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1.
Proc Natl Acad Sci U S A ; 116(45): 22556-22566, 2019 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-31624123

RESUMO

The membrane proximal external region (MPER) of HIV-1 envelope glycoprotein (gp) 41 is an attractive vaccine target for elicitation of broadly neutralizing antibodies (bNAbs) by vaccination. However, current details regarding the quaternary structural organization of the MPER within the native prefusion trimer [(gp120/41)3] are elusive and even contradictory, hindering rational MPER immunogen design. To better understand the structural topology of the MPER on the lipid bilayer, the adjacent transmembrane domain (TMD) was appended (MPER-TMD) and studied. Membrane insertion of the MPER-TMD was sensitive both to the TMD sequence and cytoplasmic residues. Antigen binding of MPER-specific bNAbs, in particular 10E8 and DH511.2_K3, was significantly impacted by the presence of the TMD. Furthermore, MPER-TMD assembly into 10-nm diameter nanodiscs revealed a heterogeneous membrane array comprised largely of monomers and dimers, as enumerated by bNAb Fab binding using single-particle electron microscopy analysis, arguing against preferential trimeric association of native MPER and TMD protein segments. Moreover, introduction of isoleucine mutations in the C-terminal heptad repeat to induce an extended MPER α-helical bundle structure yielded an antigenicity profile of cell surface-arrayed Env variants inconsistent with that found in the native prefusion state. In line with these observations, electron paramagnetic resonance analysis suggested that 10E8 inhibits viral membrane fusion by lifting the MPER N-terminal region out of the viral membrane, mandating the exposure of residues that would be occluded by MPER trimerization. Collectively, our data suggest that the MPER is not a stable trimer, but rather a dynamic segment adapted for structural changes accompanying fusion.


Assuntos
Membrana Celular/virologia , Proteína gp41 do Envelope de HIV/química , HIV-1/imunologia , Anticorpos Neutralizantes/imunologia , Membrana Celular/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp41 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/química , HIV-1/genética , Humanos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/imunologia , Domínios Proteicos
2.
Nat Methods ; 14(1): 49-52, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27869813

RESUMO

We engineered covalently circularized nanodiscs (cNDs) which, compared with standard nanodiscs, exhibit enhanced stability, defined diameter sizes and tunable shapes. Reconstitution into cNDs enhanced the quality of nuclear magnetic resonance spectra for both VDAC-1, a ß-barrel membrane protein, and the G-protein-coupled receptor NTR1, an α-helical membrane protein. In addition, we used cNDs to visualize how simple, nonenveloped viruses translocate their genomes across membranes to initiate infection.


Assuntos
Bicamadas Lipídicas/química , Nanoestruturas/química , Receptores de Neurotensina/metabolismo , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Humanos , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Poliomielite/metabolismo , Poliomielite/virologia , Poliovirus/fisiologia , Internalização do Vírus
3.
J Am Chem Soc ; 140(34): 10639-10643, 2018 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-30094995

RESUMO

Here we present a modular method for manufacturing large-sized nanodiscs using DNA-origami barrels as scaffolding corrals. Large-sized nanodiscs can be produced by first decorating the inside of DNA barrels with small lipid-bilayer nanodiscs, which open up when adding extra lipid to form large nanodiscs of diameters ∼45 or ∼70 nm as prescribed by the enclosing barrel dimension. Densely packed membrane protein arrays are then reconstituted within these large nanodiscs for potential structure determination. Furthermore, we demonstrate the potential of these nanodiscs as model membranes to study poliovirus entry.


Assuntos
DNA/química , Bicamadas Lipídicas/química , Proteínas de Membrana/química , Nanoestruturas/química , Colesterol/química , Humanos , Conformação de Ácido Nucleico , Tamanho da Partícula , Fosfatidilcolinas/química , Fosfatidilgliceróis/química , Complexo de Proteínas do Centro de Reação Fotossintética/química , Poliovirus/fisiologia , Receptores Virais/química , Rhodobacter sphaeroides/química , Internalização do Vírus , Canal de Ânion 1 Dependente de Voltagem/química
4.
Biochemistry ; 53(1): 4-6, 2014 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-24344975

RESUMO

The scintillation proximity assay is a powerful technique for measuring radioligand binding to membrane transporters and has become an integral part of high-throughput drug discovery screening efforts. Here we adapt the method for use with purified LeuT, a prokaryotic secondary transporter, reconstituted into phospholipid bilayer nanodiscs. This application surmounts potential challenges with background interference from endogenously expressed proteins, aggregation and loss of binding activity often accompanying detergent solubilization from native cell membranes, and heterogeneity in size and transporter orientation, where at least some ligand binding sites are inaccessible, associated with reconstitution into lipid vesicles.


Assuntos
Sistemas de Transporte de Aminoácidos Neutros/análise , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Proteínas de Bactérias/análise , Proteínas de Bactérias/metabolismo , Contagem de Cintilação , Ligantes , Bicamadas Lipídicas/metabolismo , Nanoestruturas/química
5.
Acta Pharm Sin B ; 14(6): 2475-2504, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38828160

RESUMO

Resistance to cancer immunotherapy is mainly attributed to poor tumor immunogenicity as well as the immunosuppressive tumor microenvironment (TME) leading to failure of immune response. Numerous therapeutic strategies including chemotherapy, radiotherapy, photodynamic, photothermal, magnetic, chemodynamic, sonodynamic and oncolytic therapy, have been developed to induce immunogenic cell death (ICD) of cancer cells and thereby elicit immunogenicity and boost the antitumor immune response. However, many challenges hamper the clinical application of ICD inducers resulting in modest immunogenic response. Here, we outline the current state of using nanomedicines for boosting ICD of cancer cells. Moreover, synergistic approaches used in combination with ICD inducing nanomedicines for remodeling the TME via targeting immune checkpoints, phagocytosis, macrophage polarization, tumor hypoxia, autophagy and stromal modulation to enhance immunogenicity of dying cancer cells were analyzed. We further highlight the emerging trends of using nanomaterials for triggering amplified ICD-mediated antitumor immune responses. Endoplasmic reticulum localized ICD, focused ultrasound hyperthermia, cell membrane camouflaged nanomedicines, amplified reactive oxygen species (ROS) generation, metallo-immunotherapy, ion modulators and engineered bacteria are among the most innovative approaches. Various challenges, merits and demerits of ICD inducer nanomedicines were also discussed with shedding light on the future role of this technology in improving the outcomes of cancer immunotherapy.

6.
Drug Discov Today ; 28(1): 103434, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36368630

RESUMO

Pancreatic cancer (PC) is a highly aggressive malignant type of cancer. Although immunotherapy has been successfully used for treatment of many cancer types, many challenges limit its success in PC. Therefore, nanomedicines were engineered to enhance the responsiveness of PC cells to immune checkpoint inhibitors (ICIs). In this review, we highlight recent advances in engineering nanomedicines to overcome PC immune resistance. Nanomedicines were used to increase the immunogenicity of PC cells, inactivate stromal cancer-associated fibroblasts (CAFs), enhance the antigen-presenting capacity of dendritic cells (DCs), reverse the highly immunosuppressive nature of the tumor microenvironment (TME), and, hence, improve the infiltration of cytotoxic T lymphocytes (CTLs), resulting in efficient antitumor immune responses.


Assuntos
Neoplasias , Neoplasias Pancreáticas , Humanos , Nanomedicina/métodos , Neoplasias Pancreáticas/tratamento farmacológico , Imunoterapia/métodos , Neoplasias/patologia , Microambiente Tumoral , Neoplasias Pancreáticas
7.
Vaccines (Basel) ; 11(11)2023 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-38005987

RESUMO

The emergence of vaccine-evading SARS-CoV-2 variants urges the need for vaccines that elicit broadly neutralizing antibodies (bnAbs). Here, we assess covalently circularized nanodiscs decorated with recombinant SARS-CoV-2 spike glycoproteins from several variants for eliciting bnAbs with vaccination. Cobalt porphyrin-phospholipid (CoPoP) was incorporated into the nanodisc to allow for anchoring and functional orientation of spike trimers on the nanodisc surface through their His-tag. Monophosphoryl-lipid (MPLA) and QS-21 were incorporated as immunostimulatory adjuvants to enhance vaccine responses. Following optimization of nanodisc assembly, spike proteins were effectively displayed on the surface of the nanodiscs and maintained their conformational capacity for binding with human angiotensin-converting enzyme 2 (hACE2) as verified using electron microscopy and slot blot assay, respectively. Six different formulations were prepared where they contained mono antigens; four from the year 2020 (WT, Beta, Lambda, and Delta) and two from the year 2021 (Omicron BA.1 and BA.2). Additionally, we prepared a mosaic nanodisc displaying the four spike proteins from year 2020. Intramuscular vaccination of CD-1 female mice with the mosaic nanodisc induced antibody responses that not only neutralized matched pseudo-typed viruses, but also neutralized mismatched pseudo-typed viruses corresponding to later variants from year 2021 (Omicron BA.1 and BA.2). Interestingly, sera from mosaic-immunized mice did not effectively inhibit Omicron spike binding to human ACE-2, suggesting that some of the elicited antibodies were directed towards conserved neutralizing epitopes outside the receptor binding domain. Our results show that mosaic nanodisc vaccine displaying spike proteins from 2020 can elicit broadly neutralizing antibodies that can neutralize mismatched viruses from a following year, thus decreasing immune evasion of new emerging variants and enhancing healthcare preparedness.

8.
Front Chem ; 10: 1047874, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36844038

RESUMO

Lipid-bilayer nanodiscs and liposomes have been developed to stabilize membrane proteins in order to study their structures and functions. Nanodiscs are detergent-free, water-soluble, and size-controlled planar phospholipid-bilayer platforms. On the other hand, liposomes are curved phospholipid-bilayer spheres with an aqueous core used as drug delivery systems and model membrane platforms for studying cellular activities. A long-standing challenge is the generation of a homogenous and monodispersed lipid-bilayer system with a very wide range of dimensions and curvatures (elongation, bending, and twisting). A DNA-origami template provides a way to control the shapes, sizes, and arrangements of lipid bilayers via enforcing the assembly of lipid bilayers within the cavities created by DNA nanostructures. Here, we provide a concise overview and discuss how to design planar and curved lipid-bilayer membranes by using DNA-origami nanostructures as templates. Finally, we will discuss the potential applications of DNA-origami nanostructures in the structural and functional studies of large membrane proteins and their complexes.

9.
medRxiv ; 2022 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-32995803

RESUMO

SARS-CoV-2 precipitates respiratory distress by infection of airway epithelial cells and is often accompanied by acute kidney injury. We report that Kidney Injury Molecule-1/T cell immunoglobulin mucin domain 1 (KIM-1/TIM-1) is expressed in lung and kidney epithelial cells in COVID-19 patients and is a receptor for SARS-CoV-2. Human and mouse lung and kidney epithelial cells express KIM-1 and endocytose nanoparticles displaying the SARS-CoV-2 spike protein (virosomes). Uptake was inhibited by anti-KIM-1 antibodies and TW-37, a newly discovered inhibitor of KIM-1-mediated endocytosis. Enhanced KIM-1 expression by human kidney tubuloids increased uptake of virosomes. KIM-1 binds to the SARS-CoV-2 Spike protein in vitro . KIM-1 expressing cells, not expressing angiotensin-converting enzyme 2 (ACE2), are permissive to SARS-CoV-2 infection. Thus, KIM-1 is an alternative receptor to ACE2 for SARS-CoV-2. KIM-1 targeted therapeutics may prevent and/or treat COVID-19.

10.
Nat Struct Mol Biol ; 28(3): 258-267, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33633398

RESUMO

G-protein-coupled receptors (GPCRs) are the largest superfamily of transmembrane proteins and the targets of over 30% of currently marketed pharmaceuticals. Although several structures have been solved for GPCR-G protein complexes, few are in a lipid membrane environment. Here, we report cryo-EM structures of complexes of neurotensin, neurotensin receptor 1 and Gαi1ß1γ1 in two conformational states, resolved to resolutions of 4.1 and 4.2 Å. The structures, determined in a lipid bilayer without any stabilizing antibodies or nanobodies, reveal an extended network of protein-protein interactions at the GPCR-G protein interface as compared to structures obtained in detergent micelles. The findings show that the lipid membrane modulates the structure and dynamics of complex formation and provide a molecular explanation for the stronger interaction between GPCRs and G proteins in lipid bilayers. We propose an allosteric mechanism for GDP release, providing new insights into the activation of G proteins for downstream signaling.


Assuntos
Microscopia Crioeletrônica , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/ultraestrutura , Bicamadas Lipídicas , Nanoestruturas/química , Receptores de Neurotensina/metabolismo , Receptores de Neurotensina/ultraestrutura , Regulação Alostérica , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/ultraestrutura , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/ultraestrutura , Subunidades gama da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/ultraestrutura , Guanosina Difosfato/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/química , Humanos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Micelas , Modelos Moleculares , Neurotensina/química , Neurotensina/metabolismo , Conformação Proteica , Receptores de Neurotensina/química , Transdução de Sinais
11.
Curr Opin Struct Biol ; 60: 150-156, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32066086

RESUMO

Covalently circularized and DNA-corralled nanodisc technologies have enabled engineering of large-sized bilayer nanodiscs up to 90nm. These large nanodiscs have the potential to extend the applicability of nanodisc technology from studying small and medium-sized membrane proteins to acting as surrogate membranes to investigate functional and structural aspects of viral entry. Here, we discuss the recent technical developments leading to construction of large circularized and DNA-corralled nanodiscs and examine their application in viral entry.


Assuntos
Nanoestruturas/química , Nanotecnologia/métodos , Internalização do Vírus , DNA/química , Humanos , Proteínas de Membrana/metabolismo
12.
Artigo em Inglês | MEDLINE | ID: mdl-32596222

RESUMO

Phospho-lipid bilayer nanodiscs have gathered much scientific interest as a stable and tunable membrane mimetic for the study of membrane proteins. Until recently the size of the nanodiscs that could be produced was limited to ~ 16 nm. Recent advances in nanodisc engineering such as covalently circularized nanodiscs (cND) and DNA corralled nanodiscs (DCND) have opened up the possibility of engineering nanodiscs of size up to 90 nm. This enables widening the application of nanodiscs from single membrane proteins to investigating large protein complexes and biological processes such as virus-membrane fusion and synaptic vesicle fusion. Another aspect of exploiting the large available surface area of these novel nanodiscs could be to engineer more realistic membrane mimetic systems with features such as membrane asymmetry and curvature. In this review, we discuss the recent technical developments in nanodisc technology leading to construction of large nanodiscs and examine some of the implicit applications.

13.
Sci Rep ; 10(1): 18531, 2020 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-33116203

RESUMO

Inhibition of human Monoacylglycerol Lipase (hMGL) offers a novel approach for treating neurological diseases. The design of inhibitors, targeting active-inactive conformational transitions of the enzyme, can be aided by understanding the interplay between structure and dynamics. Here, we report the effects of mutations within the catalytic triad on structure, conformational gating and dynamics of hMGL by combining kinetics, NMR, and HDX-MS data with metadynamics simulations. We found that point mutations alter delicate conformational equilibria between active and inactive states. HDX-MS reveals regions of the hMGL that become substantially more dynamic upon substitution of catalytic acid Asp-239 by alanine. These regions, located far from the catalytic triad, include not only loops but also rigid α-helixes and ß-strands, suggesting their involvement in allosteric regulation as channels for long-range signal transmission. The results identify the existence of a preorganized global communication network comprising of tertiary (residue-residue contacts) and quaternary (rigid-body contacts) networks that mediate robust, rapid intraprotein signal transmission. Catalytic Asp-239 controls hMGL allosteric communications and may be considered as an essential residue for the integration and transmission of information to enzymes' remote regions, in addition to its well-known role to facilitate Ser-122 activation. Our findings may assist in the identification of new druggable sites in hMGL.


Assuntos
Monoacilglicerol Lipases/genética , Monoacilglicerol Lipases/metabolismo , Monoacilglicerol Lipases/fisiologia , Regulação Alostérica , Catálise , Humanos , Cinética , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutação , Mutação de Sentido Incorreto , Conformação Proteica , Relação Estrutura-Atividade
15.
Curr Opin Struct Biol ; 51: 129-134, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29677570

RESUMO

Covalently circularized nanodiscs (cNDs) represent a significant advance in the durability and applicability of nanodisc technology. The new cNDs demonstrate higher size homogeneity and improved stability compared with that of non-circularized forms. Moreover, cNDs can be prepared at various defined sizes up to 80-nm diameter. The large cNDs can house much larger membrane proteins and their complexes than was previously possible with the conventional nanodiscs. In order to experience the full advantages of covalent circularization, high quality circularized scaffold protein and nanodisc samples are needed. Here, we give a concise overview and discuss the technical challenges that needed to be overcome in order to obtain high quality preparations. Furthermore, we review some potential new applications for the cNDs.


Assuntos
Nanoestruturas/química , Nanotecnologia , Bicamadas Lipídicas/química , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Ligação Proteica , Multimerização Proteica
16.
Nat Protoc ; 13(1): 79-98, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29215632

RESUMO

Suitable membrane mimetics are crucial to the performance of structural and functional studies of membrane proteins. Phospholipid nanodiscs (formed when a membrane scaffold protein encircles a small portion of a lipid bilayer) have native-like membrane properties. These have been used for a variety of functional studies, but structural studies by high-resolution solution-state NMR spectroscopy of membrane proteins in commonly used nanodiscs of 10-nm diameter were limited by the high molecular weight of these particles, which caused unfavorably large NMR line widths. We have recently constructed truncated versions of the membrane scaffold protein, allowing the preparation of a range of stepwise-smaller nanodiscs (6- to 8-nm diameter) to overcome this limitation. Here, we present a protocol on the assembly of phospholipid nanodiscs of various sizes for structural studies of membrane proteins with solution-state NMR spectroscopy. We describe specific isotope-labeling schemes required for working with large membrane protein systems in nanodiscs, and provide guidelines on the setup of NMR non-uniform sampling (NUS) data acquisition and high-resolution NMR spectra reconstruction. We discuss critical points and pitfalls relating to optimization of nanodiscs for NMR spectroscopy and outline a strategy for the high-resolution structure determination and positioning of isotope-labeled membrane proteins in nanodiscs using nuclear Overhauser enhancement spectroscopy (NOESY) spectroscopy, residual dipolar couplings (RDCs) and paramagnetic relaxation enhancements (PREs). Depending on the target protein of interest, nanodisc assembly and purification can be achieved within 12-24 h. Although the focus of this protocol is on protein NMR, these nanodiscs can also be used for (cryo-) electron microscopy (EM) and small-angle X-ray and neutron-scattering studies.


Assuntos
Bicamadas Lipídicas/química , Proteínas de Membrana , Nanoestruturas/química , Ressonância Magnética Nuclear Biomolecular/métodos , Fosfolipídeos/química , Proteínas de Membrana/análise , Proteínas de Membrana/química , Modelos Moleculares , Tamanho da Partícula
17.
Sci Rep ; 8(1): 1719, 2018 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-29379013

RESUMO

An understanding of how conformational dynamics modulates function and catalysis of human monoacylglycerol lipase (hMGL), an important pharmaceutical target, can facilitate the development of novel ligands with potential therapeutic value. Here, we report the discovery and characterization of an allosteric, regulatory hMGL site comprised of residues Trp-289 and Leu-232 that reside over 18 Å away from the catalytic triad. These residues were identified as critical mediators of long-range communication and as important contributors to the integrity of the hMGL structure. Nonconservative replacements of Trp-289 or Leu-232 triggered concerted motions of structurally distinct regions with a significant conformational shift toward inactive states and dramatic loss in catalytic efficiency of the enzyme. Using a multimethod approach, we show that the dynamically relevant Trp-289 and Leu-232 residues serve as communication hubs within an allosteric protein network that controls signal propagation to the active site, and thus, regulates active-inactive interconversion of hMGL. Our findings provide new insights into the mechanism of allosteric regulation of lipase activity, in general, and may provide alternative drug design possibilities.


Assuntos
Monoacilglicerol Lipases/genética , Monoacilglicerol Lipases/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Regulação Alostérica , Substituição de Aminoácidos , Análise Mutacional de DNA , Humanos , Modelos Moleculares , Monoacilglicerol Lipases/química , Proteínas Mutantes/química , Conformação Proteica
18.
Cell Rep ; 18(11): 2651-2663, 2017 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-28297669

RESUMO

During eukaryotic translation initiation, eIF3 binds the solvent-accessible side of the 40S ribosome and recruits the gate-keeper protein eIF1 and eIF5 to the decoding center. This is largely mediated by the N-terminal domain (NTD) of eIF3c, which can be divided into three parts: 3c0, 3c1, and 3c2. The N-terminal part, 3c0, binds eIF5 strongly but only weakly to the ribosome-binding surface of eIF1, whereas 3c1 and 3c2 form a stoichiometric complex with eIF1. 3c1 contacts eIF1 through Arg-53 and Leu-96, while 3c2 faces 40S protein uS15/S13, to anchor eIF1 to the scanning pre-initiation complex (PIC). We propose that the 3c0:eIF1 interaction diminishes eIF1 binding to the 40S, whereas 3c0:eIF5 interaction stabilizes the scanning PIC by precluding this inhibitory interaction. Upon start codon recognition, interactions involving eIF5, and ultimately 3c0:eIF1 association, facilitate eIF1 release. Our results reveal intricate molecular interactions within the PIC, programmed for rapid scanning-arrest at the start codon.


Assuntos
Fator de Iniciação 3 em Eucariotos/química , Fator de Iniciação 3 em Eucariotos/metabolismo , Fator de Iniciação 5 em Eucariotos/metabolismo , Iniciação Traducional da Cadeia Peptídica , RNA Mensageiro/metabolismo , Ribossomos/química , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Fator de Iniciação 1 em Eucariotos/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Mutação/genética , Ligação Proteica , Subunidades Proteicas/metabolismo , RNA Mensageiro/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
19.
Nat Commun ; 7: 12752, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27629921

RESUMO

Recent studies demonstrated that chitinase 3-like-1 (Chi3l1) binds to and signals via IL-13Rα2. However, the mechanism that IL-13Rα2 uses to mediate the effects of Chi3l1 has not been defined. Here, we demonstrate that the membrane protein, TMEM219, is a binding partner of IL-13Rα2 using yeast two-hybrid, co-immunoprecipitation, co-localization and bimolecular fluorescence complementation assays. Furthermore, fluorescence anisotropy nanodisc assays revealed a direct physical interaction between TMEM219 and IL-13Rα2-Chi3l1 complexes. Null mutations or siRNA silencing of TMEM219 or IL-13Rα2 similarly decreased Chi3l1-stimulated epithelial cell HB-EGF production and macrophage MAPK/Erk and PKB/Akt activation. Null mutations of TMEM219 or IL-13Rα2 also phenocopied one another as regards the ability of Chi3l1 to inhibit oxidant-induced apoptosis and lung injury, promote melanoma metastasis and stimulate TGF-ß1. TMEM219 also contributed to the decoy function of IL-13Rα2. These studies demonstrate that TMEM219 plays a critical role in Chi3l1-induced IL-13Rα2 mediated signalling and tissue responses.


Assuntos
Proteína 1 Semelhante à Quitinase-3/metabolismo , Subunidade alfa2 de Receptor de Interleucina-13/metabolismo , Proteínas de Membrana/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/metabolismo , Humanos , Lesão Pulmonar/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Sistema de Sinalização das MAP Quinases , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos Endogâmicos C57BL , Metástase Neoplásica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Via de Sinalização Wnt
20.
Protein Sci ; 22(6): 774-87, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23553709

RESUMO

The membrane-associated serine hydrolase, monoacylglycerol lipase (MGL), is a well-recognized therapeutic target that regulates endocannabinoid signaling. Crystallographic studies, while providing structural information about static MGL states, offer no direct experimental insight into the impact of MGL's membrane association upon its structure-function landscape. We report application of phospholipid bilayer nanodiscs as biomembrane models with which to evaluate the effect of a membrane system on the catalytic properties and conformational dynamics of human MGL (hMGL). Anionic and charge-neutral phospholipid bilayer nanodiscs enhanced hMGL's kinetic properties [apparent maximum velocity (Vmax) and substrate affinity (Km)]. Hydrogen exchange mass spectrometry (HX MS) was used as a conformational analysis method to profile experimentally the extent of hMGL-nanodisc interaction and its impact upon hMGL structure. We provide evidence that significant regions of hMGL lid-domain helix α4 and neighboring helix α6 interact with the nanodisc phospholipid bilayer, anchoring hMGL in a more open conformation to facilitate ligand access to the enzyme's substrate-binding channel. Covalent modification of membrane-associated hMGL by the irreversible carbamate inhibitor, AM6580, shielded the active site region, but did not increase solvent exposure of the lid domain, suggesting that the inactive, carbamylated enzyme remains intact and membrane associated. Molecular dynamics simulations generated conformational models congruent with the open, membrane-associated topology of active and inhibited, covalently-modified hMGL. Our data indicate that hMGL interaction with a phospholipid membrane bilayer induces regional changes in the enzyme's conformation that favor its recruiting lipophilic substrate/inhibitor from membrane stores to the active site via the lid, resulting in enhanced hMGL catalytic activity and substrate affinity.


Assuntos
Bicamadas Lipídicas/metabolismo , Monoacilglicerol Lipases/química , Monoacilglicerol Lipases/metabolismo , Fosfolipídeos/metabolismo , Humanos , Cinética , Bicamadas Lipídicas/química , Espectrometria de Massas , Simulação de Dinâmica Molecular , Monoacilglicerol Lipases/antagonistas & inibidores , Fosfolipídeos/química , Conformação Proteica/efeitos dos fármacos
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