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PubMed2006

Aberrant protein aggregation is essential for a mutant desmin to impair the proteolytic function of the ubiquitin-proteasome system in cardiomyocytes.

Aberrant protein aggregates in cardiomyocytes are frequently observed in many forms of cardiomyopathies and are often associated with impairment of proteolytic function of the ubiquitin-proteasome system (UPS). However, a causal relationship between mutant desmin (MT-des) induced aberrant protein aggregation and UPS impairment has not been established. The present study has tested the causal relationship. In cultured neonatal rat ventricular myocytes, modest overexpression of a human (cardio)myopathy-linked MT-des protein led to formation of desmin-positive aggregates and inhibited UPS proteolytic function in cardiomyocytes in a dose-dependent manner. Prevention or reduction of aberrant protein aggregation by co-expression of a heat shock protein (Hsp), alphaB-crystallin or inducible Hsp70, or by treatment of Congo red prevented and/or significantly attenuated the induction of UPS malfunction by MT-des. These findings prove for the first time that aberrant protein aggregation is not only sufficient but also required for MT-des to impair UPS proteolytic function in cardiomyocytes.

Animals↗

PubMed2001

Oxidative stress and protein aggregation during biological aging.

Biological aging is a fundamental process that represents the major risk factor with respect to the development of cancer, neurodegenerative, and cardiovascular diseases in vertebrates. It is, therefore, evident that the molecular mechanisms of aging are fundamental to understand many disease processes. In this regard, the oxidation and nitration of intracellular proteins and the formation of protein aggregates have been suggested to underlie the loss of cellular function and the reduced ability of senescent animals to withstand physiological stresses. Since oxidatively modified proteins are thermodynamically unstable and assume partially unfolded tertiary structures that readily form aggregates, it is likely that oxidized proteins are intermediates in the formation of amyloid fibrils. It is, therefore, of interest to identify oxidatively sensitive protein targets that may play a protective role through their ability to down-regulate energy metabolism and the consequent generation of reactive oxygen species (ROS). In this respect, the maintenance of cellular calcium gradients represents a major energetic expense, which links alterations in intracellular calcium levels to ATP utilization and the associated generation of ROS through respiratory control mechanisms. The selective oxidation or nitration of the calcium regulatory proteins calmodulin and Ca-ATPase that occurs in vivo during aging and under conditions of oxidative stress may represent an adaptive response to oxidative stress that functions to down-regulate energy metabolism and the associated generation of ROS. Since these calcium regulatory proteins are also preferentially oxidized or nitrated under in vitro conditions, these results suggest an enhanced sensitivity of these critical calcium regulatory proteins, which modulate signal transduction processes and intracellular energy metabolism, to conditions of oxidative stress. Thus, the selective oxidation of critical signal transduction proteins probably represents a regulatory mechanism that functions to minimize the generation of ROS through respiratory control mechanisms. The reduction of the rate of ROS generation, in turn, will promote cellular survival under conditions of oxidative stress, when reactive oxygen and nitrogen species overwhelm cellular antioxidant defense systems, by minimizing the non-selective oxidation of a range of biomolecules. Since protein aggregation occurs if protein repair and degradative systems are unable to act upon oxidized proteins and restore cellular function, the reduction of the oxidative load on the cell by the down-regulation of the electron transport chain functions to minimize protein aggregation. Thus, ROS function as signaling molecules that fine-tune cellular metabolism through the selective oxidation or nitration of calcium regulatory proteins in order to minimize wide-spread oxidative damage and protein aggregation. Oxidative damage to cellular proteins, the loss of calcium homeostasis and protein aggregation contribute to the formation of amyloid deposits that accumulate during biological aging. Critical to understand the relationship between these processes and biological aging is the identification of oxidatively sensitive proteins that modulate energy utilization and the associated generation of ROS. In this latter respect, oxidative modifications to the calcium regulatory proteins calmodulin (CaM) and the sarco/endoplasmic reticulum Ca-ATPase (SERCA) function to down-regulate ATP utilization and the associated generation of ROS associated with replenishing intracellular ATP through oxidative phosphorylation. Reductions in the rate of ROS generation, in turn, will minimize protein oxidation and facilitate intracellular repair and degradative systems that function to eliminate damaged and partially unfolded proteins. Since the rates of protein repair or degradation compete with the rate of protein aggregation, the modulation of intracellular calcium concentrations and energy metabolism through the selective oxidation or nitration of critical signal transduction proteins (i.e. CaM or SERCA) is thought to maintain cellular function by minimizing protein aggregation and amyloid formation. Age-dependent increases in the rate of ROS generation or declines in cellular repair or degradation mechanisms will increase the oxidative load on the cell, resulting in corresponding increases in the concentrations of oxidized proteins and the associated formation of amyloid.

Aging↗

PubMed1995

Infrared spectroscopic studies of lyophilization- and temperature-induced protein aggregation.

Recent studies have clearly demonstrated that Fourier transform IR spectroscopy can be a powerful tool for the study of protein stabilization during freeze-drying and for optimizing approaches to prevent lyophilization-induced protein aggregation. The purpose of the current review is to provide an overview of these topics, as well as an introduction to the study of protein secondary structure with IR spectroscopy. We will start with a general summary of the theories and practices for processing and interpreting protein IR spectra. We will then review the current literature on the use of IR spectroscopy to study protein structure and the effects of stabilizers during lyophilization. Next we will concentrate specifically on protein aggregation. The bulk of the research and the key assignments of spectral features in protein aggregates come from studies of the effects of high and low temperature on proteins. Therefore, we will first consider this topic. Finally, we will summarize the recent theoretical and applied work on lyophilization-induced aggregation.

Chymotrypsin↗

PubMed2000

Role of M protein aggregation in defective assembly of temperature-sensitive M protein mutants of vesicular stomatitis virus.

The goal of these experiments was to determine the steps in virus assembly that are defective at the nonpermissive temperature in temperature-sensitive (ts) matrix (M) protein mutants of vesicular stomatitis virus. It has been proposed that mutations in M protein either reduce the binding affinity for nucleocapsids or lead to aggregation, reducing the amount of M protein available for virus assembly. Cytosolic or membrane-derived M proteins from wild-type VSV and two ts M protein mutant viruses, tsM301 and tsO23, as well as a revertant of tsO23 virus, O23R1, were analyzed for binding to nucleocapsid-M protein (NCM) complexes and for M protein aggregation. The experiments presented here showed that ts M proteins synthesized at the nonpermissive temperature were capable of binding to nucleocapsids and that aggregation of ts M proteins did not reduce the amount of soluble M protein below the amount required for assembly of the O23R1 virus. Instead, the most pronounced defect in ts M proteins was in the ability of membrane-derived M proteins to be solubilized in the presence of the detergent Triton X-100. It is proposed that this detergent-insoluble form of M protein interferes with a step necessary to initiate assembly of NCM complexes. A similar detergent, Triton X-114, caused aggregation of membrane-derived wild-type M protein, disproving an earlier proposal that membrane-derived M protein behaves like an integral membrane protein in the presence of Triton X-114. Aggregation of wild-type M protein in the presence of Triton X-100 could be induced by incubation at 37 degrees C with a high-molecular-weight fraction isolated from uninfected cells by sucrose gradient centrifugation. These results implicate host components in inducing M protein aggregation.

Amino Acid Substitution↗

PubMed2005

Increase in the deposition of aggregated protein in the glomeruli of spontaneously diabetic mice.

The aim of this study was to investigate the disposal of aggregated protein in the glomeruli of spontaneously diabetic mice. Diabetic mice, KK-A(y) and db/db, and age-matched ICR mice were injected intravenously with aggregated bovine serum albumin (a-BSA) at 0.6 mg/g, and the glomeruli and the blood were obtained. Diabetic mice had larger amounts of a-BSA in their glomeruli than the ICR mice, threefold in KK-A(y) and twofold in db/db, at 3 h after the a-BSA injection. Additionally, the disappearance of a-BSA was retarded in the diabetic glomeruli. KK-A(y) displayed a-BSA in the glomeruli 24 h after the a-BSA injection and db/db did after 12 h, while the ICR did by 8 h. In spite of increases of insulin to similar degrees in both strains of diabetic mice after the a-BSA injection, blood glucose levels markedly decreased in KK-A(y) compared with db/db. There were no histopathological alterations in the glomeruli of the diabetic mice. Depositions of a-BSA were confirmed to be higher in the diabetic glomeruli by the immunofluorescence technique, and KK-A(y) displayed higher depositions of a-BSA than did db/db. The present study suggests that hyperglycemia is involved in the increased deposition of aggregated protein in the glomeruli and that the degradation of aggregated protein is retarded in diabetic glomeruli.

Animals↗

PubMed1983

[Study of serum protein aggregation after treatment with urea].

Gel chromatography and electrophoresis in polyacrylamide gel show that urea in vitro induces aggregation of blood proteins, with their content unchanged. The aggregation does not depend on the fibrinogen available. Comparison of the data on the effect of urea, DS-Na and thermal denaturation on the protein aggregation suggests that the aggregation is induced by local conformation transformations in the structure of certain protein molecules. At the same time, the data obtained do not permit neglecting the role of denaturation in the aggregation.

Animals↗

PubMed2005

On the quantitative treatment of donor-donor energy migration in regularly aggregated proteins.

An algorithm is presented that quantitatively accounts for donor-donor energy migration (DDEM) among fluorophore-labeled proteins forming regular aggregates. The DDEM algorithm is based on Monte Carlo and Brownian dynamics simulations and applies to calculation of fluorescence depolarisation data, such as the fluorescence anisotropy. Thereby local orientations, as well as reorienting motions of the fluorescent group are considered in the absence and presence of DDEM and among, in principle, infinitely many proteins as they form regular aggregates. Here we apply the algorithm for calculating and illustrating the DDEM and the time-resolved fluorescence anisotropy under static as well as dynamic conditions within helical, linear and circular aggregate structures. A principal approach of the DDEM algorithm for analysing protein aggregates is also outlined.

Algorithms↗

PubMed2002

The threshold for polyglutamine-expansion protein aggregation and cellular toxicity is dynamic and influenced by aging in Caenorhabditis elegans.

Studies of the mutant gene in Huntington's disease, and for eight related neurodegenerative disorders, have identified polyglutamine (polyQ) expansions as a basis for cellular toxicity. This finding has led to a disease hypothesis that protein aggregation and cellular dysfunction can occur at a threshold of approximately 40 glutamine residues. Here, we test this hypothesis by expression of fluorescently tagged polyQ proteins (Q29, Q33, Q35, Q40, and Q44) in the body wall muscle cells of Caenorhabditis elegans and show that young adults exhibit a sharp boundary at 35-40 glutamines associated with the appearance of protein aggregates and loss of motility. Surprisingly, genetically identical animals expressing near-threshold polyQ repeats exhibited a high degree of variation in the appearance of protein aggregates and cellular toxicity that was dependent on repeat length and exacerbated during aging. The role of genetically determined aging pathways in the progression of age-dependent polyQ-mediated aggregation and cellular toxicity was tested by expressing Q82 in the background of age-1 mutant animals that exhibit an extended lifespan. We observed a dramatic delay of polyQ toxicity and appearance of protein aggregates. These data provide experimental support for the threshold hypothesis of polyQ-mediated toxicity in an experimental organism and emphasize the importance of the threshold as a point at which genetic modifiers and aging influence biochemical environment and protein homeostasis in the cell.

Aging↗

PubMed1989

Effect of protein aggregation state on coat protein-mediated protection against tobacco mosaic virus using a transient protoplast assay.

To address the mechanism(s) of protection against tobacco mosaic virus (TMV) infection conferred by expression of the TMV capsid protein (CP) gene in transgenic tobacco plants, a transient protection assay has been developed. Introduction of either purified viral CP or virus inactivated by ultraviolet irradiation into tobacco protoplasts induced a transient protection to challenge virus introduced concomitantly or shortly thereafter. The transient protection was characterized and the effects of different aggregation states of TMV CP were tested in the transient assay system. Tobacco mosaic virus CP preparations composed largely of helical, virus-like, aggregates conferred a less transient protection against TMV and greater protection against a distantly related virus than did preparations composed primarily of smaller aggregates.

Capsid↗

PubMed2006

Rheopexy of synovial fluid and protein aggregation.

Bovine synovial fluid and albumin solutions of similar concentration are rheopectic (stress increases with time in steady shear). This unusual flow characteristic is caused by protein aggregation, and the total stress is enhanced by entanglement of this tenuous protein network with the long-chain polysaccharide sodium hyaluronate under physiological conditions. Neutron scattering measurements on albumin solutions demonstrate protein aggregation and all measurements are consistent with a weak dipolar attraction energy (of order 3kT) that is most likely augmented by hydrophobic interactions and/or disulfide bond formation between proteins. Protein aggregation appears to play an important role in the mechanical properties of blood and synovial fluid. We also suggest a connection between the observed rheopexy and the remarkable lubrication properties of synovial fluid.

Animals↗

PubMed2004

Protein aggregation and neurodegenerative disease.

Neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and prion diseases are increasingly being realized to have common cellular and molecular mechanisms including protein aggregation and inclusion body formation. The aggregates usually consist of fibers containing misfolded protein with a beta-sheet conformation, termed amyloid. There is partial but not perfect overlap among the cells in which abnormal proteins are deposited and the cells that degenerate. The most likely explanation is that inclusions and other visible protein aggregates represent an end stage of a molecular cascade of several steps, and that earlier steps in the cascade may be more directly tied to pathogenesis than the inclusions themselves. For several diseases, genetic variants assist in explaining the pathogenesis of the more common sporadic forms and developing mouse and other models. There is now increased understanding of the pathways involved in protein aggregation, and some recent clues have emerged as to the molecular mechanisms of cellular toxicity. These are leading to approaches toward rational therapeutics.

Amyloid beta-Peptides↗

PubMed2006

A century-old debate on protein aggregation and neurodegeneration enters the clinic.

The correlation between neurodegenerative disease and protein aggregation in the brain has long been recognized, but a causal relationship has not been unequivocally established, in part because a discrete pathogenic aggregate has not been identified. The complexity of these diseases and the dynamic nature of protein aggregation mean that, despite progress towards understanding aggregation, its relationship to disease is difficult to determine in the laboratory. Nevertheless, drug candidates that inhibit aggregation are now being tested in the clinic. These have the potential to slow the progression of Alzheimer's disease, Parkinson's disease and related disorders and could, if administered presymptomatically, drastically reduce the incidence of these diseases. The clinical trials could also settle the century-old debate about causality.

Alzheimer Disease↗

PubMed2001

Protein aggregation as bacterial inclusion bodies is reversible.

Inclusion bodies are refractile, intracellular protein aggregates usually observed in bacteria upon targeted gene overexpression. Since their occurrence has a major economical impact in protein production bio-processes, in vitro refolding strategies are under continuous exploration. In this work, we prove spontaneous in vivo release of both beta-galactosidase and P22 tailspike polypeptides from inclusion bodies resulting in their almost complete disintegration and in the concomitant appearance of soluble, properly folded native proteins with full biological activity. Since, in particular, the tailspike protein exhibits an unusually slow and complex folding pathway involving deep interdigitation of beta-sheet structures, its in vivo refolding indicates that bacterial inclusion body proteins are not collapsed into an irreversible unfolded state. Then, inclusion bodies can be observed as transient deposits of folding-prone polypeptides, resulting from an unbalanced equilibrium between in vivo protein precipitation and refolding that can be actively displaced by arresting protein synthesis. The observation that the formation of big inclusion bodies is reversible in vivo can be also relevant in the context of amyloid diseases, in which deposition of important amounts of aggregated protein initiates the pathogenic process.

Escherichia coli↗

PubMed2003

Protofibrils, pores, fibrils, and neurodegeneration: separating the responsible protein aggregates from the innocent bystanders.

Many neurodegenerative diseases, including Alzheimer's and Parkinson's and the transmissible spongiform encephalopathies (prion diseases), are characterized at autopsy by neuronal loss and protein aggregates that are typically fibrillar. A convergence of evidence strongly suggests that protein aggregation is neurotoxic and not a product of cell death. However, the identity of the neurotoxic aggregate and the mechanism by which it disables and eventually kills a neuron are unknown. Both biophysical studies aimed at elucidating the precise mechanism of in vitro aggregation and animal modeling studies support the emerging notion that an ordered prefibrillar oligomer, or protofibril, may be responsible for cell death and that the fibrillar form that is typically observed at autopsy may actually be neuroprotective. A subpopulation of protofibrils may function as pathogenic amyloid pores. An analogous mechanism may explain the neurotoxicity of the prion protein; recent data demonstrates that the disease-associated, infectious form of the prion protein differs from the neurotoxic species. This review focuses on recent experimental studies aimed at identification and characterization of the neurotoxic protein aggregates.

Amyloid beta-Peptides↗

PubMed2001

Histological evidence of protein aggregation in mutant SOD1 transgenic mice and in amyotrophic lateral sclerosis neural tissues.

The mechanisms leading to neurodegeneration in ALS (amyotrophic lateral sclerosis) are not well understood, but cytosolic protein aggregates appear to be common in sporadic and familial ALS as well as transgenic mouse models expressing mutant Cu/Zn superoxide dismutase (SOD1). In this study, we systematically evaluated the presence of these aggregates in three different mouse models (G93A, G85R, and G37R SOD1) and compared these aggregates to those seen in cases of sporadic and familial ALS. Inclusions and loss of motor neurons were observed in spinal cords of all of these three mutant transgenic lines. Since a copper-mediated toxicity hypothesis has been proposed to explain the cytotoxic gain-of-function of mutant SOD1, we sought to determine the involvement of the copper chaperone for SOD1 (CCS) in the formation of protein aggregates. Although all aggregates contained CCS, SOD1 was not uniformly found in the inclusions. Similarly, CCS-positive skein-like inclusions were rarely seen in ALS neurons. These studies do not provide strong evidence for a causal role of CCS in aggregate formation, but they do suggest that protein aggregation is a common event in all animal models of the disease. Selected proteins, such as the glutamate transporter GLT-1, were not typically observed within the inclusions. Most inclusions were positively stained with antibodies recognizing ubiquitin, proteasome, Hsc70 in transgenic lines, and some Hsc70-positive inclusions were detected in sporadic ALS cases. Overall, these observations suggest that inclusions might be sequestered into ubiquitin-proteasome pathway and some chaperone proteins such as Hsc70 may be involved in formation and/or degradation of these inclusions.

Amyotrophic Lateral Sclerosis↗

PubMed1979

Anaphylactoid reactions to infusions of plasma protein and human serum albumin. Role of aggregated proteins and of stabilizers added during production.

Six patients suffering from anaphylactoid reactions after infusion of pasteurized plasma (PP) or human serum albumin (HSA) were investigated. Clinical symptoms ranged from urticaria and hypotension to cardiac arrest. Immunoglobulin levels, especially of IgA, were normal, as were concentrations of complement factors C3, C4 and factor B. In skin and lymphocyte transformation tests patients, with the exception of one severely allergic to protein, did not react to the monomeric pure HSA. Five out of six patients reacted against HSA aggregates and three patients to the HSA modified by caprylate added as stabilizer during commercial HSA production. It is concluded that the anaphylactoid reactions developing after PP or HSA infusion result from a non-specific reaction to protein aggregates and in some cases possibly from a specific immune response to the caprylate-modified HSA.

Anaphylaxis↗

PubMed2003

Proteasomal inhibition causes the formation of protein aggregates containing a wide range of proteins, including nitrated proteins.

Mutations in Cu,Zn-superoxide dismutase (SOD-1) are associated with some familial cases of amyotrophic lateral sclerosis (ALS), but it is not known how they result in cell death. We examined effects of overexpression of wild-type SOD-1 or the G37R or G85R mutations on the accumulation of ubiquitinated and nitrated proteins, and on loss of cell viability induced by the proteasome inhibitor, lactacystin. Wild-type SOD-1 had no effect on proteasomal activity, but the mutants decreased it somewhat. Treatment with lactacystin (1 micro m) caused only limited cell viability loss, even though it induced a marked inhibition of proteasomal activities. However, viability loss due to apoptosis was substantial in response to lactacystin when cells were overexpressing a mutant SOD-1. The frequency of cells showing immunoreactivity against ubiquitinated- or nitrated-proteins was enhanced when wild-type and mutant SOD-1 s were overexpressed. Ubiquitinated or nitrated alpha-tubulin, SOD-1, alpha-synuclein and 68K neurofilaments were observed in the aggregates. Similar aggregates were observed in cells overexpressing mutant parkin (Del3-5, T240R and Q311'X). The nitric oxide synthase inhibitor, l-NAME, decreased viability loss and aggregation, suggesting that nitration of proteins may play an important role in aggregation and in the cell death accompanying it.

Acetylcysteine↗

PubMedDate not supplied

The effect of macromolecular crowding on protein aggregation and amyloid fibril formation.

Macromolecular crowding is expected to have several significant effects on protein aggregation; the major effects will be those due to excluded volume and increased viscosity. In this report we summarize data demonstrating that macromolecular crowding may lead to a dramatic acceleration in the rate of protein aggregation and formation of amyloid fibrils, using the protein alpha-synuclein. The aggregation of alpha-synuclein has been implicated as a critical factor in development of Parkinson's disease. Various types of polymers, from neutral polyethylene glycols and polysaccharides (Ficolls, dextrans) to inert proteins, are shown to accelerate alpha-synuclein fibrillation. The stimulation of fibrillation increases with increasing length of polymer, as well as increasing polymer concentration. At lower polymer concentrations (typically up to approximately 100 mg/ml) the major effect is ascribed to excluded volume, whereas at higher polymer concentrations evidence of opposing viscosity effects become apparent. Pesticides and metals, which are linked to increased risk of Parkinson's disease by epidemiological studies, are shown to accelerate alpha-synuclein fibrillation under conditions of molecular crowding.

Amyloid↗