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At least 217 records · Page 12Linked to original sources
PubMed1990

Isolation and purification of a non-A, non-B hepatitis-associated microtubular aggregates protein.

Blood-borne type non-A, non-B (NANB) hepatitis-associated microtubular aggregates protein was isolated and partially sequenced. The microtubular aggregates were isolated from the hepatocytes of NANB-infected chimpanzees and were found to have a buoyant density in sucrose solution of 1.21 to 1.23 g/ml. A single protein, recognized by our anti-microtubular aggregates monoclonal antibodies, was found to have an Mr of 44,000 (p44). This p44 protein was not found in uninfected chimpanzees. We determined a partial amino acid sequence for p44, and showed that it has no homology to any known proteins.

Amino Acid Sequence↗
PubMed2001

Chaperonin-mediated de novo generation of prion protein aggregates.

The infectious prion protein, PrP(Sc), a predominantly beta-sheet aggregate, is derived from PrP(C), the largely alpha-helical cellular isoform of PrP. Conformational conversion of PrP(C) into PrP(Sc) has been suggested to involve a chaperone-like factor. Here we report that the bacterial chaperonin GroEL, a close homolog of eukaryotic Hsp60, can catalyze the aggregation of chemically denatured and of folded, recombinant PrP in a model reaction in vitro. Aggregates form upon ATP-dependent release of PrP from chaperonin and have certain properties of PrP(Sc), including a high beta-sheet content, the ability to bind the dye Congo red, detergent-insolubility and increased protease-resistance. A conserved sequence segment of PrP (residues 90-121), critical for PrP(Sc) generation in vivo, is also required for chaperonin-mediated aggregate formation in vitro. Initial binding of refolded, alpha-helical PrP to chaperonin is mediated by the unstructured N-terminal segment of PrP (residues 23-121) and is followed by a rearrangement of the globular PrP core-domain. These results show that chaperonins of the Hsp60 class can, in principle, mediate PrP aggregation de novo, i.e. independently of a pre-existent PrP(Sc) template.

Adenosine Triphosphate↗
PubMed1992

High molecular weight protein aggregates formed in the liver of the rat following large doses of paracetamol.

Paracetamol (200 and 500 mg kg-1) was given intraperitoneally to rats pretreated with 3-methylcholanthrene for 3 days. Glutamic oxalacetic acid transaminase (GOT) activity in plasma increased in rats receiving 500 mg kg-1 paracetamol. Plasma GOT activity was low at the dose of 200 mg kg-1, but the same dose to diethyl maleate pretreated rats increased the GOT activity. High mol. wt protein aggregates were found to be formed in liver homogenates and microsomes of rats which showed high plasma GOT activity, accompanied by depletion of hepatic glutathione. The formation of protein aggregates in the liver of rats following large doses of paracetamol suggests a contribution of lipid peroxidation to paracetamol-induced hepatotoxicity.

Acetaminophen↗
PubMed2004

Active solubilization and refolding of stable protein aggregates by cooperative unfolding action of individual hsp70 chaperones.

Hsp70 is a central molecular chaperone that passively prevents protein aggregation and uses the energy of ATP hydrolysis to solubilize, translocate, and mediate the proper refolding of proteins in the cell. Yet, the molecular mechanism by which the active Hsp70 chaperone functions are achieved remains unclear. Here, we show that the bacterial Hsp70 (DnaK) can actively unfold misfolded structures in aggregated polypeptides, leading to gradual disaggregation. We found that the specific unfolding and disaggregation activities of individual DnaK molecules were optimal for large aggregates but dramatically decreased for small aggregates. The active unfolding of the smallest aggregates, leading to proper global refolding, required the cooperative action of several DnaK molecules per misfolded polypeptide. This finding suggests that the unique ATP-fueled locking/unlocking mechanism of the Hsp70 chaperones can recruit random chaperone motions to locally unfold misfolded structures and gradually disentangle stable aggregates into refoldable proteins.

Chromatography, Gel↗
PubMed1997

Protein aggregates seem to play a key role among the parameters influencing the antigenicity of interferon alpha (IFN-alpha) in normal and transgenic mice.

PURPOSE: During long-term treatment of various malignant or viral diseases with IFN-alpha up to 20% of patients develop anti-IFN-alpha antibodies for as yet unknown reasons. METHODS: To address this issue, a mouse model using Balb/C mice was established and the relevance of several potentially anti-IFN-alpha antibodies inducing factors was studied. RESULTS: The model revealed that both a higher frequency of injections and a higher dosage of IFN-alpha were more immunogenic and that the route of administration affected the antibody response to IFN-alpha. The intrinsic immunostimulatory activity of IFN-alpha itself also enhanced the immune response. IFN-alpha protein aggregates (IFN-alpha-IFN-alpha and human serum albumin (HSA)-IFN-alpha aggregates), which were recently identified in all marketed IFN-alpha products, were significantly more immunogenic than IFN-alpha monomers. These aggregates broke the tolerance against human IFN-alpha monomers in human IFN-alpha transgenic mice. CONCLUSIONS: Based on these animal studies it is proposed that the immune response to IFN-alpha in humans is most probably elicited by a combination of several factors among which IFN-alpha protein aggregates seem to play a key role.

Adjuvants, Immunologic↗
PubMed1982

Mechanism of tobacco mosaic virus assembly: role of subunit and larger aggregate protein.

Tobacco mosaic virus (TMV) was reconstituted from the RNA of a common strain (OM) and the protein of a watermelon strain of cucumber green mottle mosaic virus (CGMMV-W), which is a member of the tobamovirus group. In 0.25 M phosphate buffer at 25 degrees C, CGMMV-W protein existed mainly as 21S aggregates. When this protein was mixed with OM RNA, complexes of short rods were formed but further elongation did not occur. After the addition of subunits in 0.1 M phosphate buffer at 25 degrees C, elongation to the 5' end of the RNA proceeded as fast as in the case of reconstitution with the usual equilibrium "disk preparation" of OM protein, to give 260-nm intermediates in the first 5-7 min. The results proved that the rapid elongation we previously observed in the reconstitution of TMV-OM following the assembly initiation is the outcome of preferential incorporation of TMV subunit protein. Either preformed 21S aggregate or the subunit of CGMMV protein was added to the 260-nm intermediate. Elongation to the 3' end of the RNA was investigated in 0.1 M phosphate buffer at 25 degrees C by measuring the distribution of rod length and the RNase-resistant infectivity. The results showed that the 21S aggregate is kinetically favored as the protein source during the slow elongation process.

Kinetics↗
PubMed2004

Oligomers on the brain: the emerging role of soluble protein aggregates in neurodegeneration.

Extracellular fibrous amyloid deposits or intracellular inclusion bodies containing abnormal protein fibrils characterize many different neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), dementia with Lewy bodies, multiple system atrophy, Huntington's disease, and the transmissible 'prion' dementias. There is strong evidence from genetic, transgenic mouse and biochemical studies to support the idea that the accumulation of protein aggregates in the brain plays a seminal role in the pathogenesis of these diseases. How monomeric proteins ultimately convert to highly polymeric deposits is unknown. However, studies employing, synthetic, cell-derived and purified recombinant proteins suggest that amyloid proteins first come together to form soluble low n-oligomers. Further association of these oligomers results in higher molecular weight assemblies including so-called 'protofibrils' and 'ADDLs' and these eventually exceed solubility limits until, finally, they are deposited as amyloid fibrils. With particular reference to AD and PD, we review recent evidence that soluble oligomers are the principal pathogenic species that drive neuronal dysfunction.

Alzheimer Disease↗
PubMed1990

Cataract-related changes in protein aggregates of human lens studied by ultracentrifugation.

In human lens, cataract development causes an increase in the amount of protein aggregates. Their specific density of 1.25 g/ml is much higher than that of 1.14 g/ml and 1.06 g/ml obtained for water-soluble proteins. During the formation of water-insoluble aggregates their size varies up to some micrometers. Infrared spectra of water-soluble protein and in the aggregates confirm that the content of bound water in aggregates decreases.

Aged↗
PubMed2005

Influence of osmolytes on inactivation and aggregation of muscle glycogen phosphorylase b by guanidine hydrochloride. Stimulation of protein aggregation under crowding conditions.

The effects of the osmolytes trimethylamine-N-oxide (TMAO), betaine, proline, and glycine on the kinetics of inactivation and aggregation of rabbit skeletal muscle glycogen phosphorylase b by guanidine hydrochloride (GuHCl) have been studied. It is shown that the osmolytes TMAO and betaine exhibit the highest protective efficacy against phosphorylase b inactivation. A test system for studying the effects of macromolecular crowding induced by osmolytes on aggregation of proteins is proposed. TMAO and glycine increase the rate of phosphorylase b aggregation induced by GuHCl.

Animals↗
PubMed1994

Induction of the human gene for p44, a hepatitis-C-associated microtubular aggregate protein, by interferon-alpha/beta.

A hepatitis-C-associated microtubular aggregate protein, referred to as p44, has been identified as a cytoplasmic antigen in the hepatocytes of chimpanzees infected with hepatitis C virus. The production of p44 mRNA is markedly induced in the liver of chimpanzees infected with hepatitis C or hepatitis D virus. To examine the mechanism of this induction, we isolated a genomic clone for the human p44 protein and analyzed its structure. The human p44 gene spans approximately 14 kbp of DNA and consists of nine exons separated by eight introns. An interferon-stimulated response element, which confers inducibility by interferon-alpha/beta, was found in the promoter region of the gene. Northern-blot analysis revealed that the human p44 gene is inducible by interferon-alpha/beta, but not by interferon-gamma. Functional analysis demonstrated that the interferon-stimulated response element in the promoter region of the gene mediates the inducibility of the gene by interferon-alpha/beta. Thus, the human p44 gene is a member of the family of interferon-alpha/beta inducible genes. The protein p44 may be one of the mediators involved in the antiviral action of interferon.

Amino Acid Sequence↗
PubMed2005

Protein aggregation in retinal cells and approaches to cell protection.

1. Retinal dystrophies (RD) comprise a group of clinically and genetically heterogeneous retinal disorders, which typically result in the degeneration of photoreceptors followed by the impairment or loss of vision. Although age-related macular degeneration (AMD) and retinitis pigmentosa (RP) are among the most common forms of RD, currently, there is no effective treatment for either disorder. 2. Recently, abnormal protein accumulation and aggregation due to protein misfolding and proteasome inhibition have been implicated in the pathogenesis of RD. In this paper we describe effects of several factors on protein aggregation and survival of photoreceptor cells. 3. Expression of rhodopsin carrying P23H mutation causes its accumulation in intracellular inclusion bodies in a perinuclear area of photoreceptor cells. beta- and gamma-synucleins and heat shock protein Hsp-70, but not alpha-synuclein, protect cultured ocular cells from mutant opsin accumulation. This effect might be explained by their chaperonic activity. 4. Knock-out of alpha- and gamma-synucleins does not affect gross retinal morphology, but induces tyrosine hydroxylase in the inner prexiform layer of the retina. Selegiline-a monoamine oxidase inhibitor used for the treatment of Parkinson's disease, reduces apoptosis and increases viability in cultured retinal pigment epithelium cells (APRE-19). 5. These results suggest that chaperones and selegiline may be considered promising candidates for the protection of ocular cells from the accumulation of misfolded and aggregated proteins.

Animals↗
PubMed2001

VCP/p97 in abnormal protein aggregates, cytoplasmic vacuoles, and cell death, phenotypes relevant to neurodegeneration.

Neuronal cell death, abnormal protein aggregates, and cytoplasmic vacuolization are major pathologies observed in many neurodegenerative disorders such as the polyglutamine (polyQ) diseases, prion disease, Alzheimer disease, and the Lewy body diseases, suggesting common mechanisms underlying neurodegeneration. Here, we have identified VCP/p97, a member of the AAA+ family of ATPase proteins, as a polyQ-interacting protein in vitro and in vivo, and report on its characterization. Endogenous VCP co-localized with expanded polyQ (ex-polyQ) aggregates in cultured cells expressing ex-polyQ, with nuclear inclusions in Huntington disease patient brains, and with Lewy bodies in patient samples. Moreover, the expression of VCP mutants with mutations in the 2nd ATP binding domain created cytoplasmic vacuoles, followed by cell death. Very similar vacuoles were also induced by ex-polyQ expression or proteasome inhibitor treatment. These results suggest that VCP functions not only as a recognition factor for abnormally folded proteins but also as a pathological effector for several neurodegenerative phenotypes. VCP may thus be an ideal molecular target for the treatment of neurodegenerative disorders.

Adenosine Triphosphatases↗
PubMed2001

Conformational changes of pancreatitis-associated protein (PAP) activated by trypsin lead to insoluble protein aggregates.

Pancreatitis-associated protein (PAP), a secretory acute-phase protein of the pancreatic acinar cell, is highly up-regulated early in acute pancreatitis. PAP expression returns to undetectable levels when the pancreas recovers. In the rat, three isoforms of PAP are known, all of which are upregulated during acute pancreatitis. Their functions remain obscure. Pancreatic stone protein (PSP/reg), which shows strong sequence homology to PAP, is secreted into pancreatic juice under physiologic and pathologic conditions. PSP/reg is highly susceptible to trypsin cleavage at its ARG11-ILE12 bond. Cleavage results in an N-terminal undecapeptide and a C-terminal peptide called pancreatic thread protein (PTP). PTP forms oligomeric fibrillar structures, which spontaneously sediment in vitro. PTP can be found in protein plugs or stones from patients with chronic pancreatitis. Rat PAP contains a trypsin cleavage site at the same position as PSP/reg. We hypothesize that PAP is susceptible to tryptic cleavage, and that the C-terminal cleavage product of PAP spontaneously precipitates at neutral pH. To test our hypothesis, we generated and purified recombinant PAP. Here we report the production of rat PAP I, II, and III in a yeast expression system using Pichia pastoris. We demonstrate in vitro the tryptic cleavage of rat PAP and the formation of a spontaneously precipitating peptide, which we call pancreatitis-associated thread protein (PATP). PATP displays pH-dependent solubility characteristics very similar to those of PTP.

Acute-Phase Proteins↗
PubMed2004

Acid-induced cold gelation of globular proteins: effects of protein aggregate characteristics and disulfide bonding on rheological properties.

The process of cold gelation of ovalbumin and the properties of the resulting cold-set gels were compared to those of whey protein isolate. Under the chosen heating conditions, most protein was organized in aggregates. For both protein preparations, the aggregates consisted of covalently linked monomers. Both types of protein aggregates had comparable numbers of thiol groups exposed at their surfaces but had clearly different shapes. During acid-induced gelation, the characteristic ordering caused by the repulsive character disappeared and was replaced by a random distribution. This process did not depend on aggregate characteristics and probably applies to any type of protein aggregate. Covalent bonds are the main determinants of the gel hardness. The formation of additional disulfide bonds during gelation depended on the number and accessibility of thiol groups and disulfide bonds in the molecule and was found to clearly differ between the proteins studied. However, upon blocking of the thiol groups, long fibrillar structures of ovalbumin contribute significantly to gel hardness, demonstrating the importance of aggregate shape.

Cold Temperature↗
PubMed1989

Quantitation of high molecular weight protein aggregates in opaque and transparent parts from the same human cataractous lens.

Water soluble proteins from clear and opaque parts of the cortex of the same human cataractous lens were analyzed by high pressure liquid chromatography using a TSK 3000 SW gel filtration column. Studies of the 42 different microdissected parts from 7 different lenses demonstrated that the opaque parts possessed a higher percentage of high molecular weight material eluting with the void volume than did the corresponding clear part from the same lens. These results suggest that increased amounts of high molecular weight protein aggregates accompany the process of opacification in the cortex of the human cataractous lens.

Aged↗
PubMed1998

I. Study of protein aggregation due to heat denaturation: A structural approach using circular dichroism spectroscopy, nuclear magnetic resonance, and static light scattering.

The objective of this study was to investigate the relationship between oxidized RNase A protein structure and the occurrence of protein aggregation using several spectroscopic techniques. Circular dichroism spectroscopy (CD) measurements taken at small temperature intervals were used to determine the protein's melting temperature, Tm, of approximately 65 degrees C in deionized water. A more detailed examination of the protein structure was undertaken at several temperatures around Tm using near- and far-UV CD and one-dimensional nuclear magnetic resonance (NMR) measurements. These measurements revealed the presence of folded structures at 55 degrees C and below, while denatured structures appeared at 65 degrees C and above. Concurrent static light scattering (SLS) measurements, employed to detect the presence of RNase A aggregates, showed that RNase A aggregation was observed at 65 degrees C and above, when much of the protein was denatured. Subsequent NMR time-course data demonstrated that aggregates forming at 75 degrees C and pH 7.8 were indeed derived from heat-denatured protein. However, aggregation was also detected at 55 degrees C when the spectroscopic data suggested the protein was present predominantly in the folded configuration. In contrast, heat denaturation did not lead to RNase A aggregation in a very acidic environment. We attribute this phenomenon to the effect of charge-charge repulsion between the highly protonated RNase A molecules in very acidic pH.

Circular Dichroism↗
PubMed1989

[Purification and some properties of platelet aggregating protein (PAP) from the plasma of a patient with thrombotic thrombocytopenic purpura].

A new platelet aggregating protein (PAP) was purified from the plasma of a patient with acute thrombotic thrombocytopenic purpura (TTP) using A1 (OH)3 adsorption, PEG fractionation, DEAE cellulose chromatography, lentil lectin Sepharose 4B chromatography and gel filtration HPLC. The M. W. of this PAP was estimated to be 59,000. It induced the aggregation of washed platelets from normal subjects and the same patient after recovery. The antigenic material was present in 2 out of 6 other TTP patients but was absent in 7 normal subjects, two patients with DIC and two patients with ITP. The aggregation of washed platelets by PAP was concentration dependent and required energy metabolism, Ca2+ and fibrinogen. PAP was present in the plasma of a subset of TTP patients and was likely the cause of platelet thrombi in the microvessels in these patients.

Blood Coagulation Factors↗
PubMed2003

Role of the soluble and micelle-bound heat-induced protein aggregates on network formation in acid skim milk gels.

Gel formation was monitored by low amplitude rheometry during acidification at 40 degrees C with 1.5% glucono-delta-lactone in combined milk systems containing soluble and/or micelle-bound heat-induced (95 degrees C/10 min) aggregates of denatured whey proteins and kappa-casein and in heated dairy mixes with varying micellar casein/whey protein ratio (CN/WP). Both soluble and micelle-bound aggregates increased gelation pH and gel strength. Micelle-bound aggregates seemed to modify the micelle surface so that micelles were destabilized at a pH of 5.1 (instead of 4.7), while soluble aggregates precipitated at their calculated pI of approximately 5.3, and initiated an early gelation by interacting with the micelles. Decreasing the CN/WP ratio produced larger aggregates with higher whey protein: kappa-casein ratio, which gave more elastic gels. The specific effects of the micellar and soluble aggregates on gel strength are discussed with respect to their relative proportions in the heated milk.

Animals↗