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Results for “Protein Aggregation, Pathological”

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PubMed2006

Merger of laser capture microdissection and mass spectrometry: a window into the amyloid plaque proteome.

The occurrence of protein accumulation and aggregation in the brain is one of the pathological hallmarks of neurodegenerative diseases such as Alzheimer's disease (AD). Although it is instructive to analyze the aggregated proteins in the brain, biochemical purification and identification of these proteins have been challenging. Recent developments in laser capture microdissection (LCM) and mass spectrometry (MS) enable large-scale protein profiling of captured tissue samples. We present here the method of analyzing senile plaques from postmortem AD brains by coupling LCM and highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS). First, the senile plaques were stained with thioflavin-S and precisely isolated by adjusted laser beams under a microscope. Total proteins in the isolated tissues were extracted and resolved on an SDS gel. To identify all proteins in the samples, the gel was excised into multiple pieces followed by trypsin digestion. The resulting peptides were further separated by reverse-phase chromatography and analyzed by tandem mass spectrometry. A database search of acquired MS/MS spectra allowed the identification of hundreds to thousands of peptides/proteins in the original samples. Moreover, quantitative comparison of protein composites in different LCM samples could be achieved by MS strategies. For instance, the comparison between plaques and surrounding nonplaque tissues from the same specimen revealed tens of proteins specifically enriched in the plaques. Finally, the data were corroborated by independent experiments using the approach of immunohistochemistry. Taken together, the merger of LCM and MS is a powerful tool to probe the proteome of any given pathological lesions.

Animals↗

PubMed1999

Tissue transglutaminase: an enzyme with a split personality.

Tissue transglutaminase (tTG) belongs to the family of transglutaminase enzymes that catalyze the posttranslational modification of proteins via Ca(2+)-dependent cross-linking reactions. The catalytic action of tTG results in the formation of an isopeptide bond that is of great physiological significance since it is highly resistant to proteolysis and denaturants. Although tTG-mediated cross-linking reactions have been implicated to play a role in diverse biological processes, the precise physiological function of the enzyme remains unclear. Recent data, however, suggest that the protein polymers resulting from tTG-catalyzed reactions may play a role in commitment of cells to undergo apoptosis. On the same token, tTG-mediated formation of insoluble protein aggregates may underlie the markers of numerous pathological conditions, such as the senile plaques in Alzheimer's disease and the Lewy bodies in Parkinson's disease. In addition to catalyzing Ca(2+)-dependent cross-linking reactions, tTG can also bind and hydrolyze guanosine triphosphate and adenosine triphosphate. By virtue of this ability, tTG has been identified as a novel G-protein that interacts and activates phospholipase C following stimulation of the alpha-adrenergic receptor. The ability of tTG to mediate signal transduction may contribute to its involvement in the regulation of cell cycle progression. The following review summarizes the important features of this multifunctional enzyme that have emerged as a result of recent work from different laboratories.

Alzheimer Disease↗

PubMed2001

Dysregulation of human brain microtubule-associated tau mRNA maturation in myotonic dystrophy type 1.

Intraneuronal aggregates of hyperphosphorylated tau proteins, referred to as pathological tau, are found in brain areas of demented patients affected by numerous different neurodegenerative disorders. We previously described a particular biochemical profile of pathological tau proteins in myotonic dystrophy type 1 (DM1). This multisystemic disorder is characterized by an unstable CTG repeat expansion in the 3'-untranslated region of the DM protein kinase gene. In the human central nervous system, tau proteins consist of six isoforms that differ by the presence or absence of the alternatively spliced exons 2, 3 and 10. Here we show that the pattern of tau isoforms aggregated in DM1 brain lesions is characteristic. It consists mainly of the aggregation of the shortest human tau isoform. A disruption in normal tau isoform expression consisting of a reduced expression of tau isoforms containing the exon 2 was observed at both the mRNA and protein levels. Large expanded CTG repeats were detected and showed marked somatic heterogeneity between DM1 cases and in cortical brains regions analysed. Our data suggest a relationship between the CTG repeat expansion and the alteration of tau expression showing that DM1 is a peculiar tauopathy.

Adult↗

PubMed2004

Mitochondrial injury: a hot spot for parkinsonism and Parkinson's disease?

The recent identification of genes (parkin, DJ-1, and PINK1) involved in recessive autosomal parkinsonism, and the indications that these proteins may have protective effects on the mitochondria, has led to the reemergence of the notion that mitochondrial dysfunction might play a central role in the etiology of sporadic Parkinson's disease (PD). This idea has previously been supported by biochemical analyses showing reduced mitochondrial activity in PD patients and in animal models of PD generated by the selective inhibition of mitochondria activity. However, the involvement of DJ-1 or PINK1 loss of function in classical idiopathic PD, characterized by pathological inclusions composed of aggregated alpha-synuclein protein, has still not been evaluated. More detailed studies of the possible interactions between parkin, DJ-1, PINK1, and alpha-synuclein and their effects on mitochondria are needed to more adequately define the biological pathways that may convergently or independently lead to parkinsonism.

Animals↗

PubMed2006

Signaling, polyubiquitination, trafficking, and inclusions: sequestosome 1/p62's role in neurodegenerative disease.

Aggregated misfolded proteins are hallmarks of most neurodegenerative diseases. In a chronic disease state, including pathologic situations of oxidative stress, these proteins are sequestered into inclusions. Accumulation of aggregated proteins can be prevented by chaperones, or by targeting their degradation to the UPS. If the accumulation of these proteins exceeds their degradation, they may impair the function of the proteasome. Alternatively, the function of the proteasome may be preserved by directing aggregated proteins to the autophagy-lysosome pathway for degradation. Sequestosome 1/p62 has recently been shown to interact with polyubiquitinated proteins through its UBA domain and may direct proteins to either the UPS or autophagosome. P62 is present in neuronal inclusions of individuals with Alzheimer's disease and other neurodegenerative diseases. Herein, we review p62's role in signaling, aggregation, and inclusion formation, and specifically as a possible contributor to Alzheimer's disease. The use of p62 as a potential target for the development of therapeutics and as a disease biomarker is also discussed.

Journal Article↗

PubMed2000

Follicular dendritic cells and dissemination of Creutzfeldt-Jakob disease.

The contribution of immune system cells to the propagation of transmissible encephalopathies is not well understood. To determine how follicular dendritic cells (FDC) may act, we challenged lymphotoxin beta null and wild-type (wt) controls with a Creutzfeldt-Jakob disease (CJD) agent. There was only a small difference in incubation time to clinical disease even after peripheral challenge with low infectious doses (31 in a total of 410 days). Brain pathology with extensive microglial infiltration, identified by keratan sulfate, as well as astrocytic hypertrophy, was also equivalent in all groups despite the fact that null mice had neither FDC nor splenic metallophilic macrophages that filter particulate antigen. Because FDC accumulate pathologic prion protein (PrP) in infected but not wt mice, we studied the cellular distribution of PrP by confocal microscopy. The majority of pathologic PrP collected on the plasma membrane of FDC, as identified by the Ca(+2)-binding protein S100A. This surface distribution suggested that agent aggregated with pathologic PrP might spread by cell-to-cell contacts. While several types of leukocytes may be involved in agent dissemination, cells of myeloid lineage were found to be infectious. Moreover, perivascular tracks of microglia and abnormal PrP after intraperitoneal inoculation were consistent with hematogenous spread. In summary, FDC are not required for CJD agent spread from the periphery, although FDC may enhance spread through surface accumulation of pathologic PrP. While it is still not clear where the infectious agent replicates, macrophages can sequester appreciable levels of infectivity and hence act as reservoirs for prolonged latent infection.

Animals↗

PubMed2003

Amphotericin B binds to amyloid fibrils and delays their formation: a therapeutic mechanism?

The membrane-active antifungal agent amphotericin B (AmB) is one of the few agents shown to slow the course of prion diseases in animals. Congo Red and other small molecules have been reported to directly inhibit amyloidogenesis in both prion and Alzheimer peptide model systems via specific binding. We propose that it is possible that AmB may act similarly to physically prevent conversion of the largely alpha-helical prion protein (PrP) to the pathological beta-sheet aggregate protease-resistant isoform (PrP(res)) in prion disease and by analogy prevent fibrillization in amyloid diseases. To assess whether AmB is capable of binding specifically to amyloid fibrils as does Congo Red, we have used the insulin fibril and Abeta 25-35 amyloid model fibril system. We find that AmB does bind strongly to both insulin (K(d) = 1.1 microM) and Abeta 25-35 amyloid (K(d) = 6.4 microM) fibrils but not to native insulin. Binding is characterized by a red-shifted AmB spectrum indicative of a more hydrophobic environment. Thus AmB seems to have a complementary face for amyloid fibrils but not the native protein. In addition, AmB interacts specifically with Congo Red, a known fibril-binding agent. In kinetic fibril formation studies, AmB was able to significantly kinetically delay the formation of Abeta 25-35 fibrils at pH 7.4 but not insulin fibrils at pH 2.

Alzheimer Disease↗

PubMed1999

Assembly of paired helical filaments from mouse tau: implications for the neurofibrillary pathology in transgenic mouse models for Alzheimer's disease.

In Alzheimer's disease and related dementias, human tau protein aggregates into paired helical filaments and neurofibrillary tangles. However, such tau aggregates have not yet been demonstrated in transgenic mouse models of the disease. One of the possible explanations would be that mouse tau has different properties which prevents it from aggregating. We have cloned several murine tau isoforms, containing three or four repeats and different combinations of inserts, expressed them in Escherichia coli and show here that they can all be assembled into paired helical filaments similar to those in Alzheimer's disease, using the same protocols as with human tau. Therefore, the absence of pathologically aggregated tau in transgenic mice cannot be explained by intrinsic differences in mouse tau protein and instead must be explained by other as yet unknown factors.

Alzheimer Disease↗

PubMed2004

Aggregation of the Acylphosphatase from Sulfolobus solfataricus: the folded and partially unfolded states can both be precursors for amyloid formation.

Protein aggregation is associated with a number of human pathologies including Alzheimer's and Creutzfeldt-Jakob diseases and the systemic amyloidoses. In this study, we used the acylphosphatase from the hyperthermophilic Archaea Sulfolobus solfataricus (Sso AcP) to investigate the mechanism of aggregation under conditions in which the protein maintains a folded structure. In the presence of 15-25% (v/v) trifluoroethanol, Sso AcP was found to form aggregates able to bind specific dyes such as thioflavine T, Congo red, and 1-anilino-8-naphthalenesulfonic acid. The presence of aggregates was confirmed by circular dichroism and dynamic light scattering. Electron microscopy revealed the presence of small aggregates generally referred to as amyloid protofibrils. The monomeric form adopted by Sso AcP prior to aggregation under these conditions retained enzymatic activity; in addition, folding was remarkably faster than unfolding. These observations indicate that Sso AcP adopts a folded, although possibly distorted, conformation prior to aggregation. Most important, aggregation appeared to be 100-fold faster than unfolding under these conditions. Although aggregation of Sso AcP was faster at higher trifluoroethanol concentrations, in which the protein adopted a partially unfolded conformation, these findings suggest that the early events of amyloid fibril formation may involve an aggregation process consisting of the assembly of protein molecules in their folded state. This conclusion has a biological relevance as globular proteins normally spend most of their lifetime in folded structures.

Acid Anhydride Hydrolases↗

PubMed1994

Pathogenic potential of human monoclonal immunoglobulin light chains: relationship of in vitro aggregation to in vivo organ deposition.

The deposition of certain Bence Jones proteins as tubular casts, basement membrane precipitates, or amyloid fibrils results in the human light-chain-associated renal and systemic diseases--myeloma (cast) nephropathy, light-chain deposition disease, and immunocyte-derived (primary or AL) amyloidosis. To determine if light-chain nephrotoxicity or amyloidogenicity is related to the propensity of these components to form high molecular weight aggregates under physiological conditions, we used a size-exclusion chromatographic system to study 40 different Bence Jones proteins. Each samples was tested over a wide range of protein concentration in three different buffers varying in pH, osmolality, and the presence or absence of low concentrations of urea. Thirty-three of the 35 proteins found clinically and/or experimentally to form in vivo pathologic light-chain deposits were shown to undergo high-order self-association and form high molecular weight aggregates. In contrast, of five nonpathologic proteins, one showed polymerization under the chromatographic conditions used. The correlation between the in vivo results achieved by size-exclusion chromatography and that found in vivo provides (i) a rapid diagnostic method to identify potential nephrotoxic or amyloidogenic Bence Jones proteins and (ii) an experimental means to gain new insight into the physicochemical basis of light-chain aggregation and the treatment of those invariably fatal disorders associated with pathologic light-chain deposition.

Bence Jones Protein↗

PubMed1999

Properties of polyglutamine expansion in vitro and in a cellular model for Huntington's disease.

Eight neurodegenerative diseases have been shown to be caused by the expansion of a polyglutamine stretch in specific target proteins that lead to a gain in toxic property. Most of these diseases have some features in common. A pathological threshold of 35-40 glutamine residues is observed in five of the diseases. The mutated proteins (or a polyglutamine-containing subfragment) form ubiquitinated aggregates in neurons of patients or mouse models, in most cases within the nucleus. We summarize the properties of a monoclonal antibody that recognizes specifically, in a Western blot, polyglutamine stretches longer than 35 glutamine residues with an affinity that increases with polyglutamine length. This indicates that the pathological threshold observed in five diseases corresponds to a conformational change creating a pathological epitope, most probably involved in the aggregation property of the carrier protein. We also show that a fragment of a normal protein carrying 38 glutamine residues is able to aggregate into regular fibrils in vitro. Finally, we present a cellular model in which the induced expression of a mutated full-length huntingtin protein leads to the formation of nuclear inclusions that share many characteristics with those observed in patients: those inclusions are ubiquitinated and contain only an N-terminal fragment of huntingtin. This model should thus be useful in studying a processing step that is likely to be important in the pathogenicity of mutated huntingtin.

Animals↗

PubMed2005

Regulation of tau isoform expression and dementia.

In the central nervous system (CNS), aberrant changes in tau mRNA splicing and consequently in protein isoform ratios cause abnormal aggregation of tau and neurodegeneration. Pathological tau causes neuronal loss in Alzheimer's disease (AD) and a diverse group of disorders called the frontotemporal dementias (FTD), which are two of the most common forms of dementia and afflict more than 10% of the elderly population. Autosomal dominant mutations in the tau gene cause frontotemporal dementia with parkinsonism-chromosome 17 type (FTDP-17). Just over half the mutations affect tau protein function and decrease its affinity for microtubules (MTs) or increase self-aggregation. The remaining mutations occur within exon 10 (E10) and intron 10 sequences and alter complex regulation of E10 splicing by multiple mechanisms. FTDP-17 splicing mutations disturb the normally balanced levels of distinct protein isoforms that result in altered biochemical and structural properties of tau. In addition to FTDP-17, altered tau isoform levels are also pathogenically associated with other FTD disorders such as progressive supranuclear palsy (PSP), corticobasal degeneration and Pick's disease; however, the mechanisms remain undefined and mutations in tau have not been detected. FTDP-17 highlights the association between splicing mutations and the pronounced variability in pathology as well as phenotype that is characteristic of inherited disorders.

Alternative Splicing↗

PubMed2003

Early and reversible neuropathology induced by tetracycline-regulated lentiviral overexpression of mutant huntingtin in rat striatum.

The ability to overexpress full-length huntingtin or large fragments represents an important challenge to mimic Huntington's pathology and reproduce all stages of the disease in a time frame compatible with rodent life span. In the present study, tetracycline-regulated lentiviral vectors leading to high expression levels were used to accelerate the pathological process. Rats were simultaneously injected with vectors coding for the transactivator and wild type (WT) or mutated huntingtin (TRE-853-19Q/82Q) in the left and right striatum, respectively, and analyzed in the 'on' and 'off' conditions. Overexpression of TRE-853-19Q protein or residual expression of TRE-853-82Q in 'off' condition did not cause any significant neuronal pathology. Overexpressed TRE-853-82Q protein led to proteolytic release of N-terminal htt fragments, nuclear aggregation, and a striatal dysfunction as revealed by decrease of DARPP-32 staining but absence of NeuN down-regulation. The differential effect on the DARPP-32/NeuN neuronal staining was observed as early as 1 month after injection and maintained at 3 months. In contrast, expression of a shorter htt form (htt171-82Q) did not require processing prior formation of nuclear aggregates and caused decrease of both DARPP-32 and NeuN neuronal markers at one month post-injection suggesting that polyQ pathology may be dependent on protein context. Finally, the reversibility of the pathology was assessed. Huntingtin expression was turn 'on' for 1 month and then shut 'off' for 2 months. Recovery of DARPP-32 immunoreactivity and clearance of huntingtin aggregates were observed in animals treated with doxycycline. These results suggest that a tetracycline-regulated system may be particularly attractive to model Huntington's disease and induce early and reversible striatal neuropathology in vivo.

Animals↗

PubMed2005

Protein aggregation, metals and oxidative stress in neurodegenerative diseases.

There is clear evidence implicating oxidative stress in the pathology of many different neurodegenerative diseases. ROS (reactive oxygen species) are the primary mediators of oxidative stress and many of the aggregating proteins and peptides associated with neurodegenerative disease can generate hydrogen peroxide, a key ROS, apparently through interactions with redox-active metal ions. Our recent results suggest that ROS are generated during the very early stages of protein aggregation, when protofibrils or soluble oligomers are present, but in the absence of mature amyloid fibrils. The generation of ROS during early-stage protein aggregation may be a common, fundamental molecular mechanism underlying the pathogenesis of oxidative damage, neurodegeneration and cell death in several different neurodegenerative diseases. Drugs that specifically target this process could be useful in the future therapy of these diseases.

Humans↗

PubMed2006

Calpain-resistant fragment(s) of alpha-synuclein regulates the synuclein-cleaving activity of 20S proteasome.

Alpha-synuclein is a pathological component of Parkinson's disease by participating in Lewy body formation. Imbalance in protein turnover could result in the abnormal protein aggregation responsible for eventual neuronal cell death. This in vitro digestion study showed that both m-calpain and 20S proteasome preferentially hydrolyzed the N-terminal half of alpha-synuclein, which made the hydrophobic NAC and following acidic C-terminal region resistant against the proteolyses. Since the acidic C-terminal region contains the PEST segment-a protein degradation signal enriched with amino acids of proline (P), glutamate (E), serine (S), and threonine (T)-, the PEST segment has not been processed or even required for the proteolyses. Alpha-synuclein would be recognized primarily by m-calpain since the common substrate was processed by m-calpain five times more effectively than 20S proteasome with k(cat)/K(m) of 1.64 x 10(4)M(-1)s(-1) and 0.32 x 10(4) M(-1)s(-1), respectively. The N-terminally truncated protease-resistant C-terminal fragment of alpha-syn61-140 was demonstrated to stimulate the 20S proteasome-mediated breakdown of alpha-synuclein and its mutant forms of Ala53Thr and Ala30Pro. The stimulation for Ala53Thr, however, was noticeably less efficient than those for the other proteins, which might support the previous observation of the prolonged intracellular life span of Ala53Thr by 1.5-fold compared to that of wild-type form. We have hypothesized that the N-terminally truncated C-terminal fragment derived from the abundant alpha-synuclein through intracellular proteolyses could be involved in various physiological or pathological effects which might be related to the formation of abnormal protein aggregation and subsequent neuronal degeneration by influencing the intracellular protein turnover or directly participating in the aggregate formation.

Amino Acid Sequence↗

PubMed2001

Protein oxidation and 20S proteasome-dependent proteolysis in mammalian cells.

The generation of reactive oxygen species is an inevitable aspect of aerobic life. In addition to being exposed to free radicals in the environment, aerobic organisms must also deal with oxygen radicals generated as byproducts of a number of physiological mechanisms for example, by the mitochondrial and endoplasmic reticulum electron transport chains, and by cells of the immune system. Although most organisms are equipped with several lines of defense against oxidative stress, these defensive mechanisms are not 100% effective, and oxidatively modified forms of proteins accumulate during aging, and in many pathological conditions. Oxidatively modified proteins can form large aggregates due to covalent cross-linking or increased surface hydrophobicity. Unless repaired or removed from cells, these oxidized proteins are often toxic and can threaten cell viability. Mammalian cells exhibit only limited direct repair mechanisms, and oxidatively damaged proteins appear to undergo selective proteolysis, primarily by the major cytosolic proteinase, the proteasome. Interestingly, it appears that the 20S 'core' proteasome conducts the recognition and elimination of oxidized proteins in an ATP-independent and ubiquitin-independent pathway.

Adenosine Triphosphate↗

PubMed2005

NMR analysis of synthetic human serum albumin alpha-helix 28 identifies structural distortion upon amadori modification.

The non-enzymatic reaction between reducing sugars and long-lived proteins in vivo results in the formation of glycation and advanced glycation end products, which alter the properties of proteins including charge, helicity, and their tendency to aggregate. Such protein modifications are linked with various pathologies associated with the general aging process such as Alzheimer disease and the long-term complications of diabetes. Although it has been suggested that glycation and advanced glycation end products altered protein structure and helicity, little structural data and information currently exist on whether or not glycation does indeed influence or change local protein secondary structure. We have addressed this problem using a model helical peptide system containing a di-lysine motif derived from human serum albumin. We have shown that, in the presence of 50 mm glucose and at 37 degrees C, one of the lysine residues in the di-lysine motif within this peptide is preferentially glycated. Using NMR analysis, we have confirmed that the synthetic peptide constituting this helix does indeed form a alpha-helix in solution in the presence of 30% trifluoroethanol. Glycation of the model peptide resulted in the distortion of the alpha-helix, forcing the region of the helix around the site of glycation to adopt a 3(10) helical structure. This is the first reported evidence that glycation can influence or change local protein secondary structure. The implications and biological significance of such structural changes on protein function are discussed.

Amino Acid Motifs↗

PubMed2003

Subcellular localisation, secretion, and post-translational processing of normal cochlin, and of mutants causing the sensorineural deafness and vestibular disorder, DFNA9.

Five missense mutations in the FCH/LCCL domain of the COCH gene, encoding the protein cochlin, are pathogenic for the autosomal dominant hearing loss and vestibular dysfunction disorder, DFNA9. To date, the function of cochlin and the mechanism of pathogenesis of the mutations are unknown. We have used the biological system of transient transfections of the entire protein coding region of COCH into several mammalian cell lines, to investigate various functional properties of cochlin. By western blot analysis of lysates prepared from transfected cells, we show that cochlin is a secreted protein. Immunocytochemistry shows concentrated localisation of cochlin in perinuclear structures consistent with the Golgi apparatus and endoplasmic reticulum, showing intracellular passage through these secretory compartments. We detected that cochlin is proteolytically cleaved between the FCH/LCCL domain and the downstream vWFA domains, resulting in a smaller cochlin isoform of approximately 50 kDa. Interestingly, this isoform lacks the entire mutation bearing FCH/LCCL domain. We have also shown that cochlin is N-glycosylated in its mature secreted form. Previous studies of the FCH/LCCL domain alone, expressed in bacteria, have demonstrated that three of four DFNA9 mutations cause misfolding of this domain. Characteristic eosinophilic deposits in DFNA9 affected inner ear structures could be the result of aberrant folding, secretion, or solubility of mutated cochlins, as in certain other pathological states in which misfolded proteins accumulate and aggregate causing toxicity. To examine the biological consequences of cochlin misfolding, we made separate constructs with three of the DFNA9 mutations and performed parallel studies of the mutated and wild type cochlins. We detected that mutated cochlins are not retained intracellularly, and are able to be secreted adequately by the cells, through the Golgi/ER secretory pathway, and also undergo proteolytic cleavage and glycosylation. These results suggest that DFNA9 mutations may manifest deleterious effects beyond the point of secretion, in the unique environment of the extracellular matrix of the inner ear by disrupting cochlin function or interfering with protein-protein interactions involving the FCH/LCCL domain. It is also possible that the mutations may result in aggregation of cochlin in vivo over a longer time course, as supported by the late onset and progressive nature of this disorder.

3T3 Cells↗