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PubMed2006

High-pressure studies on protein aggregates and amyloid fibrils.

High hydrostatic pressure (HHP) modulates protein-protein and protein-solvent interactions through volume changes and thereby affects the equilibrium of protein conformational species between native and denatured forms as well as monomeric, oligomeric, and aggregated forms without the addition of chemicals or use of high temperature. Because of this unique property, HHP has provided deep insights into the thermodynamics and kinetics of protein folding and aggregation, including amyloid fibril formation. In particular, HHP is a useful tool to stabilize and populate specific folding intermediates, the characterization of which provides thorough understanding of protein folding and aggregation pathways. Furthermore, recent application of HHP for dissociation of protein aggregates, such as inclusion bodies (IBs), into native proteins in a single step facilitates protein preparation for structural and functional studies. This chapter overviews recent HHP studies on the population and characterization of folding intermediates associated with protein aggregation and protein refolding from protein aggregates of amyloid fibrils and IBs. Finally, we describe overall experimental procedures of HHP-mediated protein refolding and provide a detailed discussion of each operating parameter to optimize the refolding.

Amyloid↗

PubMed1996

Facilitated protein aggregation. Effects of calcium on the chaperone and anti-chaperone activity of protein disulfide-isomerase.

Protein disulfide-isomerase (PDI) catalyzes the formation and isomerization of disulfides during oxidative protein folding in the eukaryotic endoplasmic reticulum. At high concentrations, it also serves as a chaperone and inhibits aggregation. However, at lower concentrations, PDI can display the unusual ability to facilitate aggregation, termed anti-chaperone activity (Puig, A., and Gilbert, H. F. (1994) J. Biol. Chem. 269, 7764-7771). Under reducing conditions (10 mM dithiothreitol) and at a low concentration (0.1-0. 3 microM) relative to the unfolded protein substrate, PDI facilitates aggregation of alcohol dehydrogenase (11 microM) that has been denatured thermally or chemically. But at higher concentrations (>0.8 microM), PDI inhibits aggregation under the same conditions. With denatured citrate synthase, PDI does not facilitate aggregation, but higher concentrations do inhibit aggregation. Anti-chaperone behavior is associated with the appearance of both PDI and substrate proteins in insoluble complexes, while chaperone behavior results in the formation of large (>500 kDa) but soluble complexes that contain both proteins. Physiological concentrations of calcium and magnesium specifically increase the apparent rate of PDI-dependent aggregation and shift the chaperone activity to higher PDI concentrations. However, calcium has no effect on the Km or Vmax for PDI-catalyzed oxidative folding, suggesting that the interactions that lead to chaperone/anti-chaperone behavior are distinct from those required for catalytic activity. To account for this unusual behavior of a folding catalyst, a model with analogy to classic immunoprecipitation is proposed; multivalent interactions between PDI and a partially aggregated protein stimulate further aggregate formation by noncovalently cross-linking smaller aggregates. However, at high ratios of PDI to substrate, cross-linking may be inhibited by saturation of the sites with PDI. The effects of PDI concentration on substrate aggregation and the modulation of the behavior by physiological levels of calcium may have implications for the involvement of PDI in protein folding, aggregation, and retention in the endoplasmic reticulum.

Alcohol Dehydrogenase↗

PubMed2004

Investigating the effects of mutations on protein aggregation in the cell.

The conversion of peptides and proteins into highly ordered and intractable aggregates is associated with a range of debilitating human diseases and represents a widespread problem in biotechnology. Protein engineering studies carried out in vitro have shown that mutations promote aggregation when they either destabilize the native state of a globular protein or accelerate the conversion of unfolded or partially folded conformations into oligomeric structures. We have extended such studies to investigate protein aggregation in vivo where a number of additional factors able to modify dramatically the aggregation behavior of proteins are present. We have expressed, in Escherichia coli cells, an E. coli protein domain, HypF-N. The results for a range of mutational variants indicate that although mutants with a conformational stability similar to that of the wild-type protein are soluble in the E. coli cytosol, variants with single point mutations predicted to destabilize the protein invariably aggregate after expression. We show, however, that aggregation of destabilized variants can be prevented by incorporating multiple mutations designed to reduce the intrinsic propensity of the polypeptide chain to aggregate; in the cases discussed here, this is achieved by an increase in the net charge of the protein. These results suggest that the principles being established to rationalize aggregation behavior in vitro have general validity for situations in vivo where aggregation has both biotechnological and medical relevance.

Animals↗

PubMed1988

Isolation and characterization of the hemichrome-stabilized membrane protein aggregates from sickle erythrocytes. Major site of autologous antibody binding.

Because the interaction of denatured hemoglobins (i.e. hemichromes) with the red cell membrane has been associated with several abnormalities commonly observed in hemichrome-containing erythrocytes, we have undertaken to isolate and characterize the hemichrome-rich membrane protein aggregates from sickle cells. The aggregates were isolated by two procedures: one at low ionic strength by centrifugation of detergent-solubilized spectrin-depleted inside-out vesicles, and the other at physiological ionic strength by detergent solubilization of whole cells followed by cytoskeletal disruption and centrifugation. The extensively washed aggregates obtained by both methods yielded similar results. These insoluble complexes were found to be highly cross-linked by predominantly intermolecular disulfide bonds; however, other nonreducible covalent linkages were also observed. Both in the presence and absence of reducing agents, the aggregate disintegrated when the hemichromes were removed by high ionic strength, suggesting that the aggregate depended heavily on the cohesive properties of the hemichromes for stability. Protein assays demonstrated that the aggregates comprised approximately 1.3% of the total membrane protein, roughly two-thirds of which appeared to be globin chains. Other major components identified in the aggregate were band 3, ankyrin, bands 4.1, 4.9, and 5, glycophorins A and B, and autologous IgG. Quantitative analysis of the IgG content demonstrated that three-fourths of the surface-bound IgG on washed sickle cells was clustered at these aggregate sites, representing an enrichment of approximately 250-fold over nonaggregated regions of the membrane. Since clustered cell surface IgG is thought to trigger removal of erythrocytes from circulation, the hemichrome-induced membrane reorganization at these aggregate sites may be an important cause of the greatly shortened life span of sickle cells.

Anemia, Sickle Cell↗

PubMed1997

Analysis of protein aggregates by combination of cross-linking reactions and chromatographic separations.

Chemical cross-linking provides a method that covalently bridges near-neighbour associations within proteins and protein aggregates. Combined with chromatographic separations and protein-chemical methods, it may be used to localize and to investigate three-dimensional relations as present under natural conditions. This paper reviews the chemistry and application of cross-linking reagents and the development of combination experimental approaches in view of chromatographic separations and cross-linking reactions. Investigations of homooligomeric and heterooligomeric protein associations as well as conformational analysis are presented.

Chromatography, Liquid↗

PubMed1975

Glomerular localization of aggregated protein in mice; effect of strain difference and relationship to systemic macrophage function.

Glomerular uptake of aggregated bovine serum albumin has been studied in 3 different mouse strains. Systemic macrophage activity in the same 3 strains has been estimated by measuring the uptake of carbon particles. The amount of aggregated protein taken up by the glomerular mesangium in each strain appears to be inversely related to the systemic macrophage activity. The possible significance of these findings is discussed.

Animals↗

PubMed2001

IbpA/B small heat-shock protein of marine bacterium Vibrio harveyi binds to proteins aggregated in a cell during heat shock.

The IbpA and IbpB are 16-kDa Escherichia coli proteins belonging to a family of small heat-shock proteins (sHsps). According to the present model, based on the in vitro experiments, sHsps are molecular chaperones that bind and prevent aggregation of nonnative proteins during heat shock. Previously, we have shown that IbpA and IbpB bind to endogenous E. coli proteins aggregated intracellularly by heat shock, which can be separated from soluble proteins and membranes in sucrose density gradients (fraction S). In this work we have found that marine bacterium Vibrio harveyi contains a single sHsp which is strongly induced by heat shock and reacts with the anti-IbpA/B serum. The 26 amino-terminal amino acids of this sHsp bear high homology to E. coli IbpA and IbpB proteins (73% and 54% identity, respectively). Fraction S was prepared from heat-shocked cells of V. harveyi, it contained high amounts of the IbpA/B protein. This result indicates that the IbpA/B protein of V. harveyi binds to the proteins that aggregate in V. harveyi cells during heat shock.

Journal Article↗

PubMed2005

Disassembling protein aggregates in the yeast cytosol. The cooperation of Hsp26 with Ssa1 and Hsp104.

In all organisms studied, elevated temperatures induce the expression of a variety of stress proteins, among them small Hsps (sHsp). sHsps are chaperones that prevent the unspecific aggregation of proteins by forming stable complexes with unfolded polypeptides. Reactivation of captured proteins requires the assistance of other ATP-dependent chaperones. How sHsps and ATP-dependent chaperones work together is poorly understood. Here, we analyzed the interplay of chaperones present in the cytosol of Saccharomyces cerevisiae. Specifically, we characterized the influence of Hsp104 and Ssa1 on the disassembly of Hsp26 x substrate complexes in vitro and in vivo. We show that recovery of proteins from aggregates in the cell requires the chaperones to work together with defined but overlapping functions. During reactivation, proteins are transferred from a stable complex with Hsp26 to Hsp104 and Hsp70. The need for ATP-dependent chaperones depends on the type of sHsp x substrate complex. Although Ssa1 is able to release substrate proteins from soluble Hsp26 x substrate complexes, Hsp104 is essential to dissociate substrate proteins from aggregates with incorporated sHsps. Our results are consistent with a model of several interrelated defense lines against protein aggregation.

Adenosine Triphosphatases↗

PubMedDate not supplied

Transglutaminase-dependent formation of protein aggregates as possible biochemical mechanism for polyglutamine diseases.

Transglutaminases (Enzyme Commission 2.3.2.13) are a large family of enzymes that show the common capacity to catalyze cross-linking of protein substrates. Some members of this family of enzymes are also capable of catalyzing other reactions important for the cell life. The distribution and the role of these enzymes have been widely studied in numerous cell types and tissues, but only recently their expression and functions started to be investigated in the central nervous system. One of the main biochemical properties of the transglutaminase enzymes is to form large protein aggregates that are insoluble in all known protein detergents, such as urea, guanidinium, and sodium dodecyl sulfate. Recently, the transglutaminase activity has been hypothesized to be involved in the pathogenetic mechanisms responsible for the formation of cellular inclusions present in Huntington disease and in all the other polyglutamine (polyQ) diseases hitherto identified, such as spinobulbar muscular atrophy or Kennedy disease, spinocerebellar ataxias (SCA-1, SCA-2, SCA-3 or Machado-Joseph disease, SCA-6 and SCA-7) and dentatorubropallidoluysian atrophy. In this review we describe the biochemical properties of the transglutaminase enzymes and some recent findings about the physiopathological roles played by these enzymes in the central nervous system.

Brain↗

PubMed1988

Effect of epinephrine on rapid ADP-induced aggregation, protein phosphorylation, and cytoplasmic calcium dynamics of platelets: a quenched-flow study.

We have used a general quenched-flow approach to study platelet function as early as 0.3 seconds after stimulation with a low concentration of adenosine 5'-diphosphate (ADP) (0.5 mumol/L), epinephrine (15 mumol/L), or a combination of the two. Compared with ADP alone, the combination nearly doubled the rate of aggregation as measured by the loss of single particles. Our aim was to determine whether the aggregation, protein phosphorylation, and cytoplasmic calcium responses to this potentiating combination of ADP and epinephrine were analogous to those caused by a maximal concentration of ADP (10 mumol/L) and also to determine whether cyclooxygenase was involved in this potentiation. Phosphorylation of myosin light chain (MLC) and the 47 kd protein were analyzed during the first 5 seconds of platelet response and were related to the progress of aggregation. Unlike aggregation, ADP-induced phosphorylations of both MLC and the 47 kd protein were inhibited rather than potentiated by epinephrine. Pretreatment of the platelets with indomethacin did not affect aggregation or MLC phosphorylation, but increased 47 kd phosphorylation at 0.6 seconds and eliminated it at 3 seconds. Cytoplasmic free calcium ([Ca2+]i) of indo-1-loaded platelets were monitored with our continuous-flow fluorescence approach. Although epinephrine itself did not change [Ca2+]i, it potentiated the [Ca2+]i rise induced by a low dose of ADP. A comparison of these results with platelet activation caused by 10 mumol/L ADP indicates that MLC and 47 kd phosphorylations are not correlated with [Ca2+]i dynamics and are not required or directly involved in platelet aggregation.

Adenosine Diphosphate↗

PubMed2006

pH-Dependent behaviour of soluble protein aggregates formed during heat-treatment of milk at pH 6.5 or 7.2.

The pH-dependent behaviour of soluble protein aggregates produced by the pre-heating of reconstituted skim milk at 90 degrees C for 10 min was studied, in order to understand the role of these aggregates in acid gelation of heated milk. The following milk samples were prepared: (1) control (unheated reconstituted milk, pH 6.5); (2) milk heat-treated at pH 6.5 (mHtd6.5) and (3) milk heat-treated at pH 7.2 (mHtd7.2). They were centrifuged and the supernatants (SPNT 1) pH-adjusted to yield a series of pH values ranging from 6.5 or 7.2 to 4.6 using HCl at 20 degrees C or GDL at 20 and 38 degrees C. pH-Adjusted SPNTs 1 were re-centrifuged. The resulting supernatants (SPNTs 2) were analysed by OD (at 600 and 280 nm) and SDS-PAGE in order to characterise proteins still soluble as a function of pH. Particle size in SPNTs 1 was analysed by Steric Exclusion Chromatography. The OD600 nm revealed that during acidification soluble casein in both control and heat-treated samples exhibits variations in its optical properties or size as previously shown with micellar casein. In heat-treated samples, soluble casein and heat-induced covalent soluble aggregates precipitate at the same pH value. A progressive acidification of the soluble phase did not separate them. Increasing the temperature of acidification from 20 to 38 degrees C resulted in an increase in the precipitation pH of the proteins. However choice of acidifier did not have a significant effect on OD profiles. The soluble covalent aggregates from mHtd7.2 were smaller, more numerous, and had a higher content of kappa-casein than mHtd6.5. Both types of aggregates began to precipitate at the same pH value but precipitation occurred over a narrower pH-range for soluble aggregates prepared from mHtd7.2. This may explain the higher gelation pH of mHtd7.2 compared with mHtd6.5.

Animals↗

PubMed1984

Vitamin A metabolism in chick liver: some properties of the cytosolic lipid-protein aggregate.

Incubation of hepatic microsomes with retinol resulted in formation of retinyl esters and glucuronides. The presence of the cytosolic lipid-protein aggregate (LPA) in the system in addition induced release of holo-retinol-binding protein from the microsomes. The extent of these reactions was influenced by the addition of coenzyme A and ATP, or uridine diphosphate glucuronic acid. Incubation of hepatic microsomes containing labelled retinyl esters with the LPA resulted in the appearance of the labelled retinyl esters in the LPA. Small amounts of retinoic acid were formed on incubation of retinol with microsomes (approximately 1% of added retinol); this was found to be associated with a protein of approximately 14 500 molecular weight, and less than 10% was associated with the LPA. This is in contrast to retinol, which was found to be almost completely associated with the LPA. The cytosolic LPA was associated both with carotene-cleavage activity and alcohol dehydrogenase (NAD(P)+) (EC 1.1.1.71) activity. These findings lend some support to the concept of a specific role for hepatic LPA.

Animals↗

PubMed1993

The development of stable protein formulations: a close look at protein aggregation, deamidation, and oxidation.

The biochemical literature has been surveyed to present an overview of the three most common protein degradation pathways: protein aggregation, deamidation, and oxidation. The mechanisms for each of these degradation routes are discussed with particular attention given to the effect of formulation conditions such as pH, ionic strength, temperature, and buffer composition. Strategies to reduce protein degradation are also discussed. These strategies are based on an understanding of the degradation mechanisms and the effect of changes in the storage conditions and formulation components on the degradation. The effects of each of the degradation routes on pharmaceutically relevant properties such as biological activity, metabolic half-life, and immunogenicity are summarized. Predicting a priori the alteration of pharmaceutical properties caused by the three degradation routes is difficult, and must be determined on a case-by-case basis for each protein. The difficulty in predicting the effect of degradation and analyzing the temperature dependence of reaction rates in proteins results in longer development times for protein formulations than for small molecule formulations. Although the use of accelerated stability to predict protein shelf life is difficult, conditions are discussed whereby the Arrhenius equation can be used to shorten formulation development time.

Amides↗

PubMed2004

Reversal of protein aggregation provides evidence for multiple aggregated States.

Observations that prefibrillar aggregates from different amyloidogenic proteins can be solubilised under some conditions have raised questions as to the generality of this phenomenon and the nature of the factors that influence it. By studying aggregates formed from human muscle acylphosphatase (AcP) under mild denaturing conditions, and by using a battery of techniques, we demonstrate that disaggregation is possible under conditions close to physiological where the protein is stable in its native state. In the presence of 25% (v/v) trifluoroethanol (TFE) AcP undergoes partial unfolding and globular aggregates (60-200 nm in diameter) that can assemble further into clusters (400-800 nm in diameter) develop progressively. Yet larger superstructures (>5 microm) are formed when the concentration of the globular aggregates exceeds a critical concentration. After diluting the sample to give a solution containing 5% TFE, the fraction of partially unfolded monomeric protein refolds very rapidly, with a rate constant of approximately 1s(-1). The 60-200 nm globular aggregates disaggregate with an apparent rate constant of approximately 2.5 x 10(-3)s(-1) while the 400-800 nm clusters disassembly more slowly with a rate constant of approximately 3.1 x 10(-4)s(-1). The larger (>5 microm) superstructures are not disrupted under the conditions used here. These results suggest that amyloid formation occurs in discrete steps whose reversibility is increasingly difficult, and dependent on the size of the aggregates, and that disaggregation experiments can provide a powerful method of detecting different types of species within the complex process of aggregation. In addition, our work suggests that destabilization of amyloid aggregates resulting in the conversion of misfolded proteins back to their native states could be an important factor in both the onset and treatment of diseases associated with protein aggregation.

Acid Anhydride Hydrolases↗

PubMed2002

Protein aggregation in Huntington's disease.

The presence of an expanded polyglutamine produces a toxic gain of function in huntingtin. Protein aggregation resulting from this gain of function is likely to be the cause of neuronal death. Two main mechanisms of aggregation have been proposed: hydrogen bonding by polar-zipper formation and covalent bonding by transglutaminase-catalyzed cross-linking. In cell culture models of Huntington's disease, aggregates are mostly stabilized by hydrogen bonds, but covalent bonds are also likely to occur. Nothing is known about the nature of the bonds that stabilize the aggregates in the brain of patients with Huntington's disease. It seems that the nature of the bond stabilizing the aggregates is one of the most important questions, as the answer would condition the therapeutic approach to Huntington's disease.

Animals↗

PubMed1991

Protein aggregation and inclusion body formation in Escherichia coli rpoH mutant defective in heat shock protein induction.

Mutations in the rpoH gene, encoding sigma 32, an alternative factor required for transcription of the heat shock genes, result in the extensive aggregation of virtually all cellular proteins and formation of inclusion bodies both under stress and non-stress conditions. Inhibitors of protein synthesis suppress this aggregation, suggesting that newly synthesized proteins preferentially aggregate in rpoH mutants. These data suggest that the heat shock proteins are involved in acquisition of the soluble state (i.e. correct conformation) of the bulk of intracellular proteins after their translation.

Escherichia coli↗

PubMed2004

Solubilization of aggregated proteins by ClpB/DnaK relies on the continuous extraction of unfolded polypeptides.

The AAA+ chaperone ClpB solubilizes in cooperation with the DnaK chaperone system aggregated proteins. The mechanistic features of the protein disaggregation process are poorly understood. Here, we investigated the mechanism of ClpB/DnaK-dependent solubilization of heat-aggregated malate dehydrogenase (MDH) by following characteristics of MDH aggregates during the disaggregation reaction. We demonstrate that disaggregation is achieved by the continuous extraction of unfolded MDH molecules and not by fragmentation of large MDH aggregates. These findings support a ClpB-dependent threading mechanism as an integral part of the disaggregation reaction.

Adenosine Triphosphatases↗

PubMed2006

Mice with targeted disruption of neurofilament light subunit display formation of protein aggregation in motoneurons and downregulation of complement receptor type 3 alpha subunit in microglia in the spinal cord at their earlier age: a possible feature in pre-clinical development of neurodegenerative diseases.

The pathogenesis of neurodegenerative diseases prior to the onset of symptoms is generally not clear. The present study has employed a mouse model with a lack of the low-molecular-weight neurofilament subunit (NFL-/-), in which formation of protein aggregates occurs in neurons, to investigate glial cellular reactions in the lumbar cord segments of NFL-/- mice at ages from 1 to 6 months. Age-matched C57BL/6 mice serve as the control. Apparent neurofilament positive aggregates in the cytoplasm of motoneurons have been observed in NFL-/- mice. However, there were no noticeable changes in microglial numbers and GFAP staining of astrocytes. Unexpectedly, a downregulation in expression of complement receptor type 3 alpha subunit (CD11b) was detected in the spinal cord of NFL-/- mice, while there was no obvious difference between NFL-/- and C57BL/6 mice in the CD11b staining intensity of macrophages from livers and spleens. In addition, retardation in morphological transformation from activated to amoeboid microglia in response to sciatic nerve injury, differential expressions of some cytokines in the lumbar cord segments and induction of Iba-1 (ionized calcium-binding adaptor molecule-1) expression in microglia were observed in NFL-/- mice. Our results suggest not only the existence of an inhibitory niche for CD11b expression in microglia in the lumbar cord segments of NFL-/- mice but also differential microglial reactions between earlier and later stages of neuropathogenesis. Although the real cause for such inhibition is still unknown, this effect might play a particular role in the survival of the abnormal protein aggregate-bearing motoneurons in the early development stage of neurodegeneration in the NFL-/- mice.

Age Factors↗