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Results for “Protein Aggregation, Pathological”

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PubMed2005

Unfolding of heptameric co-chaperonin protein follows "fly casting" mechanism: observation of transient nonnative heptamer.

Protein recognition and binding play a fundamental role in living systems but sometimes also result in pathological aggregates. To probe the coupling between folding and binding in a homoheptameric system, we have characterized the time-resolved unfolding/disassembly mechanism of human co-chaperonin protein 10 (cpn10) by a combination of experimental and computational methods. The results from both approaches are in excellent agreement and make obvious that the kinetic process is three-state: an initial polypeptide-unfolding step, resulting in a non-native heptamer, is followed by a slower heptamer-dissociation step. This demonstrates that the barriers on the kinetic free-energy landscape are defined by thermodynamic stability. cpn10 is one of few, and the only heptameric, experimentally characterized system that follows the "fly casting scenario" of molecular recognition.

Chaperonin 10↗

PubMed2005

Prediction of aggregation rate and aggregation-prone segments in polypeptide sequences.

The reliable identification of beta-aggregating stretches in protein sequences is essential for the development of therapeutic agents for Alzheimer's and Parkinson's diseases, as well as other pathological conditions associated with protein deposition. Here, a model based on physicochemical properties and computational design of beta-aggregating peptide sequences is shown to be able to predict the aggregation rate over a large set of natural polypeptide sequences. Furthermore, the model identifies aggregation-prone fragments within proteins and predicts the parallel or anti-parallel beta-sheet organization in fibrils. The model recognizes different beta-aggregating segments in mammalian and nonmammalian prion proteins, providing insights into the species barrier for the transmission of the prion disease.

Alzheimer Disease↗

PubMed2002

Tau and alpha-synuclein pathology in amygdala of Parkinsonism-dementia complex patients of Guam.

Amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC) is a progressive neurodegenerative disorder of Chamorro residents of Guam and the Mariana Islands, characterized by abundant neuron loss and tau neurofibrillary pathology similar to that observed in Alzheimer's disease (AD). A variety of neurodegenerative diseases with tau pathology including ALS/PDC also have alpha-synuclein positive pathology, primarily in the amygdala. We further characterized the tau and alpha-synuclein pathology in the amygdala of a large series of 30 Chamorros using immunohistochemical and biochemical techniques. Tau pathology was readily detected in both affected and unaffected Chamorros. In contrast, alpha-synuclein pathology was detected in 37% of patients with PDC but not detected in Chamorros without PDC or AD. The alpha-synuclein aggregates often co-localized within neurons harboring neurofibrillary tangles suggesting a possible interaction between the two proteins. Tau and alpha-synuclein pathology within the amygdala is biochemically similar to that observed in AD and synucleinopathies, respectively. Thus, the amygdala may be selectively vulnerable to developing both tau and alpha-synuclein pathology or tau pathology may predispose it to synuclein aggregation. Furthermore, in PDC, tau and alpha-synuclein pathology occurs independent of beta-amyloid deposition in amygdala thereby implicating the aggregation of these molecules in the severe neurodegeneration frequently observed in this location.

Aged↗

PubMed2005

Polyglutamine and polyalanine expansions in ataxin7 result in different types of aggregation and levels of toxicity.

Spinocerebellar ataxia type 7 (SCA7) is caused by expansion of a (CAG)n repeat in the ataxin7 gene, resulting in an abnormally long polyglutamine polyQ tract in the translated protein that aggregates in the form of neuronal intranuclear inclusions. Polyalanine (polyA) stretches, implicated in several genetic disorders, also appear to aggregate. To investigate the role of the aggregates in the pathologies, we compared the effects of ataxin7 containing a polyA (ataxin7 - 90A) or polyQ (ataxin7 - 100Q) expansion in HEK 293 cells and in primary cultures of rat mesencephalon. Both proteins formed nuclear and perinuclear aggregates that contained molecular chaperones and components of the ubiquitin-proteasome system, suggesting that they were abnormally folded. Ataxin-90A aggregates differed morphologically from ataxin7 - 100Q aggregates, consisted of small and amorphous rather than fibrillar inclusions and were more toxic to mesencephalic neurons, suggesting that toxicity was determined by the type of aggregate rather than the cellular misfolding response.

Animals↗

PubMed2004

Trehalose alleviates polyglutamine-mediated pathology in a mouse model of Huntington disease.

Inhibition of polyglutamine-induced protein aggregation could provide treatment options for polyglutamine diseases such as Huntington disease. Here we showed through in vitro screening studies that various disaccharides can inhibit polyglutamine-mediated protein aggregation. We also found that various disaccharides reduced polyglutamine aggregates and increased survival in a cellular model of Huntington disease. Oral administration of trehalose, the most effective of these disaccharides, decreased polyglutamine aggregates in cerebrum and liver, improved motor dysfunction and extended lifespan in a transgenic mouse model of Huntington disease. We suggest that these beneficial effects are the result of trehalose binding to expanded polyglutamines and stabilizing the partially unfolded polyglutamine-containing protein. Lack of toxicity and high solubility, coupled with efficacy upon oral administration, make trehalose promising as a therapeutic drug or lead compound for the treatment of polyglutamine diseases. The saccharide-polyglutamine interaction identified here thus provides a new therapeutic strategy for polyglutamine diseases.

Animals↗

PubMed2003

Protein topology determines binding mechanism.

Protein recognition and binding, which result in either transient or long-lived complexes, play a fundamental role in many biological functions, but sometimes also result in pathologic aggregates. We use a simplified simulation model to survey a range of systems where two highly flexible protein chains form a homodimer. In all cases, this model, which corresponds to a perfectly funneled energy landscape for folding and binding, reproduces the macroscopic experimental observations on whether folding and binding are coupled in one step or whether intermediates occur. Owing to the minimal frustration principle, we find that, as in the case of protein folding, the native topology is the major factor that governs the choice of binding mechanism. Even when the monomer is stable on its own, binding sometimes occurs fastest through unfolded intermediates, thus showing the speedup envisioned in the fly-casting scenario for molecular recognition.

Dimerization↗

PubMed1999

Mechanisms of immune regulation in Alzheimer's disease: a viewpoint.

The immune system may play an important role in the neurodegenerative process in Alzheimer's disease (AD). Complement components, eicosanoids and cytokines are found in cerebral amyloid plaques. These inflammatory proteins may stimulate the amyloid beta (Abeta) production, support its aggregation and increase its cytotoxicity, thus aggrevating the pathology of AD. Abeta may trigger their release from activated microglia and astrocytes which are the main sources of these proteins. However, there are also indications for a protective role of the immune system against the development of AD. Microglial cells have been shown to degrade Abeta and recent evidence suggests a role of autoreactive Abeta-specific T cells in the elimination of the peptide. This mechanism seems to be impaired in the majority of patients with AD. An Abeta-specific immune reaction may thus represent a natural defence mechanism directed against the accumulation of dangerous amyloidogenic substances. Impairment of the immune system and the failure to eliminate a toxic metabolite can be the basis for a chronic non-specific inflammatory process in the brain, as described above. AD is a good example how an immune response may lead to tissue destruction and neuronal loss instead of maintaining the integrity of the body.

Alzheimer Disease↗

PubMed2004

The G336S variant in the human neurofilament-M gene does not affect its assembly or distribution: importance of the functional analysis of neurofilament variants.

The human neurofilament medium (hNFM) subunit is one of the 3 neurofilament (NF) polypeptides, which are the most abundant intermediate filament (IF) proteins in post-mitotic neurons. The formation of neurofilamentous aggregates is a pathological hallmark of many neurodegenerative diseases, including the Lewy bodies found in Parkinson disease (PD). A Gly336Ser (G336S) variant in the rod domain of hNFM has recently been described in a patient with early-onset autosomal-dominant PD. In this study, we have generated a mammalian expression vector encoding the variant hNFM cDNA and characterized its effects on the formation of heteropolymeric IFs in heterologous cell lines. We have also investigated the distribution of the (G336S) hNFM variant protein in neuronal CAD cells, as well as the effects of the variant on the distribution of other cellular organelles and proteins. Our results demonstrate that the G336S variant does not affect the formation of IF networks nor the distribution of the variant hNFM protein. Our data suggest that if the G336S variant is involved in the development of PD, it does not appear to be due to defects in the assembly and distribution of NFs.

Amino Acid Substitution↗

PubMed1993

Protein kinase C is activated in platelets subjected to pathological shear stress.

High levels of fluid shear stress at the blood vessel wall directly stimulate von Willebrand factor (vWF)-mediated platelet adhesion and aggregation and thereby contribute to the pathogenesis of arterial thrombosis. We have found that a pathological level of arterial wall shear stress (90 dynes/cm2) induces platelet aggregation that is associated with the phosphorylation of pleckstrin, a M(r) 47,000 protein kinase C substrate (p47). Shear-induced p47 phosphorylation depends entirely on vWF binding to platelet glycoprotein (Gp) Ib and GpIIb-IIIa, and the specific inhibition of protein kinase C with the staurosporine analogue Ro 31-7549 inhibits the full aggregation response to shear. Shear stress-induced platelet p47 phosphorylation occurs independent of any measurable change in diacylglycerol mass or hydrolysis of phosphatidylinositol 4,5-bisphosphate. These results indicate that mechanical shear stress induces vWF to bind to platelet GpIb and GpIIb-IIIa, stimulating a diacylglycerol-independent pathway of protein kinase C activation that contributes to platelet aggregation in response to shear.

Blood Platelets↗

PubMed2005

Transgenic mice over-expressing human beta-amyloid have functional nicotinic alpha 7 receptors.

A potentially major factor in the development of Alzheimer's disease is the enhanced production of soluble beta-amyloid peptide fragments amyloid beta peptide(1-40) and amyloid beta peptide(1-42). These amyloid peptides are generated by cleavage of the amyloid-precursor protein and aggregate spontaneously to form amyloid plaques, which are a classical pathological hallmark in Alzheimer's disease. Although the precise mechanisms are unknown, it is widely believed that amyloid peptides initiate the degenerative process, resulting in subsequent cognitive decline. One interaction of amyloid beta peptide that may contribute to an impairment of cognition is its high affinity binding to the alpha 7 nicotinic receptor; a receptor shown to be important for cognition in a number of studies. There is some controversy, however, whether amyloid beta peptide inhibits or activates this receptor. We have cloned and stably expressed the human alpha 7 receptor and investigated its interaction with amyloid beta peptide using patch clamp electrophysiology. Human alpha 7 was activated in a concentration-dependent fashion by nicotine, acetylcholine and choline and potently inhibited by methyllycaconitine citrate. The responses were inwardly rectifying and exhibited rapid activation, desensitization and deactivation. Amyloid beta peptide(1-42) antagonized human alpha7 responses in a partially reversible fashion; no agonist effects of amyloid beta peptide(1-42) were detected. A similar inhibition of mouse alpha 7 was also observed. In addition, we have assessed the function of native alpha 7 receptors in hippocampal slices prepared from transgenic mice that over-express human amyloid. Despite this clear inhibition of recombinant receptors, hippocampal GABAergic interneurones in slices from beta-amyloid over-expressing mice still possess alpha 7 receptor-mediated currents.

Amyloid beta-Peptides↗

PubMed2002

Aggregation of misfolded proteins can be a selective process dependent upon peptide composition.

Intracellular aggregation of misfolded proteins is observed in a number of human diseases, in particular, neurologic disorders in which expanded tracts of polyglutamine residues play a central role. A variety of other proteins are prone to aggregation when mutated, indicating that this process is a common pathologic mechanism for inherited disorders. However, little is known about the relationship between the sequence of aggregating peptides and the specificity of intracellular accumulation. Here we demonstrate that substitution of two residues eliminates aggregation of a 111-amino acid peptide derived from the C-terminal portion of the cystic fibrosis transmembrane conductance regulator (CFTR). We also show that fusion to a reporter protein considerably alters the subcellular distribution of aggregating peptide. When fused to green fluorescent protein, the peptide containing amino acids 1370-1480 of CFTR accumulates in large perinuclear or nuclear aggregates. The same CFTR fragment devoid of green fluorescent protein localizes predominantly to discrete accumulations associated with mitochondria. Importantly, both types of accumulation are dependent on the presence of the same two amino acids within the CFTR sequence. Co-expression studies show that both CFTR-derived proteins can co-localize in large cytoplasmic/nuclear aggregates. However, neither CFTR construct accumulates in intracellular inclusions formed by N-terminal fragment of huntingtin. In addition to unique accumulation patterns, each aggregating peptide shows differences in association with chaperone proteins. Thus, our results indicate that the process of intracellular aggregation can be a selective process determined by the composition of the aggregating peptides.

Amino Acid Sequence↗

PubMed2000

Polyglutamine aggregates alter protein folding homeostasis in Caenorhabditis elegans.

Expansion of polyglutamine repeats in several unrelated proteins causes neurodegenerative diseases with distinct but related pathologies. To provide a model system for investigating common pathogenic features, we have examined the behavior of polyglutamine expansions expressed in Caenorhabditis elegans. The expression of polyglutamine repeats as green fluorescent protein (GFP)-fusion proteins in body wall muscle cells causes discrete cytoplasmic aggregates that appear early in embryogenesis and correlates with a delay in larval to adult development. The heat shock response is activated idiosyncratically in individual cells in a polyglutamine length-dependent fashion. The toxic effect of polyglutamine expression and the formation of aggregates can be reversed by coexpression of the yeast chaperone Hsp104. The altered homeostasis associated with polyglutamine aggregates causes both the sequestration of an otherwise soluble protein with shorter arrays of glutamine repeats and the relocalization of a nuclear glutamine-rich protein. These observations of induced aggregation and relocalization have implications for disorders involving protein aggregation.

Animals↗

PubMed1998

SH3GL3 associates with the Huntingtin exon 1 protein and promotes the formation of polygln-containing protein aggregates.

The mechanism by which aggregated polygins cause the selective neurodegeneration in Huntington's disease (HD) is unknown. Here, we show that the SH3GL3 protein, which is preferentially expressed in brain and testis, selectively interacts with the HD exon 1 protein (HDex1p) containing a glutamine repeat in the pathological range and promotes the formation of insoluble polyglutamine-containing aggregates in vivo. The C-terminal SH3 domain in SH3GL3 and the proline-rich region in HDex1p are essential for the interaction. Coimmunoprecipitations and immunofluorescence studies revealed that SH3GL3 and HDex1p colocalize in transfected COS cells. Additionally, an anti-SH3GL3 antibody was also able to coimmunoprecipitate the full-length huntingtin from an HD human brain extract. The characteristics of the interaction between SH3GL3 and huntingtin and the colocalization of the two proteins suggest that SH3GL3 could be involved in the selective neuronal cell death in HD.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Green tea catechins as brain-permeable, natural iron chelators-antioxidants for the treatment of neurodegenerative disorders.

Neurodegeneration in Parkinson's, Alzheimer's, or other neurodegenerative diseases appears to be multifactorial, where a complex set of toxic reactions, including oxidative stress (OS), inflammation, reduced expression of trophic factors, and accumulation of protein aggregates, lead to the demise of neurons. One of the prominent pathological features is the abnormal accumulation of iron on top of the dying neurons and in the surrounding microglia. The capacity of free iron to enhance and promote the generation of toxic reactive oxygen radicals has been discussed numerous times. The observations that iron induces aggregation of inert alpha-synuclein and beta-amyloid peptides to toxic aggregates have reinforced the critical role of iron in OS-induced pathogenesis of neurodegeneration, supporting the notion that a combination of iron chelation and antioxidant therapy may be one significant approach for neuroprotection. Tea flavonoids (catechins) have been reported to possess divalent metal chelating, antioxidant, and anti-inflammatory activities, to penetrate the brain barrier and to protect neuronal death in a wide array of cellular and animal models of neurological diseases. This review aims to shed light on the multipharmacological neuroprotective activities of green tea catechins with special emphasis on their brain-permeable, nontoxic, transitional metal (iron and copper)-chelatable/radical scavenger properties.

Animals↗

PubMed1997

Aluminum-induced structural alterations of the precursor of the non-A beta component of Alzheimer's disease amyloid.

The precursor of the non-A beta component of Alzheimer's disease amyloid (NACP) is a presynaptic protein whose function has been suspected to be tightly involved in neuronal biogenesis including synaptic regulations. NACP was suggested to seed the neuritic plaque formation in the presence of A beta during the development of Alzheimer's disease (AD). Recombinant NACP purified through heat treatment, DEAE-Sephacel anion-exchange, Sephacryl S-200 size-exclusion, and S-Sepharose cation-exchange chromatography steps appeared as a single band on SDS-PAGE with Mr of 19 kDa. Its N-terminal amino acid sequence clearly confirmed that the protein was NACP. Interestingly, however, the protein was split into a doublet on a nondenaturing (ND)-PAGE with equal intensities. The doublet was located slightly above a 45-kDa marker protein on a 12.5% ND-PAGE. In addition, the size of NACP was more carefully estimated as 53 kDa with high-performance gel-permeation chromatography using a TSK G3000sw size-exclusion column. Recently, Lansbury and his colleagues (Biochemistry 35, 13709-13715) have reported that NACP exists as an elongated "natively unfolded" structure which would make the protein more actively involved in protein-protein interactions and Kim (Mol. Cells 7, 78-83) has also shown that the natively unfolded protein is extremely sensitive to proteases. Here, we report that the structure of NACP could be altered by certain environmental factors. Aluminum, a suspected risk factor for AD, converged the doublet of NACP into a singlet with slightly lower mobility on ND-PAGE. Spectroscopic analysis employing uv absorption, intrinsic fluorescence, and circular dichroism indicated that NACP experienced the structural alterations in the presence of aluminum such as the secondary structure transition to generate about 33% alpha-helix. This altered structure of NACP became resistant to proteases such as trypsin, alpha-chymotrypsin, and calpain. Therefore, it is suggested that aluminum, which influences two pathologically critical processes in AD such as the protein turnover and the protein aggregation via the structural modifications, could participate in the disease.

Aluminum↗

PubMed2005

Amyloid beta degradation: a challenging task for brain peptidases.

Amyloid beta (Abeta) accumulates in the neuropil and within the walls of cerebral vessels in association with normal aging, dementia or stroke. Abeta is released from its precursor protein as soluble monomeric species yet, under pathological conditions, it self-aggregates to form soluble oligomers or insoluble fibrils that may be toxic to neurons and vascular cells. Abeta levels could be lowered by inhibiting its generation or by promoting its clearance by transport or degradation. Here we will summarize recent findings on brain proteases capable of degrading Abeta, with a special focus on those enzymes for which there is genetic, transgenic or biochemical evidence supporting a role in the proteolysis of Abeta in vivo.

Alzheimer Disease↗

PubMed1993

Protected endogenous retroviral sequences copurify with infectivity in experimental Creutzfeldt-Jakob disease.

Scrapie and Creutzfeldt-Jakob disease (CJD) are caused by infectious agents that are defined phenomenologically. No agent-specific molecules or particles have been identified. Biological properties, such as exponential agent replication and strain variation, as well as physical characteristics of infectivity indicate a protected viral structure. A host membrane glycoprotein of 34 kDa ("prion" protein) that aggregates at end stages of disease is clearly important in pathology and susceptibility to infection, but has no demonstrable infectivity in any purified or recombinant form. Thus a characterization of more viral-like molecules is important. In order to identify viral-like nucleic acids we previously developed methods to substantially purify the human CJD agent from experimentally infected hamster brains, and demonstrated selected retroviral-like LTR bands at pg levels that were insufficient for sequencing. To further define these and other viral-like sequences we cloned nucleic acids from highly infectious CJD fractions, and tested the efficacy of our methods using a selected retroviral probe. RNA extracted from an infectious 120 S Gaussian peak, which is reproducibly purified approximately 100,000 fold with respect to starting nucleic acids, and contains approximately 20% of the initial brain infectivity, was used to generate a cDNA library in a sequence independent amplification strategy for low levels of RNA (< 6 ng). Reconstituted strong stop experiments using several retroviral tRNA primers had indicated that Syrian hamster IAP (SHIAP) sequences should be present in both CJD and uninfected control fractions. Because SHIAP particles are extremely resistant to denaturation, their representation in a cDNA library would imply adequate extraction of other protected RNAs of viral origin. At least 900 bases of the Syrian hamster retroviral IAP genome were unambiguously identified in the cDNA library, and in independent PCR walks with selected primers, all of which were based on our cloned sequences. Sequencing confirmed the presence of protected LTR and adjacent retroviral motifs. Because these sequences were also present in control preparations they may represent normal endogenous viral contaminants that cosediment with infectivity in size and density gradients. On the other hand, LTRs can drive the expression of many diverse sequences, and it remains to be seen if CJD specific sequences are either transduced, or copackaged with, protected IAP complexes. The effective extraction and amplification of highly protected SHIAP nucleic acids of significant length sets the stage for identifying additional protected viral elements that may specify the CJD agent.

Animals↗

PubMed2004

Genetic aspects of amyloid beta-protein fibrillogenesis in Alzheimer's disease.

Although deposition of aggregated amyloid beta-protein (Abeta) in human brain is a fundamental pathological event in the development of Alzheimer's disease (AD), our knowledge of the molecular mechanisms underlying the initiation of Abeta fibril formation remains still very incomplete. Recent data indicate that genetic factors have a direct effect on Abeta fibrillogenesis. Most of pathogenic mutations identified in genes responsible for familial AD (FAD) affect activities of alpha-, beta, and gamma-secretases during amyloid precursor protein (APP) processing leading to a significant increase in the Abeta42/Abeta40 concentration ratio. The enhanced anabolism of Abeta may lead to its deposition. Recently, it was shown that the two main alloforms of Abeta have distinct biological activity and behaviour at the earliest stage of assembly. In vitro studies showed that Abeta42 monomers, but not Abeta40, form initial and minimal structures (pentamer/hexamer units called paranuclei), which can oligomerise to larger forms. This finding may explain the particularly strong association of Abeta42 with AD. We have reviewed molecular effects of APP and Presenilin mutations responsible for FAD in both Abeta metabolism and formation of Abeta fibril.

Alzheimer Disease↗