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1.
Gene ; 728: 144287, 2020 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-31843359

RESUMO

C-type lectins are a superfamily of Ca2+-dependent carbohydrate-binding proteins that play crucial roles in invertebrate immunity. In this study, a novel C-type lectin gene (ScCTL-1) was identified in razor clam Sinonovacula constricta. The ScCTL-1 gene, consisting of four C-type carbohydrate recognition domains (CRDs) with an N-terminal signal peptide and a C-terminal transmembrane region. The gene is widely expressed in almost all tissues, with the highest expression in the hepatopancreas. To explore the functional characteristics of this structurally novel gene, tests of binding specificity, agglutinating activity, and phagocytic promoting activity were included in this study. Bacterial stimulation up-regulated ScCTL-1 expression in hemocytes. The binding activity of rScCTL-1 to bacteria was tested in vitro, and bacterial agglutination was observed under the same conditions. Ca2+ was essential for carbohydrate binding. Additionally, rScCTL-1 promoted the phagocytic activity of hemocytes to varying degrees against different bacteria, unlike the classical opsonin. These results suggest ScCTL-1 is a classical immune-related C-type lectin possessing unique immune-related properties.


Assuntos
Bivalves/genética , Bivalves/imunologia , Hepatopâncreas/metabolismo , Lectinas Tipo C/genética , Fagocitose , Staphylococcus aureus/fisiologia , Vibrio/fisiologia , Aglutinação , Sequência de Aminoácidos , Animais , Bivalves/microbiologia , Carboidratos/química , Filogenia , Domínios Proteicos , Homologia de Sequência , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia
2.
Chem Commun (Camb) ; 55(86): 12920-12923, 2019 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-31608905

RESUMO

We report the direct observation of equilibrium folding-unfolding dynamics of a mechanically labile, three helix bundle protein GA using a commercial atomic force microscope (AFM). Our study extends the capability of commercial AFMs towards studying protein unfolding/refolding at equilibrium as well as increasingly complex biomolecular systems in the context of biological function.


Assuntos
Proteínas de Bactérias/química , Microscopia de Força Atômica , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Dobramento de Proteína , Desdobramento de Proteína
3.
Protein Pept Lett ; 26(10): 792-797, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31618172

RESUMO

BACKGROUND: Head-to-tail polymerising domains generating heterogeneous aggregates are generally difficult to crystallise. DIX domains, exclusively found in the Wnt signalling pathway, are polymerising factors following this head-to-tail arrangement; moreover, they are considered to play a key role in the heterotypic interaction between Dishevelled (Dvl) and Axin, which are cytoplasmic proteins also positively and negatively regulating the canonical Wnt/ß- catenin signalling pathway, but this interaction mechanism is still unknown. OBJECTIVE: This study mainly aimed to clarify whether the Dvl2 and Axin-DIX domains (Dvl2-DIX and Axin-DIX, respectively) form a helical polymer in a head-to-tail way during complexation. METHODS: Axin-DIX (DAX) and Dvl2-DIX (DIX), carrying polymerisation-blocking mutations, were expressed as a fusion protein by using a flexible peptide linker to fuse the C-terminal of the former to the N-terminal of the latter, enforcing a defined 1:1 stoichiometry between them. RESULTS: The crystal of the DAX-DIX fusion protein diffracted to a resolution of about 0.3 nm and a data set was collected at a 0.309 nm resolution. The structure was solved via the molecular replacement method by using the DIX and DAX structures. A packing analysis of the crystal revealed the formation of a tandem heterodimer in a head-to-tail way, as predicted by the Wntsignalosome model. CONCLUSION: The results demonstrated that the combination of polymerisation-blocking mutations and a fusion protein of two head-to-tail polymerising domains is effective especially for crystallising complexes among heterologous polymerising proteins or domains.


Assuntos
Proteína Axina/química , Proteína Axina/genética , Proteínas Desgrenhadas/química , Domínios Proteicos/genética , Sequência de Aminoácidos , Cristalização , Cristalografia por Raios X/métodos , Proteínas Desgrenhadas/genética , Escherichia coli , Regulação da Expressão Gênica , Humanos , Ligação Proteica , Via de Sinalização Wnt
4.
Chem Commun (Camb) ; 55(85): 12857-12860, 2019 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-31598611

RESUMO

5-Hydroxy-2-phenyl-7-(thiiran-2-ylmethoxy)-4H-chromen-4-one (compound 52) was found as a DNA non-intercalative topo II specific catalytic inhibitor by targeting its ATP-binding domain. Showing changes in interaction with Mg2+, it exhibited highly selective properties against the α-isoform with less toxicity, unlike other topo II poisons, such as etoposide.


Assuntos
Trifosfato de Adenosina/química , DNA Topoisomerases Tipo II/química , Proteínas de Ligação a DNA/química , Inibidores da Topoisomerase II/química , Trifosfato de Adenosina/metabolismo , Biocatálise , DNA/química , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Etoposídeo/química , Humanos , Domínios Proteicos , Isoformas de Proteínas , Inibidores da Topoisomerase II/metabolismo
5.
Chem Commun (Camb) ; 55(86): 12932-12935, 2019 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-31599282

RESUMO

Here, we report the development of novel PET radiotracer ([11C]CW22) of BET proteins. In vivo imaging results in rodents and nonhuman primates (NHP) demonstrate that [11C]CW22 has excellent brain uptake, good specificity, good selectivity, suitable metabolism, appropriate kinetics and distribution in the brain. Our studies demonstrated that [11C]CW22 exhibits ideal properties as a PET imaging probe of BET proteins for further validation.


Assuntos
Encéfalo/diagnóstico por imagem , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos/química , Animais , Barreira Hematotesticular/metabolismo , Encéfalo/metabolismo , Radioisótopos de Carbono/química , Cristalografia por Raios X , Cinética , Macaca , Camundongos , Conformação Molecular , Proteínas do Tecido Nervoso/química , Neurônios , Domínios Proteicos , Compostos Radiofarmacêuticos/metabolismo , Receptores de Superfície Celular/química
6.
Photochem Photobiol Sci ; 18(11): 2740-2747, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31573014

RESUMO

Cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) is associated with memory formation and controls cell survival and proliferation via regulation of downstream gene expression in tumorigenesis. As a transcription factor, CREB binds to cAMP response elements. Phosphorylation of CREB triggers transcriptional activation of CREB downstream genes following the interaction of the kinase-inducible domain (KID) of CREB with the KID interaction domain (KIX) of CREB-binding protein. Nevertheless, because of the lack of single-cell analytical techniques, little is known about spatiotemporal regulation of CREB phosphorylation. To analyze CREB activation in single living cells, we developed genetically encoded bioluminescent sensors using luciferase-fragment complementation: the sensors are designed based on KID-KIX interaction with a single-molecule format. The luminescence intensity of the sensor, designated as CREX (a sensor of CREB activation based on KID(CREB)-KIX interaction), increased by phosphorylation of CREB. Moreover, the luminescence intensity of CREX was sufficient to detect CREB activation in live-cell bioluminescence imaging for single-cell analysis because of the higher sensitivity. CREX sensor is expected to contribute to elucidation of the spatiotemporal regulation of CREB phosphorylation by applying single-cell analysis.


Assuntos
Proteína de Ligação a CREB/análise , Medições Luminescentes/métodos , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Colforsina/química , Células HEK293 , Humanos , Luciferases/química , Luciferases/metabolismo , Fosforilação , Ligação Proteica , Domínios Proteicos/genética , Análise de Célula Única , Imagem com Lapso de Tempo
7.
J Chem Phys ; 151(12): 125103, 2019 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-31575200

RESUMO

This article reports on a frequency domain analysis of quasielastic neutron scattering spectra from free and Huperzine-A-inhibited human acetylcholinesterase, extending a recent time domain analysis of the same experimental data [M. Saouessi et al., J. Chem. Phys. 150, 161104 (2019)]. An important technical point here is the construction of a semianalytical model for the resolution-broadened dynamic structure factor that can be fitted to the experimental spectra. We find comparable parameters as in our previous study and demonstrate that our model is sensitive to subpercent changes in the experimental data, which are caused by reversible binding of the inhibitor Huperzine A.


Assuntos
Acetilcolinesterase/química , Alcaloides/química , Inibidores da Colinesterase/química , Sesquiterpenos/química , Alcaloides/farmacologia , Inibidores da Colinesterase/farmacologia , Humanos , Difração de Nêutrons , Domínios Proteicos , Sesquiterpenos/farmacologia
8.
J Chem Phys ; 151(12): 124905, 2019 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-31575216

RESUMO

The von Willebrand Factor (vWF) is a large blood glycoprotein that aids in hemostasis. Within each vWF monomer, the A2 domain hosts a cleavage site for enzyme ADAMTS13, which regulates the size of vWF multimers. This cleavage site can only be exposed when an A2 domain unfolds, and the unfolding reaction energy landscape is highly sensitive to the force conditions on the domain. Based on previous optical tweezer experimental results, we advance here a new activated A2 monomer model (AA2MM) for coarse-grained modeling of vWF that accurately represents the force-based probabilistic change between the unfolded/refolded states. A system of springs is employed to mimic the complex mechanical response of vWF monomers subject to pulling forces. AA2MM was validated by comparing monomer scale simulation results to data from prior pulling experiments on vWF monomer fragments. The model was further validated by comparing multimer scale Brownian dynamics simulation results to experiments using microfluidic chamber microscopy to visualize tethered vWF proteins subject to flow. The A2 domain unfolding reaction was studied in bulk flow simulations (pure shear and elongation flow), giving evidence that elongational flow drives the vWF size regulation process in blood. The mechanoreactive, coarse-grained AA2MM accurately describes the complex mechanical coupling between human blood flow conditions and vWF protein reactivity.


Assuntos
Modelos Químicos , Fator de von Willebrand/química , Proteína ADAMTS13/sangue , Proteína ADAMTS13/química , Fenômenos Biomecânicos , Simulação por Computador , Humanos , Domínios Proteicos , Desdobramento de Proteína
9.
Nat Cell Biol ; 21(10): 1219-1233, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31576058

RESUMO

Protein trafficking requires coat complexes that couple recognition of sorting motifs in transmembrane cargoes with biogenesis of transport carriers. The mechanisms of cargo transport through the endosomal network are poorly understood. Here, we identify a sorting motif for endosomal recycling of cargoes, including the cation-independent mannose-6-phosphate receptor and semaphorin 4C, by the membrane tubulating BAR domain-containing sorting nexins SNX5 and SNX6. Crystal structures establish that this motif folds into a ß-hairpin, which binds a site in the SNX5/SNX6 phox homology domains. Over sixty cargoes share this motif and require SNX5/SNX6 for their recycling. These include cargoes involved in neuronal migration and a Drosophila snx6 mutant displays defects in axonal guidance. These studies identify a sorting motif and provide molecular insight into an evolutionary conserved coat complex, the 'Endosomal SNX-BAR sorting complex for promoting exit 1' (ESCPE-1), which couples sorting motif recognition to the BAR-domain-mediated biogenesis of cargo-enriched tubulo-vesicular transport carriers.


Assuntos
Endossomos/metabolismo , Proteínas de Membrana/metabolismo , Nexinas de Classificação/química , Nexinas de Classificação/metabolismo , Motivos de Aminoácidos/genética , Animais , Drosophila melanogaster , Células HEK293 , Células HeLa , Humanos , Domínios Proteicos/genética , Transporte Proteico/fisiologia , Receptor IGF Tipo 2/química , Receptor IGF Tipo 2/metabolismo , Semaforinas/genética , Semaforinas/metabolismo , Nexinas de Classificação/genética
10.
Nature ; 574(7779): 565-570, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31645726

RESUMO

Co-inhibitory immune receptors can contribute to T cell dysfunction in patients with cancer1,2. Blocking antibodies against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1) partially reverse this effect and are becoming standard of care in an increasing number of malignancies3. However, many of the other axes by which tumours become inhospitable to T cells are not fully understood. Here we report that V-domain immunoglobulin suppressor of T cell activation (VISTA) engages and suppresses T cells selectively at acidic pH such as that found in tumour microenvironments. Multiple histidine residues along the rim of the VISTA extracellular domain mediate binding to the adhesion and co-inhibitory receptor P-selectin glycoprotein ligand-1 (PSGL-1). Antibodies engineered to selectively bind and block this interaction in acidic environments were sufficient to reverse VISTA-mediated immune suppression in vivo. These findings identify a mechanism by which VISTA may engender resistance to anti-tumour immune responses, as well as an unexpectedly determinative role for pH in immune co-receptor engagement.


Assuntos
Antígenos B7/química , Antígenos B7/metabolismo , Glicoproteínas de Membrana/metabolismo , Linfócitos T/metabolismo , Animais , Anticorpos Bloqueadores/imunologia , Anticorpos Bloqueadores/farmacologia , Antígenos B7/antagonistas & inibidores , Antígenos B7/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Cristalografia por Raios X , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Feminino , Histidina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Ligantes , Masculino , Glicoproteínas de Membrana/imunologia , Camundongos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Linfócitos T/citologia , Linfócitos T/imunologia , Microambiente Tumoral/imunologia
11.
Phys Chem Chem Phys ; 21(36): 19795-19804, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31482888

RESUMO

Allostery plays important roles in the regulation of many biological processes, such as signal transduction and transcriptional regulation. Although great advances have been achieved in understanding the allosteric mechanism through experimental and theoretical investigations, the details of the allosteric process are still not clear. Here, using the N-terminal domain of calmodulin (nCaM) as the model protein, we reported the atomic level characterization of the allosteric process induced by Ca2+ binding through extensive and unbiased molecular dynamics simulations. In two trajectories, it was found that Ca2+ first binds to EF-hand 2 and then induces the conformational transformation of nCaM from the Apo to Holo state assisted by second Ca2+ binding to EF-hand 1 completely. The binding order was consistent with a recent experimental result. The simulations also indicated that the two EF-hands changed conformations synergistically and the EF-hand 2 showed an earlier and more gradual conformational transition. Meanwhile, the allosteric process of nCaM triggered by Ca2+ binding might be completed within hundreds of nanoseconds in a two-state-like manner. This was validated by biased simulations, in which the Ca2+ ions were restrained near the binding sites. This work provides the molecular details of the conformational transition of nCaM triggered by Ca2+ binding.


Assuntos
Cálcio/química , Calmodulina/química , Íons/química , Simulação de Acoplamento Molecular , Domínios Proteicos , Ligação Proteica , Conformação Proteica
12.
J Chem Phys ; 151(10): 105101, 2019 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-31521094

RESUMO

The diffusion map is a dimensionality reduction method. The reduction coordinates are associated with the leading eigenfunctions of the backward Fokker-Planck operator, providing a dynamic meaning for these coordinates. One of the key factors that affect the accuracy of diffusion map embedding is the dynamic measure implemented in the Gaussian kernel. A common practice in diffusion map study of molecular systems is to approximate dynamic proximity with RMSD (root-mean-square deviation). In this paper, we present a hybrid geometry-energy based kernel. Since high energy-barriers may exist between geometrically similar conformations, taking both RMSD and energy difference into account in the kernel can better describe conformational transitions between neighboring conformations and lead to accurate embedding. We applied our diffusion map method to the ß-hairpin of the B1 domain of streptococcal protein G and to Trp-cage. Our results in ß-hairpin show that the diffusion map embedding achieves better results with the hybrid kernel than that with the RMSD-based kernel in terms of free energy landscape characterization and a new correlation measure between the cluster center Euclidean distances in the reduced-dimension space and the reciprocals of the total net flow between these clusters. In addition, our diffusion map analysis of the ultralong molecular dynamics trajectory of Trp-cage has provided a unified view of its folding mechanism. These promising results demonstrate the effectiveness of our diffusion map approach in the analysis of the dynamics and thermodynamics of molecular systems. The hybrid geometry-energy criterion could be also useful as a general dynamic measure for other purposes.


Assuntos
Proteínas de Bactérias/química , Peptídeos/química , Algoritmos , Difusão , Cadeias de Markov , Simulação de Dinâmica Molecular , Conformação Proteica em Folha beta , Domínios Proteicos , Termodinâmica
13.
Phys Chem Chem Phys ; 21(37): 20628-20640, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31495862

RESUMO

Methionine synthase (MetH) is a methylcobalamin (MeCbl)-dependent mammalian enzyme which plays a critical role in carrying out the transfer of a methyl group from methyl tetrahydrofolate to homocysteine to generate methionine and tetrahydrofolate. This catalytic cycle proceeds via cleavage of a Co-C bond which is formally heterolytic. This cleavage results in a structural change in the MeCbl cofactor bound to an enzyme. Unlike the native catalysis, upon photoexcitation, the Co-C bond in MeCbl-bound MetH generates the Co(ii)/CH3 radical pairs (RPs). Protein residues of the cap domain, particularly phenylalanine708 (F708) and leucine 715 (L715), which surrounds the upper face of the MeCbl cofactor, inhibit the photolysis of MeCbl by caging the CH3 radical and inducing the geminate recombination of the Co(ii)/CH3 RP. A molecular-level understanding of these effects requires a detailed investigation of the low-lying electronic states. Toward this, we have mutated the F708 residue with alanine (A708) and constructed the potential energy surfaces (PESs) for the low-lying S1 electronic state using a combined quantum mechanics/molecular mechanics (QM/MM) approach. The S1 PESs for the wild-type (WT) and mutant enzymes are the result of crossing of two electronic states, namely metal-to-ligand charge transfer (MLCT) and ligand field (LF) states, indicated by a seam. It is shown that the topologies of the S1 PESs are significantly modulated by introducing a mutation at the F708 position. Specifically, for the WT enzyme, the energy barrier of photoreaction and the energy difference between MLCT and LF minima are markedly higher than those of its mutant counterpart. Moreover, mutation influences the photoactivation of the Co-C bond in enzyme-bound MeCbl by decreasing the rate of geminate recombination and altering the rate of radical pair formation. This theoretical insight was also compared with transient absorption spectroscopic (TAS) studies which are in good agreement with the present findings.


Assuntos
5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , Carbono/química , Cobalto/química , Vitamina B 12/análogos & derivados , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Modelos Químicos , Estrutura Molecular , Mutação/genética , Fotólise , Domínios Proteicos/genética , Vitamina B 12/metabolismo
14.
Ann Agric Environ Med ; 26(3): 392-395, 2019 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-31559791

RESUMO

Existing research for using the protective antigen (PA) of Bacillus anthracis as a vaccine component shows that protection against anthrax may be obtained using fragments of this protein. The aim of the research is to check whether the selected protein fragment of the protective antigen (domain 4) encoded by an appropriate nucleotide sequence of gene pag of B. anthracis, was expressed in the bacterial system of E. coli. In order to examine the selected sequence of the pag gene, a PCR reaction and a highly effective TOPO cloning strategy were used, followed by purification of the recombinant proteins and their detection by a western-blot method. In the planning of the PA4 antigen expression a higher level of effectiveness in production of small protein - domain 4 - was anticipated. As a result, the 139 amino acids protein fragment of B. anthracis PA (domain 4) was isolated. The research may have found the basis for in vivo research aimed at finding potential anthrax vaccine components.


Assuntos
Vacinas contra Antraz/imunologia , Antraz/microbiologia , Antígenos de Bactérias/imunologia , Bacillus anthracis/imunologia , Toxinas Bacterianas/imunologia , Animais , Antraz/imunologia , Antraz/prevenção & controle , Vacinas contra Antraz/administração & dosagem , Vacinas contra Antraz/genética , Vacinas contra Antraz/isolamento & purificação , Anticorpos Antibacterianos/imunologia , Anticorpos Neutralizantes/imunologia , Antígenos de Bactérias/administração & dosagem , Antígenos de Bactérias/genética , Antígenos de Bactérias/isolamento & purificação , Bacillus anthracis/química , Bacillus anthracis/genética , Toxinas Bacterianas/administração & dosagem , Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Western Blotting , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Domínios Proteicos
15.
Microb Cell Fact ; 18(1): 159, 2019 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-31542050

RESUMO

BACKGROUND: Xylanases randomly cleave the internal ß-1,4-glycosidic bonds in the xylan backbone and are grouped into different families in the carbohydrate-active enzyme (CAZy) database. Although multiple xylanases are detected in single strains of many filamentous fungi, no study has been reported on the composition, synergistic effect, and mode of action in a complete set of xylanases secreted by the same microorganism. RESULTS: All three xylanases secreted by Penicillium chrysogenum P33 were expressed and characterized. The enzymes Xyl1 and Xyl3 belong to the GH10 family and Xyl3 contains a CBM1 domain at its C-terminal, whereas Xyl2 belongs to the GH11 family. The optimal temperature/pH values were 35 °C/6.0, 50 °C/5.0 and 55 °C/6.0 for Xyl1, Xyl2, and Xyl3, respectively. The three xylanases exhibited synergistic effects, with the maximum synergy observed between Xyl3 and Xyl2, which are from different families. The synergy between xylanases could also improve the hydrolysis of cellulase (C), with the maximum amount of reducing sugars (5.68 mg/mL) observed using the combination of C + Xyl2 + Xyl3. Although the enzymatic activity of Xyl1 toward xylan was low, it was shown to be capable of hydrolyzing xylooligosaccharides into xylose. Xyl2 was shown to hydrolyze xylan to long-chain xylooligosaccharides, whereas Xyl3 hydrolyzed xylan to xylooligosaccharides with a lower degree of polymerization. CONCLUSIONS: Synergistic effect exists among different xylanases, and it was higher between xylanases from different families. The cooperation of hydrolysis modes comprised the primary mechanism for the observed synergy between different xylanases. This study demonstrated, for the first time, that the hydrolysates of GH11 xylanases can be further hydrolyzed by GH10 xylanases, but not vice versa.


Assuntos
Endo-1,4-beta-Xilanases/metabolismo , Proteínas Fúngicas/metabolismo , Penicillium chrysogenum/enzimologia , Polissacarídeos/metabolismo , Biocatálise , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/genética , Estabilidade Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Glucuronatos/metabolismo , Temperatura Alta , Hidrólise , Família Multigênica , Oligossacarídeos/metabolismo , Penicillium chrysogenum/química , Penicillium chrysogenum/genética , Domínios Proteicos , Xilanos/metabolismo
16.
Nat Cell Biol ; 21(10): 1191-1205, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31548610

RESUMO

Cells of multicellular organisms need to adopt specific morphologies. However, the molecular mechanisms bringing about membrane topology changes are far from understood-mainly because knowledge of membrane-shaping proteins that can promote local membrane curvatures is still limited. Our analyses unveiled that several members of a large, previously unrecognised protein family, which we termed N-Ank proteins, use a combination of their ankyrin repeat array and an amino (N)-terminal amphipathic helix to bind and shape membranes. Consistently, functional analyses revealed that the N-Ank protein ankycorbin (NORPEG/RAI14), which was exemplarily characterised further, plays an important, ankyrin repeat-based and N-terminal amphipathic helix-dependent role in early morphogenesis of neurons. This function furthermore required coiled coil-mediated self-assembly and manifested as ankycorbin nanodomains marked by protrusive membrane topologies. In summary, here, we unveil a class of powerful membrane shapers and thereby assign mechanistic and cell biological functions to the N-Ank protein superfamily.


Assuntos
Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Morfogênese , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Animais , Repetição de Anquirina/genética , Células Cultivadas , Proteínas do Citoesqueleto/genética , Células HEK293 , Células HeLa , Humanos , Camundongos , Modelos Moleculares , Neurônios/citologia , Neurônios/metabolismo , Domínios Proteicos/genética , Ratos , Fatores de Transcrição/genética
17.
Nature ; 574(7777): 206-210, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31514202

RESUMO

Soluble guanylate cyclase (sGC) is the primary sensor of nitric oxide. It has a central role in nitric oxide signalling and has been implicated in many essential physiological processes and disease conditions. The binding of nitric oxide boosts the enzymatic activity of sGC. However, the mechanism by which nitric oxide activates the enzyme is unclear. Here we report the cryo-electron microscopy structures of the human sGCα1ß1 heterodimer in different functional states. These structures revealed that the transducer module bridges the nitric oxide sensor module and the catalytic module. Binding of nitric oxide to the ß1 haem-nitric oxide and oxygen binding (H-NOX) domain triggers the structural rearrangement of the sensor module and a conformational switch of the transducer module from bending to straightening. The resulting movement of the N termini of the catalytic domains drives structural changes within the catalytic module, which in turn boost the enzymatic activity of sGC.


Assuntos
Microscopia Crioeletrônica , Guanilil Ciclase Solúvel/metabolismo , Guanilil Ciclase Solúvel/ultraestrutura , Animais , Dissulfetos/química , Dissulfetos/metabolismo , Drosophila melanogaster , Ativação Enzimática , Células HEK293 , Heme/metabolismo , Humanos , Hidrazinas/farmacologia , Camundongos , Modelos Moleculares , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/metabolismo , Oxigênio/metabolismo , Domínios Proteicos , Multimerização Proteica , Guanilil Ciclase Solúvel/química , Guanilil Ciclase Solúvel/genética
18.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 48(3): 318-325, 2019 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-31496165

RESUMO

Proteins are the physical basis of life and perform all kinds of life activities. Proteins have different orientations and function in different tissues. The same protein, located in different subcellular regions, can perform different and even opposite functions. Both functional and structural proteins are capable of undergoing re-localization which can directly or indirectly participate in signal transduction. Due to abnormal transduction of signals during carcinogenesis, the proteins originally expressed in the cytoplasm are translocated into the nucleus and lead to functional changes in the tumor tissue. The changes of protein localization are affected by many factors, including the interaction between proteins, expression level of proteins and the cleaved intracellular domain of transmembrane protein.


Assuntos
Núcleo Celular , Citoplasma , Proteínas de Membrana , Carcinogênese/patologia , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Membrana/metabolismo , Domínios Proteicos , Transporte Proteico/fisiologia , Transdução de Sinais
19.
Plant Mol Biol ; 101(4-5): 373-387, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31422517

RESUMO

KEY MESSAGE: Polysaccharide composition of seed mucilage was successfully modified using three seed coat-specific promoters driving expression of genes encoding cell wall-modifying enzymes. Arabidopsis thaliana seed coat epidermal cells synthesize and secrete large quantities of mucilage, a specialized secondary cell wall composed of cellulose, hemicellulose, and pectin. The composition and structure of mucilage confers its unique properties of expansion, extrusion, and adherence. We are developing seed mucilage as a model to study the biochemical and biological consequences of manipulating cell wall polysaccharides in vivo using cell wall-modifying enzymes. To specifically engineer mucilage composition and avoid altering other cell types, seed coat-specific promoters are required. In this study, we investigated the ability of seed coat-specific promoters from three genes, TESTA-ABUNDANT2 (TBA2), PEROXIDASE36 (PER36), and MUCILAGE-MODIFIED4 (MUM4), to express the cell wall modifying ß-galactosidase (BGAL)-encoding gene MUCILAGE-MODIFIED2 (MUM2) and complement the mum2 mutant. The strength of the three promoters relative to one another was found to vary by two to 250 fold, and correlated with their ability to rescue the mum2 mutant phenotype. The strongest of the three promoters, TBA2p, was then used to examine the ability of three MUM2 homologs to complement the mum2 extrusion and cell wall composition phenotypes. The degree of complementation was variable and correlated with the amino acid sequence similarity between the homologous gene products and MUM2. These data demonstrate that all three seed coat-specific promoters can drive expression of genes encoding carbohydrate-active enzymes in a spatial and temporal pattern sufficiently to modify polysaccharide composition in seed mucilage without obvious negative consequences to the rest of the plant.


Assuntos
Arabidopsis/genética , Parede Celular/metabolismo , Mucilagem Vegetal/metabolismo , Sequência de Aminoácidos , Arabidopsis/metabolismo , Parede Celular/química , Regulação da Expressão Gênica de Plantas , Filogenia , Mucilagem Vegetal/genética , Regiões Promotoras Genéticas , Domínios Proteicos , Sementes/genética , Sementes/metabolismo , Alinhamento de Sequência , Análise de Sequência de Proteína
20.
Nucleic Acids Res ; 47(18): 9658-9665, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31428783

RESUMO

CRISPR-Cas systems are widespread bacterial adaptive defence mechanisms that provide protection against bacteriophages. In response, phages have evolved anti-CRISPR proteins that inactivate CRISPR-Cas systems of their hosts, enabling successful infection. Anti-CRISPR genes are frequently found in operons with genes encoding putative transcriptional regulators. The role, if any, of these anti-CRISPR-associated (aca) genes in anti-CRISPR regulation is unclear. Here, we show that Aca2, encoded by the Pectobacterium carotovorum temperate phage ZF40, is an autoregulator that represses the anti-CRISPR-aca2 operon. Aca2 is a helix-turn-helix domain protein that forms a homodimer and interacts with two inverted repeats in the anti-CRISPR promoter. The inverted repeats are similar in sequence but differ in their Aca2 affinity, and we propose that they have evolved to fine-tune, and downregulate, anti-CRISPR production at different stages of the phage life cycle. Specific, high-affinity binding of Aca2 to the first inverted repeat blocks the promoter and induces DNA bending. The second inverted repeat only contributes to repression at high Aca2 concentrations in vivo, and no DNA binding was detectable in vitro. Our investigation reveals the mechanism by which an Aca protein regulates expression of its associated anti-CRISPR.


Assuntos
Sistemas CRISPR-Cas/genética , Pectobacterium carotovorum/genética , Transcrição Genética , Proteínas Virais/genética , Bacteriófagos/genética , Escherichia coli/genética , Óperon/genética , Regiões Promotoras Genéticas/genética , Domínios Proteicos/genética , Fatores de Transcrição/genética
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