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1.
Am J Physiol Cell Physiol ; 325(1): C172-C185, 2023 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-37212546

RESUMO

Specific force (SF) has been shown to be reduced in some but not all studies of human aging using chemically skinned single muscle fibers. This may be due, in part, not only to the health status/physical activity levels of different older cohorts, but also from methodological differences in studying skinned fibers. The aim of the present study was to compare SF in fibers from older hip fracture patients (HFP), healthy master cyclists (MC), and healthy nontrained young adults (YA) using two different activating solutions. Quadriceps muscle samples and 316 fibers were obtained from HFPs (74.6 ± 4 years, n = 5), MCs (74.8 ± 1, n = 5), and YA (25.5 ± 2, n = 6). Fibers were activated (pCa 4.5, 15°C) in solutions containing either 60 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid pH buffer (TES) or 20 mM imidazole. SF was determined by normalizing force to fiber cross-sectional area (CSA) assuming either an elliptical or circular shape and to fiber myosin heavy chain content. Activation in TES resulted in significantly higher MHC-I SF in all groups and YA MHC-IIA fibers, irrespective of normalization method. Although there were no differences in SF between the participant groups, the ratio of SF between the TES and imidazole solutions was lower in HFPs compared with YAs (MHC-I P < 0.05; MHC-IIA P = 0.055). Activating solution composition, as opposed to donor characteristics, had a more notable effect on single fiber SF. However, this two-solution approach revealed an age-related difference in sensitivity in HFPs, which was not shown in MCs. This suggests further novel approaches may be required to probe age/activity-related differences in muscle contractile quality.NEW & NOTEWORTHY Whether specific force (SF) decreases with advancing age in human single skeletal muscle fibers is uncertain. Equivocal published findings may be due to the different physical activity levels of the elderly cohorts studied and/or different chemical solutions used to measure force. We compared single fiber SF between young adults, elderly cyclists, and hip fracture patients (HFP) using two solutions. The solution used significantly affected force and revealed a difference in sensitivity of HFP muscle fibers.


Assuntos
Contração Muscular , Fibras Musculares Esqueléticas , Adulto Jovem , Humanos , Idoso , Contração Muscular/fisiologia , Cadeias Pesadas de Miosina , Envelhecimento , Músculo Quadríceps , Músculo Esquelético/fisiologia
2.
Bioorg Med Chem Lett ; 42: 128044, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-33865971

RESUMO

Glutamate carboxypeptidase II (GCP(II)), also known as the prostate-specific membrane antigen (PSMA), is a transmembrane zinc(II) metalloenzyme overexpressed in prostate cancer. Inhibitors of this receptor are used to target molecular imaging agents and molecular radiotherapy agents to prostate cancer and if the affinity of inhibitors for GCP(II)/PSMA could be improved, targeting might also improve. Compounds containing the dipeptide OH-Lys-C(O)-Glu-OH (compound 3), incorporating a urea motif, have high affinity for GCP(II)/PSMA. We hypothesized that substituting the zinc-coordinating urea group for a thiourea group, thus incorporating a sulfur atom, could facilitate stronger binding to zinc(II) within the active site, and thus improve affinity for GCP(II)/PSMA. A structurally analogous urea and thiourea pair (HO-Glu-C(O)-Glu-OH - compound 5 and HO-Glu-C(S)-Glu-OH - compound 6) were synthesized and the inhibitory concentration (IC50) of each compound measured with a cell-based assay, allowing us to refute the hypothesis: the thiourea analogue showed 100-fold weaker binding to PSMA than the urea analogue.


Assuntos
Dipeptídeos/farmacologia , Inibidores Enzimáticos/farmacologia , Glutamato Carboxipeptidase II/antagonistas & inibidores , Ureia/farmacologia , Antígenos de Superfície/metabolismo , Dipeptídeos/síntese química , Dipeptídeos/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Glutamato Carboxipeptidase II/metabolismo , Humanos , Estrutura Molecular , Relação Estrutura-Atividade , Ureia/análogos & derivados , Ureia/química
3.
Nucleic Acids Res ; 47(8): 4272-4291, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-30820564

RESUMO

LARP4A belongs to the ancient RNA-binding protein superfamily of La-related proteins (LARPs). In humans, it acts mainly by stabilizing mRNAs, enhancing translation and controlling polyA lengths of heterologous mRNAs. These activities are known to implicate its association with mRNA, protein partners and translating ribosomes, albeit molecular details are missing. Here, we characterize the direct interaction between LARP4A, oligoA RNA and the MLLE domain of the PolyA-binding protein (PABP). Our study shows that LARP4A-oligoA association entails novel RNA recognition features involving the N-terminal region of the protein that exists in a semi-disordered state and lacks any recognizable RNA-binding motif. Against expectations, we show that the La module, the conserved RNA-binding unit across LARPs, is not the principal determinant for oligoA interaction, only contributing to binding to a limited degree. Furthermore, the variant PABP-interacting motif 2 (PAM2w) featured in the N-terminal region of LARP4A was found to be important for both RNA and PABP recognition, revealing a new role for this protein-protein binding motif. Our analysis demonstrates the mutual exclusive nature of the PAM2w-mediated interactions, thereby unveiling a tantalizing interplay between LARP4A, polyA and PABP.


Assuntos
Autoantígenos/química , Poli A/química , Proteínas de Ligação a Poli(A)/química , RNA Mensageiro/química , Proteínas de Ligação a RNA/química , Ribonucleoproteínas/química , Motivos de Aminoácidos , Autoantígenos/genética , Autoantígenos/metabolismo , Sítios de Ligação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cinética , Modelos Moleculares , Poli A/genética , Poli A/metabolismo , Proteínas de Ligação a Poli(A)/genética , Proteínas de Ligação a Poli(A)/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Especificidade por Substrato , Termodinâmica , Antígeno SS-B
4.
Nucleic Acids Res ; 43(1): 645-60, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25488812

RESUMO

The La-related proteins (LARPs) form a diverse group of RNA-binding proteins characterized by the possession of a composite RNA binding unit, the La module. The La module comprises two domains, the La motif (LaM) and the RRM1, which together recognize and bind to a wide array of RNA substrates. Structural information regarding the La module is at present restricted to the prototypic La protein, which acts as an RNA chaperone binding to 3' UUUOH sequences of nascent RNA polymerase III transcripts. In contrast, LARP6 is implicated in the regulation of collagen synthesis and interacts with a specific stem-loop within the 5' UTR of the collagen mRNA. Here, we present the structure of the LaM and RRM1 of human LARP6 uncovering in both cases considerable structural variation in comparison to the equivalent domains in La and revealing an unprecedented fold for the RRM1. A mutagenic study guided by the structures revealed that RNA recognition requires synergy between the LaM and RRM1 as well as the participation of the interdomain linker, probably in realizing tandem domain configurations and dynamics required for substrate selectivity. Our study highlights a considerable complexity and plasticity in the architecture of the La module within LARPs.


Assuntos
Regiões 5' não Traduzidas , Autoantígenos/química , Colágeno/genética , Ribonucleoproteínas/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Autoantígenos/genética , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Ribonucleoproteínas/genética , Alinhamento de Sequência , Antígeno SS-B
5.
Amino Acids ; 48(8): 1969-81, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27143170

RESUMO

Mice over-expressing the creatine transporter have elevated myocardial creatine levels [Cr] and are protected against ischaemia/reperfusion injury via improved energy reserve. However, mice with very high [Cr] develop cardiac hypertrophy and dysfunction. To investigate these contrasting effects, we applied a non-biased hypothesis-generating approach to quantify global protein and metabolite changes in the LV of mice stratified for [Cr] levels: wildtype, moderately elevated, and high [Cr] (65-85; 100-135; 160-250 nmol/mg protein, respectively). Male mice received an echocardiogram at 7 weeks of age with tissue harvested at 8 weeks. RV was used for [Cr] quantification by HPLC to select LV tissue for subsequent analysis. Two-dimensional difference in-gel electrophoresis identified differentially expressed proteins, which were manually picked and trypsin digested for nano-LC-MS/MS. Principal component analysis (PCA) showed efficient group separation (ANOVA P ≤ 0.05) and peptide sequences were identified by mouse database (UniProt 201203) using Mascot. A total of 27 unique proteins were found to be differentially expressed between normal and high [Cr], with proteins showing [Cr]-dependent differential expression, chosen for confirmation, e.g. α-crystallin B, a heat shock protein implicated in cardio-protection and myozenin-2, which could contribute to the hypertrophic phenotype. Nuclear magnetic resonance (¹H-NMR at 700 MHz) identified multiple strong correlations between [Cr] and key cardiac metabolites. For example, positive correlations with α-glucose (r² = 0.45; P = 0.002), acetyl-carnitine (r² = 0.50; P = 0.001), glutamine (r² = 0.59; P = 0.0002); and negative correlations with taurine (r² = 0.74; P < 0.0001), fumarate (r² = 0.45; P = 0.003), aspartate (r² = 0.59; P = 0.0002), alanine (r² = 0.66; P < 0.0001) and phosphocholine (r² = 0.60; P = 0.0002). These findings suggest wide-ranging and hitherto unexpected adaptations in substrate utilisation and energy metabolism with a general pattern of impaired energy generating pathways in mice with very high creatine levels.


Assuntos
Creatina/metabolismo , Metabolismo Energético , Regulação da Expressão Gênica , Proteínas Musculares/biossíntese , Contração Miocárdica , Traumatismo por Reperfusão Miocárdica/metabolismo , Animais , Masculino , Proteínas de Membrana Transportadoras/biossíntese , Proteínas de Membrana Transportadoras/genética , Metabolômica , Camundongos , Camundongos Transgênicos , Proteínas Musculares/genética , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio , Proteômica , Coelhos
6.
Biochemistry ; 54(6): 1327-37, 2015 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-25590897

RESUMO

PDZ domains are highly abundant protein-protein interaction modules commonly found in multidomain scaffold proteins. The PDZ1 domain of MAGI-1, a protein present at cellular tight junctions that contains six PDZ domains, is targeted by the E6 oncoprotein of the high-risk human papilloma virus. Thermodynamic and dynamic studies using complementary isothermal titration calorimetry and nuclear magnetic resonance (NMR) (15)N heteronuclear relaxation measurements were conducted at different temperatures to decipher the molecular mechanism of this interaction. Binding of E6 peptides to the MAGI-1 PDZ1 domain is accompanied by an unusually large and negative change in heat capacity (ΔC(p)) that is attributed to a disorder-to-order transition of the C-terminal extension of the PDZ1 domain upon E6 binding. Analysis of temperature-dependent thermodynamic parameters and (15)N NMR relaxation data of a PDZ1 mutant in which this disorder-to-order transition was abolished allows the unusual thermodynamic signature of E6 binding to be correlated to local folding of the PDZ1 C-terminal extension. Comparison of the exchange contributions observed for wild-type and mutant proteins explains how variation in the solvent-exposed area may compensate for the loss of conformational entropy and further designates a distinct set of a few residues that mediate this local folding phenomena.


Assuntos
Domínios PDZ , Peptídeos/química , Sequência de Aminoácidos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Conformação Proteica , Termodinâmica , Junções Íntimas/química
7.
J Biol Chem ; 287(10): 7146-58, 2012 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-22130672

RESUMO

Retinoblastoma-binding protein-6 (RBBP6) plays a facilitating role, through its RING finger-like domain, in the ubiquitination of p53 by Hdm2 that is suggestive of E4-like activity. Although the presence of eight conserved cysteine residues makes it highly probable that the RING finger-like domain coordinates two zinc ions, analysis of the primary sequence suggests an alternative classification as a member of the U-box family, the members of which do not bind zinc ions. We show here that despite binding two zinc ions, the domain adopts a homodimeric structure highly similar to those of a number of U-boxes. Zinc ions could be replaced by cadmium ions without significantly disrupting the structure or the stability of the domain, although the rate of substitution was an order of magnitude slower than any previous measurement, suggesting that the structure is particularly stable, a conclusion supported by the high thermal stability of the domain. A hallmark of U-box-containing proteins is their association with chaperones, with which they cooperate in eliminating irretrievably unfolded proteins by tagging them for degradation by the proteasome. Using a yeast two-hybrid screen, we show that RBBP6 interacts with chaperones Hsp70 and Hsp40 through its N-terminal ubiquitin-like domain. Taken together with the structural similarities to U-box-containing proteins, our data suggest that RBBP6 plays a role in chaperone-mediated ubiquitination and possibly in protein quality control.


Assuntos
Proteínas de Transporte/química , Proteínas de Ligação a DNA/química , Cádmio/química , Cádmio/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico HSP40/química , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Ligação Proteica/fisiologia , Domínios RING Finger , Saccharomyces cerevisiae/genética , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina-Proteína Ligases , Ubiquitinação/fisiologia , Zinco/química , Zinco/metabolismo
8.
J Biol Chem ; 287(41): 34120-33, 2012 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-22869378

RESUMO

We used a combination of fluorescence, circular dichroism (CD), and NMR spectroscopies in conjunction with size exclusion chromatography to help rationalize the relative antibacterial, antiplasmodial, and cytotoxic activities of a series of proline-free and proline-containing model antimicrobial peptides (AMPs) in terms of their structural properties. When compared with proline-free analogs, proline-containing peptides had greater activity against Gram-negative bacteria, two mammalian cancer cell lines, and intraerythrocytic Plasmodium falciparum, which they were capable of killing without causing hemolysis. In contrast, incorporation of proline did not have a consistent effect on peptide activity against Mycobacterium tuberculosis. In membrane-mimicking environments, structures with high α-helix content were adopted by both proline-free and proline-containing peptides. In solution, AMPs generally adopted disordered structures unless their sequences comprised more hydrophobic amino acids or until coordinating phosphate ions were added. Proline-containing peptides resisted ordering induced by either method. The roles of the angle subtended by positively charged amino acids and the positioning of the proline residues were also investigated. Careful positioning of proline residues in AMP sequences is required to enable the peptide to resist ordering and maintain optimal antibacterial activity, whereas varying the angle subtended by positively charged amino acids can attenuate hemolytic potential albeit with a modest reduction in potency. Maintaining conformational flexibility improves AMP potency and selectivity toward bacterial, plasmodial, and cancerous cells while enabling the targeting of intracellular pathogens.


Assuntos
Antibacterianos , Antimaláricos , Peptídeos Catiônicos Antimicrobianos , Antineoplásicos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Plasmodium falciparum/crescimento & desenvolvimento , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Antimaláricos/química , Antimaláricos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Transformada , Linhagem Celular Tumoral , Humanos , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Estrutura Secundária de Proteína
9.
Nat Commun ; 14(1): 2740, 2023 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-37217519

RESUMO

Cell migration is crucial for cancer dissemination. We find that AMP-activated protein kinase (AMPK) controls cell migration by acting as an adhesion sensing molecular hub. In 3-dimensional matrices, fast-migrating amoeboid cancer cells exert low adhesion/low traction linked to low ATP/AMP, leading to AMPK activation. In turn, AMPK plays a dual role controlling mitochondrial dynamics and cytoskeletal remodelling. High AMPK activity in low adhering migratory cells, induces mitochondrial fission, resulting in lower oxidative phosphorylation and lower mitochondrial ATP. Concurrently, AMPK inactivates Myosin Phosphatase, increasing Myosin II-dependent amoeboid migration. Reducing adhesion or mitochondrial fusion or activating AMPK induces efficient rounded-amoeboid migration. AMPK inhibition suppresses metastatic potential of amoeboid cancer cells in vivo, while a mitochondrial/AMPK-driven switch is observed in regions of human tumours where amoeboid cells are disseminating. We unveil how mitochondrial dynamics control cell migration and suggest that AMPK is a mechano-metabolic sensor linking energetics and the cytoskeleton.


Assuntos
Proteínas Quinases Ativadas por AMP , Dinâmica Mitocondrial , Neoplasias , Humanos , Trifosfato de Adenosina/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Adesão Celular , Movimento Celular/fisiologia , Miosina Tipo II/metabolismo , Fosforilação Oxidativa , Fosforilação
10.
EMBO Rep ; 11(8): 612-8, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20634802

RESUMO

SAGA (Spt-Ada-Gcn5 acetyltransferase), a coactivator complex involved in chromatin remodelling, harbours both histone acetylation and deubiquitination activities. ATXN7/Sgf73 and ATXN7L3, two subunits of the SAGA deubiquitination module, contain an SCA7 domain characterized by an atypical zinc-finger. We show that the yeast Sgf73-SCA7 domain is not required to recruit Sgf73 into SAGA. Instead, it binds to nucleosomes, a property that is conserved in the human ATXN7-SCA7 domain but is lost in the ATXN7L3 domain. The solution structures of the SCA7 domain of both ATXN7 and ATXN7L3 reveal a new, common zinc-finger motif at the heart of two distinct folds, providing a molecular basis for the observed functional differences.


Assuntos
Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Nucleossomos/metabolismo , Estrutura Secundária de Proteína , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Ataxina-7 , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Fatores de Transcrição/genética , Ubiquitinação , Dedos de Zinco
11.
Dalton Trans ; 50(44): 16156-16165, 2021 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-34704995

RESUMO

Radiotracers labelled with technetium-99m (99mTc) enable accessible diagnostic imaging of disease, provided that radiotracer preparation is simple. Whilst 99mTc radiopharmaceuticals for imaging perfusion are routinely prepared from kits, and regularly used in healthcare, there are no 99mTc-labelled receptor-targeted radiopharmaceuticals in widespread clinical use. This is in part due to the multistep radiosyntheses required for the latter. We demonstrate that the diphosphine, 2,3-bis(diphenylphosphino)maleic anhydride (BMA), is an excellent platform for preparation of kit-based, receptor-targeted 99mTc-labelled radiotracers: its conjugates are simple to prepare and can be easily labelled with 99mTc using one-step, kit-based protocols. Here, reaction of BMA with the αvß3-integrin receptor targeted cyclic peptide, Arg-Gly-Asp-DPhe-Lys (RGD), provided the first diphosphine-peptide conjugate, DP-RGD. DP-RGD was incorporated into a "kit", and addition of a saline solution containing 99mTcO4- to this kit, followed by heating, furnished the radiotracer [99mTcO2(DP-RGD)2]+ in consistently high radiochemical yields (>90%). The analogous [ReO2(DP-RGD)2]+ compound was prepared and characterised, revealing that both [99mTcO2(DP-RGD)2]+ and [ReO2(DP-RGD)2]+ consist of a mixture of cis and trans geometric isomers. Finally, [99mTcO2(DP-RGD)2]+ exhibited high metabolic stability, and selectively targeted αvß3-integrin receptors, enabling in vivo SPECT imaging of αvß3-integrin receptor expression in mice.


Assuntos
Quelantes , Peptídeos Cíclicos , Fosfinas , Compostos Radiofarmacêuticos , Tecnécio , Animais , Artrite Reumatoide/diagnóstico por imagem , Artrite Reumatoide/metabolismo , Quelantes/administração & dosagem , Quelantes/química , Quelantes/farmacocinética , Feminino , Humanos , Integrina alfaVbeta3/química , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Peptídeos Cíclicos/administração & dosagem , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacocinética , Fosfinas/administração & dosagem , Fosfinas/química , Fosfinas/farmacocinética , Compostos Radiofarmacêuticos/administração & dosagem , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Tecnécio/administração & dosagem , Tecnécio/química , Tecnécio/farmacocinética , Tomografia Computadorizada de Emissão de Fóton Único
12.
Commun Biol ; 3(1): 697, 2020 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-33247193

RESUMO

Antimicrobial peptides (AMPs) are a potential alternative to classical antibiotics that are yet to achieve a therapeutic breakthrough for treatment of systemic infections. The antibacterial potency of pleurocidin, an AMP from Winter Flounder, is linked to its ability to cross bacterial plasma membranes and seek intracellular targets while also causing membrane damage. Here we describe modification strategies that generate pleurocidin analogues with substantially improved, broad spectrum, antibacterial properties, which are effective in murine models of bacterial lung infection. Increasing peptide-lipid intermolecular hydrogen bonding capabilities enhances conformational flexibility, associated with membrane translocation, but also membrane damage and potency, most notably against Gram-positive bacteria. This negates their ability to metabolically adapt to the AMP threat. An analogue comprising D-amino acids was well tolerated at an intravenous dose of 15 mg/kg and similarly effective as vancomycin in reducing EMRSA-15 lung CFU. This highlights the therapeutic potential of systemically delivered, bactericidal AMPs.


Assuntos
Antibacterianos/farmacologia , Proteínas de Peixes/farmacologia , Pneumopatias/tratamento farmacológico , Proteínas Citotóxicas Formadoras de Poros/farmacologia , Animais , Antibacterianos/química , Antibacterianos/uso terapêutico , Modelos Animais de Doenças , Proteínas de Peixes/química , Proteínas de Peixes/uso terapêutico , Células HEK293 , Células HeLa , Humanos , Ligação de Hidrogênio , Pneumopatias/microbiologia , Masculino , Membranas Artificiais , Camundongos , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/uso terapêutico , Conformação Proteica
13.
PLoS One ; 14(2): e0210352, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30707691

RESUMO

Nek2 is a dimeric serine/ threonine protein kinase that belongs to the family of NIMA-related kinases (Neks). Its N-terminal catalytic domain and its C-terminal regulatory region are bridged by a leucine zipper, which plays an important role in the activation of Nek2's catalytic activity. Unusual conformational dynamics on the intermediary/slow timescale has thwarted all attempts so far to determine the structure of the Nek2 leucine zipper by means of X-ray crystallography and Nuclear Magnetic Resonance (NMR). Disulfide engineering, the strategic placement of non-native disulfide bonds into flexible regions flanking the coiled coil, was used to modulate the conformational exchange dynamics of this important dimerization domain. The resulting reduction in exchange rate leads to substantial improvements of important features in NMR spectra, such as line width, coherence transfer leakage and relaxation. These effects were comprehensively analyzed for the wild type protein, two single disulfide bond-bearing mutants and another double disulfide bonds-carrying mutant. Furthermore, exchange kinetics were measured across a wide temperature range, allowing for a detailed analysis of activation energy (ΔG‡) and maximal rate constant (k'ex). For one mutant carrying a disulfide bond at its C-terminus, a full backbone NMR assignment could be obtained for both conformers, demonstrating the benefits of the disulfide engineering. Our study demonstrates the first successful application of 'kinetic' disulfide bonds for the purpose of controlling the adverse effects of protein dynamics. Firstly, this provides a promising, robust platform for the full structural and functional investigation of the Nek2 leucine zipper in the future. Secondly, this work broadens the toolbox of protein engineering by disulfide bonds through the addition of a kinetic option in addition to the well-established thermodynamic uses of disulfide bonds.


Assuntos
Substituição de Aminoácidos , Dissulfetos/química , Zíper de Leucina , Quinases Relacionadas a NIMA/química , Cristalografia por Raios X , Humanos , Quinases Relacionadas a NIMA/genética , Ressonância Magnética Nuclear Biomolecular
14.
Biomol NMR Assign ; 13(1): 169-172, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30632004

RESUMO

Human LARP4A belongs to a superfamily of RNA binding proteins called La-related proteins (LARPs). Whilst being a positive regulator of protein synthesis and a promoter of mRNA stability, LARP4A also controls cell morphology and motility in human breast and prostate cancer cells. All LARPs share a characteristic RNA binding unit named the La-module, which despite a high level of primary structure conservation exhibits a great versatility in RNA target selection. Human LARP4A La-module is the most divergent compared with other LARPs and its RNA recognition properties have only recently started to be revealed. Given the key role of LARP4A protein in cancer cell biology, we have initiated a complete NMR characterisation of its La-module and here we report the assignment of 1H, 15N and 13C resonances resulting from our studies.


Assuntos
Autoantígenos/química , Ressonância Magnética Nuclear Biomolecular , Ribonucleoproteínas/química , Humanos , Estrutura Secundária de Proteína , Antígeno SS-B
15.
Commun Biol ; 2: 188, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31123712

RESUMO

Human soluble epoxide hydrolase (hsEH) is an enzyme responsible for the inactivation of bioactive epoxy fatty acids, and its inhibition is emerging as a promising therapeutical strategy to target hypertension, cardiovascular disease, pain and insulin sensitivity. Here, we uncover the molecular bases of hsEH inhibition mediated by the endogenous 15-deoxy-Δ12,14-Prostaglandin J2 (15d-PGJ2). Our data reveal a dual inhibitory mechanism, whereby hsEH can be inhibited by reversible docking of 15d-PGJ2 in the catalytic pocket, as well as by covalent locking of the same compound onto cysteine residues C423 and C522, remote to the active site. Biophysical characterisations allied with in silico investigations indicate that the covalent modification of the reactive cysteines may be part of a hitherto undiscovered allosteric regulatory mechanism of the enzyme. This study provides insights into the molecular modes of inhibition of hsEH epoxy-hydrolytic activity and paves the way for the development of new allosteric inhibitors.


Assuntos
Epóxido Hidrolases/antagonistas & inibidores , Prostaglandina D2/análogos & derivados , Regulação Alostérica , Sequência de Aminoácidos , Substituição de Aminoácidos , Domínio Catalítico/genética , Cristalografia por Raios X , Cisteína/química , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Epóxido Hidrolases/química , Epóxido Hidrolases/genética , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Prostaglandina D2/farmacologia , Domínios Proteicos , Alinhamento de Sequência , Solubilidade
16.
Sci Rep ; 9(1): 10934, 2019 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-31358802

RESUMO

Frogs such as Rana temporaria and Litoria aurea secrete numerous closely related antimicrobial peptides (AMPs) as an effective chemical dermal defence. Damage or penetration of the bacterial plasma membrane is considered essential for AMP activity and such properties are commonly ascribed to their ability to form secondary amphipathic, α-helix conformations in membrane mimicking milieu. Nevertheless, despite the high similarity in physical properties and preference for adopting such conformations, the spectrum of activity and potency of AMPs often varies considerably. Hence distinguishing apparently similar AMPs according to their behaviour in, and effects on, model membranes will inform understanding of primary-sequence-specific antimicrobial mechanisms. Here we use a combination of molecular dynamics simulations, circular dichroism and patch-clamp to investigate the basis for differing anti-bacterial activities in representative AMPs from each species; temporin L and aurein 2.5. Despite adopting near identical, α-helix conformations in the steady-state in a variety of membrane models, these two AMPs can be distinguished both in vitro and in silico based on their dynamic interactions with model membranes, notably their differing conformational flexibility at the N-terminus, ability to form higher order aggregates and the characteristics of induced ion conductance. Taken together, these differences provide an explanation of the greater potency and broader antibacterial spectrum of activity of temporin L over aurein 2.5. Consequently, while the secondary amphipathic, α-helix conformation is a key determinant of the ability of a cationic AMP to penetrate and disrupt the bacterial plasma membrane, the exact mechanism, potency and spectrum of activity is determined by precise structural and dynamic contributions from specific residues in each AMP sequence.


Assuntos
Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Membrana Celular/efeitos dos fármacos , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Transporte de Íons , Simulação de Dinâmica Molecular , Conformação Proteica em alfa-Hélice , Lipossomas Unilamelares/química
17.
Sci Rep ; 9(1): 1385, 2019 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-30718667

RESUMO

Antimicrobial peptides (AMPs) are a potential source of new molecules to counter the increase in antimicrobial resistant infections but a better understanding of their properties is required to understand their native function and for effective translation as therapeutics. Details of the mechanism of their interaction with the bacterial plasma membrane are desired since damage or penetration of this structure is considered essential for AMPs activity. Relatively modest modifications to AMPs primary sequence can induce substantial changes in potency and/or spectrum of activity but, hitherto, have not been predicted to substantially alter the mechanism of interaction with the bacterial plasma membrane. Here we use a combination of molecular dynamics simulations, circular dichroism, solid-state NMR and patch clamp to investigate the extent to which temporin B and its analogues can be distinguished both in vitro and in silico on the basis of their interactions with model membranes. Enhancing the hydrophobicity of the N-terminus and cationicity of the C-terminus in temporin B improves its membrane activity and potency against both Gram-negative and Gram-positive bacteria. In contrast, enhancing the cationicity of the N-terminus abrogates its ability to trigger channel conductance and renders it ineffective against Gram-positive bacteria while nevertheless enhancing its potency against Escherichia coli. Our findings suggest even closely related AMPs may target the same bacterium with fundamentally differing mechanisms of action.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Membrana Celular/metabolismo , Sequência de Aminoácidos , Membrana Celular/efeitos dos fármacos , Condutividade Elétrica , Bicamadas Lipídicas/química , Micelas , Testes de Sensibilidade Microbiana , Simulação de Dinâmica Molecular , Conformação Proteica , Dodecilsulfato de Sódio , Relação Estrutura-Atividade
18.
Sci Rep ; 9(1): 2297, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30783167

RESUMO

At high altitude oxygen delivery to the tissues is impaired leading to oxygen insufficiency (hypoxia). Acclimatisation requires adjustment to tissue metabolism, the details of which remain incompletely understood. Here, metabolic responses to progressive environmental hypoxia were assessed through metabolomic and lipidomic profiling of human plasma taken from 198 human participants before and during an ascent to Everest Base Camp (5,300 m). Aqueous and lipid fractions of plasma were separated and analysed using proton (1H)-nuclear magnetic resonance spectroscopy and direct infusion mass spectrometry, respectively. Bayesian robust hierarchical regression revealed decreasing isoleucine with ascent alongside increasing lactate and decreasing glucose, which may point towards increased glycolytic rate. Changes in the lipid profile with ascent included a decrease in triglycerides (48-50 carbons) associated with de novo lipogenesis, alongside increases in circulating levels of the most abundant free fatty acids (palmitic, linoleic and oleic acids). Together, this may be indicative of fat store mobilisation. This study provides the first broad metabolomic account of progressive exposure to environmental hypobaric hypoxia in healthy humans. Decreased isoleucine is of particular interest as a potential contributor to muscle catabolism observed with exposure to hypoxia at altitude. Substantial changes in lipid metabolism may represent important metabolic responses to sub-acute exposure to environmental hypoxia.


Assuntos
Lipidômica/métodos , Metabolômica/métodos , Adulto , Teorema de Bayes , Peso Corporal/fisiologia , Feminino , Humanos , Metabolismo dos Lipídeos/fisiologia , Masculino , Pessoa de Meia-Idade , Oxigênio/metabolismo , Triglicerídeos/sangue
19.
JCI Insight ; 4(24)2019 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-31751317

RESUMO

BACKGROUNDHepatic encephalopathy (HE) is associated with poor outcomes. A prior randomized, pilot trial demonstrated safety after oral capsular fecal microbial transplant (FMT) in HE, with favorable changes in microbial composition and cognition. However, microbial functional changes are unclear. The aim of this study was to determine the effect of FMT on the gut-brain axis compared with placebo, using microbial function based on bile acids (BAs), inflammation (serum IL-6, LPS-binding protein [LBP]), and their association with EncephalApp.METHODSTwenty cirrhotic patients were randomized 1:1 into groups that received 1-time FMT capsules from a donor enriched in Lachnospiraceae and Ruminococcaceae or placebo capsules, with 5-month follow-up for safety outcomes. Stool microbiota and BA; serum IL-6, BA, and LBP; and EncephalApp were analyzed at baseline and 4 weeks after FMT/placebo. Correlation networks among microbiota, BAs, EncephalApp, IL-6, and LBP were performed before/after FMT.RESULTSFMT-assigned participants had 1 HE recurrence and 2 unrelated infections. Six placebo-assigned participants developed negative outcomes. FMT, but not placebo, was associated with reduced serum IL-6 and LBP and improved EncephalApp. FMT-assigned participants demonstrated higher deconjugation and secondary BA formation in feces and serum compared with baseline. No change was seen in placebo. Correlation networks showed greater complexity after FMT compared with baseline. Beneficial taxa, such as Ruminococcaceae, Verrucomicrobiaceae, and Lachnospiraceae, were correlated with cognitive improvement and decrease in inflammation after FMT. Fecal/serum secondary/primary ratios and PiCRUST secondary BA pathways did not increase in participants who developed poor outcomes.CONCLUSIONGut microbial function in cirrhosis is beneficially affected by capsular FMT, with improved inflammation and cognition. Lower secondary BAs in FMT recipients could select for participants who develop negative outcomes.TRIAL REGISTRATIONClinicaltrials.gov NCT03152188.FUNDINGNational Center for Advancing Translational Sciences NIH grant R21TR002024, VA Merit Review grant 2I0CX001076, the United Kingdom National Institute for Health Research Biomedical Facility at Imperial College London, the British Heart Foundation, Wellcome Trust, and King's College London.


Assuntos
Cognição/fisiologia , Transplante de Microbiota Fecal/métodos , Microbioma Gastrointestinal/fisiologia , Encefalopatia Hepática/terapia , Cirrose Hepática/terapia , Adulto , Idoso , Cápsulas , Fezes/microbiologia , Feminino , Encefalopatia Hepática/etiologia , Encefalopatia Hepática/microbiologia , Encefalopatia Hepática/fisiopatologia , Humanos , Cirrose Hepática/complicações , Cirrose Hepática/microbiologia , Cirrose Hepática/fisiopatologia , Masculino , Pessoa de Meia-Idade , Placebos/administração & dosagem , Resultado do Tratamento , Reino Unido , Adulto Jovem
20.
J Mol Biol ; 368(2): 473-80, 2007 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-17350038

RESUMO

Trichothiodystrophy (TTD) is a rare hereditary multi-system disorder associated with defects in nucleotide excision repair (NER) and transcription as consequences of mutations in XPB, XPD and p8/TTD-A subunits of transcription factor IIH (TFIIH). Here, we report the solution structure of the p8/TTD-A protein, a small alpha/beta protein built around an antiparallel beta-sheet that forms a homodimer with an extended interface. In order to characterize the dimer interface, we have introduced a mutation at position 44, which destabilizes the dimeric form of the protein. We have shown that this mutation has no effect on the intrinsic ability of p8/TTD-A to stimulate NER in vitro, but affects the capacity of p8/TTD-A to restore TFIIH concentration in TTD-A fibroblasts. Point mutations found in TTD-A patients are discussed on the basis of the present structure.


Assuntos
Anormalidades Múltiplas/metabolismo , Subunidades Proteicas/química , Fator de Transcrição TFIIH/química , Fatores de Transcrição/química , Sequência de Aminoácidos , Cromatografia em Gel , Reparo do DNA , Dimerização , Humanos , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Soluções , Relação Estrutura-Atividade
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