Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 13.420
Filtrar
1.
Nat Commun ; 10(1): 2399, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160585

RESUMO

Manganese superoxide dismutase (MnSOD) functions as a tumor suppressor; however, once tumorigenesis occurs, clinical data suggest MnSOD levels correlate with more aggressive human tumors, implying a potential dual function of MnSOD in the regulation of metabolism. Here we show, using in vitro transformation and xenograft growth assays that the MnSOD-K68 acetylation (Ac) mimic mutant (MnSODK68Q) functions as a tumor promoter. Interestingly, in various breast cancer and primary cell types the expression of MnSODK68Q is accompanied with a change of MnSOD's stoichiometry from a known homotetramer complex to a monomeric form. Biochemical experiments using the MnSOD-K68Q Ac-mimic, or physically K68-Ac (MnSOD-K68-Ac), suggest that these monomers function as a peroxidase, distinct from the established MnSOD superoxide dismutase activity. MnSODK68Q expressing cells exhibit resistance to tamoxifen (Tam) and cells selected for Tam resistance exhibited increased K68-Ac and monomeric MnSOD. These results suggest a MnSOD-K68-Ac metabolic pathway for Tam resistance, carcinogenesis and tumor progression.


Assuntos
Neoplasias da Mama/genética , Carcinogênese/genética , Resistencia a Medicamentos Antineoplásicos/genética , Superóxido Dismutase/genética , Acetilação , Animais , Antineoplásicos Hormonais/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Progressão da Doença , Humanos , Técnicas In Vitro , Lisina/metabolismo , Células MCF-7 , Camundongos , Mutação , Transplante de Neoplasias , Peroxidase/metabolismo , Estrutura Quaternária de Proteína/genética , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Tamoxifeno/uso terapêutico , Proteínas Supressoras de Tumor
2.
Biochemistry (Mosc) ; 84(Suppl 1): S193-S205, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31213202

RESUMO

Cytokines of the IL-17 family play a key role in the host organism defense against bacterial and fungal infections. At the same time, upregulated synthesis of IL-17 cytokines is associated with immunoinflammatory and autoimmune diseases such as psoriasis, rheumatoid arthritis, systemic lupus erythematosus, and others. The members of this family are important therapeutic targets in the treatment of various human chronic inflammatory disorders. Elucidation of signaling pathways involving IL-17 family proteins and analysis of the structure of cytokine complexes with specific antibodies, inhibitors, and receptors are essential for the development of new drugs for the therapy of immunoinflammatory rheumatic diseases.


Assuntos
Doenças Autoimunes/imunologia , Interleucina-17 , Terapia de Alvo Molecular , Linfócitos T/imunologia , Anticorpos Monoclonais/farmacologia , Humanos , Interleucina-17/antagonistas & inibidores , Interleucina-17/química , Interleucina-17/fisiologia , Estrutura Quaternária de Proteína , Transdução de Sinais
3.
Biochemistry (Mosc) ; 84(5): 453-463, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31234761

RESUMO

Amino acids undergo many covalent modifications, but only few amino acid repair enzymes have been identified. Protein-L-isoaspartate (D-aspartate) O-methyltransferase (PIMT), also known as L-isoaspartyl/D-aspartyl protein carboxyl methyltransferase (PCMT), methylates covalently modified isoaspartate (isoAsp) residues accumulated in proteins via Asn deamidation and Asp hydrolysis. This cytoplasmic reaction occurs through the formation of succinimide cyclical intermediate and generates either isoAsp or Asp from succinimide. Succinimide conversion into Asp is spontaneous, while isoAsp is restored by PIMT using S-adenosylmethionine as a methyl donor. PIMT transforms isoAsp into succinimide, thereby creating an opportunity for the later to be converted into Asp. Apart from normal cell physiology, formation of isoAsp in proteins is promoted by various stress conditions. The resulting isoAsp can form a kink or bend in the protein backbone thus making the protein conformationally and functionally distorted. Many PIMT-interacting proteins (proteins with isoAsp residues) have been reported in eukaryotes, but only few of them have been found in prokaryotes. Extensive studies in mice have shown the importance of PIMT in neurodegeneration. Detail elucidation of PIMT function can create a platform for addressing various disorders such as Alzheimer's disease and cancer.


Assuntos
Proteína D-Aspartato-L-Isoaspartato Metiltransferase/metabolismo , Animais , Ácido Aspártico/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Humanos , Ácido Isoaspártico/metabolismo , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Proteína D-Aspartato-L-Isoaspartato Metiltransferase/química , Estrutura Quaternária de Proteína , S-Adenosilmetionina/metabolismo
4.
Chem Biol Interact ; 309: 108698, 2019 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-31176713

RESUMO

Structure-guided design of novel pharmacologically active molecules relies at least in part on functionally relevant accuracy of macromolecular structures for template based drug design. Currently, about 95% of all macromolecular X-ray structures available in the PDB (Protein Data Bank) were obtained from diffraction experiments at low, cryogenic temperatures. However, it is known that functionally relevant conformations of both macromolecules and pharmacological ligands can differ at higher, physiological temperatures. We describe in this article development and properties of new human acetylcholinesterase (AChE) crystals of space group P31 and a new unit cell, amenable for room-temperature X-ray diffraction studies. We co-crystallized hAChE in P31 unit cell with the reversible inhibitor 9-aminoacridine that binds at the base of the active center gorge in addition to inhibitors that span the full length of the gorge, donepezil (Aricept, E2020) and AChE specific inhibitor BW284c51. Their new low temperature P31 space group structures appear similar to those previously obtained in the different P3121 unit cell. Successful solution of the new room temperature 3.2 Å resolution structure of BW284c51*hAChE complex from large P31 crystals enables us to proceed with studying room temperature structures of lower affinity complexes, such as oxime reactivators bound to hAChE, where temperature-related conformational diversity could be expected in both oxime and hAChE, which could lead to better informed structure-based design under conditions approaching physiological temperature.


Assuntos
Acetilcolinesterase/química , Cristalografia por Raios X , Acetilcolinesterase/genética , Acetilcolinesterase/metabolismo , Aminacrina/química , Aminacrina/metabolismo , Sítios de Ligação , Inibidores da Colinesterase/química , Inibidores da Colinesterase/metabolismo , Dimerização , Humanos , Simulação de Dinâmica Molecular , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Temperatura Ambiente
5.
Nat Commun ; 10(1): 2653, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201319

RESUMO

Ribonucleotide reductases (RNRs) use a conserved radical-based mechanism to catalyze the conversion of ribonucleotides to deoxyribonucleotides. Within the RNR family, class Ib RNRs are notable for being largely restricted to bacteria, including many pathogens, and for lacking an evolutionarily mobile ATP-cone domain that allosterically controls overall activity. In this study, we report the emergence of a distinct and unexpected mechanism of activity regulation in the sole RNR of the model organism Bacillus subtilis. Using a hypothesis-driven structural approach that combines the strengths of small-angle X-ray scattering (SAXS), crystallography, and cryo-electron microscopy (cryo-EM), we describe the reversible interconversion of six unique structures, including a flexible active tetramer and two inhibited helical filaments. These structures reveal the conformational gymnastics necessary for RNR activity and the molecular basis for its control via an evolutionarily convergent form of allostery.


Assuntos
Sítio Alostérico/genética , Proteínas de Bactérias/genética , Ribonucleotídeo Redutases/genética , Regulação Alostérica/genética , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica , Cristalografia por Raios X , Evolução Molecular , Modelos Moleculares , Estrutura Quaternária de Proteína/genética , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/metabolismo , Ribonucleotídeo Redutases/ultraestrutura , Ribonucleotídeos/metabolismo , Espalhamento a Baixo Ângulo
6.
Nat Commun ; 10(1): 2697, 2019 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-31217444

RESUMO

Atomic-resolution structure determination is crucial for understanding protein function. Cryo-EM and NMR spectroscopy both provide structural information, but currently cryo-EM does not routinely give access to atomic-level structural data, and, generally, NMR structure determination is restricted to small (<30 kDa) proteins. We introduce an integrated structure determination approach that simultaneously uses NMR and EM data to overcome the limits of each of these methods. The approach enables structure determination of the 468 kDa large dodecameric aminopeptidase TET2 to a precision and accuracy below 1 Å by combining secondary-structure information obtained from near-complete magic-angle-spinning NMR assignments of the 39 kDa-large subunits, distance restraints from backbone amides and ILV methyl groups, and a 4.1 Å resolution EM map. The resulting structure exceeds current standards of NMR and EM structure determination in terms of molecular weight and precision. Importantly, the approach is successful even in cases where only medium-resolution cryo-EM data are available.


Assuntos
Complexos Multienzimáticos/ultraestrutura , Estrutura Quaternária de Proteína , Aminopeptidases/química , Aminopeptidases/ultraestrutura , Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica/métodos , Espectroscopia de Ressonância Magnética/métodos , Simulação de Dinâmica Molecular , Peso Molecular , Complexos Multienzimáticos/química , Pyrococcus horikoshii
7.
Nat Commun ; 10(1): 2635, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201302

RESUMO

Multidrug efflux pumps actively expel a wide range of toxic substrates from the cell and play a major role in intrinsic and acquired drug resistance. In Gram-negative bacteria, these pumps form tripartite assemblies that span the cell envelope. However, the in situ structure and assembly mechanism of multidrug efflux pumps remain unknown. Here we report the in situ structure of the Escherichia coli AcrAB-TolC multidrug efflux pump obtained by electron cryo-tomography and subtomogram averaging. The fully assembled efflux pump is observed in a closed state under conditions of antibiotic challenge and in an open state in the presence of AcrB inhibitor. We also observe intermediate AcrAB complexes without TolC and discover that AcrA contacts the peptidoglycan layer of the periplasm. Our data point to a sequential assembly process in living bacteria, beginning with formation of the AcrAB subcomplex and suggest domains to target with efflux pump inhibitors.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Transporte/fisiologia , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiologia , Escherichia coli/fisiologia , Lipoproteínas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Antibacterianos/farmacologia , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/ultraestrutura , Microscopia Crioeletrônica/métodos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Tomografia com Microscopia Eletrônica/métodos , Escherichia coli/efeitos dos fármacos , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/ultraestrutura , Microscopia Intravital/métodos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Peptidoglicano/metabolismo , Periplasma/metabolismo , Ligação Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína/efeitos dos fármacos
8.
Soft Matter ; 15(21): 4326-4333, 2019 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-31070654

RESUMO

A persistent problem in the studies of membrane-active peptides, including antimicrobial peptides and pathogenic amyloidal peptides, is the lack of methods for investigating their molecular configurations in membranes. These peptides spontaneously bind to membranes from solutions, and often form oligomers that induce changes of membrane permeability. For antimicrobials, such actions appear to relate to the antimicrobial mechanisms, but for amyloidal peptides, the oligomerization has been linked to neurodegenerative diseases. In many cases, no further understanding of such oligomerization has been achieved due to the lack of structural information. In this article, we will demonstrate a method of trapping such peptide oligomers in a rhombohedral (R) phase of lipid so that the oligomers can be subjected to 3D diffraction analysis. The conditions for forming the R phase and the electron density distribution in the rhombohedral unit cell provide information about peptide-lipid interactions and the molecular size of the trapped oligomer. Such information cannot be obtained from membranes in the planar configuration. For illustration, we apply this method to daptomycin, an FDA-approved antibiotic that attacks membranes containing phosphatidylglycerol, in the presence of calcium ions. We have successfully used the brominated phosphatidylglycerol to perform bromine-atom anomalous diffraction in the rhombohedral phase containing daptomycin and calcium ions. The preliminary results apparently exhibit diffraction data related to daptomycin oligomers. We believe that this method will also be applicable to the difficult problems related to amyloidal peptides, such as amyloid beta of Alzheimer's disease.


Assuntos
Membrana Celular/química , Daptomicina/química , Multimerização Proteica , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Daptomicina/metabolismo , Estrutura Quaternária de Proteína , Água/química
9.
Nat Commun ; 10(1): 2032, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-31048734

RESUMO

The SLC26 family of transporters maintains anion equilibria in all kingdoms of life. The family shares a 7 + 7 transmembrane segments inverted repeat architecture with the SLC4 and SLC23 families, but holds a regulatory STAS domain in addition. While the only experimental SLC26 structure is monomeric, SLC26 proteins form structural and functional dimers in the lipid membrane. Here we resolve the structure of an SLC26 dimer embedded in a lipid membrane and characterize its functional relevance by combining PELDOR/DEER distance measurements and biochemical studies with MD simulations and spin-label ensemble refinement. Our structural model reveals a unique interface different from the SLC4 and SLC23 families. The functionally relevant STAS domain is no prerequisite for dimerization. Characterization of heterodimers indicates that protomers in the dimer functionally interact. The combined structural and functional data define the framework for a mechanistic understanding of functional cooperativity in SLC26 dimers.


Assuntos
Proteínas de Bactérias/metabolismo , Simulação de Dinâmica Molecular , Multimerização Proteica , Estrutura Quaternária de Proteína , Transportadores de Sulfato/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Deinococcus , Espectroscopia de Ressonância de Spin Eletrônica , Mutagênese Sítio-Dirigida , Transportadores de Ânions Orgânicos Dependentes de Sódio/química , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas SLC4A/química , Proteínas SLC4A/metabolismo , Transportadores de Sulfato/química , Transportadores de Sulfato/genética , Transportadores de Sulfato/isolamento & purificação
10.
Methods Mol Biol ; 1966: 125-135, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31041743

RESUMO

Steroid receptors are members of a subfamily of the nuclear receptor superfamily. They play a dual role of steroid hormone receptors and transcription factors. Actually, these receptors are steroid-activated transcription factors. Classical soluble receptors exist as oligomeric complexes with the Hsp90-based chaperone machinery. The steroid receptor field was born and developed along with the molecular chaperone field. Chaperones are not exclusive partners associated to these receptors, but also comprise a large variety of heterocomplexes with other proteins involved in signal transduction. By using the glucocorticoid receptor (GR) as a standard model for most Hsp90-client proteins, in this chapter we describe the functional GR·Hsp90 heterocomplex assembly system from reticulocyte lysate or purified proteins.


Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Técnicas In Vitro/métodos , Multimerização Proteica , Receptores de Glucocorticoides/metabolismo , Animais , Estrutura Quaternária de Proteína , Coelhos , Receptores de Glucocorticoides/química , Reticulócitos/metabolismo
11.
Eur Biophys J ; 48(6): 569-577, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31134309

RESUMO

Microtubule mechanical properties are essential for understanding basic cellular processes, including cell motility and division, but the forces that result in microtubule rupture or breakage have not yet been measured directly. These forces are essential to understand the mechanical properties of the cytoskeleton and responses by cells to both normal conditions and stress caused by injury or disease. Here we estimate the force required to rupture a microtubule by analyzing kinesin-14 Ncd motor-induced microtubule breakage in ensemble motility assays. We model the breakage events as caused by Ncd motors pulling or pushing on single microtubules that are clamped at one end by other motors attached to the glass surface. The number of pulling or pushing Ncd motors is approximated from the length of the microtubule bound to the surface and the forces produced by the pulling or pushing motors are estimated from forces produced by the Ncd motor in laser-trap assays, reported by others. Our analysis provides an estimate, to the first approximation, of ~ 500 pN for the minimal force required to rupture a 13-pf microtubule. The value we report is close to the forces estimated from microtubule stretching/fragmentation experiments and overlaps with the forces applied by AFM in microtubule indentation assays that destabilize microtubules and break microtubule protofilaments. It is also consistent with the forces required to disrupt protein noncovalent bonds in force spectroscopy experiments. These findings are relevant to microtubule deformation and breakage caused by cellular tension in vivo.


Assuntos
Fenômenos Mecânicos , Microtúbulos/metabolismo , Fenômenos Biomecânicos , Ligações de Hidrogênio , Cinesina/metabolismo , Modelos Moleculares , Multimerização Proteica , Estrutura Quaternária de Proteína , Tubulina (Proteína)/química
12.
Essays Biochem ; 63(1): 133-145, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30967477

RESUMO

The organization and regulation of genomic DNA as nuclear chromatin is necessary for proper DNA function inside living eukaryotic cells. While this has been extensively explored, no true consensus is currently reached regarding the exact mechanism of chromatin organization. The traditional view has assumed that the DNA is packaged into a hierarchy of structures inside the nucleus based on the regular 30-nm chromatin fiber. This is currently being challenged by the fluid-like model of the chromatin which views the chromatin as a dynamic structure based on the irregular 10-nm fiber. In this review, we focus on the recent progress in chromatin structure elucidation highlighting the paradigm shift in chromatin folding mechanism from the classical textbook perspective of the regularly folded chromatin to the more dynamic fluid-like perspective.


Assuntos
Montagem e Desmontagem da Cromatina , Nucleossomos/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Humanos , Complexos Multiproteicos/metabolismo , Conformação de Ácido Nucleico , Nucleossomos/química , Dobramento de Proteína , Estrutura Quaternária de Proteína
13.
Essays Biochem ; 63(1): 89-96, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015385

RESUMO

The dynamic structure of chromatin is linked to gene regulation and many other biological functions. Consequently, it is of importance to understand the factors that regulate chromatin dynamics. While the in vivo analysis of chromatin has verified that histone post-translational modifications play a role in modulating DNA accessibility, the complex nuclear environment and multiplicity of modifications prevents clear conclusions as to how individual modifications influence chromatin dynamics in the cell. For this reason, in vitro analyses of model reconstituted nucleosomal arrays has been pivotal in understanding the dynamic nature of chromatin compaction and the affects that specific post-translational modifications can have on the higher order chromatin structure. In this mini-review, we briefly describe the dynamic chromatin structures that have been observed in vitro and the environmental conditions that give rise to these various conformational states. Our focus then turns to a discussion of the specific histone post-translational modifications that have been shown to alter formation of these higher order chromatin structures in vitro and how this may relate to the biological state and accessibility of chromatin in vivo.


Assuntos
Histonas/metabolismo , Nucleossomos/metabolismo , Processamento de Proteína Pós-Traducional , Acetilação , DNA/química , DNA/metabolismo , Histonas/química , Humanos , Metilação , Nucleossomos/química , Dobramento de Proteína , Estrutura Quaternária de Proteína , Sumoilação
14.
Acta Crystallogr D Struct Biol ; 75(Pt 3): 296-307, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30950400

RESUMO

Suppressor of copper sensitivity protein C from Proteus mirabilis (PmScsC) is a homotrimeric disulfide isomerase that plays a role in copper tolerance, which is a key virulence trait of this uropathogen. Each protomer of the enzyme has an N-terminal trimerization stem (59 residues) containing a flexible linker (11 residues) connected to a thioredoxin-fold-containing catalytic domain (163 residues). Here, two PmScsC variants, PmScsCΔN and PmScsCΔLinker, are characterized. PmScsCΔN is an N-terminally truncated form of the protomer with two helices of the trimerization stem removed, generating a protein with dithiol oxidase rather than disulfide isomerase activity. The crystal structure of PmScsCΔN reported here reveals, as expected, a monomer that is structurally similar to the catalytic domain of native PmScsC. The second variant, PmScsCΔLinker, was designed to remove the 11-amino-acid linker, and it is shown that it generates a protein that has neither disulfide isomerase nor dithiol oxidase activity. The crystal structure of PmScsCΔLinker reveals a trimeric arrangement, with the catalytic domains packed together very closely. Small-angle X-ray scattering analysis found that native PmScsC is predominantly trimeric in solution even at low concentrations, whereas PmScsCΔLinker exists as an equilibrium between monomeric, dimeric and trimeric states, with the monomeric form dominating at low concentrations. These findings increase the understanding of disulfide isomerase activity, showing how (i) oligomerization, (ii) the spacing between and (iii) the dynamic motion of catalytic domains in PmScsC all contribute to its native function.


Assuntos
Proteínas de Bactérias/química , Isomerases de Dissulfetos de Proteínas/química , Proteus mirabilis/enzimologia , Domínio Catalítico , Modelos Moleculares , Multimerização Proteica , Estrutura Quaternária de Proteína , Espalhamento a Baixo Ângulo
15.
Artif Cells Nanomed Biotechnol ; 47(1): 1248-1255, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30945565

RESUMO

Mitochondrial dysfunction is a major contributory factor for myocardial ischemia-reperfusion (I/R) injury. It has been reported that Pink1-Parkin-mediated mitochondrial autophagy could effectively remove damaged mitochondria and excess ROS to ensure the stability of intracellular mitochondria. The present study was designed to evaluate whether the polymerized porcine haemoglobin (pPolyHb), a novel type of haemoglobin oxygen carrier, has an effect on I/R injury via regulating the Pink1-Parkin mediated mitochondrial autophagy pathway in myocardial H9C2 cells. The results revealed that pPolyHb could effectively reduce apoptosis and improve the survival rates of H9C2 cells. In addition, Pink1 and Parkin levels gradually decreased with pPolyHb reoxygenation. The inhibition of mitochondrial autophagy through mitochondrial-division inhibitor-1(mdivi-1) resulted in a decrease in anti-apoptotic protein Bcl-2 and an increase in pro-apoptotic protein Bax and CytC. In conclusion, pPolyHb has a protective effect on myocardial ischemia-reperfusion injury by regulating moderate mitochondrial autophagy.


Assuntos
Autofagia/efeitos dos fármacos , Hemoglobinas/farmacologia , Mitocôndrias/efeitos dos fármacos , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/efeitos dos fármacos , Proteínas Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citocromos c/metabolismo , Citoproteção/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Hemoglobinas/química , Humanos , L-Lactato Desidrogenase/metabolismo , Mitocôndrias/patologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Oxigênio/metabolismo , Multimerização Proteica , Estrutura Quaternária de Proteína , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Suínos , Proteína X Associada a bcl-2/metabolismo
16.
Nat Commun ; 10(1): 972, 2019 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-30814507

RESUMO

Bacterial toxin-antitoxin (TA) modules are tightly regulated to maintain growth in favorable conditions or growth arrest during stress. A typical regulatory strategy involves the antitoxin binding and repressing its own promoter while the toxin often acts as a co-repressor. Here we show that Pseudomonas putida graTA-encoded antitoxin GraA and toxin GraT differ from other TA proteins in the sense that not the antitoxin but the toxin possesses a flexible region. GraA auto-represses the graTA promoter: two GraA dimers bind cooperatively at opposite sides of the operator sequence. Contrary to other TA modules, GraT is a de-repressor of the graTA promoter as its N-terminal disordered segment prevents the binding of the GraT2A2 complex to the operator. Removal of this region restores operator binding and abrogates Gr aT toxicity. GraTA represents a TA module where a flexible region in the toxin rather than in the antitoxin controls operon expression and toxin activity.


Assuntos
Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Antitoxinas/genética , Antitoxinas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/toxicidade , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Genes Bacterianos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Óperon , Regiões Promotoras Genéticas , Ligação Proteica , Dobramento de Proteína , Estrutura Quaternária de Proteína , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Eletricidade Estática , Sistemas Toxina-Antitoxina/genética
17.
Nat Commun ; 10(1): 1068, 2019 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-30842409

RESUMO

The small heat-shock protein HSP27 is a redox-sensitive molecular chaperone that is expressed throughout the human body. Here, we describe redox-induced changes to the structure, dynamics, and function of HSP27 and its conserved α-crystallin domain (ACD). While HSP27 assembles into oligomers, we show that the monomers formed upon reduction are highly active chaperones in vitro, but are susceptible to self-aggregation. By using relaxation dispersion and high-pressure nuclear magnetic resonance (NMR) spectroscopy, we observe that the pair of ß-strands that mediate dimerisation partially unfold in the monomer. We note that numerous HSP27 mutations associated with inherited neuropathies cluster to this dynamic region. High levels of sequence conservation in ACDs from mammalian sHSPs suggest that the exposed, disordered interface present in free monomers or oligomeric subunits may be a general, functional feature of sHSPs.


Assuntos
Proteínas de Choque Térmico HSP27/metabolismo , Doenças do Sistema Nervoso Periférico/genética , Agregação Patológica de Proteínas/genética , Multimerização Proteica/genética , Desdobramento de Proteína , Proteínas de Choque Térmico HSP27/química , Proteínas de Choque Térmico HSP27/genética , Mutação , Ressonância Magnética Nuclear Biomolecular , Oxirredução , Conformação Proteica em Folha beta/genética , Estrutura Quaternária de Proteína/fisiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
18.
Yakugaku Zasshi ; 139(5): 827-835, 2019 May 01.
Artigo em Japonês | MEDLINE | ID: mdl-30842349

RESUMO

Proteolysis mediated by the ubiquitin-proteome system plays an important role in cancer. Recently, a deubiquitinating enzyme, ubiquitin-specific protease 7 (USP7) has attracted attention as a key regulator of the p53-human double minute 2 (HDM2) pathway in cancer cells. Although some USP7 enzyme inhibitors have been identified, issues related to activity and selectivity prevent their therapeutic application. In this study, we aimed to search for novel USP7-HDM2 protein-protein interaction (PPI) inhibitors that do not affect the USP7 enzyme activity. Using the fragment-mapping program Fsubsite and the canonical subsite-fragment database (CSFDB) developed in our laboratory, we mapped a variety of fragments onto USP7 protein and constructed 3D-pharmacophore models based on the arrangement patterns of the mapped fragments. Finally, we performed 3D pharmacophore-based virtual screening of a commercial compound database and successfully selected promising USP7-HDM2 PPI inhibitor candidates.


Assuntos
Antineoplásicos , Simulação por Computador , Descoberta de Drogas , Inibidores de Proteases , Mapas de Interação de Proteínas , Proteínas Proto-Oncogênicas c-mdm2 , Mapeamento por Restrição/métodos , Peptidase 7 Específica de Ubiquitina , Modelos Moleculares , Inibidores de Proteases/química , Estrutura Quaternária de Proteína , Proteólise , Proteínas Proto-Oncogênicas c-mdm2/química , Peptidase 7 Específica de Ubiquitina/química
19.
Proc Natl Acad Sci U S A ; 116(15): 7333-7342, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30918129

RESUMO

Hsp104 is a large AAA+ molecular machine that can rescue proteins trapped in amorphous aggregates and stable amyloids by drawing substrate protein into its central pore. Recent cryo-EM studies image Hsp104 at high resolution. We used hydrogen exchange mass spectrometry analysis (HX MS) to resolve and characterize all of the functionally active and inactive elements of Hsp104, many not accessible to cryo-EM. At a global level, HX MS confirms the one noncanonical interprotomer interface in the Hsp104 hexamer as a marker for the spiraled conformation revealed by cryo-EM and measures its fast conformational cycling under ATP hydrolysis. Other findings enable reinterpretation of the apparent variability of the regulatory middle domain. With respect to detailed mechanism, HX MS determines the response of each Hsp104 structural element to the different bound adenosine nucleotides (ADP, ATP, AMPPNP, and ATPγS). They are distinguished most sensitively by the two Walker A nucleotide-binding segments. Binding of the ATP analog, ATPγS, tightly restructures the Walker A segments and drives the global open-to-closed/extended transition. The global transition carries part of the ATP/ATPγS-binding energy to the somewhat distant central pore. The pore constricts and the tyrosine and other pore-related loops become more tightly structured, which seems to reflect the energy-requiring directional pull that translocates the substrate protein. ATP hydrolysis to ADP allows Hsp104 to relax back to its lowest energy open state ready to restart the cycle.


Assuntos
Nucleotídeos de Adenina/química , Proteínas de Choque Térmico/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Nucleotídeos de Adenina/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Espectrometria de Massas , Domínios Proteicos , Estrutura Quaternária de Proteína , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Relação Estrutura-Atividade
20.
Mar Drugs ; 17(3)2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30832207

RESUMO

Marine cone snails are carnivorous gastropods that use peptide toxins called conopeptides both as a defense mechanism and as a means to immobilize and kill their prey. These peptide toxins exhibit a large chemical diversity that enables exquisite specificity and potency for target receptor proteins. This diversity arises in terms of variations both in amino acid sequence and length, and in posttranslational modifications, particularly the formation of multiple disulfide linkages. Most of the functionally characterized conopeptides target ion channels of animal nervous systems, which has led to research on their therapeutic applications. Many facets of the underlying molecular mechanisms responsible for the specificity and virulence of conopeptides, however, remain poorly understood. In this review, we will explore the chemical diversity of conopeptides from a computational perspective. First, we discuss current approaches used for classifying conopeptides. Next, we review different computational strategies that have been applied to understanding and predicting their structure and function, from machine learning techniques for predictive classification to docking studies and molecular dynamics simulations for molecular-level understanding. We then review recent novel computational approaches for rapid high-throughput screening and chemical design of conopeptides for particular applications. We close with an assessment of the state of the field, emphasizing important questions for future lines of inquiry.


Assuntos
Conotoxinas/química , Caramujo Conus/química , Desenho de Drogas , Canais Iônicos/antagonistas & inibidores , Sequência de Aminoácidos/genética , Animais , Simulação por Computador , Conotoxinas/genética , Conotoxinas/farmacologia , Conotoxinas/toxicidade , Caramujo Conus/genética , Ensaios de Triagem em Larga Escala/métodos , Aprendizado de Máquina , Modelos Moleculares , Processamento de Proteína Pós-Traducional , Estrutura Quaternária de Proteína , Relação Estrutura-Atividade , Transcriptoma/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA