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PubMed2005

Protein aggregation in the pathogenesis of familial and sporadic Parkinson's disease.

Parkinson's disease (PD) is a slowly progressive, age-related, neurodegenerative disorder. The cause and mechanism of neuronal death have been elusive. However, recent genetic, postmortem and experimental evidence show that protein accumulation and aggregation are prominent occurrences in both sporadic and familial PD. The relevance of these events to other cellular and biochemical changes, and to the neurodegenerative process, is being unraveled. It is increasingly evident that one or a combination of defects, including mutations, oxidative stress, mitochondrial impairment and dysfunction of the ubiquitin-proteasome system, lead to an excess production and aggregation of abnormal proteins in PD. In this respect, altered protein handling appears to be a central factor in the pathogenic process occurring in the various hereditary and sporadic forms of PD. This suggests that manipulation of proteolytic systems is a rational approach in the development of neuroprotective therapies that could modify the pathological course of PD.

Animals↗

PubMed1995

Evidence that alpha-crystallin prevents non-specific protein aggregation in the intact eye lens.

The ocular lens is a transparent organ comprised of a highly concentrated and highly ordered matrix of structural proteins, called crystallins, which are probably the longest lived proteins of the body. Lens transparency is dependent upon maintenance of the short range order of the crystallin matrix. This transparency must be maintained for decades in the absence of normal protein synthesis or repair capacity. We present evidence here that alpha-crystallin, one of the major lens proteins, plays a central role in vivo in stabilizing the other crystallins and preventing uncontrolled aggregation of these progressively modified and aging molecules. alpha-Crystallin has previously been shown to suppress non-specific aggregation of denaturing proteins in simple binary systems through a chaperone-like activity. Our studies using soluble homogenates of monkey lenses demonstrate a strong resistance to heat induced non-specific aggregation when the complete complement of crystallins is present; in contrast, if alpha-crystallin is selectively removed prior to heating, the remaining crystallins undergo extensive non-specific aggregation as indicated by light scattering. When alpha-crystallin is present it complexes with denaturing proteins forming a soluble heavy molecular weight (HMW) fraction but no insolubilization is observed, while when alpha-crystallin is absent there is heavy insolubilization and no HMW formed. When intact monkey lenses were heated it could be demonstrated that soluble HMW was generated. Similar HMW protein appears in vivo in the human lens as a function of age. These findings suggest that the soluble HMW protein present in the human lens is the product of the chaperone-like function of alpha-crystallin and that under physiological conditions alpha-crystallin inhibits the uncontrolled aggregation of damaged proteins, thereby preventing the formation of light scattering centers and opacification of the lens.

Animals↗

PubMed2004

MAP2 prevents protein aggregation and facilitates reactivation of unfolded enzymes.

It is well established that in addition to its functional role in cell motility, cell division and intracellular transport, cytoskeletal protein tubulin also possesses significant chaperone-like activity. In vitro studies from our laboratory showed that dimeric tubulin can prevent stress induced aggregation of substrate proteins, can resist thermal deactivation of enzymes and can also refold enzymes from their fully denatured state [Manna, T., Sarkar, T., Poddar, A., Roychowdhury, M., Das, K.P. & Bhattacharyya, B. (2001) J. Biol. Chem.276, 39742-39747]. Negative charges of the C-termini of both subunits of tubulin are essential for this chaperone-like property as the deletion of only beta-C-terminus or the binding of a 14-residue basic peptide P2 to the alpha-C-terminus completely abolishes this property [Sarkar, T., Manna, T., Bhattacharyya, S., Mahapatra, P., Poddar, A., Roy, S., Pena, J., Solana, R., Tarazona, R. & Bhattacharyya, B. (2001) Proteins Struct. Funct. Genet.44, 262-269]. Based on these results, one would expect that the microtubular proteins (MTP, tubulin with microtubular-associated proteins, i.e. MAPs bound to the C-terminus) should not possess any chaperone-like activity. To our surprise we noticed excellent chaperone-like activity of MTP. MTP prevents chemical and thermal aggregation of other proteins and can enhance the extent of refolding of fully unfolded substrate enzymes. Because MTP contains tubulin as well as several MAPs bound to the C-termini of tubulin, we fractionated and purified microtubular associated protein 2 (MAP2) and tau using phosphocellulose chromatography. Experiments with purified proteins demonstrated that it is the MAP2 of MTP that exhibits significant chaperone-like activity. This has been shown by the prevention of dithiothreitol-induced aggregation of insulin, thermal aggregation of alcohol dehydrogenase and regain of enzymatic activity during refolding of unfolded substrates. Tau, which shares a homologous C-terminal domain with MAP2, possesses no such activity.

Alcohol Dehydrogenase↗

PubMed2004

Is there structural specificity in the reversible protein aggregates that are stored in secretory granules?

There are several steps that must occur for secretory granules to form: (1) Secretory proteins that make up the dense cores of the granules must be concentrated; (2) membrane proteins necessary for granule function must accumulate in the correct location; and (3) inappropriate membrane proteins and excess membrane must be removed. Reversible aggregation of secretory granule proteins provides a mechanism for concentrating and sorting these proteins. There is specificity in the way secretory granule proteins are treated in cells that make granules. The specificity has been shown in some cases to occur after the aggregation process, so that granules containing different aggregates function differently. An explanation could be that a property of the aggregate, such as a surface motif, might influence the accumulation of membrane proteins necessary for granule function. Such a conclusion implies that the aggregates are not amorphous but have structure. Use of NMR spectroscopy to investigate changes in the environment of amino acid residues in secretory granule proteins as they form oligomers by using 15N relaxation times might provide a means to determine which residues are specifically involved in aggregation.

Amino Acid Motifs↗

PubMed2006

Amyloids, prions and the inherent infectious nature of misfolded protein aggregates.

Misfolded aggregates present in amyloid fibrils are associated with various diseases known as "protein misfolding" disorders. Among them, prion diseases are unique in that the pathology can be transmitted by an infectious process involving an unprecedented agent known as a "prion". Prions are infectious proteins that can transmit biological information by propagating protein misfolding and aggregation. The molecular mechanism of prion conversion has a striking resemblance to the process of amyloid formation, suggesting that misfolded aggregates have an inherent ability to be transmissible. Intriguing recent data suggest that other protein misfolding disorders might also be transmitted by a prion-like infectious process.

Alzheimer Disease↗

PubMed2002

Identification of a new Pmp22 mouse mutant and trafficking analysis of a Pmp22 allelic series suggesting that protein aggregates may be protective in Pmp22-associated peripheral neuropathy.

We have identified and characterized a new peripheral myelin protein 22 (Pmp22) mouse mutant. The mutation results in a serine to threonine amino acid substitution at residue 72, which is a hot spot for mutation in human PMP22, leading to the peripheral neuropathy Dejerine-Sottas syndrome. We have previously described two other Pmp22 mutants, providing an allelic series for gene function analysis. Pmp22 mutations generally lead to abnormal intracellular trafficking of Pmp22, and we show that each mutant protein in the allelic series has a unique pattern of intracellular localization in transfected cell lines. The mutant protein from the less severely affected mutants occurs in large aggregates, while the mutant protein from the most severely affected mutant occurs in a diffuse perinuclear pattern that largely colocalizes with wild-type protein. This suggests that large Pmp22 aggregates may be protective in this form of peripheral neuropathy.

Alleles↗

PubMed2006

Are amyloid diseases caused by protein aggregates that mimic bacterial pore-forming toxins?

Protein fibrillization is implicated in the pathogenesis of most, if not all, age-associated neurodegenerative diseases, but the mechanism(s) by which it triggers neuronal death is unknown. Reductionist in vitro studies suggest that the amyloid protofibril may be the toxic species and that it may amplify itself by inhibiting proteasome-dependent protein degradation. Although its pathogenic target has not been identified, the properties of the protofibril suggest that neurons could be killed by unregulated membrane permeabilization, possibly by a type of protofibril referred to here as the 'amyloid pore'. The purpose of this review is to summarize the existing supportive circumstantial evidence and to stimulate further studies designed to test the validity of this hypothesis.

Aging↗

PubMed2004

Hsp70 dynamics in vivo: effect of heat shock and protein aggregation.

The molecular chaperone Hsp70 interacts with misfolded proteins and also accumulates in the nucleus during heat shock. Using GFP-Hsp70 and fluorescence recovery after photobleaching, we show that Hsp70 accumulates in the nucleus during heat shock not only because its inflow rate increases but also because of a marked decrease in its outflow rate. Dynamic imaging also shows that GFP-Hsp70 has greatly reduced mobility when it interacts with organelles such as nucleoli in heat-shocked cells or the large inclusions formed from fragments of mutant huntingtin protein. In heat-shocked cells, nucleoplasmic Hsp70 has reduced mobility relative to the cytoplasm, whereas the ATPase-deficient mutant of Hsp70, Hsp70(K71E), is almost completely immobilized both in the nucleoplasm and the cytoplasm. Moreover, the Hsp70 mutant shows reduced mobility in the presence of diffusive huntingtin fragments with expanded polyglutamine repeats. This provides strong evidence that Hsp70 interacts not only with organelles but also with diffusive proteins in the nucleoplasm and cytoplasm during heat shock as well as with diffusive huntingtin fragments.

Biological Transport↗

PubMed2003

Proteasome inhibitors induce intracellular protein aggregation and cell death by an oxygen-dependent mechanism.

Clone 9 liver cells incubated under aerobic conditions with the proteasome inhibitors lactacystin, clastro lactacystin beta-lactone, and tri-leucine vinyl sulfone, in the absence of an obvious oxidative challenge, underwent oxidative protein modifications, such as loss of solubility, formation of aggregates (predominantly by disulfide bridges), and increased carbonyl formation, similar to those seen with hydrogen peroxide treatment. These alterations were accompanied by modification of cell morphology and loss of cell viability. Remarkably, almost all of these modifications were prevented when cell incubation with proteasome inhibitors was performed under a 3% oxygen atmosphere instead of the 21% oxygen routinely used in cell culture experiments. Our results suggest an oxygen-dependent mechanism for the protein oxidation, protein aggregation, cellular dysfunction, and apoptosis induced by proteasome inhibitors.

Acetylcysteine↗

PubMed2007

Molecular crime and cellular punishment: active detoxification of misfolded and aggregated proteins in the cell by the chaperone and protease networks.

Labile or mutation-sensitised proteins may spontaneously convert into aggregation-prone conformations that may be toxic and infectious. This hazardous behavior, which can be described as a form of "molecular criminality", can be actively counteracted in the cell by a network of molecular chaperone and proteases. Similar to law enforcement agents, molecular chaperones and proteases can specifically identify, apprehend, unfold and thus neutralize "criminal" protein conformers, allowing them to subsequently refold into harmless functional proteins. Irreversibly damaged polypeptides that have lost the ability to natively refold are preferentially degraded by highly controlled ATP-consuming proteases. Damaged proteins that escape proteasomal degradation can also be "incarcerated" into dense amyloids, "evicted" from the cell, or internally "exiled" to the lysosome to be hydrolysed and recycled. Thus, remarkable parallels exist between molecular and human forms of criminality, as well as in the cellular and social responses to various forms of crime. Yet, differences also exist: whereas programmed death is the preferred solution chosen by aged and aggregation-stressed cells, collective suicide is seldom chosen by lawless societies. Significantly, there is no cellular equivalent for the role of familial care and of education in general, which is so crucial to the proper shaping of functional persons in the society. Unlike in the cell, humanism introduces a bias against radical solutions such as capital punishment, favouring crime prevention, reeducation and social reinsertion of criminals.

Aging↗

PubMed1994

Protein aggregation reactions: surface free energy model.

The energetics of protein reactions can be evaluated in terms of the surface free-energy model, an approach that estimates changes in the surface free energy of proteins from changes in the area and the charge density of the protein-water interface. This approach has given insight into the reactions of proteins in solution as well as in membranes (receptors and voltage-gated channels). The model highlights the importance of pH and thermal changes in aggregation reactions. It also enables a correct interpretation of the Hill equation describing co-operative reactions, since the Hill coefficient is related to a Gibbs surface excess that varies with the degree of reaction.

Chemical Phenomena↗

PubMed1994

Anionic and cationic components from protein aggregates in bovine seminal plasma and their effects on sperm motility.

Bovine seminal plasma proteins are in an aggregated form of high molecular weight in their native state. By immobilisation on a cation exchanger with exposure to disaggregating conditions (i.e., acetonitrile and low pH), the high-molecular-weight aggregates could be dissociated to slowly release the low-molecular-weight components. The anionic component released from the cation exchanger during disaggregation was collected by adsorption on a hydrophobic interaction column. The cationic component remaining on the cation exchanger was eluted with NaOH. Both components were found on gel permeation chromatography to be < 5 kDa. SDS-PAGE of the various fractions showed that components of low molecular weight were still in an aggregated form. These components resulting from the disaggregation process have detrimental effects on sperm motility and the effects were more substantial compared with that of whole seminal plasma. All the cationic components were significantly detrimental to sperm motility, especially the fractions of low molecular weight. The anionic fractions reduced sperm motility when in an aggregated state. The isolated anionic peptide was not detrimental in its free form. In all fractions the peptides tended to re-aggregate to a higher molecular weight under neutral conditions, however, the isolated anionic peptide (molecular weight < 1,500) failed to do so.

Animals↗

PubMed1998

Cloning of subunits of convulxin, a collagen-like platelet-aggregating protein from Crotalus durissus terrificus venom.

Convulxin (CVX) is a potent platelet-aggregating glycoprotein from the venom of the snake Crotalus durissus terrificus. It consists of two subunits, alpha and beta, joined by disulphide bridges in a hexameric structure. A cDNA library from venom gland was constructed in the vector pT3T7. The cloned cDNAs encoding the two chains of CVX were sequenced. Both are preceded by an identical 23-amino acid peptide signal sequence and encode sequences of 135 amino acids for the alpha chain and 125 amino acids for the beta chain. These polypeptides include a carbohydrate-recognition domain (CRD) in which some of the specific amino acids required for binding Ca2+ and galactose or mannose are absent. The presence of such a domain means that CVX can be included in the family of C-type lectins along with other snake venom proteins, although it is not a true lectin. Assuming that the localization of intracatenary disulphide bridges of each CVX chain is similar to that of the CRD and that an intercatenary bridge between the alpha and beta chains is similar to that of the C-type lectin botrocetin, we postulate the existence of an additional intercatenary bridge, which explains the tridimeric structure (alphabeta)3 of CVX.

Amino Acid Sequence↗

PubMed2006

Protein aggregates are transported to vacuoles by a macroautophagic mechanism in nutrient-starved plant cells.

When a fusion protein of cytochrome b5 (Cyt b5) and the red fluorescent protein (RFP) are expressed in tobacco BY-2 cells, the expressed protein forms intracellular aggregates that emit red fluorescence. When such cells are grown to the stationary phase or incubated in nutrient limited medium, RFP fluorescence can be detected in the vacuolar lumen. We investigated this transport mechanism using a limited-nitrogen model. E-64 and 3-methyladenine, which inhibit autophagic processes, blocked the transport of the RFP signal to the vacuole. We next traced the autophagic process in tobacco cells using YFP fused with the tobacco Atg8 homologue (YFP-NtAtg8) and analyzed the contribution of autophagy to the vacuolar transport of the aggregates. Under limited-nitrogen conditions, the aggregates were degraded in preference to other organelles, and the autophagosomes colocalized with the aggregates at a higher frequency than with mitochondria. This is the first demonstration that selective macroautophagic degradation can occur in plant cells.

Adenine↗

PubMed2004

Recognition of lumenal prion protein aggregates by post-ER quality control mechanisms is mediated by the preoctarepeat region of PrP.

Prion diseases are fatal transmissible neurodegenerative disorders linked to an aberrant conformation of the cellular prion protein (PrP(c)). We have shown previously that the chemical compound suramin induced aggregation of fully matured PrP(c) in post-ER compartments, thereby, activating a post-ER quality control mechanism and preventing cell surface localization of PrP by intracellular re-routing of aggregated PrP from the Golgi/TGN directly to lysosomes. Of note, drug-induced PrP aggregates were not toxic and could easily be degraded by neuronal cells. Here, we focused on determining the PrP domains mediating these effects. Using PrP deletion mutants we show that intracellular re-routing but not aggregation depends on the N-terminal PrP (aa 23-90) and, more precisely, on the preoctarepeat domain (aa 23-50). Fusion of the PrP N-terminus to the GPI-anchored protein Thy-1 did not cause aggregation or re-routing of the chimeric protein, indicating that the N-terminus is only active in re-routing when prion protein aggregation occurs. Insertion of a region with a comparable primary structure contained in the PrP paralogue prnd/doppel (aa 27-50) into N-terminally deleted PrP re-established the re-routing phenotype. Our data reveal an important role for the conserved preoctarepeat region of PrP, namely controlling the intracellular trafficking of misfolded PrP.

Animals↗

PubMed2001

SeqA protein aggregation is necessary for SeqA function.

The binding of SeqA protein to hemimethylated GATC sequences is important in the negative modulation of chromosomal initiation at oriC, and in the formation of SeqA foci necessary for Escherichia coli chromosome segregation. Using gel-filtration chromotography and glycerol gradient sedimentation, we demonstrate that SeqA exists as a homotetramer. SeqA tetramers are able to aggregate or multimerize in a reversible, concentration-dependent manner. Using a bacterial two-hybrid system, we demonstrate that the N-terminal region of SeqA, especifically the 9th amino acid residue, glutamic acid, is required for functional SeqA-SeqA interaction. Although the SeqA(E9K) mutant protein, containing lysine rather than glutamic acid at the 9th amino acid residue, exists as a tetramer, the mutant protein binds to hemimethylated DNA with altered binding patterns as compared with wild-type SeqA. Aggregates of SeqA(E9K) are defective in hemimethylated DNA binding. Here we demonstrate that proper interaction between SeqA tetramers is required for both hemimethylated DNA binding and formation of active aggregates. SeqA tetramers and aggregates might be involved in the formation of SeqA foci required for the segregation of chromosomal DNA as well as the regulation of chromosomal initiation.

Bacterial Outer Membrane Proteins↗

PubMed2003

Rice stripe virus 23.9 K protein aggregates and forms inclusion bodies in cultured insect cells and virus-infected plant cells.

The genome of Rice stripe virus (RSV, genus Tenuivirus) contains seven open reading frames (ORFs). Little is known about the products of four of these ORFs, including the 23.9 K protein encoded by the virus-sense ORF of RNA3. Western blotting revealed that the 23.9 K protein was synthesized in the host plant and also in the planthopper vector of RSV. Using a baculovirus vector, the 23.9 K protein was expressed, both unfused and fused with red-shifted green fluorescent protein, in Spodoptera frugiperda cells. Inclusion bodies were observed by light microscope in cells expressing fused or unfused proteins. Inclusion bodies in cells expressing the fused protein fluoresced under blue light. By immunoelectron microscopy, electron-dense inclusion bodies in cells expressing the unfused protein were specifically labeled with 23.9 K protein antiserum. Moreover, electron-dense masses labeled with 23.9 K protein antiserum were observed in virus-infected wheat tissue by electron microscopy. This paper thus demonstrates that RSV 23.9 K protein can aggregate in vivo and form inclusion bodies in infected plant tissue.

Animals↗

PubMed1996

Inhibition of amyloid beta protein aggregation and neurotoxicity by rifampicin. Its possible function as a hydroxyl radical scavenger.

Aggregation of physiologically produced soluble amyloid beta protein (Abeta) to insoluble, neurotoxic fibrils is a crucial step in the pathogenesis of Alzheimer's disease. Aggregation studies with synthetic Abeta1-40 peptide by the thioflavin T fluorescence assay and electron microscopy and cytotoxicity assays using rat pheochromocytoma PC12 cells showed that an antibiotic, rifampicin, and its derivatives, which possess a naphthohydroquinone or naphthoquinone structure, inhibited Abeta1-40 aggregation and neurotoxicity in a concentration-dependent manner. Hydroquinone, p-benzoquinone, and 1,4dihydroxynaphthalene, which represent partial structures of the aromatic chromophore of rifampicin derivatives, also inhibited A beta1 40 aggregation and neurotoxicity at comparable molar concentrations to rifampicin. Electron spin resonance spectrometric analysis revealed that the inhibitory activities of those agents correlated with their radical-scavenging ability on hydroxyl free radical, which was shown to be generated in cell-free incubation of Abeta1-40 peptide. These results suggest that at least one mechanism of rifampicin-mediated inhibition of A beta aggregation and neurotoxicity involves scavenging of free radicals and that rifampicin and/or appropriate hydroxyl radical scavengers may have therapeutic potential for Alzheimer's disease.

Alzheimer Disease↗