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At least 73 records · Page 4Linked to original sources
PubMed2005

Ischemic preconditioning prevents protein aggregation after transient cerebral ischemia.

Transient cerebral ischemia leads to protein aggregation mainly in neurons destined to undergo delayed neuronal death after ischemia. This study utilized a rat transient cerebral ischemia model to investigate whether ischemic preconditioning is able to alleviate neuronal protein aggregation, thereby protecting neurons from ischemic neuronal damage. Ischemic preconditioning was introduced by a sublethal 3 min period of ischemia followed by 48 h of recovery. Brains from rats with either ischemic preconditioning or sham-surgery were then subjected to a subsequent 7 min period of ischemia followed by 30 min, 4, 24, 48 and 72 h of reperfusion. Protein aggregation and neuronal death were studied by electron and confocal microscopy, as well as by biochemical analyses. Seven minutes of cerebral ischemia alone induced severe protein aggregation after 4 h of reperfusion mainly in CA1 neurons destined to undergo delayed neuronal death (which took place after 72 h of reperfusion). Ischemic preconditioning reduced significantly protein aggregation and virtually eliminated neuronal death in CA1 neurons. Biochemical analyses revealed that ischemic preconditioning decreased accumulation of ubiquitin-conjugated proteins (ubi-proteins) and reduced free ubiquitin depletion after brain ischemia. Furthermore, ischemic preconditioning also reduced redistribution of heat shock cognate protein 70 and Hdj1 from cytosolic fraction to protein aggregate-containing fraction after brain ischemia. These results suggest that ischemic preconditioning decreases protein aggregation after brain ischemia.

Animals↗
PubMed2001

Assembly of C. elegans apical junctions involves positioning and compaction by LET-413 and protein aggregation by the MAGUK protein DLG-1.

Specialised subapical junctions play a critical role in maintaining epithelial cell polarity and tissue integrity, and provide a platform for intracellular signalling. Here we analyse the roles of C. elegans genes let-413 and dlg-1, a homologue of Drosophila lethal discs large, in the assembly of the C. elegans apical junction (CeAJ), and provide the first characterisation of this structure. We have identified dlg-1 as an essential gene in an RNA interference screen against C. elegans homologues of genes encoding proteins involved in tight or septate junction formation. We show that DLG-1 colocalises with the junctional protein JAM-1 at CeAJs in a unit distinct from HMP-1/alpha-catenin, and apical to the laterally localised LET-413. Loss of dlg-1 activity leads to JAM-1 mislocalisation and the disappearance of the electron-dense component of the CeAJs, but only mild adhesion and polarity defects. In contrast, loss of let-413 activity leads to the formation of basally extended discontinuous CeAJs and strong adhesion and polarity defects. Interestingly, in LET-413-deficient embryos, CeAJ markers are localised along the lateral membrane in a manner resembling that observed in wild-type embryos at the onset of epithelial differentiation. We conclude that the primary function of LET-413 is to correctly position CeAJ components at a discrete subapical position. Furthermore, we propose that DLG-1 is required to aggregate JAM-1 and other proteins forming the electron-dense CeAJ structure. Our data suggest that epithelial adhesion is maintained by several redundant systems in C. elegans.

Animals↗
PubMed2006

Effects of protein aggregates: an immunologic perspective.

The capacity of protein aggregates to enhance immune responses to the monomeric form of the protein has been known for over a half-century. Despite the clear connection between protein aggregates and antibody mediated adverse events in treatment with early therapeutic protein products such as intravenous immune globulin (IVIG) and human growth hormone, surprisingly little is known about the nature of the aggregate species responsible for such effects. This review focuses on a framework for understanding how aggregate species potentially interact with the immune system to enhance immune responses, garnered from basic immunologic research. Thus, protein antigens presented in a highly arrayed structure, such as might be found in large nondenatured aggregate species, are highly potent in inducing antibody responses even in the absence of T-cell help. Their potency may relate to the ability of multivalent protein species to extensively cross-link B-cell receptor, which (1) activates B cells via Bt kinases to proliferate, and (2) targets protein to class II major histocompatibility complex (MHC)-loading compartments, efficiently eliciting T-cell help for antibody responses. The review further focuses on protein aggregates as they affect an immunogenicity risk assessment, the use of animal models and studies in uncovering effects of protein aggregates, and changes in product manufacture and packaging that may affect generation of protein aggregates.

Antibodies↗
PubMed2003

Time scale of protein aggregation dictated by liquid-liquid demixing.

The growing impact of protein aggregation pathologies, together with the current high need for extensive information on protein structures are focusing much interest on the physics underlying the nucleation and growth of protein aggregates and crystals. Sickle Cell Hemoglobin (HbS), a point-mutant form of normal human Hemoglobin (HbA), is the first recognized and best-studied case of pathologically aggregating protein. Here we reanalyze kinetic data on nucleation of deoxy-HbS aggregates by referring them to the (concentration-dependent) temperature T(s) characterizing the occurrence of the phase transition of liquid-liquid demixing (LLD) of the solution. In this way, and by appropriate scaling of kinetic data at different concentrations, so as to normalize their spans, the apparently disparate sets of data are seen to fall on a master curve. Expressing the master curve vs. the parameter epsilon = (T - T(s)) / T(s), familiar from phase transition theory, allows eliciting the role of anomalously large concentration fluctuations associated with the LLD phase transition and also allows decoupling quantitatively the role of such fluctuations from that of microscopic, inter-protein interactions leading to nucleation. Referring to epsilon shows how in a narrow temperature span, that is at T - T(s), nucleation kinetics can undergo orders-of-magnitude changes, unexpected in terms of ordinary chemical kinetics. The same is true for similarly small changes of other parameters (pH, salts, precipitants), capable of altering T(s) and consequently epsilon. This offers the rationale for understanding how apparently minor changes of parameters can dramatically affect protein aggregation and related diseases.

Hemoglobin, Sickle↗
PubMed2006

The role of protein aggregates in neuronal pathology: guilty, innocent, or just trying to help?

Protein aggregates such as Lewy bodies have done much for the scientists in the field of neurodegenerative diseases: They have highlighted the affected cell populations and they have trapped the mutant disease protein. Instead of a good reputation, however, protein aggregates have received incriminations, because they are consistently seen at the site of crime. Reviewing the arguments, crucial evidence has become known that (a) the specific neuronal pathology precedes the appearance of protein aggregates in mouse models of disease, (b) the neurodegenerative disease in patients occurs with comparable severity when visible protein aggregates remain absent, (c) the neurotoxicity in vitro is best reproduced by oligomers, not polymers of the mutant disease protein. Is it feasible that protein aggregates are inert byproducts of the disease protein malconformation, or that they even represent beneficial cellular efforts to sequestrate the soluble toxic disease protein, together with normal or aberrant interactor proteins? Whatever the answer will be, one positive role of protein aggregates seems clear: In contrast to earlier speculations that random cytoplasmic proteins are trapped within the aggregates, scientists now believe that the composition of the Lewy body reflects the network of interactions between crucial players in disease pathogenesis, such as the PARK1, PARK2 and PARK5 protein.

Alzheimer Disease↗
PubMed2006

p62/SQSTM1: a missing link between protein aggregates and the autophagy machinery.

In eukaryotic cells short-lived proteins are degraded in a specific process by the ubiquitin-proteasome system (UPS), whereas long-lived proteins and damaged organelles are degraded by macroautophagy (hereafter referred to as autophagy). A growing body of evidence now suggests that autophagy is important for clearance of protein aggregates that form in cells as a consequence of ageing, oxidative stress, alterations that elevate the amounts of certain aggregation-prone proteins or expression of aggregating mutant variants of specific proteins. Autophagy is generally considered to be a non-specific, bulk degradation process. However, a recent study suggests that p62/SQSTM1 may link the recognition of polyubiquitinated protein aggregates to the autophagy machinery.(1) This protein is able to polymerize via its N-terminal PB1 domain and to recognize polyubiquitin via its C-terminal UBA domain. It can also recruit the autophagosomal protein LC3 and co-localizes with many types of polyubiquitinated protein aggregates.(1) Here we discuss possible implications of these findings and raise some questions for further investigation.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Small heat shock proteins, ClpB and the DnaK system form a functional triade in reversing protein aggregation.

Small heat shock proteins (sHsps) can efficiently prevent the aggregation of unfolded proteins in vitro. However, how this in vitro activity translates to function in vivo is poorly understood. We demonstrate that sHsps of Escherichia coli, IbpA and IbpB, co-operate with ClpB and the DnaK system in vitro and in vivo, forming a functional triade of chaperones. IbpA/IbpB and ClpB support independently and co-operatively the DnaK system in reversing protein aggregation. A delta ibpAB delta clpB double mutant exhibits strongly increased protein aggregation at 42 degrees C compared with the single mutants. sHsp and ClpB function become essential for cell viability at 37 degrees C if DnaK levels are reduced. The DnaK requirement for growth is increasingly higher for delta ibpAB, delta clpB, and the double delta ibpAB delta clpB mutant cells, establishing the positions of sHsps and ClpB in this chaperone triade.

Endopeptidase Clp↗
PubMed2002

Protein aggregation in Escherichia coli: role of proteases.

Protein aggregation is involved in several human diseases, and presumed to be an important process in protein quality control. In bacteria, aggregation of proteins occurs during stress conditions, such as heat shock. We studied the protein aggregates of Escherichia coli during heat shock. Our results demonstrate that the concentration and diversity of proteins in the aggregates depend on the availability of proteases. Aggregates obtained from mutants in the Lon (La) protease contain three times more protein than wild-type aggregates and show the broadest protein diversity. The results support the assumption that protein aggregates are formed from partially unfolded proteins that were not refolded by chaperones or degraded by proteases.

ATP-Dependent Proteases↗
PubMed2000

Enzymatic removal of oxidized protein aggregates from erythrocyte membranes.

Erythrocytes oxidized or aged in the circulation undergo membrane protein aggregation and anti-band 3 autoantibody binding to the cell surface. When human erythrocytes were mildly oxidized in vitro with 0.1 mM Fe(III) at 37 degrees C for 3 h, the aggregation of nonionic detergent C(12)E(8)-insoluble membrane protein and the binding of anti-band 3 IgG to the cell surface were increased. Incubation of membranes isolated from the oxidized cells increased the amount of protein aggregates by 5-fold after 6 h, while incubation for a further 12 h sharply decreased the amount of aggregates. In the presence of diisopropyl fluorophosphate (DFP), however, the increased amount of aggregates was maintained in the subsequent incubation. Western blot analysis of the aggregates using rabbit anti-band 3 showed that band 3 protein aggregates increased in the initial stage of incubation and decreased upon subsequent incubation, whereas the increased band 3 protein aggregates did not subsequently decrease when membranes were incubated in the presence of DFP. Incubation of the oxidized cells at 37 degrees C for 18 h caused reduction of the membrane protein aggregates and the (125)I-anti-band 3 IgG binding to the cell surface, while incubation in the presence of DFP did not cause these reductions. The results suggest that the oxidation-induced cell membrane protein aggregates were probably removed by 80-kDa serine protease, namely, oxidized protein hydrolase (OPH), in the oxidized cell membranes [Fujino et al. (1998) Biochim. Biophys. Acta 1374, 47-54; (1998) J. Biochem. 124, 1077-1085; (2000) Biochim. Biophys. Acta 1478, 102-112], and as a result the increased anti-band 3 binding to the cell surface was reduced.

Anion Exchange Protein 1, Erythrocyte↗
PubMed2003

Protein aggregation and aggregate toxicity: new insights into protein folding, misfolding diseases and biological evolution.

The deposition of proteins in the form of amyloid fibrils and plaques is the characteristic feature of more than 20 degenerative conditions affecting either the central nervous system or a variety of peripheral tissues. As these conditions include Alzheimer's, Parkinson's and the prion diseases, several forms of fatal systemic amyloidosis, and at least one condition associated with medical intervention (haemodialysis), they are of enormous importance in the context of present-day human health and welfare. Much remains to be learned about the mechanism by which the proteins associated with these diseases aggregate and form amyloid structures, and how the latter affect the functions of the organs with which they are associated. A great deal of information concerning these diseases has emerged, however, during the past 5 years, much of it causing a number of fundamental assumptions about the amyloid diseases to be re-examined. For example, it is now apparent that the ability to form amyloid structures is not an unusual feature of the small number of proteins associated with these diseases but is instead a general property of polypeptide chains. It has also been found recently that aggregates of proteins not associated with amyloid diseases can impair the ability of cells to function to a similar extent as aggregates of proteins linked with specific neurodegenerative conditions. Moreover, the mature amyloid fibrils or plaques appear to be substantially less toxic than the pre-fibrillar aggregates that are their precursors. The toxicity of these early aggregates appears to result from an intrinsic ability to impair fundamental cellular processes by interacting with cellular membranes, causing oxidative stress and increases in free Ca2+ that eventually lead to apoptotic or necrotic cell death. The 'new view' of these diseases also suggests that other degenerative conditions could have similar underlying origins to those of the amyloidoses. In addition, cellular protection mechanisms, such as molecular chaperones and the protein degradation machinery, appear to be crucial in the prevention of disease in normally functioning living organisms. It also suggests some intriguing new factors that could be of great significance in the evolution of biological molecules and the mechanisms that regulate their behaviour.

Amyloid↗
PubMed1987

Photosensitized acceleration of riboflavin on the formation of lenticular HMW-protein aggregation.

The percentage of high molecular weight protein aggregates obtained by Sepharose 6B column chromatography was increased significantly at pH 9.0 as compared to that of at pH 4.0 and 7.0, especially by exposure to light. The authors suggest that aggregation of lens protein is induced by light-exposure in the presence of riboflavin at higher pH's and that riboflavin acts as a photosensitizer to form high molecular weight protein aggregates in the lens.

Animals↗
PubMed2003

Protein aggregation in recombinant bacteria: biological role of inclusion bodies.

Protein aggregation is an ordinary consequence of thermal stress. In recombinant bacteria, the over-expression of plasmid-encoded genes triggers transcription of heat-shock genes and other stress responses and often results in the aggregation of the encoded protein as inclusion bodies. The formation of these deposits represents a major obstacle for the production of biologically active polypeptides and restricts the spectrum of protein products being available for the industrial-biomedical market. Inclusion body formation was formerly considered to occur passively by the irretrievable deposition of partially-folded intermediates. Increasing evidence, however, indicates that protein aggregation in bacteria occurs as a reversible process deeply integrated in the cell mechanisms for coping with thermal stress, and that inclusion bodies are structurally dynamic structures. Inclusion body formation might actually be supported by the cellular machinery that when operated under specific stress conditions, transiently stores misfolded polypeptides until they could be further processed: either refolded or proteolysed. A better understanding of protein aggregation in cell physiology could allow not only inclusion body formation to be minimized more efficiently for a higher soluble yield, but also to comprehend in detail the intricacy of cell mechanisms committed to handling the misfolding danger.

Bacteria↗
PubMed1995

Thermotolerance and nuclear protein aggregation: protection against initial damage or better recovery?

Heat-induced nuclear protein aggregation and subsequent disaggregation were measured in nonpreheated and preheated (thermotolerant) HeLa S3 cells. The effect of thermotolerance on the formation of and recovery from heat-induced nuclear protein aggregates was related to the cellular levels of hsp27, hsp60, hsp70, hsc70, and hsp90. Cells heated at different time points after the thermotolerance trigger showed various levels of protection against heat-induced nuclear protein aggregation. This protection, however, did not parallel the development and decay of thermotolerance on cell survival. The protection was maximal when the thermotolerance level already had started to decay. The level of protection against nuclear protein aggregation did however parallel the cellular level of hsp70 indicating that hsp70 may be involved in this process. At all stages during the development and decay, thermotolerant cells showed a more rapid recovery (disaggregation) from the heat-induced nuclear protein aggregates than non-thermotolerant cells. The rates of disaggregation during development and decay of thermotolerance paralleled the cellular levels of hsp27 suggesting that hsp27 is somehow involved in this recovery process from heat-induced nuclear protein aggregates. The total cellular levels of none of the individual hsp's completely correlate with development and decay of thermotolerance, indicating that overexpression of any of these hsp's alone does not determine the level of thermotolerance. Clonogenic cell survival paralleled the rates of disaggregation, leading to the notion that recovery processes are the most important determinant for the thermotolerant state of HeLa S3 cells. The best correlation with clonogenic survival was found when both initial aggregation and subsequent disaggregation were taken into account, suggesting that the combined action of various hsp's in these two processes have to be included in thermotolerance development and decay.

HeLa Cells↗
PubMed2006

Detection and characterization of protein aggregates by fluorescence microscopy.

Aggregation may compromise the stability as well as the biological activity of protein drugs. Detection of protein aggregates is needed in the process of protein characterization and during optimization of pharmaceutical formulations. This paper describes a technique, which consists of analysing protein aggregates by fluorescence microscopy after staining with the hydrophobic probe Nile Red. Dilution, filtration or other modifications of the sample are not needed. Assessment of aggregation was possible in highly concentrated protein samples (193 mg/ml). Fluorescence microscopy observations allowed the detection and characterization of protein aggregates not easily detected by spectroscopic techniques. Nile Red was shown to be very sensitive for the detection and analysis of immunoglobulin aggregates. Nile Red, Congo red and Thioflavine T stainings were compared. Nile Red and Thioflavine T fluorescence were colocalized. The diameter of immunoglobulin aggregates was determined, and the number of aggregates was correlated with 90 degrees light scattering measurements. Studies of human calcitonin aggregates brought to light new aspects of the human calcitonin aggregation mechanisms. Thus, Nile Red staining not only allows detection of very low levels of protein aggregates, but also contributes to a better understanding of the complex mechanisms governing protein aggregation.

Antibodies↗
PubMed2005

Global impairment of the ubiquitin-proteasome system by nuclear or cytoplasmic protein aggregates precedes inclusion body formation.

The highly conserved ubiquitin-proteasome system (UPS) controls the stability of most nuclear and cytoplasmic proteins and is therefore essential for virtually all aspects of cellular function. We have previously shown that the UPS is impaired in the presence of aggregated proteins that become deposited into cytoplasmic inclusion bodies (IBs). Here, we report that production of protein aggregates specifically targeted to either the nucleus or cytosol leads to global impairment of UPS function in both cellular compartments and is independent of sequestration of aggregates into IBs. The observation of severe UPS impairment in compartments lacking detectable aggregates or aggregation-prone protein, together with the lack of interference of protein aggregates on 26S proteasome function in vitro, suggests that UPS impairment is unlikely to be a consequence of direct choking of proteasomes by protein aggregates. These data suggest a common proteotoxic mechanism for nuclear and cytoplasmic protein aggregates in the pathogenesis of neurodegenerative disease.

Cell Line↗
PubMed2002

Prevention and reversion of protein aggregation by molecular chaperones in the E. coli cytosol: implications for their applicability in biotechnology.

The amount of a native protein reflects an equilibrium of protein synthesis, de novo folding and protein stability. Stress situations, like heat shock, or overproduction of a protein can cause an imbalance in this equilibrium, resulting in protein aggregation. Molecular chaperones control protein folding processes and protect misfolded proteins from aggregation in all cells. Since protein aggregation is frequently observed upon synthesis of heterologous proteins in E. coli, molecular chaperones have been applied in biotechnology by their co-overproduction with the desired protein. While increasing protein solubility in some cases, this approach has not been generally successful. Recent findings demonstrate, that protein aggregation, even in case of inclusion bodies, must not be a dead end in the life cycle of a protein. Such resolubilization of aggregated proteins is mediated by a bi-chaperone system consisting of ClpB and DnaK, the prokaryotic representatives of the Hsp100 and Hsp70 families. The disaggregation capacity of this bi-chaperone system has now been demonstrated in vitro and in vivo for a wide variety of aggregated proteins and offers a new perspective to increase the solubility of proteins of interest.

Biotechnology↗
PubMed2004

Neurodegenerative diseases caused by protein aggregation: a phenomenon at the borderline between molecular evolution and ageing.

A process of protein aggregation that causes intracellular or extracellular accumulation of insoluble protein deposits causes many important neurodegenerative diseases associated with the ageing. The recognition that protein aggregation plays a prominent role in pathogenesis of important pathologies such as Alzheimer's and Parkinson's diseases prompted the scientific community to focus on the molecular mechanism of protein aggregation. Many proteins with sophisticated functions can self-aggregate because their folding is complicate and abnormal intermolecular contacts can predominate over the normal intramolecular interactions. The review of biochemical functional and pathogenic implications attributed to alpha synuclein, A beta peptide, presenilin and apoE highlights for these proteins a common conformational plasticity and the capacity to adapt their secondary structure to surrounding solvent as well as to the contacted ligands. Their functions are not fully elucidated but there is an elevated number of metabolic pathways in which apparently they are involved as well as they generate functional contact with a remarkable number of other proteins. The mechanism by which alpha synuclein and A beta protein make fibrils is an example of conformational plasticity because both these polypeptides can visit a coil or helical structure, but otherwise they convert into a pathogenic beta sheet structure highly suitable for polymerisation and fibril formation. The emerging question in the puzzling pathogenic basis of these diseases is if protein aggregation associated with ageing has a role in molecular evolution of the species or if it just represents a calculated drawback.

Aging↗