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1.
Signal Transduct Target Ther ; 5(1): 220, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33024075
2.
Mol Cell ; 80(1): 114-126.e8, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32916094

RESUMO

DNA replication is carried out by a multi-protein machine called the replisome. In Saccharomyces cerevisiae, the replisome is composed of over 30 different proteins arranged into multiple subassemblies, each performing distinct activities. Synchrony of these activities is required for efficient replication and preservation of genomic integrity. How this is achieved is particularly puzzling at the lagging strand, where current models of the replisome architecture propose turnover of the canonical lagging strand polymerase, Pol δ, at every cycle of Okazaki fragment synthesis. Here, we established single-molecule fluorescence microscopy protocols to study the binding kinetics of individual replisome subunits in live S. cerevisiae. Our results show long residence times for most subunits at the active replisome, supporting a model where all subassemblies bind tightly and work in a coordinated manner for extended periods, including Pol δ, redefining the architecture of the active eukaryotic replisome.


Assuntos
Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Células Eucarióticas/metabolismo , Complexos Multienzimáticos/metabolismo , Núcleo Celular/metabolismo , Cinética , Modelos Biológicos , Proteínas Nucleares/metabolismo , Subunidades Proteicas/metabolismo , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula , Fatores de Tempo
3.
Nat Commun ; 11(1): 4370, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32873792

RESUMO

BRAF kinase, a critical effector of the ERK signaling pathway, is hyperactivated in many cancers. Oncogenic BRAFV600E signals as an active monomer in the absence of active RAS, however, in many tumors BRAF dimers mediate ERK signaling. FDA-approved RAF inhibitors poorly inhibit BRAF dimers, which leads to tumor resistance. We found that Ponatinib, an FDA-approved drug, is an effective inhibitor of BRAF monomers and dimers. Ponatinib binds the BRAF dimer and stabilizes a distinct αC-helix conformation through interaction with a previously unrevealed allosteric site. Using these structural insights, we developed PHI1, a BRAF inhibitor that fully uncovers the allosteric site. PHI1 exhibits discrete cellular selectivity for BRAF dimers, with enhanced inhibition of the second protomer when the first protomer is occupied, comprising a novel class of dimer selective inhibitors. This work shows that Ponatinib and BRAF dimer selective inhibitors will be useful in treating BRAF-dependent tumors.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Sítio Alostérico/efeitos dos fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Cristalografia por Raios X , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Sistema de Sinalização das MAP Quinases/genética , Simulação de Acoplamento Molecular , Mutação , Neoplasias/genética , Neoplasias/patologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/uso terapêutico , Multimerização Proteica/efeitos dos fármacos , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas B-raf/ultraestrutura , Piridazinas/farmacologia , Piridazinas/uso terapêutico , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade
4.
Nat Commun ; 11(1): 4245, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32843629

RESUMO

Diheme-containing succinate:menaquinone oxidoreductases (Sdh) are widespread in Gram-positive bacteria but little is known about the catalytic mechanisms they employ for succinate oxidation by menaquinone. Here, we present the 2.8 Å cryo-electron microscopy structure of a Mycobacterium smegmatis Sdh, which forms a trimer. We identified the membrane-anchored SdhF as a subunit of the complex. The 3 kDa SdhF forms a single transmembrane helix and this helix plays a role in blocking the canonically proximal quinone-binding site. We also identified two distal quinone-binding sites with bound quinones. One distal binding site is formed by neighboring subunits of the complex. Our structure further reveals the electron/proton transfer pathway for succinate oxidation by menaquinone. Moreover, this study provides further structural insights into the physiological significance of a trimeric respiratory complex II. The structure of the menaquinone binding site could provide a framework for the development of Sdh-selective anti-mycobacterial drugs.


Assuntos
Proteínas de Bactérias/química , Mycobacterium smegmatis/enzimologia , Succinato Desidrogenase/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Catálise , Microscopia Crioeletrônica , Transporte de Elétrons , Modelos Moleculares , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Mycobacterium smegmatis/química , Oxirredução , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Relação Estrutura-Atividade , Succinato Desidrogenase/metabolismo , Ácido Succínico/metabolismo , Vitamina K 2/metabolismo
5.
Proc Natl Acad Sci U S A ; 117(29): 17418-17428, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32636267

RESUMO

Carboxysomes are membrane-free organelles for carbon assimilation in cyanobacteria. The carboxysome consists of a proteinaceous shell that structurally resembles virus capsids and internal enzymes including ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the primary carbon-fixing enzyme in photosynthesis. The formation of carboxysomes requires hierarchical self-assembly of thousands of protein subunits, initiated from Rubisco assembly and packaging to shell encapsulation. Here we study the role of Rubisco assembly factor 1 (Raf1) in Rubisco assembly and carboxysome formation in a model cyanobacterium, Synechococcus elongatus PCC7942 (Syn7942). Cryo-electron microscopy reveals that Raf1 facilitates Rubisco assembly by mediating RbcL dimer formation and dimer-dimer interactions. Syn7942 cells lacking Raf1 are unable to form canonical intact carboxysomes but generate a large number of intermediate assemblies comprising Rubisco, CcaA, CcmM, and CcmN without shell encapsulation and a low abundance of carboxysome-like structures with reduced dimensions and irregular shell shapes and internal organization. As a consequence, the Raf1-depleted cells exhibit reduced Rubisco content, CO2-fixing activity, and cell growth. Our results provide mechanistic insight into the chaperone-assisted Rubisco assembly and biogenesis of carboxysomes. Advanced understanding of the biogenesis and stepwise formation process of the biogeochemically important organelle may inform strategies for heterologous engineering of functional CO2-fixing modules to improve photosynthesis.


Assuntos
Organelas/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Synechococcus/metabolismo , Carbono/metabolismo , Ciclo do Carbono , Microscopia Crioeletrônica , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Fotossíntese , Subunidades Proteicas/metabolismo , Ribulose-Bifosfato Carboxilase/química , Ribulose-Bifosfato Carboxilase/genética , Synechococcus/genética , Transcriptoma
6.
PLoS Pathog ; 16(7): e1008715, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32716968

RESUMO

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels mostly located in the post-synaptic membrane of cholinergic synapses. The natural neurotransmitter is acetylcholine, but they are also the direct targets for neonicotinoids, chemicals widely used against ectoparasites, arthropod vectors and agricultural pests. There are significant concerns regarding adverse effects of neonicotinoids on beneficial insects. In arthropods, functional nAChRs made of α subunits have been expressed from Drosophila genes, and hybrid receptors (sometimes also referred to as chimeric receptors) using species-specific α subunits and vertebrate ß subunits have been expressed ex-vivo. Arthropod-specific nAChRs made of both α and ß subunits from the target species have not been expressed ex-vivo. The aim of the current study was to express such receptors in Xenopus oocytes using only genes from Lepeophtheirus salmonis, to characterize them and study their modulation. Genes encoding α and ß subunits of the nAChRs and three ancillary proteins, RIC-3, UNC-50 and UNC-74 were identified in the L. salmonis genome, subjected to RACE-PCR, cloned into an expression vector and the cRNA produced was then injected into Xenopus laevis oocytes. Co-expression of the ancillary proteins was essential for the successful expression of the L. salmonis nAChRs with both α and ß subunits. Two functional nAChRs were identified: Lsa-nAChR1 consisting of α1, α2, ß1 and ß2 subunits, reconstituted to one distinct receptor, while Lsa-nAChR2, consisting of α3, ß1 and ß2 subunits reconstitutes receptors with two distinct characteristics. Out of seven neonicotinoids tested, six worked as partial agonist of Lsa-nAChR1 while only three did so for Lsa-nAChR2. Four non-neonicotinoid compounds tested had no effect on either of the nAChRs. The study demonstrated that fully functional, non-hybrid nAChRs containing both α and ß subunits from an arthropod can be reconstituted ex-vivo by co-expression of essential ancillary proteins. Such models would be valuable for in-depth studies of effects by neonicotinoids and other compounds on target pests, as well as for studies of adverse effects on non-target arthropods.


Assuntos
Copépodes/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Copépodes/efeitos dos fármacos , Inseticidas/farmacologia , Neonicotinoides/farmacologia , Subunidades Proteicas/metabolismo , Receptores Nicotínicos/efeitos dos fármacos , Xenopus laevis
7.
Proc Natl Acad Sci U S A ; 117(29): 17011-17018, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32636271

RESUMO

Few antibiotics are effective against Acinetobacter baumannii, one of the most successful pathogens responsible for hospital-acquired infections. Resistance to chlorhexidine, an antiseptic widely used to combat A. baumannii, is effected through the proteobacterial antimicrobial compound efflux (PACE) family. The prototype membrane protein of this family, AceI (Acinetobacter chlorhexidine efflux protein I), is encoded for by the aceI gene and is under the transcriptional control of AceR (Acinetobacter chlorhexidine efflux protein regulator), a LysR-type transcriptional regulator (LTTR) protein. Here we use native mass spectrometry to probe the response of AceI and AceR to chlorhexidine assault. Specifically, we show that AceI forms dimers at high pH, and that binding to chlorhexidine facilitates the functional form of the protein. Changes in the oligomerization of AceR to enable interaction between RNA polymerase and promoter DNA were also observed following chlorhexidine assault. Taken together, these results provide insight into the assembly of PACE family transporters and their regulation via LTTR proteins on drug recognition and suggest potential routes for intervention.


Assuntos
Acinetobacter baumannii , Antibacterianos , Proteínas de Bactérias , Clorexidina , Proteínas de Membrana Transportadoras , Acinetobacter baumannii/química , Acinetobacter baumannii/enzimologia , Antibacterianos/química , Antibacterianos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Clorexidina/química , Clorexidina/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Resistência Microbiana a Medicamentos , Concentração de Íons de Hidrogênio , Espectrometria de Massas , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Ligação Proteica , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo
8.
Nat Commun ; 11(1): 3583, 2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32681005

RESUMO

The phosphatases PP1 and PP2A are responsible for the majority of dephosphorylation reactions on phosphoserine (pSer) and phosphothreonine (pThr), and are involved in virtually all cellular processes and numerous diseases. The catalytic subunits exist in cells in form of holoenzymes, which impart substrate specificity. The contribution of the catalytic subunits to the recognition of substrates is unclear. By developing a phosphopeptide library approach and a phosphoproteomic assay, we demonstrate that the specificity of PP1 and PP2A holoenzymes towards pThr and of PP1 for basic motifs adjacent to the phosphorylation site are due to intrinsic properties of the catalytic subunits. Thus, we dissect this amino acid specificity of the catalytic subunits from the contribution of regulatory proteins. Furthermore, our approach enables discovering a role for PP1 as regulator of the GRB-associated-binding protein 2 (GAB2)/14-3-3 complex. Beyond this, we expect that this approach is broadly applicable to detect enzyme-substrate recognition preferences.


Assuntos
Proteína Fosfatase 1/química , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 2/química , Proteína Fosfatase 2/metabolismo , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Motivos de Aminoácidos , Domínio Catalítico , Holoenzimas/química , Holoenzimas/genética , Holoenzimas/metabolismo , Humanos , Fosforilação , Ligação Proteica , Engenharia de Proteínas , Proteína Fosfatase 1/genética , Proteína Fosfatase 2/genética , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Especificidade por Substrato
9.
Proc Natl Acad Sci U S A ; 117(28): 16373-16382, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601233

RESUMO

In photosynthetic reaction centers from purple bacteria (PbRC) and the water-oxidizing enzyme, photosystem II (PSII), charge separation occurs along one of the two symmetrical electron-transfer branches. Here we report the microscopic origin of the unidirectional charge separation, fully considering electron-hole interaction, electronic coupling of the pigments, and electrostatic interaction with the polarizable entire protein environments. The electronic coupling between the pair of bacteriochlorophylls is large in PbRC, forming a delocalized excited state with the lowest excitation energy (i.e., the special pair). The charge-separated state in the active branch is stabilized by uncharged polar residues in the transmembrane region and charged residues on the cytochrome c 2 binding surface. In contrast, the accessory chlorophyll in the D1 protein (ChlD1) has the lowest excitation energy in PSII. The charge-separated state involves ChlD1 •+ and is stabilized predominantly by charged residues near the Mn4CaO5 cluster and the proceeding proton-transfer pathway. It seems likely that the acquirement of water-splitting ability makes ChlD1 the initial electron donor in PSII.


Assuntos
Elétrons , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Água/metabolismo , Aminoácidos , Bacterioclorofilas/química , Bacterioclorofilas/metabolismo , Transporte de Elétrons , Oxigênio/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/química , Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteobactérias/metabolismo , Água/química
10.
Proc Natl Acad Sci U S A ; 117(28): 16283-16291, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32611810

RESUMO

The difficulty of achieving robust functional expression of insect nicotinic acetylcholine receptors (nAChRs) has hampered our understanding of these important molecular targets of globally deployed neonicotinoid insecticides at a time when concerns have grown regarding the toxicity of this chemotype to insect pollinators. We show that thioredoxin-related transmembrane protein 3 (TMX3) is essential to enable robust expression in Xenopus laevis oocytes of honeybee (Apis mellifera) and bumblebee (Bombus terrestris) as well as fruit fly (Drosophila melanogaster) nAChR heteromers targeted by neonicotinoids and not hitherto robustly expressed. This has enabled the characterization of picomolar target site actions of neonicotinoids, findings important in understanding their toxicity.


Assuntos
Proteínas de Insetos/metabolismo , Inseticidas/farmacologia , Neonicotinoides/farmacologia , Agonistas Nicotínicos/farmacologia , Receptores Nicotínicos/metabolismo , Acetilcolina/farmacologia , Animais , Abelhas/metabolismo , Relação Dose-Resposta a Droga , Drosophila melanogaster/metabolismo , Proteínas de Insetos/agonistas , Proteínas de Insetos/genética , Oócitos/metabolismo , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptores Nicotínicos/genética , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Xenopus laevis
11.
Nat Commun ; 11(1): 2866, 2020 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-32513959

RESUMO

The Cullin 5 (CUL5) Ring E3 ligase uses adaptors Elongins B and C (ELOB/C) to bind different SOCS-box-containing substrate receptors, determining the substrate specificity of the ligase. The 18-member ankyrin and SOCS box (ASB) family is the largest substrate receptor family. Here we report cryo-EM data for the substrate, creatine kinase (CKB) bound to ASB9-ELOB/C, and for full-length CUL5 bound to the RING protein, RBX2, which binds various E2s. To date, no full structures are available either for a substrate-bound ASB nor for CUL5. Hydrogen-deuterium exchange (HDX-MS) mapped onto a full structural model of the ligase revealed long-range allostery extending from the substrate through CUL5. We propose a revised allosteric mechanism for how CUL-E3 ligases function. ASB9 and CUL5 behave as rigid rods, connected through a hinge provided by ELOB/C transmitting long-range allosteric crosstalk from the substrate through CUL5 to the RBX2 flexible linker.


Assuntos
Creatina Quinase/metabolismo , Microscopia Crioeletrônica , Elonguina/metabolismo , Proteínas Supressoras da Sinalização de Citocina/química , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Regulação Alostérica , Creatina Quinase/ultraestrutura , Proteínas Culina/química , Proteínas Culina/metabolismo , Elonguina/ultraestrutura , Humanos , Modelos Moleculares , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Proteínas Supressoras da Sinalização de Citocina/ultraestrutura , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo
12.
Nature ; 584(7820): 304-309, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32581365

RESUMO

The human GABAB receptor-a member of the class C family of G-protein-coupled receptors (GPCRs)-mediates inhibitory neurotransmission and has been implicated in epilepsy, pain and addiction1. A unique GPCR that is known to require heterodimerization for function2-6, the GABAB receptor has two subunits, GABAB1 and GABAB2, that are structurally homologous but perform distinct and complementary functions. GABAB1 recognizes orthosteric ligands7,8, while GABAB2 couples with G proteins9-14. Each subunit is characterized by an extracellular Venus flytrap (VFT) module, a descending peptide linker, a seven-helix transmembrane domain and a cytoplasmic tail15. Although the VFT heterodimer structure has been resolved16, the structure of the full-length receptor and its transmembrane signalling mechanism remain unknown. Here we present a near full-length structure of the GABAB receptor, captured in an inactive state by cryo-electron microscopy. Our structure reveals several ligands that preassociate with the receptor, including two large endogenous phospholipids that are embedded within the transmembrane domains to maintain receptor integrity and modulate receptor function. We also identify a previously unknown heterodimer interface between transmembrane helices 3 and 5 of both subunits, which serves as a signature of the inactive conformation. A unique 'intersubunit latch' within this transmembrane interface maintains the inactive state, and its disruption leads to constitutive receptor activity.


Assuntos
Microscopia Crioeletrônica , Receptores de GABA-B/química , Receptores de GABA-B/ultraestrutura , Cálcio/metabolismo , Etanolaminas/química , Etanolaminas/metabolismo , Humanos , Ligantes , Modelos Moleculares , Fosforilcolina/química , Fosforilcolina/metabolismo , Domínios Proteicos , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Receptores de GABA-B/metabolismo , Relação Estrutura-Atividade
13.
Nature ; 584(7821): 475-478, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32494008

RESUMO

The endoplasmic reticulum (ER) membrane complex (EMC) cooperates with the Sec61 translocon to co-translationally insert a transmembrane helix (TMH) of many multi-pass integral membrane proteins into the ER membrane, and it is also responsible for inserting the TMH of some tail-anchored proteins1-3. How EMC accomplishes this feat has been unclear. Here we report the first, to our knowledge, cryo-electron microscopy structure of the eukaryotic EMC. We found that the Saccharomyces cerevisiae EMC contains eight subunits (Emc1-6, Emc7 and Emc10), has a large lumenal region and a smaller cytosolic region, and has a transmembrane region formed by Emc4, Emc5 and Emc6 plus the transmembrane domains of Emc1 and Emc3. We identified a five-TMH fold centred around Emc3 that resembles the prokaryotic YidC insertase and that delineates a largely hydrophilic client protein pocket. The transmembrane domain of Emc4 tilts away from the main transmembrane region of EMC and is partially mobile. Mutational studies demonstrated that the flexibility of Emc4 and the hydrophilicity of the client pocket are required for EMC function. The EMC structure reveals notable evolutionary conservation with the prokaryotic insertases4,5, suggests that eukaryotic TMH insertion involves a similar mechanism, and provides a framework for detailed understanding of membrane insertion for numerous eukaryotic integral membrane proteins and tail-anchored proteins.


Assuntos
Microscopia Crioeletrônica , Retículo Endoplasmático/enzimologia , Membranas Intracelulares/enzimologia , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Saccharomyces cerevisiae , Sítios de Ligação , Retículo Endoplasmático/química , Retículo Endoplasmático/ultraestrutura , Evolução Molecular , Interações Hidrofóbicas e Hidrofílicas , Membranas Intracelulares/química , Membranas Intracelulares/ultraestrutura , Modelos Moleculares , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato
14.
PLoS One ; 15(6): e0234080, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32479525

RESUMO

In the present study we have characterized the biophysical properties of wild-type (WT) α1ß2 and α3ß2 GABAA receptors and probed the molecular basis for the observed differences. The activation and desensitization behavior and the residual currents of the receptors expressed in HEK293 cells were determined in whole-cell patch clamp recordings. Kinetic parameters of α1ß2 and α3ß2 activation differed significantly, with α1ß2 and α3ß2 exhibiting rise times (10-90%) of 24 ± 2 ms and 51 ± 7 ms, respectively. In contrast, the two receptors exhibited largely comparable desensitization behavior with decay currents that could be fitted to exponential functions with two or three components. Most notably, the two receptor compositions displayed different degrees of desentization, with the residual currents of α1ß2 and α3ß2 constituting 34 ± 2% and 21 ± 2% of the peak current, respectively. The respective contributions of the extracellular domains and the transmembrane/intracellular domains of the α-subunit to these physiological profiles were next assessed in recordings from cells expressing αß2 receptors comprising chimeric α-subunits. The rise times displayed by α1ECD/α3TMDß2 and α3ECD/α1TMDß2 receptors were intermediate to those of WT α1ß2 and WT α3ß2, and the distribution of the different components of the current decays exhibited by the two chimeric receptors followed the same pattern as the two WT receptors. The residual current exhibited by α1ECD/α3TMDß2 (23 ± 3%) was similar to that of α3ß2 but significantly different from that of α1ß2, whereas the residual current displayed by α3ECD/α1TMDß2 (27 ± 2%) was intermediate to and did not differ significantly from either of the WT receptors. This points to molecular differences in the transmembrane/intracellular domains of the α-subunit as the main determinants of the observed differences in receptor physiology between α1ß2 and α3ß2 receptors.


Assuntos
Receptores de GABA-A/metabolismo , Potenciais de Ação/efeitos dos fármacos , Células HEK293 , Humanos , Rim/fisiologia , Cinética , Técnicas de Patch-Clamp , Domínios Proteicos , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptores de GABA-A/genética , Ácido gama-Aminobutírico/farmacologia
15.
Nat Commun ; 11(1): 2799, 2020 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-32493979

RESUMO

Small molecule polyamines are abundant in all life forms and participate in diverse aspects of cell growth and differentiation. Spermidine/spermine acetyltransferase (SAT1) is the rate-limiting enzyme in polyamine catabolism and a primary genetic risk factor for suicidality. Here, using genome-wide screening, we find that SAT1 selectively controls nicotinic acetylcholine receptor (nAChR) biogenesis. SAT1 specifically augments assembly of nAChRs containing α7 or α4ß2, but not α6 subunits. Polyamines are classically studied as regulators of ion channel gating that engage the nAChR channel pore. In contrast, we find polyamine effects on assembly involve the nAChR cytosolic loop. Neurological studies link brain polyamines with neurodegenerative conditions. Our pharmacological and transgenic animal studies find that reducing polyamines enhances cortical neuron nAChR expression and augments nicotine-mediated neuroprotection. Taken together, we describe a most unexpected role for polyamines in regulating ion channel assembly, which provides a new avenue for nAChR neuropharmacology.


Assuntos
Canais Iônicos/metabolismo , Poliaminas/metabolismo , Receptores Nicotínicos/metabolismo , Acetiltransferases , Animais , Biocatálise , DNA Complementar/genética , Elementos Facilitadores Genéticos/genética , Fluorescência , Genoma Humano , Células HEK293 , Humanos , Ativação do Canal Iônico , Camundongos , Neurônios/metabolismo , Neuroproteção/efeitos dos fármacos , Estrutura Secundária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Ratos , Receptores Nicotínicos/química
16.
Nature ; 584(7820): 310-314, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32580208

RESUMO

Stimulation of the metabotropic GABAB receptor by γ-aminobutyric acid (GABA) results in prolonged inhibition of neurotransmission, which is central to brain physiology1. GABAB belongs to family C of the G-protein-coupled receptors, which operate as dimers to transform synaptic neurotransmitter signals into a cellular response through the binding and activation of heterotrimeric G proteins2,3. However, GABAB is unique in its function as an obligate heterodimer in which agonist binding and G-protein activation take place on distinct subunits4,5. Here we present cryo-electron microscopy structures of heterodimeric and homodimeric full-length GABAB receptors. Complemented by cellular signalling assays and atomistic simulations, these structures reveal that extracellular loop 2 (ECL2) of GABAB has an essential role in relaying structural transitions by ordering the linker that connects the extracellular ligand-binding domain to the transmembrane region. Furthermore, the ECL2 of each of the subunits of GABAB caps and interacts with the hydrophilic head of a phospholipid that occupies the extracellular half of the transmembrane domain, thereby providing a potentially crucial link between ligand binding and the receptor core that engages G proteins. These results provide a starting framework through which to decipher the mechanistic modes of signal transduction mediated by GABAB dimers, and have important implications for rational drug design that targets these receptors.


Assuntos
Microscopia Crioeletrônica , Receptores de GABA-B/química , Receptores de GABA-B/ultraestrutura , Sítios de Ligação , Membrana Celular/metabolismo , Antagonistas de Receptores de GABA-B/química , Antagonistas de Receptores de GABA-B/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Modelos Moleculares , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Domínios Proteicos , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Receptores de GABA-B/metabolismo , Receptores de Glutamato/química , Receptores de Glutamato/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade
17.
Clin Sci (Lond) ; 134(12): 1457-1472, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32514535

RESUMO

The chromatin remodeling complex SWI/SNF regulates the accessibility of target genes to transcription factors and plays a critical role in the tumorigenesis of hepatocellular carcinoma (HCC). The SWI/SNF complex is assembled from approximately 15 subunits, and most of these subunits have distinct roles and are often aberrantly expressed in HCC. A comprehensive exploration of the expression and clinical significance of these subunits would be of great value. In the present study, we obtained the gene expression profile of each SWI/SNF subunit and the corresponding clinical information from The Cancer Genome Atlas (TCGA). We found that 14 out of the 15 SWI/SNF subunits were significantly increased in HCC tissues compared with paired normal liver tissues, and 11 subunits were significantly associated with overall survival (OS). We identified a four-gene prognostic signature including actin-like 6A (ACTL6A), AT-rich interaction domain 1A (ARID1A), SWI/SNF related, matrix associated, actin dependent regulator of chromatin subfamily C member 1 (SMARCC1) and SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily D, member 1 (SMARCD1) that could effectively predict OS in HCC patients. Among the genes, SMARCD1 has the most prognostic value. We further conducted in vitro and in vivo experiments and revealed that SMARCD1 promotes liver cancer growth by activating the mTOR signaling pathway. In conclusion, our study has revealed that the expression of SWI/SNF complex subunits, especially SMARCD1, is highly associated with HCC development and acts as a promising prognostic predictor.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Subunidades Proteicas/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Proteínas Cromossômicas não Histona/genética , Estudos de Coortes , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Prognóstico , Subunidades Proteicas/genética , Transdução de Sinais , Resultado do Tratamento
18.
Nat Commun ; 11(1): 2481, 2020 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-32424145

RESUMO

Photosynthetic light-harvesting complexes (LHCs) play a pivotal role in collecting solar energy for photochemical reactions in photosynthesis. One of the major LHCs are fucoxanthin chlorophyll a/c-binding proteins (FCPs) present in diatoms, a group of organisms having important contribution to the global carbon cycle. Here, we report a 2.40-Å resolution structure of the diatom photosystem I (PSI)-FCPI supercomplex by cryo-electron microscopy. The supercomplex is composed of 16 different FCPI subunits surrounding a monomeric PSI core. Each FCPI subunit showed different protein structures with different pigment contents and binding sites, and they form a complicated pigment-protein network together with the PSI core to harvest and transfer the light energy efficiently. In addition, two unique, previously unidentified subunits were found in the PSI core. The structure provides numerous insights into not only the light-harvesting strategy in diatom PSI-FCPI but also evolutionary dynamics of light harvesters among oxyphototrophs.


Assuntos
Diatomáceas/metabolismo , Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/metabolismo , Complexo de Proteína do Fotossistema I/química , Complexo de Proteína do Fotossistema I/metabolismo , Clorofila/metabolismo , Proteínas de Ligação à Clorofila/química , Proteínas de Ligação à Clorofila/ultraestrutura , Transferência de Energia , Complexos de Proteínas Captadores de Luz/ultraestrutura , Modelos Moleculares , Complexo de Proteína do Fotossistema I/ultraestrutura , Ligação Proteica , Subunidades Proteicas/metabolismo , Relação Estrutura-Atividade
19.
Nucleic Acids Res ; 48(10): 5442-5456, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32365172

RESUMO

In mammalian cells, distinct H3K4 methylation states are created by deposition of methyl groups by multiple complexes of histone lysine methyltransferase 2 (KMT2) family proteins. For comprehensive analyses that directly compare the catalytic properties of all six human KMT2 complexes, we employed a biochemically defined system reconstituted with recombinant KMT2 core complexes (KMT2CoreCs) containing minimal components required for nucleosomal H3K4 methylation activity. We found that each KMT2CoreC generates distinct states and different levels of H3K4 methylation, and except for MLL3 all are stimulated by H2Bub. Notably, SET1BCoreC exhibited the strongest H3K4 methylation activity and, to our surprise, did not require H2B ubiquitylation (H2Bub); in contrast, H2Bub was required for the H3K4me2/3 activity of the paralog SET1ACoreC. We also found that WDR5, RbBP5, ASH2L and DPY30 are required for efficient H3K4 methyltransferase activities of all KMT2CoreCs except MLL3, which could produce H3K4me1 in the absence of WDR5. Importantly, deletion of the PHD2 domain of CFP1 led to complete loss of the H3K4me2/3 activities of SET1A/BCoreCs in the presence of H2Bub, indicating a critical role for this domain in the H2Bub-stimulated H3K4 methylation. Collectively, our results suggest that each KMT2 complex methylates H3K4 through distinct mechanisms in which individual subunits differentially participate.


Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Ubiquitinação , Proteínas de Ligação a DNA/metabolismo , Histona-Lisina N-Metiltransferase/química , Humanos , Metilação , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas de Neoplasias/metabolismo , Nucleossomos/enzimologia , Domínios Proteicos , Subunidades Proteicas/metabolismo
20.
Nat Commun ; 11(1): 2330, 2020 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-32393818

RESUMO

Recombinant T cell receptors (TCRs) can be used to redirect naïve T cells to eliminate virally infected or cancerous cells; however, they are plagued by low stability and uneven expression. Here, we use molecular modeling to identify mutations in the TCR constant domains (Cα/Cß) that increase the unfolding temperature of Cα/Cß by 20 °C, improve the expression of four separate α/ß TCRs by 3- to 10-fold, and improve the assembly and stability of TCRs with poor intrinsic stability. The stabilizing mutations rescue the expression of TCRs destabilized through variable domain mutation. The improved stability and folding of the TCRs reduces glycosylation, perhaps through conformational stabilization that restricts access to N-linked glycosylation enzymes. The Cα/Cß mutations enables antibody-like expression and assembly of well-behaved bispecific molecules that combine an anti-CD3 antibody with the stabilized TCR. These TCR/CD3 bispecifics can redirect T cells to kill tumor cells with target HLA/peptide on their surfaces in vitro.


Assuntos
Anticorpos Biespecíficos/imunologia , Biologia Computacional/métodos , Receptores de Antígenos de Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Biespecíficos/química , Varredura Diferencial de Calorimetria , Citotoxicidade Imunológica , Imunoglobulina G/metabolismo , Camundongos , Mutação/genética , Polissacarídeos/metabolismo , Desnaturação Proteica , Estabilidade Proteica , Subunidades Proteicas/metabolismo , Receptores de Antígenos de Linfócitos T/química , Proteínas Recombinantes/metabolismo , Solubilidade , Temperatura
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