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PubMed2002

High hydrostatic pressure as a tool to study protein aggregation and amyloidosis.

Aggregation of proteins is a serious problem, affecting both industrial production of proteins and human health. Despite recent advances in the theories and experimental techniques available to address understanding of protein aggregation processes, mechanisms of aggregate formation have proved challenging to study. This is in part because the typical irreversibility of protein aggregation processes at atmospheric conditions complicates analysis of their kinetics and thermodynamics. Because high hydrostatic pressures act to disfavor the hydrophobic and electrostatic interactions that cause protein aggregation, studies conducted under high hydrostatic pressures may allow protein aggregates to be formed reversibly, enabling thermodynamic and kinetic parameters to be measured in greater detail. Although application of high hydrostatic pressures to protein aggregation problems is rather recent, a growing literature, reviewed herein, suggests that high pressure may be a useful tool for both understanding protein aggregation and reversing it in industrial applications.

Amyloidosis↗

PubMed2002

Kinetics of protein aggregation. Quantitative estimation of the chaperone-like activity in test-systems based on suppression of protein aggregation.

The experimental data on the kinetics of irreversible aggregation of proteins caused by exposure to elevated temperatures or the action of denaturing agents (guanidine hydrochloride, urea) have been analyzed. It was shown that the terminal phase of aggregation followed, as a rule, first order kinetics. For the kinetic curves registered by an increase in the apparent absorbance (A) in time (t) the methods of estimation of the corresponding kinetic parameters A(lim) and kI (A(lim) is the limiting value of A at t --> infinity and kI is the rate constant of the first order) have been proposed. Cases are revealed when the reaction rate constant kI calculated from the kinetic curve of aggregation of the enzymes coincides with the rate constant for enzyme inactivation. Such a situation is interpreted as a case when the rate of aggregation is limited by the stage of denaturation of the enzyme. A conclusion has been made that, in order to establish the mechanism of protein aggregation, the kinetic investigations of aggregation should be carried out over a wide range of protein concentrations. The refolding experiments after denaturation of proteins by guanidine hydrochloride or urea have been also analyzed. It was shown that aggregation accompanying refolding follows first order kinetics at the final phase of the process. The model of protein refolding explaining such a kinetic regularity has been proposed. When aggregation of protein substrate follows first order kinetics, parameters A(lim) and kI may be used for the quantitative characterization of the chaperone-like activity in the test-systems based on suppression of protein aggregation.

Animals↗

PubMed2004

A chaperone network for the resolubilization of protein aggregates: direct interaction of ClpB and DnaK.

The molecular chaperones ClpB (Hsp104) and DnaK (Hsp70) co-operate in the ATP-dependent resolubilization of aggregated proteins. A sequential mechanism has been proposed for this reaction; however, the mechanism and the functional interplay between both chaperones remain poorly defined. Here, we show for the first time that complex formation of ClpB and DnaK can be detected by using various types of affinity chromatography methods. The finding that the DnaK chaperone of Escherichia coli is not co-operating with ClpB from Thermus thermophilus further strengthens the specificity of this complex. The affinity of the complex is weak and interaction between both chaperones is nucleotide-dependent. The presence of ADP, which is shown to cause dissociation of ClpB(Tth), as well as ClpB deletion mutants incapable of oligomer formation prevent ClpB-DnaK complex formation. The experiments presented indicate a correlation between the oligomeric state of ClpB and its ability to interact with DnaK. The chaperone complex described here might facilitate transfer of intermediates between ClpB and DnaK during refolding of substrates from aggregates.

Adenosine Diphosphate↗

PubMed2001

Production of reactive oxygen species from aggregating proteins implicated in Alzheimer's disease, Parkinson's disease and other neurodegenerative diseases.

The deposition of abnormal protein fibrils is a prominent pathological feature of many different 'protein conformational' diseases, including some important neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), motor neurone disease and the 'prion' dementias. Some of the fibril-forming proteins or peptides associated with these diseases have been shown to be toxic to cells in culture. A clear understanding of the molecular mechanisms responsible for this toxicity should shed light on the probable link between protein deposition and cell loss in these diseases. In the case of the beta-amyloid (Abeta), which accumulates in the brain in AD, there is good evidence that the toxic mechanism involves the production of reactive oxygen species (ROS). By means of an electron spin resonance (ESR) spin-trapping method, we have shown recently that solutions of Abeta liberate readily detectable amounts of hydroxyl radicals upon incubation in vitro followed by the addition of small amounts of Fe(II). We have also obtained similar results with alpha-synuclein, which accumulates in Lewy bodies in PD. Our data suggest that hydrogen peroxide accumulates during Abeta or alpha-synuclein incubation and that this is subsequently converted to hydroxyl radicals, on addition of Fe (II), by Fenton's reaction. Consequently, we now support the idea that one of the fundamental molecular mechanisms underlying the pathogenesis of cell death in AD, PD, and possibly some other protein conformational diseases, could be the direct production of ROS during formation of the abnormal protein aggregates. This hypothesis suggests a novel approach to the therapy of this group of diseases.

Alzheimer Disease↗

PubMed2003

Perturbed signal transduction in neurodegenerative disorders involving aberrant protein aggregation.

Aggregation of abnormal proteins, both inside and outside of cells, is a prominent feature of major neurodegenerative disorders, including Alzheimer's, Parkinson's, polyglutamine expansion, and prion diseases. Other articles in this special issue of NeuroMolecular Medicine describe the genetic and molecular factors that promote aberrant protein aggregation. In the present article, we consider how it is that pathogenic aggregation-prone proteins compromise signal transduction pathways that regulate neuronal plasticity and survival. In some cases the protein in question may have widespread and relatively nonspecific effects on signaling. For example, amyloid beta-peptide induces membrane-associated oxidative stress, which impairs the function of various receptors, ion channels and transporters, as well as downstream kinases and transcription factors. Other proteins, such as polyglutamine repeat proteins, may affect specific protein -protein interactions, including those involved in signaling pathways activated by neurotransmitters, neurotrophins, and steroid hormones. Synapses are particularly sensitive to abnormal protein aggregation and impaired synaptic signaling may trigger apoptosis and related cell death cascades. Impairment of signal transduction in protein aggregation disorders may be amenable to therapy as demonstrated by a recent study showing that dietary restriction can preserve synaptic function and protect neurons in a mouse model of Huntington's disease. Finally, emerging findings are revealing how activation of certain signaling pathways can suppress protein aggregation and/or the cytotoxicity resulting from the abnormal protein aggregation. A better understanding of how abnormal protein aggregation occurs and how it affects and is affected by specific signal transduction pathways, is leading to novel approaches for preventing and treating neurodegenerative disorders.

Animals↗

PubMed1996

Degradation by proteases Lon, Clp and HtrA, of Escherichia coli proteins aggregated in vivo by heat shock; HtrA protease action in vivo and in vitro.

Thermally aggregated, endogenous proteins of Escherichia coli form a distinct fraction, denoted S, which is separable by sucrose-density-gradient centrifugation. It was shown earlier that DnaK, DnaJ, IbpA and IbpB heat-shock proteins are associated with the S fraction. Comparison of the rise and decay of the S fraction in mutants defective for heat-shock proteases Lon (La), Clp, HtrA (DegP, Do) and in wild-type strains made studies of proteolysis and the function of the heat-shock response possible in vivo. Different timing and the extent of action of particular proteases was revealed by the initial size and decay kinetics of the S fraction. The proteases Lon, Clp, and HtrA all participated in removal of the aggregated proteins. Mutation in the gene encoding ClpB caused the most prominent effect (47% stabilization of the S fraction). The correlation between the disappearance of the S fraction and proteolytic activity was supported by the result of the in vitro reaction. Approximately one third of the isolated S fraction was converted to trichloroacetic acid-soluble products by the purified HtrA protease. Mg2+ ions stimulated the reaction, in contrast to the reaction of the HtrA protease with casein. The digestion of the aggregated proteins, unlike the digestion of casein, by HtrA protease in vitro was inhibited by added DnaJ, which might reflect protection of the aggregated proteins in vivo by DnaJ from excessive degradation. One might expect that such an activity of DnaJ would promote denatured protein renaturation versus proteolysis. Moreover, among the aggregated proteins that are discernible by electrophoresis, none could be identified as being more susceptible than any other to HtrA degradation. The separation pattern of these proteins before and after the in vitro digestion did not show a difference corresponding to the loss of about 30% of constituting proteins. This was interpreted as recognition by the HtrA protease of a state of protein denaturation rather than specific amino acid sequences in particular proteins. We conclude that the fraction consisting of proteins heat-aggregated in vivo (i.e. the S fraction) contains endogenous substrates for the heat-shock proteases tested. Their use for in vitro reaction reveals information that is in some respects different from that obtained with exogenous substrates such as casein.

ATP-Dependent Proteases↗

PubMed1968

Protein aggregation. The effect of deuterium oxide on large protein aggregates of C-phycocyanin.

The amount of 11s aggregate in phycocyanin, normally stimulated by hydrophobic forces, is dramatically increased by the presence of deuterium oxide. Proteins in which hydrophobic forces are not proposed as a mechanism for aggregation are unaffected by deuterium oxide. These observations are consistent with the lower critical micelle concentration reported for ionic detergents in deuterium oxide. Phycocyanin samples containing a majority of material sedimenting faster than 11s were also investigated in the presence of deuterium oxide with the following findings: the most rapidly sedimenting species in water buffer is 24s; in deuterium oxide more than 10% of the protein sediments at 67s and substantial amounts of other species with sedimentation coefficients larger than 24s are present. These large quantities of species sedimenting faster than 24s are found in deuterium oxide buffers from pD5.5 to 7.0. Sucrose-density-gradient studies in deuterium oxide at pD6.0 confirm the presence of large amounts of more rapidly sedimenting species. Spectrophotometric studies on fractions from the sucrose-density-gradient experiments indicate with the presence of higher aggregates a red shift of the visible-absorption maximum and an enhancement of the E(620)/E(280) ratio. Fluorescence-emission studies show a greater relative fluorescence efficiency for these higher aggregates and are consistent with the suggested enhancement of higher aggregates in deuterium oxide. The existence of phycocyanin aggregates of such a large size is suggested to be of importance in vivo, with phycocyanin playing a role as a structural protein.

Centrifugation, Density Gradient↗

PubMed2002

Polyglutamine protein aggregates are dynamic.

Protein aggregation and the formation of inclusion bodies are hallmarks of the cytopathology of neurodegenerative diseases, including Huntington's disease, Amyotropic lateral sclerosis, Parkinson's disease and Alzheimer's disease. The cellular toxicity associated with protein aggregates has been suggested to result from the sequestration of essential proteins that are involved in key cellular events, such as transcription, maintenance of cell shape and motility, protein folding and protein degradation. Here, we use fluorescence imaging of living cells to show that polyglutamine protein aggregates are dynamic structures in which glutamine-rich proteins are tightly associated, but which exhibit distinct biophysical interactions. In contrast, the interaction between wild-type, but not mutant, Hsp70 exhibits rapid kinetics of association and dissociation similar to interactions between Hsp70 and thermally unfolded substrates. These studies provide new insights into the composite organization and formation of protein aggregates and show that molecular chaperones are not sequestered into aggregates, but are instead transiently associated.

Animals↗

PubMed2005

Sequence determinants of protein aggregation: tools to increase protein solubility.

Escherichia coli is one of the most widely used hosts for the production of recombinant proteins. However, very often the target protein accumulates into insoluble aggregates in a misfolded and biologically inactive form. Bacterial inclusion bodies are major bottlenecks in protein production and are hampering the development of top priority research areas such structural genomics. Inclusion body formation was formerly considered to occur via non-specific association of hydrophobic surfaces in folding intermediates. Increasing evidence, however, indicates that protein aggregation in bacteria resembles to the well-studied process of amyloid fibril formation. Both processes appear to rely on the formation of specific, sequence-dependent, intermolecular interactions driving the formation of structured protein aggregates. This similarity in the mechanisms of aggregation will probably allow applying anti-aggregational strategies already tested in the amyloid context to the less explored area of protein aggregation inside bacteria. Specifically, new sequence-based approaches appear as promising tools to tune protein aggregation in biotechnological processes.

Journal Article↗

PubMed2002

Biophysical effect of amino acids on the prevention of protein aggregation.

Each protein folds into a unique and native structure spontaneously. However, during the unfolding or refolding process, a protein often tends to form aggregates. To establish a method to prevent undesirable protein aggregation and to increase the stability of native protein structures under deterioration conditions, two types of aggregation conditions, thermal unfolding-induced aggregation and dilution-induced aggregation from denatured state, were studied in the presence of additional amino acids and ions using lysozyme as a model protein. Among 15 amino acids tested, arginine exhibited the best results in preventing the formation of aggregates in both cases. Further biophysical studies revealed that arginine did not change the thermal denaturation temperature (T(m)) of the lysozyme. The preventive effect of arginine on aggregation was not dependent on the size or isoelectric point of eight kinds of proteins tested.

Amino Acids↗

PubMed2000

Protein aggregation after transient cerebral ischemia.

Protein aggregates containing ubiquitinated proteins are commonly present in neurodegenerative disorders and have been considered to cause neuronal degeneration. Here, we report that transient cerebral ischemia caused severe protein aggregation in hippocampal CA1 neurons. By using ethanolic phosphotungstic acid electron microscopy (EM) and ubiquitin immunogold EM, we found that protein aggregates were accumulated in CA1 neurons destined to die 72 hr after 15 min of cerebral ischemia. Protein aggregates appeared as clumps of electron-dense materials that stained heavily for ubiquitin and were associated with various intracellular membranous structures. The protein aggregates appeared at 4 hr and progressively accumulated at 24 and 48 hr of reperfusion in CA1 dying neurons. However, they were rarely observed in dentate gyrus neurons that were resistant to ischemia. At 4 hr of reperfusion, protein aggregates were mainly associated with intracellular vesicles in the soma and dendrites, and the nuclear membrane. By 24 hr of reperfusion, the aggregates were also associated with mitochondria, the Golgi apparatus, and the dendritic plasmalemma. High-resolution confocal microscopy further demonstrated that protein aggregates containing ubiquitin were persistently and progressively accumulated in all CA1 dying neurons but not in neuronal populations that survive in this model. We conclude that proteins are severely aggregated in hippocampal neurons vulnerable to transient brain ischemia. We hypothesize that the accumulation of protein aggregates cause ischemic neuronal death.

Animals↗

PubMed2001

The effect of co-overproduction of DnaK/DnaJ/GrpE and ClpB proteins on the removal of heat-aggregated proteins from Escherichia coli DeltaclpB mutant cells--new insight into the role of Hsp70 in a functional cooperation with Hsp100.

The effect of overproduction of the Hsp70 system proteins (DnaK, DnaJ, GrpE) and/or ClpB (Hsp100) from plasmids on the process of formation and removal of heat-aggregated proteins from Escherichia coli cells (the S fraction) was investigated by sucrose density gradient centrifugation. Two plasmids were employed: pKJE7 carrying the dnaK/dnaJ/grpE genes under the control of the araB promoter and pClpB carrying the clpB gene under the control of its own promoter (sigma(32)-dependent). In the wild-type cells the S fraction after 15 min of heat shock amounted to 21% of cellular insoluble proteins (IP), and disappeared 10 min after transfer of the culture to 37 degrees C. In contrast to this, in the clpB mutant the S fraction was larger (35% IP) and its elimination was retarded, nearly 60% of the aggregated proteins remained stable 30 min after heat shock. This result points to the importance of ClpB in removal of the heat-aggregated proteins from cells. Overproduction of the Hsp70 system proteins (exceeding by about 1.5-fold that of wild-type) in wild-type and DeltaclpB cells completely prevented the formation of the S fraction during heat shock. Overproduction of ClpB (exceeding by about eight-fold that of wild-type) in the same background did not prevent protein aggregation after heat shock and only partly compensated for the effect of the mutation in the clpB gene. Monitoring the S fraction during co-production of DnaK/DnaJ/GrpE and ClpB in the DeltaclpB mutant revealed that both the levels of expression and the ratios of ClpB to Hsp70 system proteins had a significant effect on the formation and removal of protein aggregates in heat-shocked E. coli cells. In the presence of excess ClpB, an increase in the levels of DnaK, DnaJ and GrpE was required to prevent aggregate formation upon heat shock or to efficiently remove protein aggregates after heat shock. Therefore, it is supposed that a high level of ClpB under some conditions, especially at insufficient levels of Hsp70 system proteins, may support protein aggregation resulting from heat shock and may lead to stabilization of hydrophobic aggregates.

Bacterial Proteins↗

PubMed2001

Encapsulation of proteins by layer-by-layer adsorption of polyelectrolytes onto protein aggregates: factors regulating the protein release.

A novel method of protein encapsulation is proposed. Preformed protein aggregates are covered with polyelectrolyte layers by means of layer-by-layer adsorption. The polyelectrolyte membrane prevents protein leakage out of the capsule. Using chymotrypsin as a model enzyme the capsule wall selective permeability was demonstrated for substrates and inhibitors of different molecular weight and solubility.

Adsorption↗

PubMed1995

Heat-induced intranuclear protein aggregation and thermal radiosensitization.

In the current study, the hypothesis that thermal radiosensitization is (indirectly) caused by heat-induced denaturation and aggregation of nuclear proteins is further investigated. Thermotolerant rodent cells showed a reduced intranuclear protein aggregation as compared with non-tolerant cells immediately after a heat treatment. This was reflected in the extent of radiosensitization when the cells were X-irradiated immediately after a heat treatment. When heat and radiation were separated in time, a faster disaggregation was found in thermotolerant cells, which was paralleled by a more rapid decline of radiosensitization. Cells transfected with hsp72 showed protection against heat-induced nuclear protein aggregation and reduced thermal radiosensitization. Transfection with hsp27 resulted in an accelerated nuclear protein disaggregation and accelerated decline of thermal radiosensitization. Despite a significant overall correlation between TER and nuclear protein aggregation, the slopes of the correlation curves for the individual cell lines deviated significantly. Yet, the experiments support the hypothesis that radiosensitization is primarily caused by inhibition of DNA repair as a result of the presence of denatured and aggregated proteins in the cell nucleus. Expression of hsps (e.g. in thermotolerant cells), by affecting nuclear protein aggregation, can have an impact on thermal radiosensitization.

Animals↗

PubMed1980

Protein aggregation in aqueous casein solution; effect of irradiation, dose level, concentration, storage, and additives (carbohydrate and lipid).

Radiation-induced aggregation of protein in aqueous solutions was studied. Different concentrations of casein solutions have been irradiated as such or in the presence of carbohydrate and/or lipid. Gel chromatography data indicated a close relation between the amounts of aggregates and protein concentration. Protein aggregation increased with the increase in radiation dose level. The addition of carbohydrate (trehalose) reduced the amount of radiation-induced aggregates, whereas the sole addition of oil caused an induction in protein aggregation when the solution was irradiated at 1 Mrad. However, insignificant changes in protein aggregation were observed when emulsified oil was added to the casein-trehalose solution.

Caseins↗

PubMed2003

Suppression of polyglutamine-induced protein aggregation in Caenorhabditis elegans by torsin proteins.

Torsion dystonia is an autosomal dominant movement disorder characterized by involuntary, repetitive muscle contractions and twisted postures. The most severe early-onset form of dystonia has been linked to mutations in the human DYT1 (TOR1A) gene encoding a protein termed torsinA. While causative genetic alterations have been identified, the function of torsin proteins and the molecular mechanism underlying dystonia remain unknown. Phylogenetic analysis of the torsin protein family indicates these proteins share distant sequence similarity with the large and diverse family of AAA+ proteins. We have established the nematode, Caenorhabditis elegans, as a model system for examining torsin activity. Using an in vivo assay for polyglutamine repeat-induced protein aggregation in living animals, we have determined that ectopic overexpression of both human and C. elegans torsin proteins results in a dramatic reduction of polyglutamine-dependent protein aggregation in a manner similar to that previously reported for molecular chaperones. The suppressive effects of torsin overexpression persisted as animals aged, whereas a mutant nematode torsin protein was incapable of ameliorating aggregate formation. Antibody staining of transgenic animals indicated that both the C. elegans torsin-related protein TOR-2 and ubiquitin were localized to sites of protein aggregation. These data represent the first functional evidence of a role for torsins in effectively managing protein folding and suggest that possible breakdown in a neuroprotective mechanism that is, in part, mediated by torsins may be responsible for the neuronal dysfunction associated with dystonia.

Aging↗

PubMed1997

Classical light scattering quantitation of protein aggregates: off-line spectroscopy versus HPLC detection.

This paper describes the development and validation of a new off-line approach to quantitate both covalent and noncovalent, in-solution aggregates present in protein formulations and compares the new assay to established HPLC methods. This off-line analysis is well suited for use in QC release testing, formulation development and stability indicating applications. An inexpensive, continuous source HPLC fluorometer has been adapted with the addition of second order filters for use as a sensitive right-angle scatterometer which can determine the molecular weight of protein aggregates in solution. When used as an HPLC detector, right-angle light scattering is a sensitive method which can determine the molecular weight of peaks separable by HPLC, thus discriminating between monomers of different conformations and aggregates. The weight-averaged molecular weight of aggregate peaks can be calculated with system calibration, yielding the average number of monomers per aggregate. If the protein concentration is high enough for an adequate signal, the off-line technique of right-angle light scattering of protein formulations has advantages of convenience and speed over the HPLC approach. Samples are placed in standard fluorometer cuvettes and toluene is used as a calibrator. Data are presented which show the off-line (static) method to be extremely rapid, rugged and precise. The accuracy of this approach is demonstrated through cross-validation to traditional GPC analysis of protein aggregate distributions. This non-invasive light scattering approach is particularly useful when non-covalent protein aggregation is reversible and readily altered by chromatographic separations typically used for characterizing aggregates.

Calibration↗

PubMed2005

How evolutionary pressure against protein aggregation shaped chaperone specificity.

As protein aggregation is potentially lethal, control of protein conformation by molecular chaperones is essential for cellular organisms. This is especially important during protein expression and translocation, since proteins are then unfolded and therefore most susceptible to form non-native interactions. Using TANGO, a statistical mechanics algorithm to predict protein aggregation, we here analyse the aggregation propensities of 28 complete proteomes. Our results show that between 10% and 20% of the residues in these proteomes are within aggregating protein segments and that this represents a lower limit for the aggregation tendency of globular proteins. Further, we show that not only evolution strongly pressurizes aggregation downwards by minimizing the amount of strongly aggregating sequences but also by selectively capping strongly aggregating hydrophobic protein sequences with arginine, lysine and proline. These residues are strongly favoured at these positions as they function as gatekeepers that are most efficient at opposing aggregation. Finally, we demonstrate that the substrate specificity of different unrelated chaperone families is geared by these gatekeepers. Chaperones face the difficulty of having to combine substrate affinity for a broad range of hydrophobic sequences and selectivity for those hydrophobic sequences that aggregate most strongly. We show that chaperones achieve these requirements by preferentially binding hydrophobic sequences that are capped by positively charged gatekeeper residues. In other words, targeting evolutionarily selected gatekeepers allows chaperones to prioritize substrate recognition according to aggregation propensity.

Algorithms↗