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1.
Duce JA Smith DP Blake RE Crouch PJ Li QX Masters CL Trounce IA 《Journal of molecular biology》2006,361(3):493-505
Alzheimer's disease (AD) and Parkinson's disease (PD) are the two most prevalent neurodegenerative diseases of the central nervous system. These two diseases share a common feature in that a normally soluble peptide (amyloid-beta) or protein (alpha-synuclein) aggregates into an ordered fibrillar structure. As well as structural similarities observed between fibrillar aggregates related to these diseases, common pathological processes of increased oxidative injury, excitotoxicity and altered cell cycle are also evident. It was the aim of this study to identify novel interacting proteins to the amyloid-like motif and therefore identify common potential pathways between neurodegenerative diseases that share biophysical properties common to classical amyloid fibrils. Optimal ageing of recombinant proteins to form amyloid-like fibrils was determined by electron microscopy, Congo red birefringement and photo-induced cross-linking. Using pull-down assays the strongest detected interacting protein to the amyloid-like motifs of amyloid-beta, alpha-synuclein and lysozyme was identified as histone H1. The interaction with the amyloid-like motif was confirmed by techniques including surface plasmon resonance and immunohistochemistry. Histone H1 is known to be an integral part of chromatin within the nucleus, with a primary role of binding DNA that enters and exits from the nucleosome, and facilitating the shift in equilibrium of chromatin towards a more condensed form. However, phosphorylated histone H1 is predominantly present in the cytoplasm and as yet the functional significance of this translocation is unknown. This study also found that histone H1 is localised within the cytoplasm of neurons and astrocytes from areas affected by disease as well as amyloid plaques, supporting the hypothesis that histone H1 favoured binding to an ordered fibrillar motif. We conclude that the binding of histone H1 to a general amyloid-like motif indicates that histone H1 may play an important common role in diseases associated with amyloid-like fibrils. 相似文献
2.
Colombo N Schroeder J Cabrele C 《Biochemical and biophysical research communications》2006,346(1):182-187
The negative regulator of DNA-binding/cell-differentiation Id2 is a small protein containing a central helix-loop-helix (HLH) motif and a C-terminal nuclear export signal (NES). Whereas the former is essential for Id2 dimerization and nuclear localization, the latter is responsible for the transport of Id2 from the nucleus to the cytoplasm. Whereas the isolated Id2 HLH motif is highly helical, large C-terminal Id2 fragments including the NES sequence are either unordered or aggregation-prone. To study the conformational properties of the isolated NES region, we synthesized the Id2 segment 103-124. The latter was insoluble in water and only temporarily soluble in water/alcohol mixtures, where it formed quickly precipitating beta-sheets. Introduction of a positively charged N-terminal tail prevented aggressive precipitation and led to aggregates consisting of long fibrils that bound thioflavin T. These results show an interesting structural aspect of the Id2 NES region, which might be of significance for both protein folding and function. 相似文献
3.
Sugimoto Y Kamada Y Tokunaga Y Shinohara H Matsumoto M Kusakabe T Ohkuri T Ueda T 《Biochimie et biologie cellulaire》2011,89(6):533-544
The interaction of egg-white lysozyme with N-ovalbumin, the native form of egg-white ovalbumin with the denaturation temperature, T(m), of 78 °C, was investigated by the inhibition of lysozyme muramidase activity, differential scanning calorimetry, and circular dichroism assay as indicators. Signals for the interaction were the most prominent when the mixture of lysozyme and N-ovalbumin was co-heated at 72 °C, slightly lower than the T(m) of N-ovalbumin. The interaction was also marked when unheated lysozyme was mixed with N-ovalbumin preheated at 72 °C. Moreover, the mixture rapidly formed fibrous precipitates, which were positive for thioflavin T fluorescent emission, a marker for the amyloid fibril formation. Also electron microscopic observation exhibited features of fibrils. The interaction potency of ovalbumin was ascribed to the tryptic fragment ILELPFASGT MSMLVLLPDE VSGLEQLESIINFEK (residues 229-263), derived from the 2B strands 2 and 3 of ovalbumin. From lysozyme, on the other hand, the chymotryptic peptide RNRCKGTDVQAW (residues 112-123), including cluster 6, and the chymotryptic/tryptic peptide GILQINSRW (residues 54-62), including cluster 3, were responsible for the interaction with N-ovalbumin. Interestingly, this nonamer peptide was found to have the ability to self-aggregate. To the authors knowledge, this may be the first report to document the possible involvement of dual proteins in the formation of amyloid-like fibrils. 相似文献
4.
The amyloid-like fibril is considered to be a macromolecular self-assemblage with a highly-ordered quaternary structure, in which numerous beta-stranded polypeptide chains align regularly. Therefore, this kind of fibril has the potential to be engineered into proteinaceous materials, although conformational alteration of proteins from their native form to the amyloid form is a misfolding and undesirable process related to amyloid diseases. In this study, we have attempted to design an artificial system to explore applicability of using the amyloid-like fibril as a construct possessing self-recognition and self-catalytic abilities. A peptide self-replicating system based on the beta-structure of the amyloid-like fibril was designed and constructed. The beta-stranded peptide was self-replicated by the native chemical ligation reaction, and the newly generated peptide was self-assembled into amyloid-like fibrils. Thus, the constructed system was of both chemical and conformational self-replicating fibrils. 相似文献
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《Biophysical journal》2022,121(11):2084-2095
Accumulation of insoluble amyloid fibrils is widely studied as a critical factor in the pathology of multiple neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease. Misfolded Cu, Zn superoxide dismutase (SOD1) was the first protein linked to ALS, and non-native SOD1 trimeric oligomers were recently linked to cytotoxicity, while larger oligomers were protective to cells. The balance between trimers and larger aggregates in the process of SOD1 aggregation is, thus, a critical determinant of potential therapeutic approaches to treat ALS. However, it is unknown whether these trimeric oligomers are a necessary intermediate for larger aggregate formation or a distinct off-pathway species competing with fibril formation. Depending on the on- or off-pathway scenario of trimer formation, we expect drastically different therapeutic approaches. Here, we show that the toxic SOD1 trimer is an off-pathway intermediate competing with protective fibril formation. We design mutant SOD1 constructs that remain in a trimeric state (super-stable trimers) and show that stabilizing the trimeric SOD1 prevents formation of fibrils in vitro and in a motor neuron-like cell model (NSC-34). Using size exclusion chromatography, we track the aggregation kinetics of purified SOD1 and show direct competition of trimeric SOD1 with larger oligomer and fibril formation. Finally, we show the trimer is structurally independent of both larger soluble oligomers and insoluble fibrils using circular dichroism spectroscopy and limited proteolysis. 相似文献
7.
Premelanosome amyloid-like fibrils are composed of only golgi-processed forms of Pmel17 that have been proteolytically processed in endosomes 总被引:1,自引:0,他引:1
Harper DC Theos AC Herman KE Tenza D Raposo G Marks MS 《The Journal of biological chemistry》2008,283(4):2307-2322
Melanin pigments are synthesized within specialized organelles called melanosomes and polymerize on intraluminal fibrils that form within melanosome precursors. The fibrils consist of proteolytic fragments derived from Pmel17, a pigment cell-specific integral membrane protein. The intracellular pathways by which Pmel17 accesses melanosome precursors and the identity of the Pmel17 derivatives within fibrillar melanosomes have been a matter of debate. We show here that antibodies that detect Pmel17 within fibrillar melanosomes recognize only the luminal products of proprotein convertase cleavage and not the remaining products linked to the transmembrane domain. Moreover, antibodies to the N and C termini detect only Pmel17 isoforms present in early biosynthetic compartments, which constitute a large fraction of detectable steady state Pmel17 in cell lysates because of slow early biosynthetic transport and rapid consumption by fibril formation. Using an antibody to a luminal epitope that is destroyed upon modification by O-linked oligosaccharides, we show that all post-endoplasmic reticulum Pmel17 isoforms are modified by Golgi-associated oligosaccharide transferases, and that only processed forms contribute to melanosome biogenesis. These data indicate that Pmel17 follows a single biosynthetic route from the endoplasmic reticulum through the Golgi complex and endosomes to melanosomes, and that only fragments encompassing previously described functional luminal determinants are present within the fibrils. These data have important implications for the site and mechanism of fibril formation. 相似文献
8.
Helicobacter pylori causes various gastric diseases, such as gastritis, peptic ulcerations, gastric cancer and mucosa-associated lymphoid tissue lymphoma. Hpn is a histidine-rich protein abundant in this bacterium and forms oligomers in physiologically relevant conditions. In this present study, Hpn oligomers were found to develop amyloid-like fibrils as confirmed by negative stain transition electron microscopy, thioflavin T and Congo red binding assays. The amyloid-like fibrils of Hpn inhibit the proliferation of gastric epithelial AGS cells through cell cycle arrest in the G2/M phase, which may be closely related to the disruption of mitochondrial bioenergetics as reflected by the significant depletion of intracellular ATP levels and the mitochondrial membrane potential. The collective data presented here shed some light on the pathologic mechanisms of H. pylori infections. 相似文献
9.
Xiaodong Yang Christopher G Adda David W Keizer Vince J Murphy Michael M Rizkalla Matthew A Perugini David C Jackson Robin F Anders Raymond S Norton 《Journal of peptide science》2007,13(12):839-848
Merozoite surface protein 2 (MSP2) from the human malaria parasite Plasmodium falciparum is expressed as a GPI-anchored protein on the merozoite surface. It has been implicated in the process of erythrocyte invasion and is a leading vaccine candidate. MSP2 is an intrinsically unstructured protein (IUP), and recombinant MSP2 forms amyloid-like fibrils upon storage. We have examined synthetic peptides corresponding to sequences in the conserved N-terminal region of MSP2 for the presence of local structure and the ability to form fibrils related to those formed by full-length MSP2. In a 25-residue peptide corresponding to the entire N-terminal region of mature MSP2, structures calculated from NMR data show the presence of nascent helical and turn-like structures. An 8-residue peptide from the central region of the N-terminal domain (residues 8-15) also formed a turn-like structure. Both peptides formed fibrils that were similar but not identical to the amyloid-like fibrils formed by full-length MSP2. Notably, the fibrils formed by the peptides bound both Congo Red and Thioflavin T, whereas the fibrils formed by full-length MSP2 bound only Congo Red. The propensity of peptides from the N-terminal conserved region of MSP2 to form amyloid-like fibrils makes it likely that this region contributes to fibril formation by the full-length protein. Thus, in contrast to the more common pathway of amyloid formation by structured proteins, which proceeds via partially unfolded intermediates that then undergo beta-aggregation, MSP2 is an example of a largely unstructured protein with at least one small structured region that has an important role in fibril formation. 相似文献
10.
Saiki M Honda S Kawasaki K Zhou D Kaito A Konakahara T Morii H 《Journal of molecular biology》2005,348(4):983-998
Various mutants of the protein fragment, barnase module-1 (1-24) were investigated in order to reveal the structural principle of amyloid-like fibrils. By means of circular dichroism spectroscopy, X-ray diffraction, electron microscopy, and thioflavin T binding assay, we found that the molecules containing two beta-strands and an intervening turn structure are assembled to form a cross-beta structure. Stabilization by both the hydrophobic interactions and hydrogen bonding between the respective paired side-chains on the coupled beta-strands was essential for fibril formation. These two types of interaction can also arrange the corresponding residues in lines on both sheet surfaces of protofilaments with a cross-beta structure. This leads to the most probable fibril structure constructed with the line-matching interactions between protofilaments. Consideration of the geometrical symmetry resulted in our finding that a limited number of essential models for molecular packing in fibril structure are stable, which would rationally explain the occurrence of two or three morphologies from an identical molecular species. The ribbon-like fibrils exhibited striped texture along the axis, which was assigned to a stacked two-sheet repeat as a structural unit. The comprehensively proposed structural model, that is, the sheet-sheet interaction between left-handed cross-beta structures, results in a slightly right-handed twist of beta-sheet stacking, which reasonably elucidates the intrinsic sizes of the fibril width and its helical period along the fibril axis, as the bias in the orientation of the hydrogen-bonded beta-strand pair at the lateral edge is larger than that at the central protofilament. 相似文献
11.
Ruiguang Ge Xuesong SunDongxian Wang Qinglu ZhouHongzhe Sun 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》2011,1813(8):1422-1427
Helicobacter pylori causes various gastric diseases, such as gastritis, peptic ulcerations, gastric cancer and mucosa-associated lymphoid tissue lymphoma. Hpn is a histidine-rich protein abundant in this bacterium and forms oligomers in physiologically relevant conditions. In this present study, Hpn oligomers were found to develop amyloid-like fibrils as confirmed by negative stain transition electron microscopy, thioflavin T and Congo red binding assays. The amyloid-like fibrils of Hpn inhibit the proliferation of gastric epithelial AGS cells through cell cycle arrest in the G2/M phase, which may be closely related to the disruption of mitochondrial bioenergetics as reflected by the significant depletion of intracellular ATP levels and the mitochondrial membrane potential. The collective data presented here shed some light on the pathologic mechanisms of H. pylori infections. 相似文献
12.
Although the formation of an alpha-helix or partial unfolding of proteins has been suggested to be important for amyloid fibrils to form in alcohols, the exact mechanism involved remains elusive. To obtain further insight into the development of amyloid fibrils, we used a 22-residue peptide, K3, corresponding to Ser20 to Lys41 of intact beta2-microglobulin. Although K3 formed an alpha-helix at high concentrations of 2,2,2-trifluoroethanol (TFE) and 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) in 10 mM HCl (pH approximately 2), the helical content was not high, indicating a low preference to do so. The partly alpha-helical conformation was converted with time into a highly ordered beta-sheet with a fibrillar morphology as revealed by atomic force microscopy. Importantly, the TFE and HFIP-induced fibrillation exhibited a concentration dependence with a maximum at approximately 20 and approximately 10% (v/v), respectively, slightly below the concentrations at which these alcohols form dynamic clusters. Focusing on the similarity of the effects of alcohol on proteins with those of sodium dodecyl sulfate (SDS), we examined the effects of SDS on K3. SDS also induced fibrils to form with a maximum at approximately 4 mM, slightly below the critical micelle concentration. These results indicate that, with an increase in the concentration of hydrophobic cosolvent (TFE, HFIP, or SDS), a delicate balance of decreasing hydrophobic interactions and increasing polar interactions (i.e. H-bonds) in and between peptides leads to the formation of ordered fibrils with a bell-shaped concentration dependence. 相似文献
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14.
Cardoso I Goldsbury CS Müller SA Olivieri V Wirtz S Damas AM Aebi U Saraiva MJ 《Journal of molecular biology》2002,317(5):683-695
Extracellular accumulation of transthyretin (TTR) variants in the form of fibrillar amyloid deposits is the pathological hallmark of familial amyloidotic polyneuropathy (FAP). The TTR Leu55Pro variant occurs in the most aggressive forms of this disease. Inhibition of TTR wild-type (WT) and particularly TTR Leu55Pro fibril formation is of interest as a potential therapeutic strategy and requires a thorough understanding of the fibril assembly mechanism. To this end, we report on the in vitro assembly properties as observed by transmission electron microscopy (TEM), atomic force microscopy (AFM) and quantitative scanning transmission electron microscopy (STEM) for both TTR WT fibrils produced by acidification, and TTR Leu55Pro fibrils assembled at physiological pH. The morphological features and dimensions of TTR WT and TTR Leu55Pro fibrils were similar, with up to 300 nm long, 8 nm wide fibrils being the most prominent species in both cases. Other species were evident; 4-5 nm wide fibrils, 9-10 nm wide fibrils and oligomers of various sizes. STEM mass-per-length (MPL) measurements revealed discrete fibril types with masses of 9.5 and 14.0(+/-1.4) KDa/nm for TTR WT fibrils and 13.7, 18.5 and 23.2(+/-1.5) kDa/nm for TTR Leu55Pro fibrils. These MPL values are consistent with a model in which fibrillar TTR structures are composed of two, three, four or five elementary protofilaments, with each protofilament being a vertical stack of structurally modified TTR monomers assembled with the 2.9 nm axial monomer-monomer spacing indicated by X-ray fibre diffraction data. Ex vivo TTR amyloid fibrils were examined. From their morphological appearance compared to these, the in vitro assembled TTR WT and Leu55Pro fibrils examined may represent immature fibrillar species. The in vitro system operating at physiological pH for TTR Leu55Pro and the model presented for the molecular arrangement of TTR monomers within fibrils may, therefore, describe early fibril assembly events in vivo. 相似文献
15.
The present review attempts to reconcile the dichotomy that exists in the literature in relation to fibromyalgia, in that it is considered either a somatic response to psychological stress or a distinct organically based syndrome. Specifically, the hypothesis explored is that the link between chronic stress and the subsequent development of fibromyalgia can be explained by one or more abnormalities in neuroendocrine function. There are several such abnormalities recognised that both occur as a result of chronic stress and are observed in fibromyalgia. Whether such abnormalities have an aetiologic role remains uncertain but should be testable by well-designed prospective studies. 相似文献
16.
Abnormally expanded polyglutamine domains in proteins are associated with several neurodegenerative diseases, including Huntington's disease. Expansion of the polyglutamine (polyQ) domain facilitates aggregation of the affected protein, and several studies directly link aggregation to neurotoxicity. Studies of synthetic polyQ peptides have contributed substantially to our understanding of the mechanism of aggregation. In this report, polyQ fibrils were immobilized onto a sensor, and their elongation by polyQ peptides of various length and conformation was examined using quartz crystal microbalance with dissipation monitoring (QCM-D). The rate of elongation increased as the peptide length increased from 8 to 24 glutamines (Q8, Q20, and Q24). Monomer conformation affected elongation rates: insertion of a β-turn template d-Pro-Gly in the center of the peptide increased elongation rates several-fold, while insertion of Pro-Pro dramatically slowed elongation. Dissipation measurements of the QCM-D provided qualitative information about mechanical properties of the elongating fibrils. These data showed clear differences in the characteristics of the elongating aggregates, depending on the specific identity of the associating polyQ peptide. Elongation rates were sensitive to the pH and ionic strength of the buffer. Comparison of QCM-D data with those obtained by optical waveguide lightmode spectroscopy revealed that very little water was associated with the elongation of fibrils by the peptide containing d-Pro-Gly, but a significant amount of water was associated when the fibrils were elongated by Q20. Together, the data indicate that elongation of polyQ fibrils can occur without full consolidation to the fibril structure, resulting in variations to the aggregate structure during elongation. 相似文献
17.
Protegrins (PG) are important in defending host tissues, preventing infection via an attack on the membrane surface of invading microorganisms. Protegrins have powerful antibiotic abilities, but the molecular-level mechanisms underlying the interactions of their beta-sheet motifs with the membrane are not known. Protegrin-1 (PG-1) is composed of 18 amino acids with a high content of basic residues and two disulfide bonds. Here we focused on the stability of PG-1 at the amphipathic interface in lipid bilayers and on the details of the peptide-membrane interactions. We simulated all-atom models of the PG-1 monomer with explicit water and lipid bilayers composed of both homogeneous POPC (palmitoyl-oleyl-phosphatidylcholine) lipids and a mixture of POPC/POPG (palmitoyl-oleyl-phosphatidylglycerol) (4:1) lipids. We observed that local thinning of the lipid bilayers mediated by the peptide is enhanced in the lipid bilayer containing POPG, consistent with experimental results of selective membrane targeting. The beta-hairpin motif of PG-1 is conserved in both lipid settings, whereas it is highly bent in aqueous solution. The conformational dynamics of PG-1, especially the highly charged beta-hairpin turn region, are found to be mostly responsible for disturbing the membrane. Even though the eventual membrane disruption requires PG-1 oligomers, our simulations clearly show the first step of the monomeric effects. The thinning effects in the bilayer should relate to pore/channel formation in the lipid bilayer and thus be responsible for further defects in the membrane caused by oligomer. 相似文献
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《生物化学与生物物理学报:生物膜》2019,1861(10):182977
Antimicrobial peptides (AMPs) are a promising class of innate host defense molecules for next-generation antibiotics, as they uniquely target and permeabilize membranes of pathogens. This selectivity has been explained by the electrostatic attraction between these predominantly cationic peptides and the bacterial membrane, which is heavily populated with anionic lipids. However, AMP-resistant bacteria have non-electrostatic countermeasures that modulate membrane rigidity and thickness. We explore how variations in physical properties affect the membrane affinity and disruption process of protegrin-1 (PG-1) in phosphatidylcholine (PC) membranes with altered lipid packing densities and thicknesses. From isothermal titration calorimetry and atomic force microscopy, our results showed that PG-1 could no longer insert into membranes of increasing cholesterol amounts nor into monounsaturated PC membranes of increasing thicknesses with similar fluidities. Prevention of PG-1’s incorporation consequently made the membranes more resistant to peptide-induced structural transformations like pore formation. Our study provides evidence that AMP affinity and activity are strongly correlated with the fluidity and thickness of the membrane. A basic understanding of how physical mechanisms can regulate cell selectivity and resistance towards AMPs will aid in the development of new antimicrobial agents. 相似文献
20.
Melanopsin forms a functional short-wavelength photopigment 总被引:5,自引:0,他引:5
Recently, melanopsin has emerged as the leading candidate for the elusive photopigment of the mammalian circadian system. This novel opsin-like protein is expressed in retinal ganglion cells that form the retinohypothalamic tract, a neuronal connection between the retina and the suprachiasmatic nucleus. These hypothalamic structures contain the circadian pacemaker, which generates daily rhythms in physiology and behavior. In mammals, proper synchronization of these rhythms to the environmental light-dark cycle requires retinal input. Surprisingly, rod and cone photoreceptors are not required. Instead, the melanopsin-containing ganglion cells are intrinsically sensitive to light, perhaps responding via a melanopsin-based signaling pathway. To test this hypothesis, we have characterized melanopsin following heterologous expression in COS cells. We found that melanopsin absorbed maximally at 424 nm after reconstitution with 11-cis-retinal. Furthermore, melanopsin activated the photoreceptor G-protein, transducin, in a light-dependent manner. In agreement with the measured absorbance spectrum, melanopsin was most efficiently excited by blue light (420-440 nm). In contrast, published action spectra suggest that the photopigment underlying the intrinsic light sensitivity of SCN-projecting RGCs has an absorption maximum near 484 nm. In summary, our experiments constitute the first direct demonstration that melanopsin forms a photopigment capable of activating a G-protein, but its spectral properties are not consistent with the action spectrum for circadian entrainment. 相似文献