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Results for “Protein Aggregation, Pathological”

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PubMed2001

Conformational change, aggregation and fibril formation induced by detergent treatments of cellular prion protein.

The conversion of protease-sensitive prion protein (PrP-sen) to a high beta-sheet, protease-resistant and often fibrillar form (PrP-res) is a central event in transmissible spongiform encephalopathies (TSE) or prion diseases. This conversion can be induced by PrP-res itself in cell-free conversion reactions. The detergent sodium N-lauroyl sarkosinate (sarkosyl) is a detergent that is widely used in PrP-res purifications and is known to stimulate the PrP-res-induced conversion reaction. Here we report effects of sarkosyl and other detergents on recombinant hamster PrP-sen purified from mammalian cells under oxidizing conditions that maintain the single native disulfide bond. Low concentrations of sarkosyl (0.001-0.1%) induced aggregation of PrP-sen molecules, increased light scattering, altered fluorescence excitation and emission spectra, and enhanced the proportion of beta-sheet secondary structure according to circular dichroism and infrared spectroscopies. An enhancement of beta-sheet content was also seen with 0.001% sodium dodecyl sulfate (SDS) but not several other types of detergents. Electron microscopy revealed that sarkosyl induced the formation of both amorphous and fibrillar aggregates. The fibrils appeared to be constructed from spherical bead-like protofibrils. Neither TSE infectivity nor the characteristic partial proteinase K resistance of PrP-res was detected in the sarkosyl-induced PrP aggregates. We conclude that certain anionic detergents can disrupt the conformation of PrP-sen and induce high beta-sheet aggregates that are distinct from scrapie-associated PrP-res in terms of protease-resistance, infrared spectrum and infectivity. These results reinforce the idea that not all high-beta aggregates of PrP are equivalent to the pathologic form, PrP-res.

Animals↗

PubMed2003

Ultraviolet radiation decreases expression and induces aggregation of corneal ALDH3A1.

Substantial reduction in corneal ALDH3A1 enzymatic activity associated with eye pathology was previously reported in C57BL/6J mice subjected to ultraviolet radiation (UVR). The aim of this study was to examine whether UVR diminishes corneal ALDH3A1 expression through modifications at the transcriptional, translational, or post-translational level. Adult C57BL/6J mice were subjected to UVR exposure (302 nm peak wavelength) for various periods of time, and corneal ALDH3A1 mRNA and protein levels were monitored by Northern and Western blot analysis, respectively. In addition, ALDH3A1 enzymatic activity was determined as a measure of post-translational modification. Mice exposed to 0.2 J/cm(2) UVB radiation demonstrated an extensive decrease, approximately 80%, in mRNA and protein levels, as well as enzymatic activity of corneal ALDH3A1. Significant reductions in corneal ALDH3A1 enzymatic activity were detected in mice 96 h after exposure to 0.05 and 0.1 J/cm(2) UVB radiation; no significant changes were observed in mRNA and protein levels. These data suggest that UVB down-regulates corneal ALDH3A1 expression at the transcriptional and/or post-translational level depending on the dose of UVB. Reduction in gene transcription requires UVB doses greater than or equal to 0.2 J/cm(2). In vitro experiments with human corneal epithelial cell lines stably transfected with human ALDH3A1 cDNA, and with purified recombinant human ALDH3A1 protein, indicated that ALDH3A1 undergoes post-translational modifications after UVR exposure. These modifications result in both covalent and non-covalent aggregation of the protein with no detectable precipitation. Such conformational changes may be associated with the function of ALDH3A1 as a chaperone-like molecule in the cornea.

Aldehyde Dehydrogenase↗

PubMed2001

Reduced protein phosphatase 2A activity induces hyperphosphorylation and altered compartmentalization of tau in transgenic mice.

Hyperphosphorylated isoforms of the microtubule-associated protein tau are the major components of neurofibrillary lesions in Alzheimer's disease (AD). Protein phosphatase (PP) 2A is a major phosphatase implicated in tau dephosphorylation in vitro. Dephosphorylation of tau can be blocked in vivo by okadaic acid, a potent inhibitor of PP2A. Moreover, activity of PP2A is reduced in AD brains. To elucidate the role of PP2A in tau phosphorylation and pathogenesis, we expressed a dominant negative mutant form of the catalytic subunit Calpha of PP2A, L199P, in mice by using a neuron-specific promoter. We obtained mice with high expression levels of Calpha L199P in cortical, hippocampal, and cerebellar neurons. PP2A activity in brain homogenates of transgenic mice was reduced to 66%. Endogenous tau protein was hyperphosphorylated at distinct sites including the AT8 epitope Ser-202/Thr-205, a major AD-associated tau phosphoepitope. AT8-positive tau aggregates accumulated in the soma and dendrites of cortical pyramidal cells and cerebellar Purkinje cells and co-localized with ubiquitin. Our data establish that PP2A plays a crucial role in tau phosphorylation. Our results also show that reduced PP2A activity is associated with altered compartmentalization and ubiquitination of tau, resembling a key pathological finding in AD.

Animals↗

PubMed1994

Existence of trichohyalin-keratohyalin hybrid granules: co-localization of two major intermediate filament-associated proteins in non-follicular epithelia.

Trichohyalin is a protein of relatively high molecular weight (approximately 200 kDa), associated with intermediate filaments, that was for many years thought to be expressed only in the inner root sheath and medulla of the hair follicle. We show here, however, that this protein is expressed in association with (pro)filaggrin in the granular layer of many non-follicular, keratinizing, stratified epithelia which also express keratins K6/K16, including those of the filiform papillae of dorsal tongue epithelia. In this epithelium, which elaborates morphologically heterogeneous keratohyalin granules in its upper cell layers, trichohyalin forms hybrid granules with filaggrin, the major intermediate filament associated protein found in keratohyalin granules, which is normally expressed in advanced epidermal differentiation. These two intermediate filament-associated proteins remain physically segregated in the hybrid granules, but they share the same fate, as they both become dispersed in transitional cells, and are undetectable in cornified cells. Trichohyalin was also detected in nail matrix epithelia, the epithelium of Hassal's corpuscles of the thymus, and newborn foreskin epidermis. It is essentially absent from normal trunk and scalp epidermis, but is expressed in a few scattered cells of the granular layer that are also filaggrin-positive. In addition, trichohyalin is expressed in the epidermis in a number of hyperplastic skin diseases. These findings demonstrate that trichohyalin is not peculiar to a small number of hair follicle cells, but is expressed in a number of normal and pathological epithelia where it is uniquely associated with filaggrin. In addition, since all these trichohyalin-expressing keratinocytes also synthesize keratins K6 and K16 (the markers for an "alternative" pathway of keratinocyte differentiation), this raises the possibility that the trichohyalin protein is specifically (or preferentially) involved in aggregating intermediate filaments containing the K6/K16 keratins.

Animals↗

PubMed2005

Is platelet adhesion assay able to quantify drug-induced platelet dysfunction?

The platelet adhesion assay (PADA) is an innovative method for the detection of both normal, pathologically increased or decreased platelet adhesiveness. Adhesion is the first important phase of platelet activation, followed by shape change and aggregation. Adhesion is triggered by glycoproteins (GP) on the platelet surface, mainly by GPIIb/IIIa, and to a lesser extent also by GPIb/V/IX. Since fibrinogen serves as adhesive protein for GPIIb/IIIa receptors, and since the PADA uses polymer particles that become coated with fibrinogen, the PADA is able to monitor GPIIb/IIIa receptor antagonists and to detect overdosing, potentially leading to bleeding complications. Ex vivo, citrated whole blood from healthy volunteers and patients was spiked with increasing GPIIb/IIIa inhibitor concentrations and PADA was measured. Comparing these results with GPIIb/IIIa receptor occupancy, determined by FACS, a basic consistency of the data was shown. Via intracellular signaling, the adenosine diphosphate (ADP) receptor mechanism is closely involved in the activation of GPIIb/IIIa receptors so that also ADP receptor antagonists of the thienopyridine type, especially clopidogrel, can be quantitatively determined by the PADA. In patients under clopidogrel therapy, the therapeutic effect was monitored and also individual dose adjustments were realized. Furthermore, patients having partial or full clopidogrel resistance were identified. Overdoses can be detected as well.

Abciximab↗

PubMed1990

Impaired hemostasis and platelet function in rats fed low zinc diets based on egg white protein.

Zinc-deficient rats exhibit impaired hemostasis, a pathological sign related to defective platelet function. The original observation was made in rats fed a low zinc diet based on soy protein. The present study was designed to test the effect of feeding a low zinc, egg white-based diet on bleeding time and platelet aggregation. The reversibility of the defect and the response of washed platelets from rats fed low zinc (less than 1 mg/kg) and control (100 mg/kg) diets were also assessed. Immature male rats were fed the respective diets for 11 d, the controls being both ad libitum- and pair-fed. To test reversibility, rats depleted for 11 d were then fed the control diet for an additional 7 d. Saline bleeding time was increased, and aggregation of platelet-rich plasma (PRP) from rats of low zinc status was impaired. The rate of the secondary phase of aggregation was significantly less than that of controls, but it was not different from that of controls after 7 d of zinc repletion. Aggregation of washed platelets was also impaired by low zinc status, showing that the defect is associated with the platelet, not the plasma. Egg white as a source of protein in the low zinc diet resulted in abnormal hemostasis and platelet aggregation, including a defective response of washed platelets.

Animals↗

PubMed2001

Role of chaperones in nuclear translocation and transactivation of steroid receptors.

Molecular chaperones assist proteins to reach their mature and functional conformation. It has become apparent in recent years that chaperones function as part of a multiprotein heterocomplex that is potentially involved not only in protein folding, but also in intracellular trafficking and in targeting proteins for degradation. In the case of steroid receptors, the activity of the chaperone heterocomplex, as well as the proteins comprising the heterocomplex, has an effect on the observed ligand-dependent transcriptional activity of the receptor. The direct interaction between chaperones and steroid receptors makes them potential therapeutic targets in a number of pathologic conditions. In the case of cancers with steroid receptor involvement, such as breast and prostate cancer, the inhibition of chaperone activity may inhibit tumor cell growth. Conversely, enhancement of chaperone activity may be beneficial in disorders of protein misfolding, as in the case of androgen receptor aggregates found in Spinal and Bulbar Muscular Atrophy.

Biological Transport↗

PubMed2006

The brain-specific protein TPPP/p25 in pathological protein deposits of neurodegenerative diseases.

Immunohistochemical detection of protein components of pathological inclusions is widely used for neuropathological diagnosis of neurodegenerative disorders. However, different antibodies and antigen unmasking methods may account for variability between research studies and thus may affect diagnostic accuracy. Using two different antibodies raised against either a segment (184-200 aa) or the full length of human recombinant brain-specific tubulin polymerization promoting protein TPPP/p25, we immunohistochemically screened neurodegenerative disorders, both with and without pathological alpha-synuclein structures. We tested three different epitope unmasking methods, we applied laser confocal microscopy to evaluate double immunolabelling, and we compared the amount of structures exhibiting TPPP/p25 and alpha-synuclein immunoreactivity. We demonstrate that there are a variety of staining patterns depending on the epitope retrieval method and antibody used. The antibody raised against aa 184-200 segment of TPPP/p25 is better in immunolabelling the majority of alpha-synuclein immunopositive neuronal and glial pathological profiles detectable in Parkinson's disease, diffuse Lewy-body disease, and multiple system atrophy, in addition to immunostaining some extracellular huntingtin immunoreactive structures, lipofuscin, and neuromelanin particles. In contrast, the one raised against the full-length human recombinant TPPP/p25 is more suitable to immunodetect normal oligodendrocytes. Exposition of the segment aa 184-200 of TPPP/p25 in the aggregates of pathological inclusions renders this antibody a reliable marker of all types of alpha-synucleinopathies and suggests a role for TPPP/p25 in the aggregation process of some neurodegenerative conditions.

Adult↗

PubMed2004

A single-chain Fv intrabody provides functional protection against the effects of mutant protein in an organotypic slice culture model of Huntington's disease.

Huntington's disease (HD) is a progressive, hereditary, neurodegenerative disorder caused by an expanded polyglutamine tract in huntingtin protein, leading to misfolding and abnormal protein-protein interactions. Reducing the initial misfolding should lead to decreased pathogenesis. We show that malonate stress increases the number of dead or dying cells when organotypic slice cultures are transduced to express pathological-length huntingtin fragments. Co-transfected anti-HD single-chain Fv (sFv) intrabodies can reverse this HD-specific increase in malonate-induced morbidity.

Animals↗

PubMed1984

Microtubule-associated protein 2: monoclonal antibodies demonstrate the selective incorporation of certain epitopes into Alzheimer neurofibrillary tangles.

Neurofibrillary tangles (NFT) are the principal structural alteration of neuronal cell bodies in Alzheimer disease as well as in normal aging of the human brain. While the ultrastructure of these intraneuronal lesions has been extensively studied, the biochemical composition of the fibers comprising the NFT is unknown. We report the production of three monoclonal antibodies against the microtubule-associated protein 2 (MAP-2), one of which intensely labels Alzheimer NFT. All three antibodies specifically recognize MAP-2 on immunoblots and stain brain tissue in a characteristic dendritic pattern. The three antibodies are directed against at least two different antigenic sites on the MAP-2 molecule, and one appears to recognize a phosphorylation site on MAP-2. That only one of the three antibodies immunolabels NFT suggests that the formation of the tangle involves some modification of the MAP-2 molecule. Our findings suggest that one aspect of Alzheimer-type neurofibrillary pathology is an aggregation of MAP-2 or MAP-2 fragments with altered neurofilamentous elements present in NFT. Normal interactive function, which putatively occurs between neurofilaments and MAP-2, may thus be disrupted in Alzheimer disease.

Alzheimer Disease↗

PubMed2003

Stress proteins and glial functions: possible therapeutic targets for neurodegenerative disorders.

Recent findings suggest that unfolded or misfolded proteins participate in the pathology of several neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease. Usually, several stress proteins and glial cells act as intracellular molecular chaperones and show chaperoning neuronal function, respectively. In the brains of patients with neurodegenerative disorders, however, stress proteins are expressed and frequently associated with protein aggregates, and glial cells are activated around degenerative regions. In addition, several stress proteins and glial cells may also regulate neuronal cell death and loss. Therefore, some types of stress proteins and glial cells are considered to be neuroprotective targets. We summarize the current findings regarding the neuroprotective effects of stress proteins and glial cells, and discuss the possibility of using this knowledge to develop new therapeutic strategies to treat neurodegeneration.

Alzheimer Disease↗

PubMed2002

Gamma-secretase inhibition.

Development of Alzheimer's disease (AD) pathology appears to be causally related to age-dependent changes in the metabolism of the amyloid-beta peptide (A beta), leading to its enhanced aggregation and deposition. gamma-Secretase is a crucial enzyme for the generation of A beta from the amyloid-beta precursor protein and thus represents a valid potential therapeutic target for the treatment or prevention of AD. Enzyme activity has been shown to be dependent on the expression of presenilins and the identification of inhibitors containing transition-state analogue mimics, together with mutagenesis and knockout studies, confirms that presenilins may provide at least a component of the catalytic site for this putative aspartyl protease. Considerable effort has been expended to identify compounds which specifically reduce gamma-secretase activity in the central nervous system, and those with the appropriate properties are being utilized in on-going proof-of-concept studies in animals and humans, to determine the extent and duration of gamma-secretase inhibition required to elicit therapeutic benefits. gamma-Secretase-mediated substrate cleavage appears to fall into the category of 'regulated intramembrane proteolysis'. By virtue of its mechanistic similarities, the effects of gamma-secretase inhibitors on proteolysis and signalling through other substrates, such as Notch, has to be determined carefully, since this is likely to impact on the clinically safe dose of these compounds.

Alzheimer Disease↗

PubMed2000

[Familial myopathy with desmin storage seen as a granulo-filamentar, electron-dense material with mutation of the alphaB-cristallin gene].

Two familial cases of a myopathy remarkable by the presence of a granulo-filamentar, electron dense material were reported in 1978. In a second step, in 1988, it was demonstrated that this material contained an abnormally-phosphorylated desmin. During the last twenty years, the occurrence of new cases in this family confirmed the autosomal dominant inheritance of the disease, and made it potentially informative for molecular genetics studies. This allowed first to map the disease on chromosome11q21-23, and afterwards to identify a mutation within a gene coding for a chaperone protein, alphaBcrystallin. An extensive clinical, pathological and genetic study of this princeps family is herein reported in detail. First, it showed the possible detection of histopathological changes in presymptomatic patients. Second, it allowed to demonstrate the simultaneous occurrence of both alphaBcrystallin and desmin in the granulo-filamentar aggregates. Third, this study provided a precise knowledge of the evolution rate of the disease. The analysis of similar observations reported in the literature clearly shows the clinical, pathological and genetic heterogeneity of this new neuro-muscular disorder.

Actin Cytoskeleton↗

PubMed2002

Lentiviral-mediated delivery of mutant huntingtin in the striatum of rats induces a selective neuropathology modulated by polyglutamine repeat size, huntingtin expression levels, and protein length.

A new strategy based on lentiviral-mediated delivery of mutant huntingtin (htt) was used to create a genetic model of Huntington's disease (HD) in rats and to assess the relative contribution of polyglutamine (CAG) repeat size, htt expression levels, and protein length on the onset and specificity of the pathology. Lentiviral vectors coding for the first 171, 853, and 1520 amino acids of wild-type (19 CAG) or mutant htt (44, 66, and 82 CAG) driven by either the phosphoglycerate kinase 1 (PGK) or the cytomegalovirus (CMV) promoters were injected in rat striatum. A progressive pathology characterized by sequential appearance of ubiquitinated htt aggregates, loss of dopamine- and cAMP-regulated phosphoprotein of 32 kDa staining, and cell death was observed over 6 months with mutant htt. Earlier onset and more severe pathology occurred with shorter fragments, longer CAG repeats, and higher expression levels. Interestingly, the aggregates were predominantly located in the nucleus of PGK-htt171-injected rats, whereas they were present in both the nucleus and processes of CMV-htt171-injected animals expressing lower transgene levels. Finally, a selective sparing of interneurons was observed in animals injected with vectors expressing mutant htt. These data demonstrate that lentiviral-mediated expression of mutant htt provides a robust in vivo genetic model for selective neural degeneration that will facilitate future studies on the pathogenesis of cell death and experimental therapeutics for HD.

Animals↗

PubMedDate not supplied

[Thrombotic thrombocytopenic purpura: clinico-pathologic characteristics and therapy].

Thrombotic thrombocytopenic purpura (TTP) is disseminated form of thrombotic microangiopathy. Classic clinical presentation is characterized by microangiopathic hemolytic anemia, thrombocytopenia, fluctuating neurologic symptoms, fever and impaired renal function. In the pathogenesis of TTP important role have changes in endothelial cells of small blood vessels, apoptosis, elevated endothelial derived proteins, anti-endothelial cell antibodies and platelet aggregating factors. In vast majority of patients with TTP deficient protease activity for von Willebrand factor (vWF), due to the presence of IgG antibodies is reported. Predisposing factors for development of TTP are infections, collagen vascular diseases, pregnancy, cancer, drugs, bone marrow transplantation, and genetic susceptability to TTP syndrome. Plasma exchange transfusions is the standard way of treatment. Additional treatment modalities with corticosteroids, cytotoxic drugs, antiplatelet drugs, intravenous immunoglobulins and splenectomy are applied.

Humans↗

PubMed2001

Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding.

Prions are the infectious agents responsible for transmissible spongiform encephalopathies. The principal component of prions is the glycoprotein PrP(Sc), which is a conformationally modified isoform of a normal cell-surface protein called PrP(C) (ref. 1). During the time between infection and the appearance of the clinical symptoms, minute amounts of PrP(Sc) replicate by conversion of host PrP(C), generating large amounts of PrP(Sc) aggregates in the brains of diseased individuals. We aimed to reproduce this event in vitro. Here we report a procedure involving cyclic amplification of protein misfolding that allows a rapid conversion of large excess PrP(C) into a protease-resistant, PrP(Sc)-like form in the presence of minute quantities of PrP(Sc) template. In this procedure, conceptually analogous to polymerase chain reaction cycling, aggregates formed when PrP(Sc) is incubated with PrP(C) are disrupted by sonication to generate multiple smaller units for the continued formation of new PrP(Sc). After cyclic amplification more than 97% of the protease-resistant PrP present in the sample corresponds to newly converted protein. The method could be applied to diagnose the presence of currently undetectable prion infectious agent in tissues and biological fluids, and may provide a unique opportunity to determine whether PrP(Sc) replication results in the generation of infectivity in vitro.

Animals↗

PubMed1995

Thyroid epithelial cells produce large amounts of the Alzheimer beta-amyloid precursor protein (APP) and generate potentially amyloidogenic APP fragments.

The Alzheimer beta-amyloid precursor protein (APP) is a transmembrane glycoprotein from which the amyloid beta-protein is proteolytically derived. The latter is a hydrophobic peptide that can aggregate and forms the core of the senile plaques found in the brains of patients suffering from Alzheimer's disease (AD). In view of the known association between familial AD and thyroid autoimmune disease, the expression pattern and cellular processing of APP in human thyroid cells were investigated. Cultured thyroid epithelial cells and homogenized thyroid tissue from normal and pathological thyroid samples were analyzed by immunoblotting using specific N- and C-terminal APP antibodies as well as by reverse transcription-polymerase chain reaction in which two sets of oligonucleotide primers were used. The results of these studies demonstrated that APP isoforms 770 and 751 were expressed in fresh thyroid extracts as well as in cultured thyroid epithelial cells, with APP 770 being the predominant form. Compared to other types of cells, such as lymphocytes and fibroblasts, thyroid epithelial cells produced larger amounts of APP. Most of the mature protein was cleaved within the amyloid beta region, as a result of which a large N-terminal APP fragment was released into the culture medium, whereas a C-terminal nonamyloidogenic fragment of 14 kilodaltons (kDa) was retained within the cell. Interestingly, thyroid epithelial cells also contained larger C-terminal APP fragments of 21, 35, and 41 kDa. From the sizes of these fragments it could be deduced that they contained the entire amyloid beta sequence and were thus potentially amyloidogenic. The 41-kDa fragment was unique to thyroid cells. These fragments may be released into the circulation after thyroid cell damage. Increased/altered thyroid APP expression in familiar AD may induce alterations in thyroid epithelial cells and cell damage, and thus explain the frequent occurrence of thyroid autoimmunity in this disease.

Adult↗

PubMed1997

Huntingtin-encoded polyglutamine expansions form amyloid-like protein aggregates in vitro and in vivo.

The mechanism by which an elongated polyglutamine sequence causes neurodegeneration in Huntington's disease (HD) is unknown. In this study, we show that the proteolytic cleavage of a GST-huntingtin fusion protein leads to the formation of insoluble high molecular weight protein aggregates only when the polyglutamine expansion is in the pathogenic range. Electron micrographs of these aggregates revealed a fibrillar or ribbon-like morphology, reminiscent of scrapie prions and beta-amyloid fibrils in Alzheimer's disease. Subcellular fractionation and ultrastructural techniques showed the in vivo presence of these structures in the brains of mice transgenic for the HD mutation. Our in vitro model will aid in an eventual understanding of the molecular pathology of HD and the development of preventative strategies.

Amino Acid Sequence↗