim Biophys Acta ; 1793(10): 1579-87, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19654028

RESUMO

Death receptor 6 (DR6/TNFRSF21) is a death domain-containing receptor of the TNFR superfamily with an apparent regulatory function in hematopoietic and neuronal cells. In this study we document that DR6 is an extensively posttranslationally modified transmembrane protein and that N- and O-glycosylations of amino acids in its extracellular part are mainly responsible for its approximately 40 kDa mobility shift in SDS polyacrylamide gels. Site-directed mutagenesis confirmed that all six extracellular asparagines are N-glycosylated and that the Ser/Thr/Pro cluster in the "stalk" domain juxtaposed to the cysteine-rich domains (CRDs) is a major site for the likely mucine-type of O-glycosylation. Deletion of the entire linker region between CRDs and the transmembrane domain, spanning over 130 amino acids, severely compromises the plasma membrane localization of DR6 and leads to its intracellular retention. Biosynthetic labeling with radiolabeled palmitate and side-directed mutagenesis also revealed that the membrane-proximal Cys368 in the intracellular part of DR6 is, similarly as cysteines in Fas/CD95 or DR4 ICPs, S-palmitoylated. However, palmitoylation of Cys368 is apparently not required for DR6 targeting into Brij-98 insoluble lipid rafts. In contrast, we show that N-glycosylation of the extracellular part might participate in directing DR6 into these membrane microdomains.


Assuntos
Receptores do Fator de Necrose Tumoral/química , Receptores do Fator de Necrose Tumoral/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Glicosilação , Células HL-60 , Células HeLa , Humanos , Células Jurkat , Lipoilação , Masculino , Microdomínios da Membrana/metabolismo , Peso Molecular , Mutagênese Sítio-Dirigida , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Receptores do Fator de Necrose Tumoral/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência
19.
Apoptosis ; 13(3): 423-36, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18165900

RESUMO

TRAIL, a ligand of the TNFalpha family, induces upon binding to its pro-death receptors TRAIL-R1/DR4 and TRAIL-R2/DR5 the apoptosis of cancer cells. Activated receptors incite the formation of the Death-Inducing Signaling Complex followed by the activation of the downstream apoptotic signaling. TRAIL-induced apoptosis is regulated at multiple levels, one of them being the presence and relative number of TRAIL pro- and anti-apoptotic receptors on the cytoplasmic membrane. In a yeast two-hybrid search for proteins that interact with the intracellular part (ICP) of DR4, we picked ARAP1, an adapter protein with ArfGAP and RhoGAP activities. In yeast, DR4(ICP) interacts with the alternatively spliced ARAP1 lacking 11 amino acids from the PH5 domain. Transfected ARAP1 co-precipitates with DR4 and co-localizes with it in the endoplasmic reticulum/Golgi, at the cytoplasmic membrane and in early endosomes of TRAIL-treated cells. ARAP1 knockdown significantly compromises the localization of DR4 at the cell surface of several tumor cell lines and slows down their TRAIL-induced death. ARAP1 overexpressed in HEL cells does not affect their TRAIL-induced apoptosis or the membrane localization of DR4, but it enhances the cell-surface presentation of phosphatidyl serine. Our data indicate that ARAP1 is likely involved in the regulation of the cell-specific trafficking of DR4 and might thus affect the efficacy of TRAIL-induced apoptosis.


Assuntos
Proteínas de Transporte/fisiologia , Membrana Celular/metabolismo , Proteínas Ativadoras de GTPase/fisiologia , Receptores do Fator de Necrose Tumoral/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , Mapeamento de Interação de Proteínas , Transporte Proteico/fisiologia , RNA Interferente Pequeno/farmacologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Técnicas do Sistema de Duplo-Híbrido
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