Further studies on the sub-units of alpha-crystallin.
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ABSTRACT:
SUBMITTER: Wisse JH
PROVIDER: S-EPMC1264974 | biostudies-other | 1966 Apr
REPOSITORIES: biostudies-other
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Project description:1. alpha(A)-Chain sub-units have been isolated from neutral haemolysates containing certain unstable beta-chain haemoglobin variants. They are identical, in electrophoretic properties, tryptic peptide analyses and their ability to recombine with Hb-beta(A) (4) at neutral pH, with the alpha(A)-chain sub-units isolated from Hb-A at acid pH. Abnormal alpha(G)-chain sub-units have been prepared from haemolysates containing the alpha-chain variant Hb-G. 2. alpha(A)-Chain sub-units prepared by either method are eluted from Sephadex at low haemoglobin concentration as monomer sub-units. The elution volume, sedimentation coefficient and apparent molecular weight are, however, concentration-dependent, suggesting an equilibrium between monomers and higher polymers with the former as the predominant species. Elevated temperatures cause the alpha(A)-chain sub-unit to aggregate and denature. 3. The product of the reaction between the alpha(A)-chain sub-unit and Hb-beta(A) (4) has been characterized as Hb-A. Only Hb-A and Hb-G can be detected as the products when a mixture of alpha(A)- and alpha(G)-chain sub-units reacts with Hb-beta(A) (4). The absence of the species alpha(A)alpha(G)beta(A) (2) can be explained in terms of the rapid equilibrium between whole and half haemoglobin molecules and the reassortment of alphabeta sub-units during the separation process.
| S-EPMC1270193 | biostudies-other
Project description:1. alpha-Crystallin has been isolated from the cortex of ox lens by isoelectric precipitation followed by chromatography on DEAE-cellulose. The amino acid composition is in agreement with that reported for alpha-crystallin prepared by a different method. There is one thiol group/20000g. of protein (20000 is the order of magnitude of the sub-unit molecular weight), and disulphide bonds are absent. 2. The thiol group has been alkylated with radioactive iodoacetate in the presence of urea. 3. Partial acid hydrolysis of the alkylated protein gives, according to the conditions, mainly three radioactive peptides or nearly exclusively one radioactive dipeptide. The dipeptide is N-seryl-(S-carboxymethyl)cysteine, Ser-CMCys. The two other peptides are probably the tripeptides related to Ser-CMCys. 4. The simplest interpretation of these results is that the sequence around the cysteine residue is a common structural feature of the sub-units of alpha-crystallin.
| S-EPMC1207209 | biostudies-other
Project description:αH-Crystallin, a high molecular weight form of α-crystallin, is one of the major proteins in the lens nucleus. This high molecular weight aggregate (HMWA) plays an important role in the pathogenesis of cataracts. We have shown that the chaperone-like activity of HMWA is 40% of that of α-crystallin from the lens cortex. Refolding with urea significantly increased-up to 260%-the chaperone-like activity of α-crystallin and slightly reduced its hydrodynamic diameter (Dh). HMWA refolding resulted in an increase in chaperone-like activity up to 120% and a significant reduction of Dh of protein particles compared with that of α-crystallin. It was shown that the chaperone-like activity of HMWA, α-crystallin, and refolded α-crystallin but not refolded HMWA was strongly correlated with the denaturation enthalpy measured with differential scanning calorimetry (DSC). The DSC data demonstrated a significant increase in the native protein portion of refolded α-crystallin in comparison with authentic α-crystallin; however, the denaturation enthalpy of refolded HMWA was significantly decreased in comparison with authentic HMWA. The authors suggested that the increase in the chaperone-like activity of both α-crystallin and HMWA could be the result of the correction of misfolded proteins during renaturation and the rearrangement of protein supramolecular structures.
| S-EPMC10487585 | biostudies-literature
Project description:The sub-unit structure of rabbit muscle triose phosphate isomerase was studied by determination of the number of unique cysteine peptides. Alkylation of the thiol groups with radioactive iodoacetate in the presence of guanidine hydrochloride gave the S-carboxy[(14)C]methyl derivative of the protein. This was digested with trypsin, and the radioactive peptides were fractionated by ion-exchange chromatography; four main radioactive peaks were obtained, one of which contained two radioactive peptides. Peptide ;maps' of the tryptic digest showed five main spots. The relationship between the members of both sets of five peptides was established. The radioactive peptides were characterized, and the results indicated the presence of five unique cysteine residues in the protein. Since there are approximately ten thiol groups/molecule, there are two closely related or identical sub-units. Studies of the terminal residues bear out this suggestion; only one kind of N-terminal residue (alanine) and one kind of C-terminal residue (glutamine) were detected. These results are in accord with the evidence from crystallography.
| S-EPMC1198745 | biostudies-other
Project description:Elucidating the top of the mammary epithelial cell hierarchy is highly important for understanding its regeneration capabilities and identifying target cells for transformation. Aiming for enriched mammary epithelial stem cell population, CD200highCD200R1high epithelial cells were identified. These cells represent ~50% of the mammary repopulating units (MRUs, CD49fhigh CD24med ) and termed MRUCD200/CD200R1. Gene expression of these cells was compared to all other MRU cells, termed MRUnot CD200/CD200R1, as well as individual CD200+ population (MRU-CD200R1-) and CD200R1+ population (MRU-CD200-).
2018-12-21 | GSE93961 | GEO
Project description:Small heat-shock proteins (sHSPs) are a widely expressed family of ATP-independent molecular chaperones that are among the first responders to cellular stress. Mechanisms by which sHSPs delay aggregation of client proteins remain undefined. sHSPs have high intrinsic disorder content of up to ~60% and assemble into large, polydisperse homo- and hetero-oligomers, making them challenging structural and biochemical targets. Two sHSPs, HSPB4 and HSPB5, are present at millimolar concentrations in eye lens, where they are responsible for maintaining lens transparency over the lifetime of an organism. Together, HSPB4 and HSPB5 compose the hetero-oligomeric chaperone known as α-crystallin. To identify the determinants of sHSP function, we compared the effectiveness of HSPB4 and HSPB5 homo-oligomers and HSPB4/HSPB5 hetero-oligomers in delaying the aggregation of the lens protein γD-crystallin. In chimeric versions of HSPB4 and HSPB5, chaperone activity tracked with the identity of the 60-residue disordered N-terminal regions (NTR). A short 10-residue stretch in the middle of the NTR ("Critical sequence") contains three residues that are responsible for high HSPB5 chaperone activity toward γD-crystallin. These residues affect structure and dynamics throughout the NTR. Abundant interactions involving the NTR Critical sequence reveal it to be a hub for a network of interactions within oligomers. We propose a model whereby the NTR critical sequence influences local structure and NTR dynamics that modulate accessibility of the NTR, which in turn modulates chaperone activity.
| S-EPMC9963673 | biostudies-literature
Project description:Aging of the lens is accompanied by extensive deamidation of the lens specific proteins, the crystallins. Deamidated crystallins are increased in the insoluble proteins and may contribute to cataracts. Deamidation has been shown in vitro to alter the structure and decrease the stability of human lens betaB1, betaB2 and betaA3-crystallin. Of particular interest, betaB2 mutants were constructed to mimic the effect of in vivo deamidations at the interacting interface between domains, at Q70 in the N terminal domain and at Q162, its C-terminal homologue. The double mutant was also constructed. We previously reported that deamidation at the critical interface sites decreased stability, while preserving the dimeric 3D structure. In the present study, dynamic light scattering, differential scanning calorimetry and small angle X-ray scattering were used to investigate the effect of deamidation on stability, thermal unfolding and aggregation. The bovine betaLb fraction was used for comparative analysis. The chaperone requirements of the various samples were determined using bovine alpha-crystallins as the chaperone. Deamidation at both interface Gln residues or at Q70, but not Q162, significantly lowered the temperature for unfolding and aggregation, which was rapidly followed by precipitation. This deamidation-induced aggregation and precipitation was not completely prevented by alpha-crystallin chaperone. A potential mechanism for cataract formation in vivo involving accumulation of deamidated beta-crystallin aggregates is discussed.
| S-EPMC2904749 | biostudies-literature
Project description:The demonstration of chaperone-like activity in peptides (mini-chaperones) derived from ?-crystallin's chaperone region has generated significant interest in exploring the therapeutic potential of peptide chaperones in diseases of protein aggregation. Recent studies in experimental animals show that mini-chaperones could reach intended targets and alter the disease phenotype. Although mini-chaperones show potential benefits against protein aggregation diseases, they do tend to form aggregates on storage. There is thus a need to fine-tune peptide chaperones to increase their solubility, pharmacokinetics, and biological efficacy.This review summarizes the properties and the potential therapeutic roles of mini-chaperones in protein aggregation diseases and highlights some of the refinements needed to increase the stability and biological efficacy of mini-chaperones while maintaining or enhancing their chaperone-like activity against precipitation of unfolding proteins.Mini-chaperones suppress the aggregation of proteins, block amyloid fibril formation, stabilize mutant proteins, sequester metal ions, and exhibit antiapoptotic properties. Much work must be done to fine-tune mini-chaperones and increase their stability and biological efficacy. Peptide chaperones could have a great therapeutic value in diseases associated with protein aggregation and apoptosis.Accumulation of misfolded proteins is a primary cause for many age-related diseases, including cataract, macular degeneration, and various neurological diseases. Stabilization of native proteins is a logical therapeutic approach for such diseases. Mini-chaperones, with their inherent antiaggregation and antiapoptotic properties, may represent an effective therapeutic molecule to prevent the cascade of protein conformational disorders. Future studies will further uncover the therapeutic potential of mini-chaperones. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.
| S-EPMC4673008 | biostudies-literature
Project description:Alpha-crystallin, a large lenticular protein complex made up of two related subunits (alphaA- and alphaB-crystallin), is known to associate increasingly with fiber cell plasma membranes with age and/or the onset of cataract. To understand better the binding mechanism, we developed a sensitive membrane binding assay using lens plasma membranes and recombinant human alphaA- and alphaB-crystallins conjugated to a small fluorescent tag (Alexa350). Both alphaA and alphaB homopolymer complexes, as well as a reconstituted 3:1 heteromeric complex, bind to lens membranes in a specific, saturable, and partially irreversible manner that is sensitive to both time and temperature. The amount of alpha-crystallin that binds to the membrane increases under acidic pH conditions and upon removal of exposed intrinsic membrane protein domains but is not affected at high ionic strength, suggesting that alpha-crystallin binds to the fiber cell plasma membranes mainly through hydrophobic interactions. The binding capacity and affinity for the reconstituted 3:1 heteromeric complex were measured to be 3. 45 +/- 0.11 ng/microg of membrane and 4.57 +/- 0.50 x 10(-4) microg(-1) of membrane, respectively. The present membrane binding data support the hypothesis that the physical properties of a mixed alpha-crystallin complex may hold particular relevance for the function of alpha-crystallin within the lens.
| S-EPMC2902967 | biostudies-literature
Project description:Γ-Crystallins play a major role in age-related lens transparency. Their destabilization by mutations and physical chemical insults are associated with cataract formation. Therefore, drugs that increase their stability should have anticataract properties. To this end, we screened 2560 Federal Drug Agency-approved drugs and natural compounds for their ability to suppress or worsen H2O2 and/or heat-mediated aggregation of bovine γ-crystallins. The top two drugs, closantel (C), an antihelminthic drug, and gambogic acid (G), a xanthonoid, attenuated thermal-induced protein unfolding and aggregation as shown by turbidimetry fluorescence spectroscopy dynamic light scattering and electron microscopy of human or mouse recombinant crystallins. Furthermore, binding studies using fluorescence inhibition and hydrophobic pocket-binding molecule bis-8-anilino-1-naphthalene sulfonic acid revealed static binding of C and G to hydrophobic sites with medium-to-low affinity. Molecular docking to HγD and other γ-crystallins revealed two binding sites, one in the "NC pocket" (residues 50-150) of HγD and one spanning the "NC tail" (residues 56-61 to 168-174 in the C-terminal domain). Multiple binding sites overlap with those of the protective mini αA-crystallin chaperone MAC peptide. Mechanistic studies using bis-8-anilino-1-naphthalene sulfonic acid as a proxy drug showed that it bound to MAC sites, improved Tm of both H2O2 oxidized and native human gamma D, and suppressed turbidity of oxidized HγD, most likely by trapping exposed hydrophobic sites. The extent to which these drugs act as α-crystallin mimetics and reduce cataract progression remains to be demonstrated. This study provides initial insights into binding properties of C and G to γ-crystallins.
| S-EPMC9525908 | biostudies-literature