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PubMed1993

High-performance gel-permeation chromatographic analysis of protein aggregation. Application to bovine carbonic anhydrase.

Protein association in bovine carbonic anhydrase (bCA) was investigated by high-performance gel-permeation chromatography (HPGPC). It was shown that bCA undergoes a time-dependent aggregation after freezing and storing at -20 degrees C. The addition of 50% ethylene glycol or glycerol prevented the aggregation. The HPGPC data were confirmed by the use of cross-linking with subsequent sodium dodecyl sulphate-polyacrylamide gel electrophoretic analysis. The esterase activity of the aggregated bCA was comparable to that of the monomer, suggesting that the observed protein association does not affect the esterase catalytic centre of bCA. This study illustrates how HPGPC can be a convenient and reliable tool for monitoring storage-induced aggregation of proteins.

Animals↗
PubMed2001

Review: mechanisms of disaggregation and refolding of stable protein aggregates by molecular chaperones.

Molecular chaperones are essential for the correct folding of proteins in the cell under physiological and stress conditions. Two activities have been traditionally attributed to molecular chaperones: (1) preventing aggregation of unfolded polypeptides and (2) assisting in the correct refolding of chaperone-bound denatured polypeptides. We discuss here a novel function of molecular chaperones: catalytic solubilization and refolding of stable protein aggregates. In Escherichia coli, disaggregation is carried out by a network of ATPase chaperones consisting of a DnaK core, assisted by the cochaperones DnaJ, GrpE, ClpB, and GroEL-GroES. We suggest a sequential mechanism in which (a) ClpB exposes new DnaK-binding sites on the surface of the stable protein aggregates; (b) DnaK binds the aggregate surfaces and, by doing so, melts the incorrect hydrophobic associations between aggregated polypeptides; (c) ATP hydrolysis and DnaK release allow local intramolecular refolding of native domains, leading to a gradual weakening of improper intermolecular links; (d) DnaK and GroEL complete refolding of solubilized polypeptide chains into native proteins. Thus, active disaggregation by the chaperone network can serve as a central cellular tool for the recovery of native proteins from stress-induced aggregates and actively remove disease-causing toxic aggregates, such as polyglutamine-rich proteins, amyloid plaques, and prions.

Adenosine Triphosphatases↗
PubMed1995

Protein aggregation kinetics in an Escherichia coli strain overexpressing a Salmonella typhimurium CheY mutant gene.

The tendency of recombinant protein in bacteria to partition into soluble and insoluble forms is attributed, in general, to a kinetic competition between protein folding and aggregation. However, little experimental work has actually been performed in vivo on the kinetics and mechanisms of protein folding and aggregation. Results are presented here from radiolabeling experiments which monitored the kinetics of recombinant protein aggregation in actively growing cultures. The strain used was an Escherichia coli strain overexpressing a Salmonella typhimurium CheY mutant gene. The rate of CheY aggregation was found to be time dependent in that the tendency of CheY to aggregate was greater for newly translated molecules, i.e., those translated within the previous several minutes, than for molecules translated less recently. CheY protein molecules that were translated less recently continued to aggregate for several hours but at a lower rate. The movement of soluble CheY to the insoluble form was enhanced at elevated growth temperatures and inhibited by the presence of chloramphenicol. The latter observation suggests that ongoing translation facilitates the movement of soluble CheY to the insoluble form. The implications of these results for the mechanism of protein aggregation in vivo, i.e., inclusion body formation, are discussed.

Bacterial Proteins↗
PubMed2003

AlphaB-crystallin modulates protein aggregation of abnormal desmin.

AlphaB-crystallin (CryAB) is the most abundant small heat shock protein in the heart. Upregulation of CryAB in desmin-related myopathy and its downregulation in end-stage congestive heart failure have both been reported. We previously demonstrated via cardiac-specific transgenesis that modest increases in normal CryAB are not detrimental to the heart, whereas expression of the R120G mutation of CryAB caused a desminopathy. It is generally believed that CryAB plays an important role in protecting the intermediate filaments, but the underlying mechanism is unclear. We hypothesized that CryAB protects the desmin filaments via preventing abnormal desmin protein from aggregating adversely. To test this hypothesis in vivo, mice expressing a desmin mutation that causes a desmin-related cardiomyopathy (D7) were bred into the R120G-CryAB transgenic (TG) background to examine the accumulation and aberrant aggregation of desmin protein. Despite lower mRNA expression of D7-des than in the D7-des TG hearts, the double-TG myocardium exhibited significantly higher desmin protein levels and dramatically more aberrant desmin aggregates than the D7-des TG hearts. The double-TG mice displayed a significantly stronger cardiac hypertrophic response, with the mice dying of congestive heart failure before 7 weeks. To explore the ability of wild-type (WT) CryAB to protect against mutant desmin, a desmin mutant was expressed in both the conventional and WT-CryAB stably transfected HEK cells. Significantly less aberrant desmin aggregation was observed in the WT-CryAB-overexpressing cells than in the HEK cells. The results suggest that CryAB modulates abnormal desmin aggregation and can serve a cardioprotective role.

Animals↗
PubMed2004

Prefibrillar amyloid protein aggregates share common features of cytotoxicity.

The intracellular free Ca(2+) concentration and redox status of murine fibroblasts exposed to prefibrillar aggregates of the HypF N-terminal domain have been investigated in vitro and in vivo using a range of fluorescent probes. Aggregate entrance into the cytoplasm is followed by an early rise of reactive oxygen species and free Ca(2+) levels and eventually by cell death. Such changes correlate directly with the viability of the cells and are not observed when cell are cultured in the presence of reducing agents or in Ca(2+)-free media. In addition, moderate cell stress following exposure to the aggregates was found to be fully reversible. The results show that the cytotoxicity of prefibrillar aggregates of HypF-N, a protein not associated with clinical disease, has the same fundamental origin as that produced by similar types of aggregates of proteins linked with specific amyloidoses. These findings suggest that misfolded proteinaceous aggregates stimulate generic cellular responses as a result of the exposure of regions of the structure (such as hydrophobic residues and the polypeptide main chain) that are buried in the normally folded proteins. They also support the idea that a higher number of degenerative pathologies than previously known might be considered as protein deposition diseases.

Amyloid↗
PubMed1989

Anomalous pressure dissociation of large protein aggregates. Lack of concentration dependence and irreversibility at extreme degrees of dissociation of extracellular hemoglobin.

The effects of hydrostatic pressure on the extracellular hemoglobin of Glossoscolex paulistus were investigated by studies of light scattering, intrinsic protein fluorescence, filtration chromatography, and oxygen binding. Pressure promoted a large decrease of light scattering consistent with the dissociation of the hemoglobin. Pressures up to 1.7 kbar caused dissociation with reversibility of the light scattering and fluorescence properties after return to atmospheric pressure. Higher pressures provoked additional dissociation with increasing loss of reversibility. After complete dissociation by incubation at 2.5 kbar followed by decompression, the protein continued mostly dissociated. The dissociated forms were distributed in two populations as based on size exclusion chromatography, one corresponding to small dissociated units (average Mr = 33,000) and the other population corresponding to the one-twelfth subunit (260,000 Mr). The pressure dissociation curves showed no significant dependence on protein concentration suggesting that the native hemoglobin population exists in a distribution of free-energies of association. Both the decrease of concentration dependence and the loss of ability to reassemble seem to increase with the complexity and size of the protein aggregate. These findings permit the conclusion that increased heterogeneity of free-energies of association with the size of the aggregate may result in the molecular individuality of large protein complexes such as subcellular particles and viruses.

Animals↗
PubMed2000

Size-dependent disaggregation of stable protein aggregates by the DnaK chaperone machinery.

Classic in vitro studies show that the Hsp70 chaperone system from Escherichia coli (DnaK-DnaJ-GrpE, the DnaK system) can bind to proteins, prevent aggregation, and promote the correct refolding of chaperone-bound polypeptides into native proteins. However, little is known about how the DnaK system handles proteins that have already aggregated. In this study, glucose-6-phosphate dehydrogenase was used as a model system to generate stable populations of protein aggregates comprising controlled ranges of particle sizes. The DnaK system recognized the glucose-6-phosphate dehydrogenase aggregates as authentic substrates and specifically solubilized and refolded the protein into a native enzyme. The efficiency of disaggregation by the DnaK system was high with small aggregates, but the efficiency decreased as the size of the aggregates increased. High folding efficiency was restored by either excess DnaK or substoichiometric amounts of the chaperone ClpB. We suggest a mechanism whereby the DnaK system can readily solubilize small aggregates and refold them into active proteins. With large aggregates, however, the binding sites for the DnaK system had to be dynamically exposed with excess DnaK or the catalytic action of ClpB and ATP. Disaggregation by the DnaK machinery in the cell can solubilize early aggregates that formed accidentally during chaperone-assisted protein folding or that escaped the protection of "holding" chaperones during stress.

Chromatography, Gel↗
PubMed1981

Effect of protein aggregation on murine splenic prostaglandin F2 alpha levels.

Intravenous injection of deaggregated human gamma globulin and deaggregated keyhole limpet hemocyanin into C57Bl/6 mice does not result in an increase in splenic prostaglandin F2 alpha. However, when aggregated human gamma globulin or aggregated keyhole limpet hemocyanin were injected, significant increases in splenic PGF2 alpha were observed. Mice which had been rendered immunologically tolerant to human gamma globulin (HGG) also demonstrated increases in splenic PGF2 alpha and PGE2 5 min after boosting with aggregated HGG with no increases in antibody against HGG. These results imply that the property of protein aggregation will engender increases in splenic prostaglandin levels and that these increases don't appear to affect the tolerant state.

Animals↗
PubMed2000

Inhibition of polyglutamine protein aggregation and cell death by novel peptides identified by phage display screening.

Proteins with expanded polyglutamine domains cause eight inherited neurodegenerative diseases, including Huntington's, but the molecular mechanism(s) responsible for neuronal degeneration are not yet established. Expanded polyglutamine domain proteins possess properties that distinguish them from the same proteins with shorter glutamine repeats. Unlike proteins with short polyglutamine domains, proteins with expanded polyglutamine domains display unique protein interactions, form intracellular aggregates, and adopt a novel conformation that can be recognized by monoclonal antibodies. Any of these polyglutamine length-dependent properties could be responsible for the pathogenic effects of expanded polyglutamine proteins. To identify peptides that interfere with pathogenic polyglutamine interactions, we screened a combinatorial peptide library expressed on M13 phage pIII protein to identify peptides that preferentially bind pathologic-length polyglutamine domains. We identified six tryptophan-rich peptides that preferentially bind pathologic-length polyglutamine domain proteins. Polyglutamine-binding peptide 1 (QBP1) potently inhibits polyglutamine protein aggregation in an in vitro assay, while a scrambled sequence has no effect on aggregation. QBP1 and a tandem repeat of QBP1 also inhibit aggregation of polyglutamine-yellow fluorescent fusion protein in transfected COS-7 cells. Expression of QBP1 potently inhibits polyglutamine-induced cell death. Selective inhibition of pathologic interactions of expanded polyglutamine domains with themselves or other proteins may be a useful strategy for preventing disease onset or for slowing progression of the polyglutamine repeat diseases.

Amino Acid Sequence↗
PubMed2005

LEA proteins prevent protein aggregation due to water stress.

LEA (late embryogenesis abundant) proteins in both plants and animals are associated with tolerance to water stress resulting from desiccation and cold shock. However, although various functions of LEA proteins have been proposed, their precise role has not been defined. Recent bioinformatics studies suggest that LEA proteins might behave as molecular chaperones, and the current study was undertaken to test this hypothesis. Recombinant forms of AavLEA1, a group 3 LEA protein from the anhydrobiotic nematode Aphelenchus avenae, and Em, a group 1 LEA protein from wheat, have been subjected to functional analysis. Heat-stress experiments with citrate synthase, which is susceptible to aggregation at high temperatures, suggest that LEA proteins do not behave as classical molecular chaperones, but they do exhibit a protective, synergistic effect in the presence of the so-called chemical chaperone, trehalose. In contrast, both LEA proteins can independently protect citrate synthase from aggregation due to desiccation and freezing, in keeping with a role in water-stress tolerance; similar results were obtained with lactate dehydrogenase. This is the first evidence of anti-aggregation activity of LEA proteins due to water stress. Again, a synergistic effect of LEA and trehalose was observed, which is significant given that non-reducing disaccharides are known to accumulate during dehydration in plants and nematodes. A model is proposed whereby LEA proteins might act as a novel form of molecular chaperone, or 'molecular shield', to help prevent the formation of damaging protein aggregates during water stress.

Animals↗
PubMed1985

Tobacco mosaic virus protein aggregates in solution: structural comparison of 20S aggregates with those near conditions for disk crystallization.

Previous X-ray studies (2.8-A resolution) on the crystals of tobacco mosaic virus protein (TMVP) grown from solutions containing high salt have characterized the structure of the protein aggregate as a bilayered cylindrical disk formed by 34 identical subunits [Bloomer, A.C., Champness, J.N., Bricogne, G., Staden, R., & Klug, A. (1978) Nature (London) 276, 362-368]. Under low-salt conditions, 20S aggregates are in equilibrium with 4S species and involved in the efficient nucleation of TMV assembly in vitro [Butler, P.J.G. (1984) J. Gen. Virol. 65, 253-279]. We have investigated by sedimentation velocity and near-UV circular dichroism (CD) measurements the structure of 20S aggregates in low salt (I = 0.1 potassium phosphate at pH 7.0 and 20 degrees C) and the aggregates in high salt [0.2 M (NH4)2SO4 in I = 0.1 tris(hydroxymethyl)aminomethane hydrochloride at pH 8.0 and 20 degrees C, close to the conditions under which TMVP crystallizes as disk aggregates]. At high salt, we observe structures (presumably stacks of disks) having s20,w values around 40, 45, and 50 S, but not the 20S species present in low-salt buffers. The near-UV CD spectrum of 20S aggregates has been obtained for the first time, using computer techniques, from the spectra of the 4S-20S equilibrium mixture and the 4S species. This spectrum of 20S aggregates differs dramatically from that of the stacks of disks examined at both high and low salt (into which the stacks can be returned by dialysis), indicating that the difference is not a solvent effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Circular Dichroism↗
PubMed2006

X-34 labeling of abnormal protein aggregates during the progression of Alzheimer's disease.

Postmortem pathological diagnosis and basic research investigations of neurodegenerative disorders rely on histochemical staining procedures developed specifically to visualize abnormal protein conformation. In Alzheimer's disease (AD), two major pathological hallmarks are required to confirm the clinical diagnosis. Both consist of abnormally aggregated proteins that share the structural and histological properties common to all amyloid deposits. Amyloid-beta peptide (Abeta) of extracellular senile plaques (SP) and hyperphosphorylated tau of intracellular neurofibrillary tangles (NFT) are assembled in the abnormal beta-pleated sheet (amyloid-like) structural conformation that can be visualized with histological staining procedures using Congo red or its derivatives. These histochemical dyes bind amyloid with high affinity and allow easy detection of amyloid structure in postmortem brain samples. This chapter focuses on the development and application of a histological protocol using the compound X-34, a highly fluorescent derivative of Congo red, for sensitive detection of pathological amyloid structures in histopathological investigations of postmortem brain tissue. This procedure provides a simple and effective method for detailed fluorescent visualization of the localization and distribution of the majority of currently known major histopathological structures in AD, including compact cored, neuritic, and diffuse-appearing SP, NFT, dystrophic neurites, neuropil threads, and cerebrovascular amyloidosis.

Alkenes↗
PubMed1979

An extrinsic membrane polypeptide associated with high-molecular-weight protein aggregates in human cataract.

A 43,000-dalton polypeptide has been isolated from the high-molecular-weight disulfide-rich fraction of the water-insoluble protein of human cataractous lenses. On the basis of immunochemical reactivity and fluorescent antibody binding, this polypeptide is localized in the membrane region of the lens cell. This observation suggests an interaction between the soluble lens proteins and membrane-associated polypeptides in the formation of large protein aggregates which may cause cataract.

Antibody Specificity↗
PubMed2005

Single particle detection and characterization of synuclein co-aggregation.

Protein aggregation is the key event in a number of human diseases such as Alzheimer's and Parkinson's disease. We present a general method to quantify and characterize protein aggregates by dual-colour scanning for intensely fluorescent targets (SIFT). In addition to high sensitivity, this approach offers a unique opportunity to study co-aggregation processes. As the ratio of two fluorescently labelled components can be analysed for each aggregate separately in a homogeneous assay, the molecular composition of aggregates can be studied even in samples containing a mixture of different types of aggregates. Using this method, we could show that wild-type alpha-synuclein forms co-aggregates with a mutant variant found in familial Parkinson's disease. Moreover, we found a striking increase in aggregate formation at non-equimolar mixing ratios, which may have important therapeutic implications, as lowering the relative amount of aberrant protein may cause an increase of protein aggregation leading to adverse effects.

Amyloid↗
PubMed2025

Benchmark for Quantitative Global and Redox Proteomics Analysis by Combining Protein-Aggregation Capture and Data Independent Acquisition.

Oxidative damage plays a critical role in various diseases including cardiovascular and neurological disorders. Thiol redox reactions, acting as oxidative stress sensors, influence protein structure and function. Redox proteomics, based on the differential alkylation of cysteine sites followed by mass spectrometry, enables the comprehensive analysis of thiol redox status in cells and tissues. However, these approaches require extensive sample manipulation and are not compatible with data-independent acquisition techniques. Here, we introduce PACREDOX, an innovative strategy based on protein aggregation capture (PAC), and demonstrate its compatibility with library-free DIA. Compared with traditional methods such as FASILOX, PACREDOX reduces preparation time and costs while maintaining thiol and proteome coverage. To enable library-free DIA, we corrected in silico spectral libraries in DIA-NN using experimental retention time data from methylthiolated-Cys peptides. PACREDOX with DIA was benchmarked against FASILOX in a myocardial infarction model, yielding the same biological insights, while enhancing peptide and protein coverage. Our results underscore the potential and efficiency of this methodology for studying oxidative damage. Overall, PACREDOX offers an automatable, high-throughput, and cost-effective strategy for redox proteomics.

Proteomics↗
PubMed2000

Ultrasensitive detection of pathological prion protein aggregates by dual-color scanning for intensely fluorescent targets.

A definite diagnosis of prion diseases such as Creutzfeldt-Jakob disease (CJD) relies on the detection of pathological prion protein (PrP(Sc)). However, no test for PrP(Sc) in cerebrospinal fluid (CSF) has been available thus far. Based on a setup for confocal dual-color fluorescence correlation spectroscopy, a technique suitable for single molecule detection, we developed a highly sensitive detection method for PrP(Sc). Pathological prion protein aggregates were labeled by specific antibody probes tagged with fluorescent dyes, resulting in intensely fluorescent targets, which were measured by dual-color fluorescence intensity distribution analysis in a confocal scanning setup. In a diagnostic model system, PrP(Sc) aggregates were detected down to a concentration of 2 pM PrP(Sc), corresponding to an aggregate concentration of approximately 2 fM, which was more than one order of magnitude more sensitive than Western blot analysis. A PrP(Sc)-specific signal could also be detected in a number of CSF samples from patients with CJD but not in control samples, providing the basis for a rapid and specific test for CJD and other prion diseases. Furthermore, this method could be adapted to the sensitive detection of other disease-associated amyloid aggregates such as in Alzheimer's disease.

Animals↗
PubMedDate not supplied

Rapid detection of protein aggregates in the brains of Alzheimer patients and transgenic mouse models of amyloidosis.

Extracellular and/or intracellular aggregates are pathological features of many, if not all, neurodegenerative diseases. In Alzheimer disease (AD), extracellular aggregates of beta-amyloid (Abeta) and intracellular aggregates of tau or a-synuclein are key diagnostic markers of the disease. We report here a method to rapidly detect these protein aggregates that relies on size exclusion filtration and immunostaining of trapped material, a method termed filter trapping. We demonstrate that aggregated forms of Abeta and tau are readily trapped in 0.22 microm cellulose acetate filter membranes, which can then be immunostained with specific antibodies in a manner similar to the standard immunoblot. Coupling this method with serial dilution permits a rapid assessment of relative aggregate burden.

Alzheimer Disease↗