RESUMO
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 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
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
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
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
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
Lactoferrin (LF) has several biological effects ranging from ribonuclease activity to antiangiogenic activity. It thus serves as a potential target protein for studies related to ribonucleolytic activity in association with its antiangiogenic activity. We have isolated buffalo LF and checked the ribonucleolytic activity via an agarose gel-based assay and precipitation assay. The ribonucleolytic activity of LF is lower compared to RNase A and the pH profile is a bell-shaped curve, with a pK1 value of 5.43 and pK2 of 7.65. The ribonuclease inhibitor that inhibits many ribonuclease-type proteins by forming a tight complex is unable to inhibit the ribonucleolytic property of LF. Fe(III) behaves as a noncompetitive inhibitor for the ribonucleolytic activity of protein. The superoxide-scavenging activity of the protein has also been measured. Histidine modification by diethylpyrocarbonate was monitored by UV-Vis spectroscopy at pH 7 and pH 8 and the effect towards the ribonucleolytic activity was determined. The antiangiogenic property of LF was investigated by the chorioallantoic membrane assay. Finally, the possible active site was analyzed via docking studies and correlated with the experimental study.
Assuntos
Inibidores da Angiogênese/farmacologia , Lactoferrina/metabolismo , Lactoferrina/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , RNA/metabolismo , Ribonucleases/metabolismo , Algoritmos , Inibidores da Angiogênese/química , Animais , Búfalos , Embrião de Galinha , Membrana Corioalantoide/irrigação sanguínea , Membrana Corioalantoide/efeitos dos fármacos , Eletroforese em Gel de Poliacrilamida , Sequestradores de Radicais Livres/química , Sequestradores de Radicais Livres/farmacologia , Concentração de Íons de Hidrogênio , Cinética , Lactoferrina/química , Modelos Moleculares , Peso Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Superóxidos/antagonistas & inibidores , Superóxidos/metabolismoRESUMO
Human serum albumin (HSA), the most abundant plasma protein in the human body is known to form fibrils under partial denaturing conditions. Natural polyphenols are known to interact with HSA and some polyphenols have been shown to be potent inhibitors of amyloid fibrillation. (-)-Epigallocatechin gallate (EGCG), the major component of green tea is known to inhibit amyloid fibrillation. In this report, we have investigated the effect of EGCG on native HSA as well as on the fibrillation process of HSA from amide III band analysis of their respective visible Raman spectra. The differential role of the tryptophan (Trp214) residue present in domain II of HSA in the absence and presence of EGCG has been pointed out using fluorescence anisotropy and visible Raman spectroscopy.
Assuntos
Catequina/análogos & derivados , Albumina Sérica/química , Catequina/química , Catequina/metabolismo , Catequina/farmacologia , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Albumina Sérica/metabolismo , Chá/químicaRESUMO
Green tea polyphenols (GTPs) are found to be potent inhibitors of amyloid fibril formation. We report the effective inhibitory property of (-)-epicatechin gallate (ECG) during the alkali-salt induced fibrillogenesis of hen egg white lysozyme (HEWL) at 37 °C. Spectroscopic techniques such as fluorescence, circular dichroism and microscopic images show that (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and (-)-epigallocatechin gallate (EGCG) show moderate inhibition of fibrillation with ECG as the most potent polyphenol. Aromatic interactions, hydrophobic interactions, the radical scavenging activity and autoxidation of polyphenols are likely to be the major reasons for ECG being the most effective inhibitor.