Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Mutations in the human parkin gene encoding an E3 ubiquitin ligase have been associated with early-onset recessive forms of Parkinson's disease (PD). However, the molecular mechanisms by which mutations in the parkin gene cause PD are still under debate. Here, we identified and characterized the Caenorhabditis elegans parkin homolog, pdr-1. PDR-1 protein physically associates and cooperates with a conserved degradation machinery to mediate ubiquitin conjugation. Strikingly, in contrast to pdr-1 loss-of-function mutants, an in-frame deletion variant with altered solubility and intracellular localization properties is hypersensitive toward different proteotoxic stress conditions. Both endoplasmic reticulum-derived folding stress and cytosolic stress conferred by expression of mutant human alpha-synuclein resulted in severe developmental defects and lethality in pdr-1(lg103) mutant background. Furthermore, we show that the corresponding truncated protein PDR-1(Deltaaa24-247) aggregates in cell culture, but still interacts with its ubiquitylation co-enzymes. Thus, it might block the cellular degradation/detoxification machinery and therefore renders worms highly vulnerable to protein folding stress. In contrast to other complete gene knockouts or RNAi models of Parkin function, this C. elegans model recapitulates Parkin insolubility and aggregation similar to several autosomal recessive juvenile parkinsonism (AR-JP)-linked Parkin mutations. We suggest that such Parkin variants that either confer a neomorphic function or a partial loss-of-function may help to further elucidate the biological function of Parkin in vivo and the pathogenic mechanisms resulting in AR-JP. Due to high-throughput capacity of C. elegans, this model is particularly well suited to identify genetic and chemical modifiers of toxicity.
Hum Mol Genet 2005 Nov 15
PMID:A Caenorhabditis elegans Parkin mutant with altered solubility couples alpha-synuclein aggregation to proteotoxic stress. 1620 51

A non-natural 16-residue "degron" peptide has been reported to convey proteasome-dependent degradation when fused to proteins expressed in yeast (Gilon, T., Chomsky, O., and Kulka, R. (2000) Mol. Cell. Biol. 20, 7214-7219) or when fused to green fluorescent protein (GFP) and expressed in mammalian cells (Bence, N. F., Sampat, R. M., and Kopito, R. R. (2001) Science 292, 1552-1555). We find that expression of the GFP::degron in Caenorhabditis elegans muscle or neurons results in the formation of stable perinuclear deposits. Similar perinuclear deposition of GFP::degron was also observed upon transfection of primary rat hippocampal neurons or mouse Neuro2A cells. The generality of this observation was supported by transfection of HEK 293 cells with both GFP::degron and DsRed(monomer)::degron constructs. GFP::degron expressed in C. elegans is less soluble than unmodified GFP and induces the small chaperone protein HSP-16, which co-localizes and co-immunoprecipitates with GFP::degron deposits. Induction of GFP::degron in C. elegans muscle leads to rapid paralysis, demonstrating the in vivo toxicity of this aggregating variant. This paralysis is suppressed by co-expression of HSP-16, which dramatically alters the subcellular distribution of GFP::degron. Our results suggest that in C. elegans, and perhaps in mammalian cells, the degron peptide is not a specific proteasome-targeting signal but acts instead by altering GFP secondary or tertiary structure, resulting in an aggregation-prone form recognized by the chaperone system. This altered form of GFP can form toxic aggregates if its expression level exceeds the capacity of chaperone-based degradation pathways. GFP::degron may serve as an instructive "generic" aggregating control protein for studies of disease-associated aggregating proteins, such as huntingtin, alpha-synuclein, and the beta-amyloid peptide.
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PMID:Conversion of green fluorescent protein into a toxic, aggregation-prone protein by C-terminal addition of a short peptide. 1623 15

Parkinson's disease (PD) is a neurodegenerative movement disorder characterized by selective loss of dopaminergic neurons and the presence of Lewy bodies. Alpha-synuclein is a major component of Lewy bodies in sporadic PD, and mutations in alpha-synuclein cause autosomal-dominant hereditary PD. Here, we generated A53T mutant alpha-synuclein-inducible PC12 cell lines using the Tet-off regulatory system. Inducing expression of A53T alpha-synuclein in differentiated PC12 cells decreased proteasome activity, increased the intracellular ROS level and caused up to approximately 40% cell death, which was accompanied by mitochondrial cytochrome C release and elevation of caspase-9 and -3 activities. Cell death was partially blocked by cyclosporine A [an inhibitor of the mitochondrial permeability transition (MPT) process], z-VAD (a pan-caspase inhibitor) and inhibitors of caspase-9 and -3 but not by a caspase-8 inhibitor. Furthermore, induction of A53T alpha-synuclein increased endoplasmic reticulum (ER) stress and elevated caspase-12 activity. RNA interference to knock down caspase-12 levels or salubrinal (an ER stress inhibitor) partially protected against cell death and further reduced A53T toxicity after treatment with z-VAD. Our results indicate that both ER stress and mitochondrial dysfunction contribute to A53T alpha-synuclein-induced cell death. This study sheds light into the pathogenesis of alpha-synuclein cellular toxicity in PD and provides a cell model for screening PD therapeutic agents.
Hum Mol Genet 2005 Dec 15
PMID:Endoplasmic reticulum stress and mitochondrial cell death pathways mediate A53T mutant alpha-synuclein-induced toxicity. 1623 41

The presynaptic protein alpha-synuclein, implicated in Parkinson disease (PD), binds phospholipids and has a role in brain fatty acid (FA) metabolism. In mice lacking alpha-synuclein (Snca-/-), total brain steady-state mass of the mitochondria-specific phospholipid, cardiolipin, is reduced 22% and its acyl side chains show a 51% increase in saturated FAs and a 25% reduction in essential n-6, but not n-3, polyunsaturated FAs. Additionally, 23% reduction in phosphatidylglycerol content, the immediate biosynthetic precursor of cardiolipin, was observed without alterations in the content of other brain phospholipids. Consistent with these changes, more ordered lipid head group and acyl chain packing with enhanced rotational motion of diphenylhexatriene (DPH) about its long axis were demonstrated in time-resolved DPH fluorescence lifetime experiments. These abnormalities in mitochondrial membrane properties were associated with a 15% reduction in linked complex I/III activity of the electron transport chain, without reductions in mitochondrial number, complex II/III activity, or individual complex I, II, III, or IV activity. Reduced complex I activity is thought to be a critical factor in the development of PD. Thus, altered membrane composition and structure and impaired complex I/III function in Snca-/- brain suggest a relationship between alpha-synuclein's role in brain lipid metabolism, mitochondrial function, and PD.
Mol Cell Biol 2005 Nov
PMID:Mitochondrial lipid abnormality and electron transport chain impairment in mice lacking alpha-synuclein. 1626 Jun 31

Protein misfolding is conformational transition dramatically facilitating the assembly of protein molecules into aggregates of various morphologies. Spontaneous formation of specific aggregates, mostly amyloid fibrils, was initially believed to be limited to proteins involved in the development of amyloidoses. However, recent studies show that, depending on conditions, the majority of proteins undergo structural transitions leading to the appearance of amyloidogenic intermediates followed by aggregate formation. Various techniques have been used to characterize the protein misfolding facilitating the aggregation process, but no direct evidence as to how such a conformational transition increases the intermolecular interactions has been obtained as of yet. We have applied atomic force microscopy (AFM) to follow the interaction between protein molecules as a function of pH. These studies were performed for three unrelated and structurally distinctive proteins, alpha-synuclein, amyloid beta-peptide (Abeta) and lysozyme. It was shown that the attractive force between homologous protein molecules is minimal at physiological pH and increases dramatically at acidic pH. Moreover, the dependence of the pulling forces is sharp, suggesting a pH-dependent conformational transition within the protein. Parallel circular dichroism (CD) measurements performed for alpha-synuclein and Abeta revealed that the decrease in pH is accompanied by a sharp conformational transition from a random coil at neutral pH to the more ordered, predominantly beta-sheet, structure at low pH. Importantly, the pH ranges for these conformational transitions coincide with those of pulling forces changes detected by AFM. In addition, protein self-assembly into filamentous aggregates studied by AFM imaging was shown to be facilitated at pH values corresponding to the maximum of pulling forces. Overall, these results indicate that proteins at acidic pH undergo structural transition into conformations responsible for the dramatic increase in interprotein interaction and promoting the formation of protein aggregates.
J Mol Biol 2005 Dec 16
PMID:Protein interactions and misfolding analyzed by AFM force spectroscopy. 1629 Sep 1

Formation of alpha-synuclein aggregates is proposed to be a crucial event in the pathogenesis of Parkinson's disease. Large soluble oligomeric species are observed as probable intermediates during fibril formation and these, or related aggregates, may constitute the toxic element that triggers neurodegeneration. Unfortunately, there is a paucity of information regarding the structure and composition of these oligomers. Here, the morphology and the conformational characteristics of the oligomers and filaments are investigated by a combined atomic force microscopy (AFM) and Raman microscopic approach on a common mica surface. AFM showed that in vitro early stage oligomers were globular with variable heights, while prolonged incubation caused the oligomers to become elongated as protofilaments. The height of the subsequently formed alpha-synuclein filaments was similar to that of the protofilaments. Analysis of the Raman amide I band profiles of the different alpha-synuclein oligomers establishes that the spheroidal oligomers contain a significant amount of alpha-helical secondary structure (47%), which decreases to about 37% in protofilaments. At the same time, when protofilaments form, beta-sheet structure increases to about 54% from the approximately 29% observed in spheroidal oligomers. Upon filament formation, the major conformation is beta-sheet (66%), confirmed by narrowing of the amide I band and the profile maximum shifting to 1667 cm(-1). The accumulation of spheroidal oligomers of increasing size but unchanged vibrational spectra during the fibrillization process suggests that a cooperative conformational change may contribute to the kinetic control of fibrillization.
J Mol Biol 2006 Jan 06
PMID:Secondary structure of alpha-synuclein oligomers: characterization by raman and atomic force microscopy. 1630 37

The natively disordered protein alpha-synuclein is the primary component of Lewy bodies, the cellular hallmark of Parkinson's disease. Most studies of this protein are performed in dilute solution, but its biologically relevant role is performed in the crowded environment inside cells. We addressed the effects of macromolecular crowding on alpha-synuclein by combining NMR data acquired in living Escherichia coli with in vitro NMR data. The crowded environment in the E.coli periplasm prevents a conformational change that is detected at 35 degrees C in dilute solution. This change is associated with an increase in hydrodynamic radius and the formation of secondary structure in the N-terminal 100 amino acid residues. By preventing this temperature-induced conformational change, crowding in the E.coli periplasm stabilizes the disordered monomer. We obtain the same stabilization in vitro upon crowding alpha-synuclein with 300 g/l of bovine serum albumin, indicating that crowding alone is sufficient to stabilize the disordered, monomeric protein. Two disease-associated variants (A30P and A53T) behave in the same way in both dilute solution and in the E.coli periplasm. These data reveal the importance of approaching the effects of macromolecular crowding on a case-by-case basis. Additionally, our work shows that discrete structured protein conformations may not be achieved by alpha-synuclein inside cells, implicating the commonly overlooked aspect of macromolecular crowding as a possible factor in the etiology of Parkinson's disease.
J Mol Biol 2006 Feb 03
PMID:Macromolecular crowding in the Escherichia coli periplasm maintains alpha-synuclein disorder. 1634 31

Mutations in the gene encoding human myocilin are associated with some cases of juvenile and early-onset glaucoma. Glaucomatous mutations prevent myocilin from being secreted. The analysis of the defects associated with mutations point to the existence of factor(s) in addition to mutations that might be implicated in the development of glaucoma. In the present paper, we found that interaction of myocilin with one of the members of the synuclein family alters its properties, including its ability to be secreted. Results of immunoprecipitation show that myocilin is a gamma-synuclein-interacting protein. Further analysis demonstrated that both myocilin and gamma-synuclein are expressed in human TM cells, immortalized rat ganglion (RGC-5) cells, and HT22 hippocampal neurons. According to Western blotting, in addition to monomeric form with molecular weight 17 kDa gamma-synuclein is present as higher molecular weight forms ( approximately 35 and 68 KDa), presumably dimer and tetramer. Myocilin and gamma-synuclein have partially overlapping perinuclear localization. Dexamethasone upregulates myocilin expression in RGC-5 cells and HT22 hippocampal neurons. We found alterations of myocilin properties as a result of its interaction with gamma-synuclein. In cultured cells, gamma-synuclein upregulates myocilin expression, inhibits its secretion and prevents the formation of high molecular weight forms of myocilin. Although both alpha-synuclein and gamma-synuclein are expressed in HTM cells, only gamma-synuclein interacts with myocilin and alters its properties. We conclude that myocilin and gamma-synuclein interact and as a result, myocilin's properties are changed. Since myocilin and gamma-synuclein have partially overlapping intracellular localization in cell types that are implicated in glaucoma development, their interaction may play an important role in glaucoma.
Cell Mol Neurobiol 2005 Sep
PMID:Interaction of myocilin with gamma-synuclein affects its secretion and aggregation. 1639 33

1. Retinal dystrophies (RD) comprise a group of clinically and genetically heterogeneous retinal disorders, which typically result in the degeneration of photoreceptors followed by the impairment or loss of vision. Although age-related macular degeneration (AMD) and retinitis pigmentosa (RP) are among the most common forms of RD, currently, there is no effective treatment for either disorder. 2. Recently, abnormal protein accumulation and aggregation due to protein misfolding and proteasome inhibition have been implicated in the pathogenesis of RD. In this paper we describe effects of several factors on protein aggregation and survival of photoreceptor cells. 3. Expression of rhodopsin carrying P23H mutation causes its accumulation in intracellular inclusion bodies in a perinuclear area of photoreceptor cells. beta- and gamma-synucleins and heat shock protein Hsp-70, but not alpha-synuclein, protect cultured ocular cells from mutant opsin accumulation. This effect might be explained by their chaperonic activity. 4. Knock-out of alpha- and gamma-synucleins does not affect gross retinal morphology, but induces tyrosine hydroxylase in the inner prexiform layer of the retina. Selegiline-a monoamine oxidase inhibitor used for the treatment of Parkinson's disease, reduces apoptosis and increases viability in cultured retinal pigment epithelium cells (APRE-19). 5. These results suggest that chaperones and selegiline may be considered promising candidates for the protection of ocular cells from the accumulation of misfolded and aggregated proteins.
Cell Mol Neurobiol 2005 Sep
PMID:Protein aggregation in retinal cells and approaches to cell protection. 1639 36

DJ-1 has been reported to have chaperone activity by preventing the aggregation of some proteins, and by structural analogy to Hsp31. The L166P mutation has been linked to a familial early onset form of Parkinson's disease (PD). Since the aggregation of alpha-synuclein is believed to be a critical step in the etiology of PD, we have investigated the interaction of wild-type DJ-1 and its oxidized forms with alpha-synuclein. Native (unoxidized) DJ-1 did not inhibit alpha-synuclein fibrillation, and no evidence for stable interactions between alpha-synuclein and native DJ-1 was observed. However, DJ-1 is very susceptible to oxidation by the addition of two oxygen atoms to form the sulfinic acid of Cys106 (2O DJ-1) (no 1O oxidized state is detectable). 2O DJ-1 was readily prepared by the addition of H(2)O(2) at concentrations up to a 20-fold molar excess. The oxidation of Cys106 to the sulfinic acid had minimal effect on the structural properties of DJ-1. However, 2O DJ-1 was very effective in preventing the fibrillation of alpha-synuclein, and only this form of DJ-1 appears to have significant anti-aggregation properties against alpha-synuclein. Further oxidation of DJ-1 leads to loss of some secondary structure, and to loss of the ability to inhibit alpha-synuclein fibrillation. Our observations confirm the suggestion that DJ-1 may act as an oxidative-stress-induced chaperone to prevent alpha-synuclein fibrillation. Since oxidative stress has been associated with PD, this observation may explain why mutations of DJ-1 could be a contributing factor in PD, and also indicates that excess oxidative stress could also lead to enhanced alpha-synuclein aggregation and hence PD.
J Mol Biol 2006 Mar 03
PMID:The oxidation state of DJ-1 regulates its chaperone activity toward alpha-synuclein. 1640 19


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