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PubMed2003

Detection and prevention of protein aggregation before, during, and after purification.

The use of proteins for in vitro studies or as therapeutic agents is frequently hampered by protein aggregation during expression, purification, storage, or transfer into requisite assay buffers. A large number of potential protein stabilizers are available, but determining which are appropriate can take days or weeks. We developed a solubility assay to determine the best cosolvent for a given protein that requires very little protein and only a few hours to complete. This technique separates native protein from soluble and insoluble aggregates by filtration and detects both forms of protein by SDS-PAGE or Western blotting. Multiple buffers can be simultaneously screened to determine conditions that enhance protein solubility. The behavior of a single protein in mixtures and crude lysates can be analyzed with this technique, allowing testing prior to and throughout protein purification. Aggregated proteins can also be assayed for conditions that will stabilize native protein, which can then be used to improve subsequent purifications. This solubility assay was tested using both prokaryotic and eukaryotic proteins that range in size from 17 to 150 kDa and include monomeric and multimeric proteins. From the results presented, this technique can be applied to a variety of proteins.

Bacterial Proteins↗
PubMed2002

Polyglutamine expansion, protein aggregation, proteasome activity, and neural survival.

Huntington's disease (HD) is one of eight established triplet repeat neurodegenerative disorders, which are collectively caused by the genetic expansion of polyglutamine repeats. While the mechanism(s) by which polyglutamine expansion causes neurodegeneration in each of these disorders is being intensely investigated, the underlying cause of polyglutamine toxicity has not been fully elucidated. A number of studies have focused on the potential role of protein aggregation and disruption of the proteasome proteolytic pathway in polyglutamine-mediated neurodegeneration. However, at present it is not clear whether polyglutamine-mediated protein aggregation is sufficient to induce cell death, nor has it been clearly determined whether proteasome inhibition precedes, coincides, or occurs as the result of the formation of polyglutamine-associated protein aggregation. To address these important components of polyglutamine toxicity, in the present study we utilized neural SH-SY5Y cells stably transfected with polyglutamine-green fluorescent protein constructs to examine the effects of polyglutamine expansion on protein aggregation, proteasome activity, and neural cell survival. Data from the present study demonstrate that polyglutamine expansion does not dramatically impair proteasome activity or elevate protein aggregate formation under basal conditions, but does significantly impair the ability of the proteasome to respond to stress, and increases stress-induced protein aggregation following stress, all in the absence of neural cell death.

Blotting, Northern↗
PubMed2001

Oligomerization of polyalanine expanded PABPN1 facilitates nuclear protein aggregation that is associated with cell death.

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by progressive eyelid drooping, swallowing difficulties and proximal limb weakness. The autosomal dominant form of this disease is caused by short expansions of a (GCG)(6) repeat to (GCG)(8-13) in the PABPN1 gene, which results in the expansion of a polyalanine stretch from 10 to 12-17 alanines in the N-terminus of the protein. Mutated PABPN1 (mPABPN1) is able to induce nuclear protein aggregation and form filamentous nuclear inclusions, which are the pathological hallmarks of OPMD. PABPN1, when bound to poly(A) RNA, forms both linear filaments and discrete-sized, compact oligomeric particles in vitro. In the absence of poly(A) RNA, PABPN1 can form oligomers. Here we report that: (i) oligomerization of PABPN1 is mediated by two potential oligomerization domains (ODs); (ii) inactivating oligomerization of mPABPN1 by deletions of 6-8 amino acids in either of the ODs prevents nuclear protein aggregation; (iii) expression of mPABPN1 in COS-7 cells is associated with cell death; and (iv) preventing nuclear protein aggregation by inactivating oligomerization of mPABPN1 significantly reduces cell death. These findings suggest that oligomerization of PABPN1 plays a crucial role in the formation of OPMD nuclear protein aggregation, while the expanded polyalanine stretch is necessary but not sufficient to induce OPMD protein aggregation, and that the nuclear protein aggregation might be toxic and cause cell death. These observations also imply that inactivation of oligomerization of mPABPN1 might be a useful therapeutic strategy for OPMD.

Animals↗
PubMed1995

Thermal protein denaturation and protein aggregation in cells made thermotolerant by various chemicals: role of heat shock proteins.

Thermotolerance (TT) induced by sodium arsenite (A-TT: 100 microM, 1 h, 37 degrees C) was compared to heat-induced thermotolerance (H-TT: 15 min, 44 degrees C) using HeLa S3 cells. All four pretreatments led to comparable levels of thermotolerance and also induced resistance to arsenite-, ethanol-, and diamide-induced toxicity (clonogenic ability). Stress-induced expression of the major heat shock proteins (hsp27, hsc70(p73), hsp70(p72), and hsp90) was generally highest in H-TT cells and lowest in A-TT cells. Interestingly, the four types of TT cells showed distinct differences in certain aspects of resistance against thermal protein damage. Thermal protein denaturation and aggregation determined in isolated cellular membrane fractions was found to be attenuated when they were isolated from H-TT and A-TT cells but not when isolated from E-TT and D-TT cells. The heat resistance in the proteins of the membrane fraction corresponded with elevated levels of hsp70(p72) associated with the isolated membrane fractions. In the nuclear fraction, only marginal (not significant) attenuation of the formation of protein aggregates (as determined by TX-100 (in)solubility) was observed. However, the postheat recovery from heat-induced protein aggregation in the nucleus was faster in H-TT, E-TT, and D-TT cells, but not in A-TT cells. Despite the fact that elevated levels of hsp27, hsp70(p73), and hsp70(p72) were found in the TX-100 insoluble nuclear fraction derived from all TT cells, no correlation was found with the degree of resistance in terms of the accelerated recovery from nuclear protein aggregation. The only correlation between accelerated recovery from nuclear protein aggregates was that with total cellular levels of hsp27. The data indicate that heat-induced loss of clonogenic ability may be a multitarget rather than a single target event. A threshold of damage may exist in cells after exposure to heat; multiple sets of proteins in (different compartments of) the cell need to be damaged before this threshold is exceeded and the cell dies. As a consequence, stabilization of only one of these sets of proteins is already sufficient to render cells thermotolerant at the clonogenic level.

Arsenites↗
PubMed1988

Some characteristics of a high molecular weight lipid-protein aggregate and its possible role in intracellular fatty acid metabolism.

Several physical aspects of a high molecular weight lipid-protein aggregate separated by gel chromatography from chick and rat liver cytosol and its possible role in intracellular fatty acid metabolism were investigated. Electron microscopic examination of the high molecular weight lipid-protein aggregate indicated spherical particles with a diameter range of 200-600 A. This structure is consistent with a microemulsion particle of triglyceride encapsulated by phospholipid and protein. Uptake of fatty acids by microsomes occurred from the same lipid-protein aggregate, and the triglycerides synthesized in microsomes also became associated with these particles in the cytosol. The lipid-protein aggregate prepared by different homogenization methods showed identical ratios of components, but these ratios changed following incubation. These findings lend support to the concept that this aggregate plays a physiological role in intracellular lipid metabolism, and may be identifiable with previously reported subcellular fatty acid and triglyceride pools.

Animals↗
PubMed2005

Protein aggregation in Alzheimer's disease and other neoropathological disorders.

A conspicuous feature shared by Alzheimer's disease as well as a variety of highly prevalent, clinically unrelated neurodegenerative disorders is the occurrence of protein aggregates both intra- and extracellularly. Most of these conditions are characterized at autopsy by the presence of such deposits, typically of fibrillar structure and accompanying extensive neuronal cell loss, displaying a selective brain distribution. The recently discovered similarities of a number of these aggregates with a novel type of experimentally induced protein deposit, formed as a general response to discrepancies in protein turnover and designated the "aggresome", has prompted speculations about the involvement of the ubiquitin-proteasome system in a process fundamental to neurodegeneration. Consistent with this view, protein aggregates have been regarded in a pathogenic connotation, with most aspects of neurologic pathogenesis being largely attributed to their presence in nerve tissues. However, the neurotoxicity of protein aggregates remains ambiguous as direct evidence substantiating it have long remained elusive. A convergence of evidence now support the notion that the actual culprits might comprise the oligomeric, non-fibrillar intermediates that arise early during the aggregation process, termed protofibrils and that the fibrillar end-stage aggregates themselves might actually serve a neuroprotective function. These intermediates ostensibly resolve many puzzling aspects of neurodegeneration and there is evidence that neurotoxicity is one key operational property they may possess. The above attest to the fact that protein aggregation remains a complex issue with a role far more enigmatic than originally thought but nonetheless important for the understanding of the pathological basis of neurodegenerative disorders.

Alzheimer Disease↗
PubMed1998

ATP releases HSP-72 from protein aggregates after renal ischemia.

The pattern of 72-kDa heat-shock protein (HSP-72) induction after renal ischemia suggests a role in restoring cell structure. HSP-72 activity in the repair and release from denatured and aggregated proteins requires ATP. Protein aggregates were purified from normal and ischemic rat renal cortex. The addition of ATP to cortical homogenates reduced HSP-72, Na(+)-K(+)-ATPase, and actin in aggregates subsequently isolated, suggesting that their interaction is ATP dependent. Altering ATP hydrolysis in the purified aggregates, however, had different effects. ATP released HSP-72 during reflow and preserved Na(+)-K(+)-ATPase association with aggregates at 2 h but had no effect in controls or at 6 h reflow and caused no change in actin. These results indicate that HSP-72 complexes with aggregated cellular proteins in an ATP-dependent manner and suggests that enhancing HSP-72 function after ischemic renal injury assists refolding and stabilization of Na(+)-K(+)-ATPase or aggregated elements of the cytoskeleton, allowing reassembly into a more organized state.

Actins↗
PubMed2002

Molecular chaperones as modulators of polyglutamine protein aggregation and toxicity.

The formation of insoluble protein aggregates in neurons is a hallmark of neurodegenerative diseases caused by proteins with expanded polyglutamine (polyQ) repeats. However, the mechanistic relationship between polyQ aggregation and its toxic effects on neurons remains unclear. Two main hypotheses have been put forward for how polyQ expansions may cause cellular dysfunction. In one model neurotoxicity results from the ability of polyQ-expanded proteins to recruit other important cellular proteins with polyQ stretches into the aggregates. In the other model, aggregating polyQ proteins partially inhibit the ubiquitin-proteasome system for protein degradation. These two mechanisms are not exclusive but may act in combination. In general, protein misfolding and aggregation are prevented by the machinery of molecular chaperones. Some chaperones such as the members of the Hsp70 family also modulate polyQ aggregation and suppress its toxicity. These recent findings suggest that an imbalance between the neuronal chaperone capacity and the production of potentially dangerous polyQ proteins may trigger the onset of polyQ disease.

Animals↗
PubMed2001

Usefulness of the Kohlrausch-Williams-Watts stretched exponential function to describe protein aggregation in lyophilized formulations and the temperature dependence near the glass transition temperature.

PURPOSE: We studied the feasibility of using the Kohlrausch-Williams-Watts stretched exponential function (KWW equation) to describe protein aggregation in lyophilized formulations during storage. Parameters representing "mean aggregation time" (taua) and stretched exponential constant (betaa) were calculated according to the KWW equation by assuming that the time required for protein molecules to aggregate (tau) varies because of the fact that protein aggregation occurs at a rate that depends on the degree of protein deformation resulting from stresses created during freeze-drying. The temperature dependence of the parameters near the glass transition temperature was examined to discuss the possibility of predicting protein aggregation by accelerated testing. METHODS: Protein aggregation in lyophilized bovine serum gamma-globulin (BGG) formulations containing dextran or methylcellulose, at temperatures ranging from 10 to 80 degrees C, was followed by size-exclusion chromatography. RESULTS: Non-exponential BGG aggregation in lyophilized formulations could be described by the KWW equation. The taua and betaa, parameters changed abruptly around the NMR relaxation-based critical mobility temperature for formulations containing dextran and methylcellulose. In the glassy state, in contrast, the taua parameter of these formulations exhibited continuous temperature dependence. The parameter taur, as calculated from taua, and betaa, reflected differences in tau values between the two excipients. CONCLUSIONS: The results indicate that the parameter betaa is reflective of physical changes wihtin lyophilized formulations. Within the temperature range, during which no abrupt changes in betaa were observed, knowledge regarding the taua and betaa parameters allows the rate of protein aggregation to be predicted. The parameter taur was found to be useful in comparing the protein aggregation behavior of formulations having different taua and betaa values.

Algorithms↗
PubMed2000

Increased band 3 protein aggregation and anti-band 3 binding of erythrocyte membranes on treatment with sesamol.

The influence of sesamol, an antioxidant in processed sesame oil, on oxidative modification of human erythrocyte membrane proteins was investigated. Human erythrocytes were incubated with sesamol at various concentrations up to 10 mM at 37 degrees C for 1 h. The amounts of hemoglobin bound to the membranes and detergent C12E8-insoluble membrane protein aggregates were increased as the concentration of sesamol increased. Western blot analysis indicated that aggregates of band 3 protein were increased by the treatment. Binding of anti-band 3 antibody to the erythrocytes was increased by the treatment. Isolated cell membranes were incubated with sesamol similarly. Aggregates of band 3 protein were also increased, indicating that the band 3 protein aggregation was little affected by hemoglobin bound to the membranes. Aggregation of band 3 protein in the treatment of isolated cell membranes was partially prevented when the treatment was conducted under anaerobic conditions, suggesting that augmentation of the protein aggregation by sesamol involved both oxygen-dependent and oxygen-independent pathways. Among phenolics, sesamol showed a distinctive feature to increase band 3 protein aggregation in erythrocyte membranes and to enhance anti-band 3 binding to erythrocytes.

Anion Exchange Protein 1, Erythrocyte↗
PubMed2004

Involvement of protein radical, protein aggregation, and effects on NO metabolism in the hypochlorite-mediated oxidation of mitochondrial cytochrome c.

Cytochrome c (cyt c)-derived protein radicals, radical adduct aggregates, and protein tyrosine nitration have been implicated in the pro-apoptotic event connecting inflammation to the development of diseases. During inflammation, one of the reactive oxygen species metabolized via neutrophil activation is hypochlorite (HOCl); destruction of the mitochondrial electron transport chain by hypochlorite is considered to be a damaging factor. Previous study has shown that HOCl induces the site-specific oxidation of cyt c at met-80. In this work, we have assessed the hypothesis that exposure of cyt c to physiologically relevant concentrations of HOCl leads to protein-derived radical and consequent protein aggregation, which subsequently affects cyt c's regulation of nitric oxide metabolism. Reaction intermediates, chemical pathways available for protein aggregation, and protein nitration were examined. A weak ESR signal for immobilized nitroxide derived from the protein was detected when a high concentration of cyt c was reacted with hypochlorite in the presence of the nitroso spin trap 2-methyl-2-nitrosopropane. When a low concentration of cyt c was exposed to the physiologically relevant levels of HOCl in the presence of 5,5-dimethyl-pyrroline N-oxide (DMPO), we detected DMPO nitrone adducts derived from both protein and protein aggregate radicals as assessed by Western blot using an antibody raised against the DMPO nitrone adduct. The cyt c-derived protein radicals formed by HOCl were located on lysine and tyrosine residues, with lysine predominating. Cyt c-derived protein aggregates induced by HOCl involved primarily lysine residues and hydrophobic interaction. In addition, HOCl-oxidized cyt c (HOCl-cyt c) exhibited a higher affinity for NO and enhancement of nonenzymatic NO synthesis from nitrite reduction. Furthermore, HOCl-mediated cyt c oxidation also resulted in a significant elevation of cyt c nitration derived from either NO trapping of the cyt c-derived tyrosyl radical or cyt c-catalyzed one-electron oxidation of nitrite.

Animals↗
PubMed1997

Limited in vivo proteolysis of aggregated proteins.

Degradation pathways of insoluble proteins have been analyzed in Escherichia coli by using a N-terminal beta-galactosidase fusion protein (VP1LAC) that aggregates immediately after its synthesis. In recombinant E. coli cells, lower molecular mass products, antigenically related to the entire fusion, accumulate together with the entire fusion. In absence of protein synthesis, the insoluble intact protein declines, suggesting that degradation of the recombinant protein also affects aggregated protein. Time course analysis of both soluble and insoluble cell fractions has revealed a limited proteolysis of the insoluble protein that removes the heterologous domain and permits the resulting beta-galactosidase fragments to refold and solubilize. Further extensive degradation occurs exclusively on soluble protein. The restricted proteolysis of misfolded, insoluble protein is the initiating event of a subsequent degradative pathway in which rate-limiting steps permit the accumulation of stable degradative intermediates.

Escherichia coli↗
PubMed2005

A chaperone pathway in protein disaggregation. Hsp26 alters the nature of protein aggregates to facilitate reactivation by Hsp104.

Cellular protein folding is challenged by environmental stress and aging, which lead to aberrant protein conformations and aggregation. One way to antagonize the detrimental consequences of protein misfolding is to reactivate vital proteins from aggregates. In the yeast Saccharomyces cerevisiae, Hsp104 facilitates disaggregation and reactivates aggregated proteins with assistance from Hsp70 (Ssa1) and Hsp40 (Ydj1). The small heat shock proteins, Hsp26 and Hsp42, also function in the recovery of misfolded proteins and prevent aggregation in vitro, but their in vivo roles in protein homeostasis remain elusive. We observed that after a sublethal heat shock, a majority of Hsp26 becomes insoluble. Its return to the soluble state during recovery depends on the presence of Hsp104. Further, cells lacking Hsp26 are impaired in the disaggregation of an easily assayed heat-aggregated reporter protein, luciferase. In vitro, Hsp104, Ssa1, and Ydj1 reactivate luciferase:Hsp26 co-aggregates 20-fold more efficiently than luciferase aggregates alone. Small Hsps also facilitate the Hsp104-mediated solubilization of polyglutamine in yeast. Thus, Hsp26 renders aggregates more accessible to Hsp104/Ssa1/Ydj1. Small Hsps partially suppress toxicity, even in the absence of Hsp104, potentially by sequestering polyglutamine from toxic interactions with other proteins. Hence, Hsp26 plays an important role in pathways that defend cells against environmental stress and the types of protein misfolding seen in neurodegenerative disease.

Heat-Shock Proteins↗
PubMed1998

Fatal attractions: abnormal protein aggregation and neuron death in Parkinson's disease and Lewy body dementia.

The abnormal aggregation of proteins into fibrillar lesions is a neuropathological hallmark of several sporadic and hereditary neurodegenerative diseases. For example, Lewy bodies (LBs) are intracytoplasmic filamentous inclusions that accumulate primarily in subcortical neurons of patients with Parkinson's disease (PD), or predominantly in neocortical neurons in a subtype of Alzheimer's disease (AD) known as the LB variant of AD (LBVAD) and in dementia with LBs (DLB). Aggregated neurofilament subunits and alpha-synuclein are major protein components of LBs, and these inclusions may contribute mechanistically to the degeneration of neurons in PD, DLB and LBVAD. Here we review recent studies of the protein building blocks of LBs, as well as the role LBs play in the onset and progression of PD, DLB and LBVAD. Increased understanding of the protein composition and pathological significance of LBs may provide insight into mechanisms of neuron dysfunction and death in other neurodegenerative disorders characterized by brain lesions containing massive deposits of proteinacious fibrils.

Alzheimer Disease↗
PubMed1988

Acetylcholine receptor-aggregating proteins are associated with the extracellular matrix of many tissues in Torpedo.

The synaptic basal lamina, a component of extracellular matrix (ECM) in the synaptic cleft at the neuromuscular junction, directs the formation of new postsynaptic specializations, including the aggregation of acetylcholine receptors (AChRs), during muscle regeneration in adult animals. Although the molecular basis of this phenomenon is unknown, it is mimicked by AChR-aggregating proteins in ECM-enriched fractions from muscle and the synapse-rich electric organ of the ray Torpedo californica. Molecules immunologically similar to these proteins are concentrated in the synaptic basal lamina at neuromuscular junctions of the ray and frog. Here we demonstrate that immunologically, chemically, and functionally similar AChR-aggregating proteins are also associated with the ECM of several other tissues in Torpedo. Monoclonal antibodies against the AChR-aggregating proteins from electric organ intensely stained neuromuscular junctions and the ventral surfaces of electrocytes, structures with a high density of AChRs. However, they also labeled many other structures which have basal laminae, including the extrajunctional perimeters of skeletal muscle fibers, smooth and cardiac muscle cells, Schwann cell sheaths in peripheral nerves, walls of some blood vessels, and epithelial basement membranes in the gut, skin, and heart. Some structures with basal laminae did not stain with the antibodies; e.g., the dorsal surfaces of electrocytes. Bands of similar molecular weight were detected by the antibodies on Western blots of extracts of ECM-enriched fractions from electric organ and several other tissues. Proteins from all tissues examined, enriched from these extracts by affinity chromatography with the monoclonal antibodies, aggregated AChRs on cultured myotubes. Thus, similar AChR-aggregating proteins are associated with the extracellular matrix of many Torpedo tissues. The broad distribution of these proteins suggests they have functions in addition to AChR aggregation.

Agrin↗
PubMed2006

Protein aggregation as a cause for disease.

The ability of proteins to fold into a defined and functional conformation is one of the most fundamental processes in biology. Certain conditions, however, initiate misfolding or unfolding of proteins. This leads to the loss of functional protein or it can result in a wide range of diseases. One group of diseases, which includes Alzheimer's, Parkinson's, Huntington's disease, and the transmissible spongiform encephalopathies (prion diseases), involves deposition of aggregated proteins. Normally, such protein aggregates are not found in properly functioning biological systems, because a variety of mechanisms inhibit their formation. Understanding the nature of these protective mechanisms together with the understanding of factors reducing or deactivating the natural protection machinery will be crucial for developing strategies to prevent and treat these disastrous diseases.

Alzheimer Disease↗
PubMed2006

Theoretical approaches to protein aggregation.

The process of protein misfolding and aggregation has been associated with an increasing number of pathological conditions that include Alzheimer's and Parkinson's diseases, and type II diabetes. In addition, the discovery that proteins unrelated to any known disorder can be converted into aggregates of morphologies similar to those found in diseased tissue has lead to the recognition that this type of assemblies represents a generic state of polypeptide chains. Therefore, despite the enormous complexity of the in vivo mechanisms that have evolved in living organisms to prevent and control the formation of protein aggregates, the process of aggregation itself appears ultimately to be caused by intrinsic properties of polypeptide chains, in particular by the tendency of the backbone to form hydrogen bonds, and be modulated by the presence of specific patterns of hydrophobic and charged residues. Theoreticians have just recently started to respond to the challenge of identifying the determinants of the aggregation process. In this review, we provide an account of the theoretical results obtained so far.

Alzheimer Disease↗
PubMed2003

The unfolded protein response in protein aggregating diseases.

For many genetic diseases, clinical phenotypes arise through the dysfunction of the gene products encoded by mutant genes. Effective treatment entails providing a source of the gene product in the diet or circulation, as has been achieved for type I diabetes and hemophilia, or in cases of enzyme deficiency by supplementation with metabolites synthesized by the defective protein, as in adrenoleukodystrophy. However, a growing list of diseases do not appear to be amenable to such treatment strategies. In these instances, defective gene products acquire novel properties that disrupt normal cell function, even in the presence of proteins encoded by the normal allele. One class of such diseases, collectively termed "conformational diseases," is composed of clinically unrelated disorders that share a common pathophysiology because the mutant proteins cannot adopt stable three-dimensional conformations. These mutant proteins aggregate in various subcellular compartments and may even cause cell death. Some of these diseases are associated with inclusion bodies containing the aggregating proteins whereas others do not exhibit such pathology; however, all appear to activate cell stress signaling pathways. Herein, we highlight one such disorder, Pelizaeus-Merzbacher disease, that disrupts formation of whiter matter in the brain. Accumulation of the mutant protein in oligodendrocytes activates the unfolded protein response. The well-characterized genetics and large number of animal models available for Pelizaeus-Merzbacher disease enables this disease to serve as an important model for conformational diseases, both in terms of defining molecular components of the unfolded protein response signaling pathway as well as testing therapeutic approaches to ameliorate disease.

Animals↗