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PubMed1968

Protein aggregation. Studies of larger aggregates of C-phycocyanin.

Aggregates of phycocyanin sedimenting at 17s, 22s and 27s are demonstrated to constitute more than 40% of crude blue-green-algal extracts, pH6.0 and I0.1, and are retained in highly purified preparations. Sedimentation-velocity studies of the large aggregates as a function of pH are reported. Sucrose-density-gradient experiments performed as a function of time of sedimentation indicate that: (1) with increasing time of sedimentation, the largest aggregates are dissipated at the leading protein boundary and the several phycocyanin species present are not completely resolved; (2) phycocyanin fractions with the largest aggregates exhibit the highest E(620)/E(280) ratio and the largest relative fluorescence efficiency. Gel-filtration experiments with Sephadex G-200 do not resolve the species completely, and reapplication of phycocyanin gel-filtration fractions to the column results in an elution pattern similar to the original, except that there is an enhancement of the allophycocyanin fraction and the amount of denatured protein. Increasing the sedimentation times in a sucrose density gradient also enhances the allophycocyanin fraction. Fluorescence results demonstrate that there are possibly three excitation maxima, one corresponding to the monomer (approx. 600mmu), one for higher aggregates (625-630mmu) and one for the allophycocyanin fraction (approx. 650mmu). Only a single fluorescence-emission band is detected, which is fairly symmetrical and which has a red shift with higher aggregation and with the appearance of allophycocyanin. The appearance of allophycocyanin may be correlated with the irreversible disaggregation of the largest phycocyanin species. It is suggested that the largest protein aggregates are in the size range of the biliprotein aggregates reported in electron microscopy of algal cells.

Chemical Phenomena↗

PubMed2001

Activation of the Ras-cAMP signal transduction pathway inhibits the proteasome-independent degradation of misfolded protein aggregates in the endoplasmic reticulum lumen.

Many kinds of misfolded secretory proteins are known to be degraded in the endoplasmic reticulum (ER). Dislocation of misfolded proteins from the ER to the cytosol and subsequent degradation by the proteasome have been demonstrated. Using the yeast Saccharomyces cerevisiae, we have been studying the secretion of a heterologous protein, Rhizopus niveus aspartic proteinase-I (RNAP-I). Previously, we found that the pro sequence of RNAP-I is important for the folding and secretion, and that Deltapro, a mutated derivative of RNAP-I in which the entire region of the pro sequence is deleted, forms gross aggregates in the yeast ER. In this study, we show that the degradation of Deltapro occurs independently of the proteasome. Its degradation was not inhibited either by a potent proteasome inhibitor or in a proteasome mutant. We also show that neither the export from the ER nor the vacuolar proteinase is required for the degradation of Deltapro. These results raise the possibility that the Deltapro aggregates are degraded in the ER lumen. We have isolated a yeast mutant in which the degradation of Deltapro is delayed. We show that the mutated gene is IRA2, which encodes a GTPase-activating protein for Ras. Because Ira2 protein is a negative regulator of the Ras-cAMP pathway, this result suggests that hyperactivation of the Ras-cAMP pathway inhibits the degradation of Deltapro. Consistently, down-regulation of the Ras-cAMP pathway in the ira2 mutant suppressed the defect of the degradation of Deltapro. Thus, the Ras-cAMP signal transduction pathway seems to control the proteasome-independent degradation of the ER misfolded protein aggregates.

Aspartic Acid Endopeptidases↗

PubMed2006

Identification of anti-prion compounds as efficient inhibitors of polyglutamine protein aggregation in a zebrafish model.

Several neurodegenerative diseases, including Huntington disease (HD), are associated with aberrant folding and aggregation of polyglutamine (polyQ) expansion proteins. Here we established the zebrafish, Danio rerio, as a vertebrate HD model permitting the screening for chemical suppressors of polyQ aggregation and toxicity. Upon expression in zebrafish embryos, polyQ-expanded fragments of huntingtin (htt) accumulated in large SDS-insoluble inclusions, reproducing a key feature of HD pathology. Real time monitoring of inclusion formation in the living zebrafish indicated that inclusions grow by rapid incorporation of soluble htt species. Expression of mutant htt increased the frequency of embryos with abnormal morphology and the occurrence of apoptosis. Strikingly, apoptotic cells were largely devoid of visible aggregates, suggesting that soluble oligomeric precursors may instead be responsible for toxicity. As in nonvertebrate polyQ disease models, the molecular chaperones, Hsp40 and Hsp70, suppressed both polyQ aggregation and toxicity. Using the newly established zebrafish model, two compounds of the N'-benzylidene-benzohydrazide class directed against mammalian prion proved to be potent inhibitors of polyQ aggregation, consistent with a common structural mechanism of aggregation for prion and polyQ disease proteins.

Animals↗

PubMed2006

A novel function of 14-3-3 protein: 14-3-3zeta is a heat-shock-related molecular chaperone that dissolves thermal-aggregated proteins.

The 14-3-3 proteins are highly conserved molecules that function as intracellular adaptors in a variety of biological processes, such as signal transduction, cell cycle control, and apoptosis. Here, we show that a 14-3-3 protein is a heat-shock protein (Hsp) that protects cells against physiological stress as its new cellular function. We have observed that, in Drosophila cells, the 14-3-3zeta is up-regulated under heat stress conditions, a process mediated by a heat shock transcription factor. As the biological action linked to heat stress, 14-3-3zeta interacted with apocytochrome c, a mitochondrial precursor protein of cytochrome c, in heat-treated cells, and the suppression of 14-3-3zeta expression by RNA interference resulted in the formation of significant amounts of aggregated apocytochrome c in the cytosol. The aggregated apocytochrome c was converted to a soluble form by the addition of 14-3-3zeta protein and ATP in vitro. 14-3-3zeta also resolubilized heat-aggregated citrate synthase and facilitated its reactivation in cooperation with Hsp70/Hsp40 in vitro. Our observations provide the first direct evidence that a 14-3-3 protein functions as a stress-induced molecular chaperone that dissolves and renaturalizes thermal-aggregated proteins.

14-3-3 Proteins↗

PubMed1993

The application of size exclusion chromatography and computer simulation to study the thermodynamic and kinetic parameters for short-lived dissociable protein aggregates.

We describe a method to study the monomer-dimer equilibrium of human growth hormone (hGH) making use of a very short size exclusion high-performance liquid chromatographic column and rapid flow rates. By adjusting the flow rate and thus the retention time on the column, the dissociation of the hGH dimer can be observed. Using computer simulation, both the equilibrium constant for dissociation and the dissociation rate constant can be determined directly, followed by the indirect determination of the association rate constant. This method is potentially useful for determining the thermodynamic the kinetic parameters of aggregation of many protein-protein (homodimers and heterodimers) and protein-ligand systems whose rates of interaction are too rapid to be studied by conventional techniques.

Chromatography, High Pressure Liquid↗

PubMed2026

Influence of protein aggregates, extracellular vesicles, and lipoprotein fusion on ionizable lipid nanoparticles protein corona analysis.

Since 2018, ionizable lipid nanoparticles (LNPs) have revolutionized nucleic acid therapeutics. However, achieving potent extrahepatic delivery remains a formidable challenge, primarily due to rapid hepatic uptake driven by apolipoprotein adsorption. While analyzing the LNP protein corona is essential for engineering organ-specific tropism, these soft materials present unique analytical hurdles. Co-isolation of blood-borne contaminants, such as extracellular vesicles and lipoproteins, often masks the true corona composition. This perspective examines the critical need for refined proteomic strategies to distinguish genuine corona proteins from impurities. We propose tailored investigative approaches, suggesting the LNP protein corona significantly differs from the rigid shells observed on inorganic nanoparticles.

Nanoparticles↗

PubMed1995

Amyloid beta-protein aggregation nullifies its pathologic properties in cultured cerebrovascular smooth muscle cells.

Alzheimer's disease and related disorders are characterized by deposition of aggregated amyloid beta-protein (A beta) and accompanying pathologic changes in the neuropil and in the walls of cerebral blood vessels. A beta induces neurotoxicity in vitro, and this effect is markedly enhanced when the peptide is preaggregated. Recently, we reported that freshly solubilized A beta 1-42 can induce cellular degeneration and a striking increase in the levels of cellular amyloid beta-protein precursor and soluble A beta peptide in cultured cerebrovascular smooth muscle cells (Davis-Salinas, J., Saporito-Irwin, S. M., Cotman, C. W., and Van Nostrand, W. E. (1995) J. Neurochem. 65, 931-934). In the present study, we show that preaggregation of A beta 1-42 abolishes the ability of the peptide to induce these cellular pathologic responses in these cells in vitro. These findings suggest that distinct mechanisms for A beta-induced cytotoxicity exist for cultured neurons and cerebrovascular smooth muscle cells, supporting that different processes may be involved in the parenchymal and cerebrovascular pathology of Alzheimer's disease and related disorders.

Alzheimer Disease↗

PubMed1999

A cytoplasmic structure resembling large protein aggregates induced by interferons.

IFP 35 is an interferon (IFN)-regulated leucine zipper protein, expression of which is observed in a variety of cell types including monocytes/macrophages, epithelial cells and fibroblasts. Using immunofluorescence studies, we demonstrate that IFP 35 is found in characteristic punctate cytoplasmic structures after IFN treatment. Co-localization experiments using double immunofluorescence and confocal laser scanning microscopy failed to show association of IFP 35 with known organelles (mitochondria, peroxisomes, endoplasmic reticulum, lysosomes, endosomes, Golgi complex), ribosomes, or actin filaments. Subcellular fractionation to separate membrane-associated from cytoplasmic proteins demonstrated that IFP 35 localizes to the cytoplasm. Separation of postnuclear supernatant from HeLa cells by gel filtration revealed that IFP 35 eluted at a molecular mass of 200-440 kD, suggesting that IFP 35 is part of protein complexes. Electron microscopic studies showed cytoplasmic clusters of a few aggregates of IFP 35 in IFN-treated cells which were neither associated with nor surrounded by a membrane. A combination of immunoprecipitation and immunofluorescence studies of cells transfected with a hemagglutinin epitope-tagged IFP 35 expression construct demonstrated complex formation and co-localization of endogenous and transfected IFP 35. Taken together, our studies demonstrate that IFP 35 associates with unique cytoplasmic structures that are distinct from known organelles and resemble large protein aggregates.

Blotting, Western↗

PubMed1998

Study of the influence of salt concentration on Newcastle disease virus matrix protein aggregation.

The aggregation process of Newcastle disease virus matrix protein (M protein) has been studied using light scattering. We observed that the aggregation of M protein is inversely correlated with ionic strength and that this process can be reversed by high salt concentrations. It was found that the oligomeric structure of NDV matrix protein is different from those described earlier for other matrix proteins of enveloped viruses.

Light↗

PubMed2005

Tau protein aggregation in the frontal and entorhinal cortices as a function of aging.

OBJECTIVES: The abnormal accumulation of tau protein is increasingly recognized as the neuropathological hallmark of a number of dementing illness in which frontotemporal lobar degeneration occurs. In this paper we examined the age-dependant deposition of tau protein in the frontal and entorhinal neocortices. METHODS: We examined autopsy records from 1997 to 2002 and selected 87 cases (10 in each decade from 0 to 79 years of age, 7 in 80-89 decade) with no history of dementia or other neurodegenerative diseases, and for which neurodegenerative diseases were excluded neuropathologically. Archival paraffin-embedded frontal and entorhinal cortices were examined by both Gallyas-Braak silver staining and a panel of antibodies recognizing tau protein accumulation. RESULTS: Tau neuronal aggregates were observed in both frontal and entorhinal cortices in the third decade. While the frontal neuronal tau aggregates remained infrequent in the remaining decades, the number and extent ofneuronal tau aggregates in the entorhinal cortex increased such that by the 7th decade the majority of cases showed extensive tau aggregate formation. The most consistent morphological observation was of dense, perikaryal neuronal tau-immunoreactive aggregates, similar to the total tau distribution, firstly presenting in cortical layers II and III and subsequently involving in layers IV-VI. Neuropil threads became maximal in the 9th decade in both frontal and entorhinal cortices. Astrocytic tau accumulation was first observed in both frontal and entorhinal cortices in the 6th decade, predominantly in layer I and subcortical white matter, and increased in number with aging. Extraneuronal tau reactive aggregates and coiled bodies were rarely observed in the entorhinal cortex, and when present, were scattered through layer II to VI. CONCLUSIONS: We have observed an age-dependant pattern of neuronal, extraneuronal and glial tau protein accumulation in the entorhinal cortex in individuals without neurodegenerative diseases. In contrast, tau protein aggregation is infrequently observed in the frontal cortex as a function of aging.

Adolescent↗

PubMed1979

Further investigation of the role of calcium in human lens protein aggregation.

High-molecular-weight (HMW) protein from human cataractous lenses, isolated by differential centrifugation, was deaggregated in 7M urea and then reaggregated in either the presence or absence of 10 mM CaCl2. Over 90% of the material reaggregated in the presence of calcium appears to have a size greater than 50 X 10(6) daltons. By contrast, only 20% to 25% of the material reaggregated in the absence of calcium has molecular weight greater than 50 X 10(6) daltons. Disulfide formation during reaggregation is unlikely in the latter experiment, since the addition of 50 mM mercaptoethanol caused no change in results. About 60% to 70% of the low-molecular-weight (LMW) protein fraction deaggregated in 7M urea buffer can be converted to HMW species in the presence of 10 mM CaCl2, when the deaggregating agent is removed. However, only 5% to 10% of this protein is converted to HMW species if the deaggregation step is eliminated. Experiments with 45 Ca indicate that whereas calcium is necessary for the formation of the HMW aggregates, only one calcium per approximately 5 X 10(5) daltons remains bound in the reaggregated material. The data suggest that although calcium may be required to induce aggregation to HMW species, it is not required to stabilize such macromolecules. SDS-polyacrylamide gel electrophoresis of the HMW species formed upon reaggregation of the dissociated HMW species with calcium indicates the presence of all the major polypeptide subunits of the original HMW species present in the lens; however, reaggregation in the absence of calcium yields HMW species lacking in the 9600 dalton component.

Calcium Chloride↗

PubMed2003

Rationalization of the effects of mutations on peptide and protein aggregation rates.

In order for any biological system to function effectively, it is essential to avoid the inherent tendency of proteins to aggregate and form potentially harmful deposits. In each of the various pathological conditions associated with protein deposition, such as Alzheimer's and Parkinson's diseases, a specific peptide or protein that is normally soluble is deposited as insoluble aggregates generally referred to as amyloid. It is clear that the aggregation process is generally initiated from partially or completely unfolded forms of the peptides and proteins associated with each disease. Here we show that the intrinsic effects of specific mutations on the rates of aggregation of unfolded polypeptide chains can be correlated to a remarkable extent with changes in simple physicochemical properties such as hydrophobicity, secondary structure propensity and charge. This approach allows the pathogenic effects of mutations associated with known familial forms of protein deposition diseases to be rationalized, and more generally enables prediction of the effects of mutations on the aggregation propensity of any polypeptide chain.

Amyloidosis↗

PubMed1998

Hsp104, Hsp70, and Hsp40: a novel chaperone system that rescues previously aggregated proteins.

Hsp104 is a stress tolerance factor that promotes the reactivation of heat-damaged proteins in yeast by an unknown mechanism. Herein, we demonstrate that Hsp104 functions in this process directly. Unlike other chaperones, Hsp104 does not prevent the aggregation of denatured proteins. However, in concert with Hsp40 and Hsp70, Hsp104 can reactivate proteins that have been denatured and allowed to aggregate, substrates refractory to the action of other chaperones. Hsp104 cooperates with the chaperones present in reticulocyte lysates but not with DnaK of E. coli. We conclude that Hsp104 has a protein remodeling activity that acts on trapped, aggregated proteins and requires specific interactions with conventional chaperones to promote refolding of the intermediates it produces.

Adenosine Triphosphatases↗

PubMedDate not supplied

Continuous removal of protein aggregates by annular chromatography.

The removal of polymeric proteins from their monomers is a frequently encountered separation task, especially in the polishing step of therapeutic proteins. Continuous separation of protein polymers from monomers by annular chromatography using size exclusion chromatography has been studied regarding the resolution, recovery, fouling, and productivity and has been compared to conventional chromatography. An IgG preparation rich in aggregates was used as a model protein mixture. Under conditions that maximized the throughput, the polymers could be separated from the monomers, but baseline separation could not be achieved. Baseline separation was also not possible in batch mode using equivalent conditions, which was also confirmed by computer simulation. For separation of the aggregates from the product the entire available separation space (360 degrees ) was indispensable. Therefore only cyclic, discontinuous regeneration could be carried out. Loading was identified as a critical step, since the concentrated protein solution evaded into the headspace instead of migrating into the gel where viscous fingering often occurs in conventional chromatography. The productivity of annular chromatography was two times higher than that of the conventional batch chromatography, and the buffer consumption was reduced to half the conventional value. These two benefits are especially important for protein separation processes that suffer from low loadability, such as size exclusion chromatography. We have demonstrated that size exclusion can be performed on an industrial scale when it is run continuously with the aid of a pressurized annular chromatograph.

Chromatography, Gel↗

PubMed1997

Prion protein aggregation reverted by low temperature in transfected cells carrying a prion protein gene mutation.

Prion diseases are characterized by the conversion of the normal cellular prion protein (PrPC), a glycoprotein that is anchored to the cell membrane by a glycosylphosphatidylinositol moiety, into an isoform that is protease-resistant (PrPres) and pathogenic. In inherited prion diseases, mutations in the prion protein (PrPM) engender the conversion of PrPM into PrPres. We developed a cell model of Gerstmann-Sträussler-Scheinker disease, a neurodegenerative condition characterized by PrPM-containing amyloid deposits and neuronal loss, by expressing the Gerstmann-Sträussler-Scheinker haplotype Q217R-129V in human neuroblastoma cells. By comparison to PrPC, this genotype results in the following alterations of PrPM: 1) expression of an aberrant form lacking the glycosylphosphatidylinositol anchor, 2) increased aggregation and protease resistance, and 3) impaired transport to the cell surface. Most of these alterations are temperature-sensitive, indicating that they are due to misfolding of PrPM.

Cell Membrane↗

PubMed1989

Determination of the fractal dimension of membrane protein aggregates using fluorescence energy transfer.

It is demonstrated that fluorescence resonance energy transfer may be used to determine the fractal dimension of aggregates of membrane-bound proteins. Theoretical and experimental results are presented for two different experimental designs: energy transfer between proteins and energy transfer from lipids to proteins. For energy transfer between proteins the lattice spacing must be known independently for a fractal dimension to be uniquely determined, and this represents a disadvantage to this experimental design. Results are presented for the calcium ATPase and a fractal dimension of 1.9 is estimated for ATPase aggregates by assuming a lattice spacing of 50 A. Energy transfer from lipids to protein provides a means of estimating the length of the "coast-line" of the aggregate. In this case the fractal dimension is uniquely determined from a log-log plot. An analysis of data for bacteriohodopsin reconstituted in phospholipid vesicles gives a fractal dimension of 1.6. The structural basis of the value for the fractal dimension is discussed for these two systems. These techniques provide a means of assessing the nature of protein-protein interactions in membranous systems.

Calcium-Transporting ATPases↗

PubMed2006

Simultaneous determination of protein aggregation, degradation, and absolute molecular weight by size exclusion chromatography-multiangle laser light scattering.

The feasibility of size exclusion chromotography (SEC)-multiangle laser-light scattering as a technique to investigate aggregation and degradation of glycosylated and nonglycosylated proteins, and antibodies under various conditions such as addition of detergent, changes in pH, and variation of protein concentration and heat stress temperature was examined. Separation of proteins and their aggregates was performed using SEC-high-performance liquid chromatography. Detection of analytes was carried out with on-line UV, refractive index, and multiangle laser light-scattering detectors. Quantification and molecular weight determination were performed using commercial software. Aggregation and degradation were examined under various conditions and quantitative results are presented for bovine serum albumin, choriogonadotropin, glyceraldehyde-3-phosphate dehydrogenase, Herceptin, and ReoPro. This method can simultaneously determine both the quantities and the molecular weights of macromolecules from a single injection. The determination of molecular weight is absolute which avoids misleading results caused by molecular shape or interactions with the column matrix. This technique is valuable not only for assessing the extent of aggregation but also for effectively monitoring molecule degradation as evidenced by molecular weight reduction and change in monomer amount.

Abciximab↗

PubMed2005

Mass spectrometry-based screening for inhibitors of beta-amyloid protein aggregation.

Alzheimer's disease is the most common cause of the loss of cognitive function among the elderly, and the aggregation and deposition of misfolded beta-amyloid protein (Abeta) contribute to this progressive central nervous system decline. Therefore, compounds that inhibit or even reverse Abeta aggregation might be useful for the treatment or prevention of Alzheimer's disease. To identify potential therapeutic agents for the treatment of Alzheimer's disease, a mass spectrometry-based screening assay was developed to identify and rank order compounds that inhibit the aggregation of Abeta. To carry out this assay, Abeta was incubated with a test compound at 37 degrees C for 20 h followed by ultrafiltration to separate the monomeric Abeta from its aggregates. Aliquots of the ultrafiltrate were analyzed for monomeric Abeta using positive ion electrospray mass spectrometry based on the abundance the quadruply protonated molecule of Abeta at m/z 1083. The calibration curve for Abeta was linear with a correlation coefficient (r2) of >0.99 over the range of at least 11-110 microM. The limit of detection was 0.224 ng (5.18 nM, 10-microL injection), and the limit of quantitation was 0.747 ng (17.2 nM, 10-microL injection). Based on previous reports of compounds that either bind to Abeta or are useful in treating Alzheimer's disease, melatonin, methysticin, 3-indolepropionic acid, and daunomycin were assayed and ranked in order of inhibition of Abeta aggregation. The most effective inhibitor of aggregation of Abeta protein was daunomycin followed in descending order by 3-indolepropionic acid, melatonin, and then methysticin. These data suggest that this ultrafiltration LC-MS screening assay may be used to identify potential therapeutic agents for the treatment of Alzheimer's disease based on the prevention of Abeta aggregation.

Amyloid beta-Peptides↗