共查询到20条相似文献,搜索用时 15 毫秒
1.
We undertook an unfolding and refolding study of αL-crystallin in presence of urea to explore the breakdown and formation of various levels of structure and to find out whether
the breakdown of various levels of structure occurs simultaneously or in a hierarchal manner. We used various techniques such
as circular dichroism, fluorescence spectroscopy, light scattering, polarization to determine the changes in secondary, tertiary,
and quaternary structure. Unfolding and refolding occurred through a number of intermediates. The results showed that all
levels of structure in αL-crystallin collapsed or reformed simultaneously. The intermediates that occurred in the 2–4 M urea concentration range during
unfolding and refolding differed from each other in terms of the polarity of the tryptophan environment. The ANS binding experiments
revealed that refolded αL-crystallin had higher number of hydrophobic pockets compared to native one. On the other hand, polarity of these pockets
remained same as that of the native protein. Both light scattering and polarization measurements showed smaller oligomeric
size of refolded αL-crystallin. Thus, although the secondary structural changes were almost reversible, the tertiary and quaternary structural
changes were not. The refolded αL-crystallin had more exposed hydrophobic sites with increased binding affinity. The refolded form also showed higher chaperone
activity than native one. Since the refolded form was smaller in oligomeric size, some buried hydrophobic sites were available.
The higher chaperone activity of lower sized oligomer of αL-crystallin again revealed that chaperone activity was dependent on hydrophobicity and not on oligomeric size. 相似文献
2.
《Biochemical and biophysical research communications》1997,239(1):217-222
α-Crystallin, the predominant eye lens protein with sequence homology to small heat shock proteins, acts like a molecular chaperone by suppressing the aggregation of damaged crystallins and proteins. To gain an insight into the amino acid sequences in α-crystallin involved in chaperone-like function, we used a cleavable, fluorescent, photoactive, crosslinking agent, sulfosuccinimidyl-2(7-azido-4-methylcoumarin-3-acetamido)-ethyl-1,3′ dithiopropionate (SAED), to derivatize yeast alcohol dehydrogenase (ADH) and allowed it to complex with bovine α-crystallin at 48°C. The complex was photolyzed and reduced with DTT and the subunits of α-crystallin, αA- and αB-, were separated. Fluorescence analysis showed that both αA- and αB-crystallins interacted with ADH during chaperone-like function. Tryptic digestion, amino acid sequencing, and mass spectral analysis of αB-crystallin revealed that APSWIDTGLSEMR (57-69) and VLGDVIEVHGKHEER (93-107) sequences were involved in binding with ADH. 相似文献
3.
Alpha crystallin is an eye lens protein with a molecular weight of approximately 800 kDa. It belongs to the class of small heat shock proteins. Besides its structural role, it is known to prevent the aggregation of β- and γ-crystallins and several other proteins under denaturing conditions and is thus believed to play an important role in maintaining lens transparency. In this communication, we have investigated the effect of 2,2,2-trifluoroethanol (TFE) on the structural and functional features of the native α-crystallin and its two constituent subunits. A conformational change occurs from the characteristic β-sheet to the α-helix structure in both native α-crystallin and its subunits with the increase in TFE levels. Among the two subunits, αA-crystallin is relatively stable and upon preincubation prevents the characteristic aggregation of αB-crystallin at 20% and 30% (v/v) TFE. The hydrophobicity and chaperone-like activity of the crystallin subunits decrease on TFE treatment. The ability of αA-crystallin to bind and prevent the aggregation of αB-crystallin, despite a conformational change, could be important in protecting the lens from external stress. The loss in chaperone activity of αA-crystallin exposed to TFE and the inability of peptide chaperone—the functional site of αA-crystallin—to stabilize αB-crystallin at 20–30% TFE suggest that the site(s) involved in subunit interaction and chaperone-like function are quite distinct. 相似文献
4.
α-Crystallin functions as a molecular chaperone and maintains transparency of eye lens by protecting other lens-proteins.
Non-enzymatic glycation of α-crystallin by methylglyoxal, plays a crucial role on its chaperone function and structural stability.
Our studies showed that methylglyoxal modification even in lower concentration caused significant decrease in chaperone function
of α-crystallin as reflected both in thermal aggregation assay and enzyme refolding assay. Thermal denaturation studies showed
drastic reduction of denaturation temperature with increase in the degree of modification. Thermodynamic stability studies
by urea denaturation assay reflected a decrease of transition midpoint. Quantitatively we found that ΔG° of native α-crystallin
decreased from 21.6 kJ/mol to 10.4 kJ/mol due to 72 h modification by 10 mM methylglyoxal. The surface hydrophobicity of α-crystallin
after MG modification, was found to be decreased. Circular dichroism spectroscopy revealed conversion of β-sheet structure
to random coil structure. Significant cross-linking was also observed due to methylglyoxal modification of human α-crystallin. 相似文献
5.
αA-crystallin and αB-crystallin are members of the small heat shock protein family and function as molecular chaperones and major lens structural proteins. Although numerous studies have examined their chaperone-like activities in vitro, little is known about the proteins they protect in vivo. To elucidate the relationships between chaperone function, substrate binding, and human cataract formation, we used proteomic and mass spectrometric methods to analyze the effect of mutations associated with hereditary human cataract formation on protein abundance in αA-R49C and αB-R120G knock-in mutant lenses. Compared with age-matched wild type lenses, 2-day-old αA-R49C heterozygous lenses demonstrated the following: increased crosslinking (15-fold) and degradation (2.6-fold) of αA-crystallin; increased association between αA-crystallin and filensin, actin, or creatine kinase B; increased acidification of βB1-crystallin; increased levels of grifin; and an association between βA3/A1-crystallin and αA-crystallin. Homozygous αA-R49C mutant lenses exhibited increased associations between αA-crystallin and βB3-, βA4-, βA2-crystallins, and grifin, whereas levels of βB1-crystallin, gelsolin, and calpain 3 decreased. The amount of degraded glutamate dehydrogenase, α-enolase, and cytochrome c increased more than 50-fold in homozygous αA-R49C mutant lenses. In αB-R120G mouse lenses, our analyses identified decreased abundance of phosphoglycerate mutase, several β- and γ-crystallins, and degradation of αA- and αB-crystallin early in cataract development. Changes in the abundance of hemoglobin and histones with the loss of normal α-crystallin chaperone function suggest that these proteins also play important roles in the biochemical mechanisms of hereditary cataracts. Together, these studies offer a novel insight into the putative in vivo substrates of αA- and αB-crystallin. 相似文献
6.
Purpose
To investigate the expression of αA- and αB-crystallin and the unfolded protein response in the lens epithelium of patients with high myopia-related cataracts.Methods and Materials
The central portion of the human anterior lens capsule together with the adhering epithelial cells, approximately 5 mm in diameter, were harvested and processed within two hours after cataract surgery from high myopia-related (spherical equivalent ≥-10.00 diopters) and age-related cataract patients or from high myopia but non-cataractous patients (tissue were collected from ocular trauma patients with high myopia and lens trauma). Anterior lens samples from fresh cadaver normal human eyes were used as normal control (collected within 6 hours from death). Real-time PCR was performed to detect the mRNA levels of α-crystallins as well as unfolded protein response (UPR)-related GRP78, spliced-XBP1, ATF4 and ATF6. Western blot analysis was used to determine the protein level of α-crystallin, GRP78, p-IRE1α, p-eIF2α and ATF6.Results
In the lens epithelium of the high myopia-related cataract group and the age related cataract group, the mRNA and soluble protein expression of αA- and αB-crystallin were both decreased; additionally, the protein levels of ATF6, p-eIF2α and p-IRE1α and the gene expression levels of spliced XBP1, GRP78, ATF6 and ATF4 were greatly increased relative to the normal control.Conclusion
These results suggest the significant loss of soluble α-crystallin and the activation of the UPR in the lens epithelium of patients with high myopia-related cataract, which may be associated with the cataractogenesis of high myopia-related cataract. 相似文献7.
Derya R. Shimshek Matthias Mueller Christoph Wiessner Tatjana Schweizer P. Herman van der Putten 《PloS one》2010,5(4)
Background
Aggregation and misfolded α-synuclein is thought to be central in the pathogenesis of Parkinson''s disease (PD). Heat-shock proteins (HSPs) that are involved in refolding and degradation processes could lower the aggregate load of α-synuclein and thus be beneficial in α-synucleinopathies.Methodology/Principal Findings
We co-overexpressed human A53T point-mutated α-synuclein and human HSP70 in mice, both under the control of Thy1 regulatory sequences. Behavior read-outs showed no beneficial effect of HSP70 expression in mice. In contrast, motor coordination, grip strength and weight were even worse in the α-synucleinopathy model in the presence of HSP70 overexpression. Biochemical analyses revealed no differences in α-synuclein oligomers/aggregates, truncations and phosphorylation levels and α-synuclein localization was unchanged in immunostainings.Conclusion/Significance
Overexpressing HSP70 in a mouse model of α-synucleinopathy did not lower the toxic load of α-synuclein species and had no beneficial effect on α-synuclein-related motor deficits. 相似文献8.
Background
The eye lens is composed of fiber cells that are filled with α-, β- and γ-crystallins. The primary function of crystallins is to maintain the clarity of the lens through ordered interactions as well as through the chaperone-like function of α-crystallin. With aging, the chaperone function of α-crystallin decreases, with the concomitant accumulation of water-insoluble, light-scattering oligomers and crystallin-derived peptides. The role of crystallin-derived peptides in age-related lens protein aggregation and insolubilization is not understood.Methodology/Principal Findings
We found that αA-crystallin-derived peptide, 66 SDRDKFVIFLDVKHF 80, which accumulates in the aging lens, can inhibit the chaperone activity of α-crystallin and cause aggregation and precipitation of lens crystallins. Age-related change in the concentration of αA-(66-80) peptide was estimated by mass spectrometry. The interaction of the peptide with native crystallin was studied by multi-angle light scattering and fluorescence methods. High molar ratios of peptide-to-crystallin were favourable for aggregation and precipitation. Time-lapse recordings showed that, in the presence of αA-(66-80) peptide, α-crystallin aggregates and functions as a nucleus for protein aggregation, attracting aggregation of additional α-, β- and γ-crystallins. Additionally, the αA-(66-80) peptide shares the principal properties of amyloid peptides, such as β-sheet structure and fibril formation.Conclusions/Significance
These results suggest that crystallin-derived peptides such as αA-(66-80), generated in vivo, can induce age-related lens changes by disrupting the structure and organization of crystallins, leading to their insolubilization. The accumulation of such peptides in aging lenses may explain a novel mechanism for age-related crystallin aggregation and cataractogenesis. 相似文献9.
Ira Surolia Sharmistha Sinha Debi Prasad Sarkar P. Yadagiri Reddy G. Bhanuprakash Reddy Avadhesha Surolia 《PloS one》2008,3(8)
Familial Danish Dementia (FDD) is an autosomal disease, which is distinguished by gradual loss of vision, deafness, progressive ataxia and dementia. Cataract is the first manifestation of the disease. In this article, we demonstrate a specific correlation between the poisoning of the chaperone activity of the rat eye lens α-crystallins, loss of lens transparency in organ culture by the pathogenic form of the Danish dementia peptide, i.e. the reduced Danish dementia peptide (redADan peptide), by a combination of ex vivo, in vitro, biophysical and biochemical techniques. The interaction of redADan peptide and lens crystallins are very specific when compared with another chaperone, HSP-70, underscoring the specificity of the pathogenic form of Danish dementia peptide, redADan, for the early onset of cataract in this disease. 相似文献
10.
An enzymatic reaction within a mesh-like structure constructed using hyaluronan was investigated in order to understand the influence of specific reaction environments in a living body on the reaction. This mesh-like structure, which mimicked extracellular matrix conditions, was found to accelerate glycohydrolysis by Jack bean α-mannosidase. 相似文献
11.
Keiichi Shimazaki Shintaro Sugai Ryoya Niki Shunrokuro Arima 《Bioscience, biotechnology, and biochemistry》2013,77(13):1995-2002
The secondary structure of bovine αs-casein and chemically modified αs-casein in various solvents was investigated by infrared absorption spectrum and optical rotatory dispersion measurements. Amino groups of αs-casein were either succinylated or acetylated, and carboxyl groups were either methylated or ethylated. Acetylated- and ethylated-αs-caseins are insoluble in water. Water-soluble samples have unordered structure in water. In organic solvents, such as 2-chloroethanol and ethylene glycol, they have about 50% α-helical fraction. On the other hand, it was found that methylated-αs-casein had two infrared absorption peaks centered at 1625 and 1643 cm?1 in D2O-CH3OD mixed solvent. This fact may be connected with the presence of β-structure. In the case of solid film of this sample, cast from solution containing CH3OH, the presence of β-structure was indicated, too. The authors attempted to explain the formation of β-structure in methylated-αs-casein in terms of the electrostatic interactions due to the differences in the net charge between methylated and unmodified αs-caseins. 相似文献
12.
Marco Fichera Angela Spalletta Flavia Fiorenza Turi Lombardo Gino Schilirò Ryad Tamouza Claudine Lapouméroulie Dominique Labie A. Ragusa 《Human genetics》1997,99(3):381-386
To evaluate the allelic frequency and genetic diversity of α-thalassemia defects in Sicily, both epidemiological and patient-oriented
studies were carried out. For the epidemiological study, phenotypic data were collected on more than 1000 Sicilian individuals.
Among them, 427 were explored at the molecular level for nine α-thalassemic variants known to be common in the Mediterranean
region. Our data reveal an allele frequency of 4.1% for α+-thalassemia matching that of β-thalassemia in this region. The presence of α°-thalassemia (––MEDI and ––CAL) was observed only in the group of referred patients. Newly acquired nucleotide sequence data on the deletional breakpoint
of ––CAL allowed us to design a simple PCR-based procedure for exploring this allele. The data also provide additional information
concerning the genetic mechanisms involved in such large deletions.
Received: 8 August 1996 / Revised: 16 October 1996 相似文献
13.
Transgenic mouse technology has been very valuable for the study of lens fiber cells since they can not be propagated in cell culture. The targeting of transgenes to the lens has traditionally been done with the A-crystallin promoter. However, while lens-specific, transgenic lines made with the A-crystallin promoter express the transgene at levels 100–300-fold lower than endogenous A-crystallin. Here we propose an alternative, the chicken B1-crystallin promoter (–432/+30). Transgenic mice made with this promoter have successfully expressed CAT, d/n m-calpain, Wee1, and B2-crystallin mRNA at levels comparable to the endogenous B1-crystallin gene and no eye abnormalities such as cataracts, have resulted. All of the transgenic lines made with the chicken B1-crystallin promoter have expressed the transgene in the lens fiber cells, and the best lines express at levels close to endogenous B1-crystallin. While RNA expression is very high, only moderate protein expression has been achieved, implying that the high protein expression of the crystallins is partially controlled at the level of translation. Thus, the chicken B1-crystallin promoter directs high level RNA expression to lens fiber cells, which may be especially useful for the expression of ribozyme and anti-sense RNAs in addition to ectopic proteins. 相似文献
14.
Functional α7 nicotinic acetylcholine receptors (AChRs) do not assemble efficiently in cells transfected with α7 subunits unless the cells are also transfected with the chaperone protein RIC-3. Despite the presence of RIC-3, large amounts of these subunits remain improperly assembled. Thus, additional chaperone proteins are probably required for efficient assembly of α7 AChRs. Cholinergic ligands can act as pharmacological chaperones to promote assembly of mature AChRs and upregulate the amount of functional AChRs. In addition, we have found that the chemical chaperones 4-phenylbutyric acid (PBA) and valproic acid (VPA) greatly increase the amount of functional α7 AChRs produced in a cell line expressing both α7 and RIC-3. Increased α7 AChR expression allows assay of drug action using a membrane potential-sensitive fluorescent indicator. Both PBA and VPA also increase α7 expression in the SH-SY5Y neuroblastoma cell line that endogenously expresses α7 AChRs. VPA increases expression of endogenous α7 AChRs in hippocampal neurons but PBA does not. RIC-3 is insufficient for optimal assembly of α7 AChRs, but provides assay conditions for detecting additional chaperones. Chemical chaperones are a useful pragmatic approach to express high levels of human α7 AChRs for drug selection and characterization and possibly to increase α7 expression in vivo. 相似文献
15.
Heterologous <Emphasis Type="Italic">in vitro</Emphasis> Synthesis of Lens α-Crystallin Polypeptide 总被引:12,自引:0,他引:12
MESSENGER RNAs coding for α and β globin chains1, for myosin2 and for the mouse immunoglobin light chains3 have been shown to be translated in a reticulocyte cell-free system. Messengers are also translated faithfully in the ascites tumour cell-free system4 and in oocytes from Xenopus laevis5. 相似文献
16.
Guido Kappé Andrew G. Purkiss Siebe T. van Genesen Christine Slingsby Nicolette H. Lubsen 《Journal of molecular evolution》2010,71(3):219-230
In jawed vertebrates, βγ-crystallins are restricted to the eye lens and thus excellent markers of lens evolution. These βγ-crystallins
are four Greek key motifs/two domain proteins, whereas the urochordate βγ-crystallin has a single domain. To trace the origin
of the vertebrate βγ-crystallin genes, we searched for homologues in the genomes of a jawless vertebrate (lamprey) and of
a cephalochordate (lancelet). The lamprey genome contains orthologs of the gnathostome βB1-, βA2- and γN-crystallin genes
and a single domain γN-crystallin-like gene. It contains at least two γ-crystallin genes, but lacks the gnathostome γS-crystallin
gene. The genome also encodes a non-lenticular protein containing βγ-crystallin motifs, AIM1, also found in gnathostomes but
not detectable in the uro- or cephalochordate genome. The four cephalochordate βγ-crystallin genes found encode two-domain
proteins. Unlike the vertebrate βγ-crystallins but like the urochordate βγ-crystallin, three of the predicted proteins contain
calcium-binding sites. In the cephalochordate βγ-crystallin genes, the introns are located within motif-encoding region, while
in the urochordate and in the vertebrate βγ-crystallin genes the introns are between motif- and/or domain encoding regions.
Coincident with the evolution of the vertebrate lens an ancestral urochordate type βγ-crystallin gene rapidly expanded and
diverged in the ancestral vertebrate before the cyclostomes/gnathostomes split. The β- and γN-crystallin genes were maintained
in subsequent evolution, and, given the selection pressure imposed by accurate vision, must be essential for lens function.
The γ-crystallin genes show lineage specific expansion and contraction, presumably in adaptation to the demands on vision
resulting from (changes in) lifestyle. 相似文献
17.
To infer the molecular evolution of yeast Saccharomyces sensu stricto from analysis of the -galactosidase MEL gene family, two new genes were cloned and sequenced from S. bayanus var. bayanus and S. pastorianus. Nucleotide sequence homology of the MEL genes of S. bayanus var. bayanus (MELb), S. pastorianus (MELpt), S. bayanus var. uvarum (MELu), and S. carlsbergensis (MELx) was rather high (94.1–99.3%), comparable with interspecific homology (94.8–100%) of S. cerevisiae
MEL1-MEL11. Homology of the MEL genes of sibling species S. cerevisiae (MEL1), S. bayanus (MELb), S. paradoxus (MELp), and S. mikatae(MELj) was 76.2–81.7%, suggesting certain species specificity. On this evidence, the -galactosidase gene of hybrid yeast S. pastorianus (S. carlsbergensis) was assumed to originate from S. bayanus rather than from S. cerevisiae. 相似文献
18.
Shuichi Kaminogawa Shunichi Dosako Kunio Yamauchi 《Bioscience, biotechnology, and biochemistry》2013,77(12):2337-2341
Some molecular properties of αs1-κ-casein complex, αs1- and κ-casein polymers were examined by gel filtration, ultracentrifugation, and viscometry at pH 7.1. The Stoke’s radii of αs1-κ-casein complex, αs1- and κ-casein polymers were 99, 44 and 108 Å, respectively. The molecular weight of the above proteins were approximately 45 × 104, 10 × 104 and 80 × 104, respectively. The stokes radius of αs1-κ casein complex reduced compared with that of κ-casein polymer and the molecular weight of the complex was about half that of κ-casein polymer. These results suggest that κ-casein polymer dissociates into 4 smaller particles when αs1-κ-casein complex is formed. The frictional coefficient and Scheraga-Mandelkern constants for each protein suggest that the molecular shape of αs1-casein polymer is globular and that of αs1-κ-casein complex and κ-casein polymer is rod-like. 相似文献
19.
Morten Bertz Jin Chen Matthias J. Feige Titus M. Franzmann Johannes Buchner Matthias Rief 《Journal of molecular biology》2010,400(5):1046-14144
In biological systems, proteins rarely act as isolated monomers. Association to dimers or higher oligomers is a commonly observed phenomenon. As an example, small heat shock proteins form spherical homo-oligomers of mostly 24 subunits, with the dimeric α-crystallin domain as the basic structural unit. The structural hierarchy of this complex is key to its function as a molecular chaperone. In this article, we analyze the folding and association of the basic building block, the α-crystallin domain dimer, from the hyperthermophilic archaeon Methanocaldococcus jannaschii Hsp16.5 in detail. Equilibrium denaturation experiments reveal that the α-crystallin domain dimer is highly stable against chemical denaturation. In these experiments, protein dissociation and unfolding appear to follow an “all-or-none” mechanism with no intermediate monomeric species populated. When the mechanical stability was determined by single-molecule force spectroscopy, we found that the α-crystallin domain dimer resists high forces when pulled at its termini. In contrast to bulk denaturation, stable monomeric unfolding intermediates could be directly observed in the mechanical unfolding traces after the α-crystallin domain dimer had been dissociated by force. Our results imply that for this hyperthermophilic member of the small heat shock protein family, assembly of the spherical 24mer starts from folded monomers, which readily associate to the dimeric structure required for assembly of the higher oligomer. 相似文献
20.
Shruti Arya Avinash K. Singh Karishma Bhasne Priyanka Dogra Anindya Datta Payel Das Samrat Mukhopadhyay 《Biophysical journal》2018,114(11):2540-2551
Protein hydration water plays a fundamentally important role in protein folding, binding, assembly, and function. Little is known about the hydration water in intrinsically disordered proteins that challenge the conventional sequence-structure-function paradigm. Here, by combining experiments and simulations, we show the existence of dynamical heterogeneity of hydration water in an intrinsically disordered presynaptic protein, namely α-synuclein, implicated in Parkinson’s disease. We took advantage of nonoccurrence of cysteine in the sequence and incorporated a number of cysteine residues at the N-terminal segment, the central amyloidogenic nonamyloid-β component (NAC) domain, and the C-terminal end of α-synuclein. We then labeled these cysteine variants using environment-sensitive thiol-active fluorophore and monitored the solvation dynamics using femtosecond time-resolved fluorescence. The site-specific femtosecond time-resolved experiments allowed us to construct the hydration map of α-synuclein. Our results show the presence of three dynamically distinct types of water: bulk, hydration, and confined water. The amyloidogenic NAC domain contains dynamically restrained water molecules that are strikingly different from the water molecules present in the other two domains. Atomistic molecular dynamics simulations revealed longer residence times for water molecules near the NAC domain and supported our experimental observations. Additionally, our simulations allowed us to decipher the molecular origin of the dynamical heterogeneity of water in α-synuclein. These simulations captured the quasi-bound water molecules within the NAC domain originating from a complex interplay between the local chain compaction and the sequence composition. Our findings from this synergistic experimental simulation approach suggest longer trapping of interfacial water molecules near the amyloidogenic hotspot that triggers the pathological conversion into amyloids via chain sequestration, chain desolvation, and entropic liberation of ordered water molecules. 相似文献