RESUMO
Protein aggregation is a common feature of many neurodegenerative diseases. In Huntington's disease, mutant huntingtin is the primary aggregating protein, but the aggregation of other proteins, such as TDP43, is likely to further contribute to toxicity. Moreover, mutant huntingtin is also a risk factor for TDP pathology in ALS. Despite this co-pathology of huntingtin and TDP43, it remains unknown whether these amyloidogenic proteins directly interact with each other. Using a combination of biophysical methods, we show that the aggregation prone regions of both proteins, huntingtin exon-1 (Httex1) and the TDP43 low complexity domain (TDP43-LCD), interact in a conformationally specific manner. This interaction significantly slows Httex1 aggregation, while it accelerates TDP43-LCD aggregation. A key intermediate responsible for both effects is a complex formed by liquid TDP43-LCD condensates and Httex1 fibrils. This complex shields seeding competent surfaces of Httex1 fibrils from Httex1 monomers, which are excluded from the condensates. In contrast, TDP43-LCD condensates undergo an accelerated liquid-to-solid transition upon exposure to Httex1 fibrils. Cellular studies show co-aggregation of untagged Httex1 with TDP43. This interaction causes mislocalization of TDP43, which has been linked to TDP43 toxicity. The protection from Httex1 aggregation in lieu of TDP43-LCD aggregation is interesting, as it mirrors what has been found in disease models, namely that TDP43 can protect from huntingtin toxicity, while mutant huntingtin can promote TDP43 pathology. These results suggest that direct protein interaction could, at least in part, be responsible for the linked pathologies of both proteins.
RESUMO
Fluorescent protein tags are convenient tools for tracking the aggregation states of amyloidogenic or phase separating proteins, but the effect of the tags is often not well understood. Here, we investigated the impact of a C-terminal red fluorescent protein (RFP) tag on the phase separation of huntingtin exon-1 (Httex1), an N-terminal portion of the huntingtin protein that aggregates in Huntington's disease. We found that the RFP-tagged Httex1 rapidly formed micron-sized, phase separated states in the presence of a crowding agent. The formed structures had a rounded appearance and were highly dynamic according to electron paramagnetic resonance and fluorescence recovery after photobleaching, suggesting that the phase separated state was largely liquid in nature. Remarkably, the untagged protein did not undergo any detectable liquid condensate formation under the same conditions. In addition to strongly promoting liquid-liquid phase separation, the RFP tag also facilitated fibril formation, as the tag-dependent liquid condensates rapidly underwent a liquid-to-solid transition. The rate of fibril formation under these conditions was significantly faster than that of the untagged protein. When expressed in cells, the RFP-tagged Httex1 formed larger aggregates with different antibody staining patterns compared to untagged Httex1. Collectively, these data reveal that the addition of a fluorescent protein tag significantly impacts liquid and solid phase separations of Httex1 in vitro and leads to altered aggregation in cells. Considering that the tagged Httex1 is commonly used to study the mechanisms of Httex1 misfolding and toxicity, our findings highlight the importance to validate the conclusions with untagged protein.
Assuntos
Artefatos , Éxons , Proteína Huntingtina , Doença de Huntington , Medições Luminescentes , Separação de Fases , Agregados Proteicos , Proteína Vermelha Fluorescente , Humanos , Espectroscopia de Ressonância de Spin Eletrônica , Éxons/genética , Fluorescência , Recuperação de Fluorescência Após Fotodegradação , Proteína Huntingtina/química , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/genética , Doença de Huntington/metabolismo , Medições Luminescentes/métodos , Proteína Vermelha Fluorescente/genética , Proteína Vermelha Fluorescente/metabolismo , Reprodutibilidade dos TestesRESUMO
Huntington's disease (HD) is a genetically inherited neurodegenerative disorder caused by expansion of a polyglutamine (polyQ) repeat in the exon-1 of huntingtin protein (HTT). The expanded polyQ enhances the amyloidogenic propensity of HTT exon 1 (HTTex1), which forms a heterogeneous mixture of assemblies with a broad neurotoxicity spectrum. While predominantly intracellular, monomeric and aggregated mutant HTT species are also present in the cerebrospinal fluids of HD patients, however, their biological properties are not well understood. To explore the role of extracellular mutant HTT in aggregation and toxicity, we investigated the uptake and amplification of recombinant HTTex1 assemblies in cell culture models. We find that small HTTex1 fibrils preferentially enter human neurons and trigger the amplification of neurotoxic assemblies; astrocytes or epithelial cells are not permissive. The amplification of HTTex1 in neurons depletes endogenous HTT protein with non-pathogenic polyQ repeat, activates apoptotic caspase-3 pathway and induces nuclear fragmentation. Using a panel of novel monoclonal antibodies and genetic mutation, we identified epitopes within the N-terminal 17 amino acids and proline-rich domain of HTTex1 to be critical in neural uptake and amplification. Synaptosome preparations from the brain homogenates of HD mice also contain mutant HTT species, which enter neurons and behave similar to small recombinant HTTex1 fibrils. These studies suggest that amyloidogenic extracellular mutant HTTex1 assemblies may preferentially enter neurons, propagate and promote neurodegeneration.
Assuntos
Astrócitos/metabolismo , Células Epiteliais/metabolismo , Proteína Huntingtina/metabolismo , Doença de Huntington/metabolismo , Neurônios/metabolismo , Agregação Patológica de Proteínas/metabolismo , Proteínas Amiloidogênicas/genética , Proteínas Amiloidogênicas/metabolismo , Animais , Apoptose , Caspase 3 , Éxons , Técnicas de Introdução de Genes , Humanos , Proteína Huntingtina/genética , Camundongos , Camundongos Transgênicos , Mutação , Peptídeos/genética , Agregação Patológica de Proteínas/genética , SinaptossomosRESUMO
The N-terminal fragments of mutant huntingtin (mHTT) misfold and assemble into oligomers, which ultimately bundle into insoluble fibrils. Conformations unique to various assemblies of mHTT remain unknown. Knowledge on the half-life of various multimeric structures of mHTT is also scarce. Using a panel of four new antibodies named PHP1-4, we have identified new conformations in monomers and assembled structures of mHTT. PHP1 and PHP2 bind to epitopes within the proline-rich domain (PRD), whereas PHP3 and PHP4 interact with motifs formed at the junction of polyglutamine (polyQ) and polyproline (polyP) repeats of HTT. The PHP1- and PHP2-reactive epitopes are exposed in fibrils of mHTT exon1 (mHTTx1) generated from recombinant proteins and mHTT assemblies, which progressively accumulate in the nuclei, cell bodies and neuropils in the brains of HD mouse models. Notably, electron microscopic examination of brain sections of HD mice revealed that PHP1- and PHP2-reactive mHTT assemblies are present in myelin sheath and in vesicle-like structures. Moreover, PHP1 and PHP2 antibodies block seeding and subsequent fibril assembly of mHTTx1 in vitro and in a cell culture model of HD. PHP3 and PHP4 bind to epitopes in full-length and N-terminal fragments of monomeric mHTT and binding diminishes as the mHTTx1 assembles into fibrils. Interestingly, PHP3 and PHP4 also prevent the aggregation of mHTTx1 in vitro highlighting a regulatory function for the polyQ-polyP motifs. These newly detected conformations may affect fibril assembly, stability and intercellular transport of mHTT.
Assuntos
Proteína Huntingtina , Motivos de Aminoácidos , Animais , Humanos , Proteína Huntingtina/química , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Camundongos , Camundongos Transgênicos , Agregados Proteicos , Domínios ProteicosRESUMO
Expansion of the polyglutamine (polyQ) tract in exon 1 of the huntingtin protein (Httex1) leads to Huntington's disease resulting in fatal neurodegeneration. However, it remains poorly understood how polyQ expansions alter protein structure and cause toxicity. Using CD, EPR, and NMR spectroscopy, we found here that monomeric Httex1 consists of two co-existing structural states whose ratio is determined by polyQ tract length. We observed that short Q-lengths favor a largely random-coil state, whereas long Q-lengths increase the proportion of a predominantly α-helical state. We also note that by following a mobility gradient, Httex1 α-helical conformation is restricted to the N-terminal N17 region and to the N-terminal portion of the adjoining polyQ tract. Structuring in both regions was interdependent and likely stabilized by tertiary contacts. Although little helicity was present in N17 alone, each Gln residue in Httex1 enhanced helix stability by 0.03-0.05 kcal/mol, causing a pronounced preference for the α-helical state at pathological Q-lengths. The Q-length-dependent structuring and rigidification could be mimicked in proteins with shorter Q-lengths by a decrease in temperature, indicating that lower temperatures similarly stabilize N17 and polyQ intramolecular contacts. The more rigid α-helical state of Httex1 with an expanded polyQ tract is expected to alter interactions with cellular proteins and modulate the toxic Httex1 misfolding process. We propose that the polyQ-dependent shift in the structural equilibrium may enable future therapeutic strategies that specifically target Httex1 with toxic Q-lengths.
Assuntos
Éxons , Proteína Huntingtina/química , Proteína Huntingtina/genética , Peptídeos , Dobramento de Proteína , Humanos , Modelos Moleculares , Conformação Proteica em alfa-Hélice , TemperaturaRESUMO
Aggregation of huntingtin protein arising from expanded polyglutamine (polyQ) sequences in the exon-1 region of mutant huntingtin plays a central role in the pathogenesis of Huntington's disease. The huntingtin aggregation pathways are of therapeutic and diagnostic interest, but obtaining critical information from the physiologically relevant htt exon-1 (Httex1) protein has been challenging. Using biophysical techniques and an expression and purification protocol that generates clean, monomeric Httex1, we identified and mapped three distinct aggregation pathways: 1) unseeded in solution; 2) seeded in solution; and 3) membrane-mediated. In solution, aggregation proceeded in a highly stepwise manner, in which the individual domains (N terminus containing 17 amino acids (N17), polyQ, and proline-rich domain (PRD)) become ordered at very different rates. The aggregation was initiated by an early oligomer requiring a pathogenic, expanded Gln length and N17 α-helix formation. In the second phase, ß-sheet forms in the polyQ. The slowest step is the final structural maturation of the PRD. This stepwise mechanism could be bypassed by seeding, which potently accelerated aggregation and was a prerequisite for prion-like spreading in vivo Remarkably, membranes could catalyze aggregation even more potently than seeds, in a process that caused significant membrane damage. The N17 governed membrane-mediated aggregation by anchoring Httex1 to the membrane, enhancing local concentration and promoting collision via two-dimensional diffusion. Considering its central roles in solution and in membrane-mediated aggregation, the N17 represents an attractive target for inhibiting multiple pathways. Our approach should help evaluate such inhibitors and identify diagnostic markers for the misfolded forms identified here.
Assuntos
Membrana Celular/metabolismo , Proteína Huntingtina/química , Proteína Huntingtina/metabolismo , Doença de Huntington/metabolismo , Motivos de Aminoácidos , Membrana Celular/química , Membrana Celular/genética , Humanos , Proteína Huntingtina/genética , Doença de Huntington/genética , Cinética , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Agregados Proteicos , Conformação Proteica em alfa-Hélice , Domínios ProteicosRESUMO
STUDY DESIGN: Randomized controlled trial. OBJECTIVES: To determine the effect of zoledronic acid on bone loss in people with acute spinal cord injury (SCI) SETTINGS: Sawai Man Singh Medical College, India. METHODS: Sixty patients with acute SCI were randomized to receive either standard treatment alone or standard treatment with zoledronic acid within 3 months after injury. Areal bone mineral density (aBMD) was measured at the hip using dual-energy X-ray absorptiometry (DXA) at baseline 3, 6, and 12 months. RESULTS: Significant differences in aBMD were found between the standard treatment alone and standard treatment plus zoledronic acid group at the femoral neck (-0.13; 95% CI, -0.18 to -0.09, p < 0.0001), and total hip (-0.16; 95% CI, -0.19 to -0.12, p < 0.0001), respectively, at 1 year and bone loss was reduced in the zoledronic acid treated group as compared to the standard treatment group. Significant differences in aBMD between the groups at 6 months post infusion was also observed at these sites. [Femoral neck -0.08; 95% CI, -0.12 to -0.03; p = 0.002 and total hip -0.12; 95% CI, -0.15 to -0.08; p < 0.0001] CONCLUSION: A zoledronic acid 5 mg infusion given within 3 month significantly reduces bone loss at the hip after 6 months post infusion in patients with acute SCI.
Assuntos
Conservadores da Densidade Óssea/uso terapêutico , Doenças Ósseas Metabólicas/etiologia , Doenças Ósseas Metabólicas/prevenção & controle , Traumatismos da Medula Espinal/complicações , Traumatismos da Medula Espinal/tratamento farmacológico , Ácido Zoledrônico/uso terapêutico , Absorciometria de Fóton , Adulto , Densidade Óssea/efeitos dos fármacos , Doenças Ósseas Metabólicas/diagnóstico por imagem , Feminino , Fêmur/diagnóstico por imagem , Fêmur/efeitos dos fármacos , Quadril/diagnóstico por imagem , Humanos , Masculino , Traumatismos da Medula Espinal/diagnóstico por imagem , Fatores de Tempo , Resultado do TratamentoRESUMO
The fact that the heritable neurodegenerative disorder Huntington's disease (HD) is autosomal dominant means that there is one wild type and one mutant allele in most HD patients. The CAG repeat expansion in the exon 1 of the protein huntingtin (HTTex1) that causes the disease leads to the formation of HTT fibrils in vitro and vivo. An important question for understanding the molecular mechanism of HD is which role wild type HTT plays for the formation, propagation, and structure of these HTT fibrils. Here we report that fibrils of mutant HTTex1 are able to seed the aggregation of wild type HTTex1 into amyloid fibrils, which in turn can seed the fibril formation of mutant HTTex1. Solid-state NMR and electron paramagnetic resonance data showed that wild type HTTex1 fibrils closely resemble the structure of mutant fibrils, with small differences indicating a less extended fibril core. These data suggest that wild type fibrils can faithfully perpetuate the structure of mutant fibrils in HD. However, wild type HTTex1 monomers have a much higher equilibrium solubility compared to mutant HTTex1, and only a small fraction incorporates into fibrils.
Assuntos
Amiloide/genética , Proteína Huntingtina/genética , Doença de Huntington/genética , Doença de Huntington/patologia , Amiloide/química , Amiloide/ultraestrutura , Éxons , Humanos , Proteína Huntingtina/química , Proteína Huntingtina/ultraestrutura , Doença de Huntington/metabolismo , Mutação , Ressonância Magnética Nuclear Biomolecular , Agregados Proteicos , SolubilidadeRESUMO
Background: Pneumonia, the leading infectious cause of child mortality globally, mainly afflicts developing countries. This prospective observational study aimed to assess the microorganisms associated with pneumonia in children aged <5 years in developing and emerging countries. Methods: A multicenter, case-control study by the GABRIEL (Global Approach to Biological Research, Infectious diseases and Epidemics in Low-income countries) network was conducted between 2010 and 2014 in Cambodia, China, Haiti, India (2 sites), Madagascar, Mali, Mongolia, and Paraguay. Cases were hospitalized children with radiologically confirmed pneumonia; controls were children from the same setting without any features suggestive of pneumonia. Nasopharyngeal swabs were collected from all subjects; 19 viruses and 5 bacteria were identified by reverse-transcription polymerase chain reaction. Associations between microorganisms and pneumonia were quantified by calculating the adjusted population attributable fraction (aPAF) after multivariate logistic regression analysis adjusted for sex, age, time period, other pathogens, and site. Results: Overall, 888 cases and 870 controls were analyzed; ≥1 microorganism was detected in respiratory samples in 93.0% of cases and 74.4% of controls (P < .001). Streptococcus pneumoniae, Mycoplasma pneumoniae, human metapneumovirus, rhinovirus, respiratory syncytial virus (RSV), parainfluenza virus 1, 3, and 4, and influenza virus A and B were independently associated with pneumonia; aPAF was 42.2% (95% confidence interval [CI], 35.5%-48.2%) for S. pneumoniae, 18.2% (95% CI, 17.4%-19.0%) for RSV, and 11.2% (95% CI, 7.5%-14.7%) for rhinovirus. Conclusions: Streptococcus pneumoniae, RSV, and rhinovirus may be the major microorganisms associated with pneumonia infections in children <5 years of age from developing and emerging countries. Increasing S. pneumoniae vaccination coverage may substantially reduce the burden of pneumonia among children in developing countries.
Assuntos
Pneumonia Bacteriana/epidemiologia , Pneumonia Bacteriana/microbiologia , Ásia/epidemiologia , Estudos de Casos e Controles , Pré-Escolar , Países em Desenvolvimento , Feminino , Haiti/epidemiologia , Humanos , Lactente , Masculino , Mali/epidemiologia , Estudos ProspectivosRESUMO
BACKGROUND: Data on the etiologies of pneumonia among children are inadequate, especially in developing countries. The principal objective is to undertake a multicenter incident case-control study of <5-year-old children hospitalized with pneumonia in developing and emerging countries, aiming to identify the causative agents involved in pneumonia while assessing individual and microbial factors associated with the risk of severe pneumonia. METHODS/DESIGN: A multicenter case-control study, based on the GABRIEL network, is ongoing. Ten study sites are located in 9 countries over 3 continents: Brazil, Cambodia, China, Haiti, India, Madagascar, Mali, Mongolia, and Paraguay. At least 1,000 incident cases and 1,000 controls will be enrolled and matched for age and date. Cases are hospitalized children <5 years with radiologically confirmed pneumonia, and the controls are children without any features suggestive of pneumonia. Respiratory specimens are collected from all enrolled subjects to identify 19 viruses and 5 bacteria. Whole blood from pneumonia cases is being tested for 3 major bacteria. S. pneumoniae-positive specimens are serotyped. Urine samples from cases only are tested for detection of antimicrobial activity. The association between procalcitonin, C-reactive protein and pathogens is being evaluated. A discovery platform will enable pathogen identification in undiagnosed samples. DISCUSSION: This multicenter study will provide descriptive results for better understanding of pathogens responsible for pneumonia among children in developing countries. The identification of determinants related to microorganisms associated with pneumonia and its severity should facilitate treatment and prevention.
Assuntos
Protocolos Clínicos , Países em Desenvolvimento , Pneumonia/etiologia , Antibacterianos/urina , Bactérias/isolamento & purificação , Brasil , Proteína C-Reativa/metabolismo , Calcitonina/sangue , Peptídeo Relacionado com Gene de Calcitonina , Camboja , Estudos de Casos e Controles , Pré-Escolar , China , Feminino , Haiti , Humanos , Índia , Lactente , Madagáscar , Masculino , Mali , Mongólia , Paraguai , Derrame Pleural/microbiologia , Pneumonia/sangue , Pneumonia/metabolismo , Pneumonia/urina , Precursores de Proteínas/sangue , Vírus/isolamento & purificaçãoRESUMO
We have investigated the binding of the biologically important flavonoid fisetin with the carrier protein bovine serum albumin using multi-spectroscopic and molecular docking methods. The binding constants were found to be in the order of 10(4) M(-1) and the number of binding sites was determined as one. MALDI-TOF analyses showed that one fisetin molecule binds to a single bovine serum albumin (BSA) molecule which is also supported by fluorescence quenching studies. The negative Gibbs free energy change (∆G°) values point to a spontaneous binding process which occurs through the presence of electrostatic forces with hydrophobic association that results in a positive entropy change (+51.69 ± 1.18 J mol(-1) K(-1)). The unfolding and refolding of BSA in urea have been studied in absence and presence of fisetin using steady-state fluorescence and lifetime measurements. Urea denaturation studies indicate that fisetin is gradually released from its binding site on the protein. In the absence of urea, an increase in temperature that causes denaturation of the protein results in the release of fisetin from its bound state indicating that fisetin binds only to the native state of the protein. The circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopic studies showed an increase in % α-helix content of BSA after binding with fisetin. Site marker displacement studies in accordance with the molecular docking results suggested that fisetin binds in close proximity of the hydrophobic cavity in site 1 (subdomain IIA) of the protein. The PEARLS (Program of Energetic Analysis of Receptor Ligand System) has been used to estimate the interaction energy of fisetin with BSA and the results are in good correlation with the experimental findings.
Assuntos
Flavonoides/química , Simulação de Acoplamento Molecular , Soroalbumina Bovina/química , Animais , Sítios de Ligação , Bovinos , Dicroísmo Circular , Entropia , Flavonóis , Ligação Proteica , Conformação Proteica , Espectroscopia de Infravermelho com Transformada de Fourier , Análise Espectral , TermodinâmicaRESUMO
The first exon of the huntingtin protein (HTTex1) important in Huntington's disease (HD) can form cross-ß fibrils of varying toxicity. We find that the difference between these fibrils is the degree of entanglement and dynamics of the C-terminal proline-rich domain (PRD) in a mechanism analogous to polyproline film formation. In contrast to fibril strains found for other cross-ß fibrils, these HTTex1 fibril types can be interconverted. This is because the structure of their polyQ fibril core remains unchanged. Further, we find that more toxic fibrils of low entanglement have higher affinities for protein interactors and are more effective seeds for recombinant HTTex1 and HTTex1 in cells. Together these data show how the structure of a framing sequence at the surface of a fibril can modulate seeding, protein-protein interactions, and thereby toxicity in neurodegenerative disease.
Assuntos
Proteína Huntingtina/metabolismo , Doença de Huntington/metabolismo , Doenças Neurodegenerativas/metabolismo , Humanos , Proteína Huntingtina/genética , Doença de Huntington/genética , Doenças Neurodegenerativas/genética , Peptídeos/química , Peptídeos/metabolismo , Mapas de Interação de ProteínasRESUMO
OBJECTIVE: Endometriosis is a leading cause of infertility, and recent studies suggest that leptin and adiponectin may have a role in its causation and progression. This study assessed levels of leptin and adiponectin in serum and peritoneal fluid (PF) in patients with endometriosis and infertility. DESIGN AND SETTING: This cross-sectional study included women undergoing diagnostic and/or therapeutic laparoscopy for endometriosis with chief complaint of infertility. Following laparoscopy, patients diagnosed with endometriosis served as cases while patients with no endometriosis served as controls. Patients with polycystic ovarian syndrome, diabetes, thyroiditis and patients on prior therapy with danazol or leuprolide were excluded from the study. Leptin and adiponectin levels were analysed in blood and PF using commercially available ELISA kits. RESULTS: Of the 50 patients (aged 22-41 years), 15 had endometriosis (cases) while 35 had no endometriosis (controls). The median PF leptin level was significantly higher in cases as compared to controls (27.7 vs. 15.6 ng/ml, p = 0.019), and this remained significant even when PF leptin was BMI-normalised (p = 0.004). However, median serum leptin and adiponectin levels remained comparable between the two groups. CONCLUSIONS: This study confirmed the role of PF leptin in causation and progression of endometriosis. However, this would have been definitive if healthy fertile females were included in this study.
Assuntos
Líquido Ascítico/química , Endometriose/metabolismo , Infertilidade Feminina/metabolismo , Leptina/análise , Pelve , Adiponectina/análise , Adiponectina/sangue , Adulto , Biomarcadores/sangue , Biomarcadores/metabolismo , Índice de Massa Corporal , Estudos de Casos e Controles , Estudos Transversais , Dieta Vegetariana , Progressão da Doença , Endometriose/sangue , Endometriose/diagnóstico , Endometriose/fisiopatologia , Feminino , Humanos , Infertilidade Feminina/sangue , Infertilidade Feminina/etiologia , Laparoscopia , Leptina/sangue , Ciclo Menstrual , Adulto JovemRESUMO
Huntington's disease (HD) is the most common inherited neurodegenerative disorder and one of the nine polyglutamine (polyQ) diseases. HD is characterized by the pathological aggregation of the misfolded huntingtin exon 1 protein (Httex1) with abnormally long polyQ expansion due to genetic mutation. While there is currently no effective treatment for HD, inhibition of aggregate formation represents a direct approach in mediating the toxicity associated with Httex1 misfolding. To exploit this therapeutic window, we engineered two fluorescence resonance energy transfer (FRET) based biosensors that monitor the aggregation of Httex1 with different expanded Q-lengths (Q39 and Q72) in living cells. These FRET biosensors, together with a high-precision fluorescence lifetime detection platform, enable high-throughput screening of small molecules that target Httex1 aggregation. We found six small molecules that decreased the FRET of the biosensors and reduced Httex1-Q72-induced neuronal cytotoxicity in N2a cells with nanomolar potency. Using advanced SPR and EPR techniques, we confirmed that the compounds directly bind to Httex1 fibrils and inhibit aggregate formation. This strategy in targeting the Httex1 aggregates can be applicable to other proteins involved in polyQ related diseases.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Doença de Huntington , Éxons , Ensaios de Triagem em Larga Escala , Humanos , Proteína Huntingtina/genética , Doença de Huntington/tratamento farmacológico , Doença de Huntington/genética , MutaçãoRESUMO
Huntington's disease is caused by a polyQ expansion in the first exon of huntingtin (Httex1). Membrane interaction of huntingtin is of physiological and pathological relevance. Using electron paramagnetic resonance and Overhauser dynamic nuclear polarization, we find that the N-terminal residues 3-13 of wild-type Httex1(Q25) form a membrane-bound, amphipathic α helix. This helix is positioned in the interfacial region, where it is sensitive to membrane curvature and electrostatic interactions with head-group charges. Residues 14-22, which contain the first five residues of the polyQ region, are in a transition region that remains in the interfacial region without taking up a stable, α-helical structure. The remaining C-terminal portion is solvent exposed. The phosphomimetic S13D/S16D mutations, which are known to protect from toxicity, inhibit membrane binding and attenuate membrane-mediated aggregation of mutant Httex1(Q46) due to electrostatic repulsion. Targeting the N-terminal membrane anchor using post-translational modifications or specific binders could be a potential means to reduce aggregation and toxicity in vivo.
Assuntos
Membrana Celular/metabolismo , Proteína Huntingtina/química , Proteína Huntingtina/metabolismo , Mutação , Sítios de Ligação , Espectroscopia de Ressonância de Spin Eletrônica , Éxons , Humanos , Proteína Huntingtina/genética , Modelos Moleculares , Peptídeos/genética , Agregados Proteicos , Ligação Proteica , Estrutura Secundária de ProteínaRESUMO
Bcl-xL is a member of the Bcl-2 family of apoptotic regulators, responsible for inhibiting the permeabilization of the mitochondrial outer membrane, and a promising anti-cancer target. Bcl-xL exists in the following conformations, each believed to play a role in the inhibition of apoptosis: (a) a soluble folded conformation, (b) a membrane-anchored (by its C-terminal α8 helix) form, which retains the same fold as in solution and (c) refolded membrane-inserted conformations, for which no structural data are available. Previous studies established that in the cell Bcl-xL exists in a dynamic equilibrium between soluble and membranous states, however, no direct evidence exists in support of either anchored or inserted conformation of the membranous state in vivo. In this in vitro study, we employed a combination of fluorescence and EPR spectroscopy to characterize structural features of the bilayer-inserted conformation of Bcl-xL and the lipid modulation of its membrane insertion transition. Our results indicate that the core hydrophobic helix α6 inserts into the bilayer without adopting a transmembrane orientation. This insertion disrupts the packing of Bcl-xL and releases the regulatory N-terminal BH4 domain (α1) from the rest of the protein structure. Our data demonstrate that both insertion and refolding of Bcl-xL are modulated by lipid composition, which brings the apparent pKa of insertion to the threshold of physiological pH. We hypothesize that conformational rearrangements associated with the bilayer insertion of Bcl-xL result in its switching to a so-called non-canonical mode of apoptotic inhibition. Presented results suggest that the alteration in lipid composition before and during apoptosis can serve as an additional factor regulating the permeabilization of the mitochondrial outer membrane.
Assuntos
Bicamadas Lipídicas/química , Lipídeos de Membrana/química , Proteína bcl-X/química , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Bicamadas Lipídicas/metabolismo , Lipídeos de Membrana/metabolismo , Domínios Proteicos , Proteína bcl-X/metabolismoRESUMO
Pneumonia is the leading cause of death in children. The objectives were to evaluate the microbiological agents linked with hypoxemia in hospitalized children with pneumonia from developing countries, to identify predictors of hypoxemia, and to characterize factors associated with in-hospital mortality. A multicenter, observational study was conducted in five hospitals, from India (Lucknow, Vadu), Madagascar (Antananarivo), Mali (Bamako), and Paraguay (San Lorenzo). Children aged 2-60 months with radiologically confirmed pneumonia were enrolled prospectively. Respiratory and whole blood specimens were collected, identifying viruses and bacteria by real-time multiplex polymerase chain reaction (PCR). Microbiological agents linked with hypoxemia at admission (oxygen saturation < 90%) were analyzed by multivariate logistic regression, and factors associated with 14-day in-hospital mortality were assessed by bivariate Cox regression. Overall, 405 pneumonia cases (3,338 hospitalization days) were analyzed; 13 patients died within 14 days of hospitalization. Hypoxemia prevalence was 17.3%. Detection of human metapneumovirus (hMPV) and respiratory syncytial virus (RSV) in respiratory samples was independently associated with increased risk of hypoxemia (adjusted odds ratio [aOR] = 2.4, 95% confidence interval [95% CI] = 1.0-5.8 and aOR = 2.5, 95% CI = 1.1-5.3, respectively). Lower chest indrawing and cyanosis were predictive of hypoxemia (positive likelihood ratios = 2.3 and 2.4, respectively). Predictors of death were Streptococcus pneumoniae detection by blood PCR (crude hazard ratio [cHR] = 4.6, 95% CI = 1.5-14.0), procalcitonin ≥ 50 ng/mL (cHR = 22.4, 95% CI = 7.3-68.5) and hypoxemia (cHR = 4.8, 95% CI = 1.6-14.4). These findings were consistent on bivariate analysis. hMPV and RSV in respiratory samples were linked with hypoxemia, and S. pneumoniae in blood was associated with increased risk of death among hospitalized children with pneumonia in developing countries.
Assuntos
Bactérias/isolamento & purificação , Criança Hospitalizada/estatística & dados numéricos , Países em Desenvolvimento/estatística & dados numéricos , Pneumonia/mortalidade , Vírus/isolamento & purificação , Causas de Morte , Pré-Escolar , Feminino , Humanos , Hipóxia/epidemiologia , Hipóxia/microbiologia , Hipóxia/virologia , Índia/epidemiologia , Lactente , Madagáscar , Masculino , Mali/epidemiologia , Paraguai/epidemiologia , Pneumonia/epidemiologia , Pneumonia/microbiologia , Pneumonia/virologia , Prevalência , Estudos Prospectivos , Fatores de RiscoRESUMO
The interaction of baicalein with bovine serum albumin (BSA) was investigated with the help of spectroscopic and molecular docking studies. The binding affinity of baicalein towards BSA was estimated to be in order of 105 M-1 from fluorescence quenching studies. Negative ΔH° (-5.66±0.14 kJ/mol) and positive (ΔS°) (+79.96±0.65 J/mol K) indicate the presence of electrostatic interactions along with the hydrophobic forces that result in a positive ΔS°. The hydrophobic association of baicalein with BSA diminishes in the presence of sodium dodecyl sulfate (SDS) due to probable hydrophobic association of baicalein with SDS, resulting in a negative ΔS° (-40.65±0.87 J/mol K). Matrix-assisted laser desorption ionization/time of flight (MALDI--TOF) experiments indicate a 1:1 complexation between baicalein and BSA. The unfolding and refolding phenomena of BSA were investigated in the absence and presence of baicalein using steady-state and fluorescence lifetime measurements. It was observed that the presence of urea ruptured the non-covalent interaction between baicalein and BSA. The presence of metal ions (Ag+, Mg2+, Ni2+, Mn2+, Co2+and Zn2+) increased the binding affinity of ligand towards BSA. The changes in conformational aspects of BSA after ligand binding were also investigated using circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopic techniques. Site selectivity studies following molecular docking analyses indicated the binding of baicalein to site 1 (subdomain IIA) of BSA.