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1.
Acta Neuropathol Commun ; 12(1): 84, 2024 05 31.
Article in English | MEDLINE | ID: mdl-38822421

ABSTRACT

Alpha-synuclein (αsyn) is an intrinsically disordered protein that aggregates in the brain in several neurodegenerative diseases collectively called synucleinopathies. Phosphorylation of αsyn at serine 129 (PSER129) was considered rare in the healthy human brain but is enriched in pathological αsyn aggregates and is used as a specific marker for disease inclusions. However, recent observations challenge this assumption by demonstrating that PSER129 results from neuronal activity and can be readily detected in the non-diseased mammalian brain. Here, we investigated experimental conditions under which two distinct PSER129 pools, namely endogenous-PSER129 and aggregated-PSER129, could be detected and differentiated in the mammalian brain. Results showed that in the wild-type (WT) mouse brain, perfusion fixation conditions greatly influenced the detection of endogenous-PSER129, with endogenous-PSER129 being nearly undetectable after delayed perfusion fixation (30-min and 1-h postmortem interval). Exposure to anesthetics (e.g., Ketamine or xylazine) before perfusion did not significantly influence endogenous-PSER129 detection or levels. In situ, non-specific phosphatase calf alkaline phosphatase (CIAP) selectively dephosphorylated endogenous-PSER129 while αsyn preformed fibril (PFF)-seeded aggregates and genuine disease aggregates (Lewy pathology and Papp-Lantos bodies in Parkinson's disease and multiple systems atrophy brain, respectively) were resistant to CIAP-mediated dephosphorylation. The phosphatase resistance of aggregates was abolished by sample denaturation, and CIAP-resistant PSER129 was closely associated with proteinase K (PK)-resistant αsyn (i.e., a marker of aggregation). CIAP pretreatment allowed for highly specific detection of seeded αsyn aggregates in a mouse model that accumulates non-aggregated-PSER129. We conclude that αsyn aggregates are impervious to phosphatases, and CIAP pretreatment increases detection specificity for aggregated-PSER129, particularly in well-preserved biological samples (e.g., perfusion fixed or flash-frozen mammalian tissues) where there is a high probability of interference from endogenous-PSER129. Our findings have important implications for the mechanism of PSER129-accumulation in the synucleinopathy brain and provide a simple experimental method to differentiate endogenous-from aggregated PSER129.


Subject(s)
Brain , Mice, Inbred C57BL , alpha-Synuclein , Animals , Humans , Male , Mice , Alkaline Phosphatase/metabolism , alpha-Synuclein/metabolism , Brain/metabolism , Brain/pathology , Mice, Transgenic , Phosphoric Monoester Hydrolases/metabolism , Phosphorylation , Protein Aggregates/physiology , Protein Aggregation, Pathological/metabolism , Protein Aggregation, Pathological/pathology , Synucleinopathies/metabolism , Synucleinopathies/pathology
2.
bioRxiv ; 2024 Apr 09.
Article in English | MEDLINE | ID: mdl-38645137

ABSTRACT

Alpha-synuclein (αsyn) is an intrinsically disordered protein that aggregates in the brain in several neurodegenerative diseases collectively called synucleinopathies. Phosphorylation of αsyn at serine 129 (PSER129) was considered rare in the healthy human brain but is enriched in pathological αsyn aggregates and is used as a specific marker for disease inclusions. However, recent observations challenge this assumption by demonstrating that PSER129 results from neuronal activity and can be readily detected in the non-diseased mammalian brain. Here, we investigated experimental conditions under which two distinct PSER129 pools, namely endogenous-PSER129 and aggregated-PSER129, could be detected and differentiated in the mammalian brain. Results showed that in the wild-type (WT) mouse brain, perfusion fixation conditions greatly influenced the detection of endogenous-PSER129, with endogenous-PSER129 being nearly undetectable after delayed perfusion fixation (30-minute and 1-hour postmortem interval). Exposure to anesthetics (e.g., Ketamine or xylazine) before perfusion did not significantly influence endogenous-PSER129 detection or levels. In situ, non-specific phosphatase calf alkaline phosphatase (CIAP) selectively dephosphorylated endogenous-PSER129 while αsyn preformed fibril (PFF)-seeded aggregates and genuine disease aggregates (Lewy pathology and Papp-Lantos bodies in Parkinson's disease and multiple systems atrophy brain, respectively) were resistant to CIAP-mediated dephosphorylation. The phosphatase resistance of aggregates was abolished by sample denaturation, and CIAP-resistant PSER129 was closely associated with proteinase K (PK)-resistant αsyn (i.e., a marker of aggregation). CIAP pretreatment allowed for highly specific detection of seeded αsyn aggregates in a mouse model that accumulates non-aggregated-PSER129. We conclude that αsyn aggregates are impervious to phosphatases, and CIAP pretreatment increases detection specificity for aggregated-PSER129, particularly in well-preserved biological samples (e.g., perfusion fixed or flash-frozen mammalian tissues) where there is a high probability of interference from endogenous-PSER129. Our findings have important implications for the mechanism of PSER129-accumulation in the synucleinopathy brain and provide a simple experimental method to differentiate endogenous-from aggregated PSER129.

3.
Rev Neurol (Paris) ; 174(9): 644-652, 2018 Nov.
Article in English | MEDLINE | ID: mdl-30201422

ABSTRACT

Protein intracellular inclusions within the central nervous system are hallmarks of several progressive neurodegenerative disorders in man. The protein constituents of those deposits and the affected regions within the brain differ from one neurodegenerative disorder to another. Until recently, the vicious circle consisting of spread, seeded assembly and accumulation over time within the central nervous system of misfolded proteins aggregates was thought to be restricted to the prion protein PrP. Recent reports suggest that other protein aggregates spread and amplify within the central nervous system leading to distinct diseases. How alpha-synuclein protein assemblies traffic between cells, amplify by recruiting endogenous monomeric alpha-synuclein and cause distinct synucleinopathies is unclear. I review here the experimental evidence supporting the propagation of alpha-synuclein mega-dalton assemblies in a manner similar to prion protein aggregates. I also describe how alpha-synuclein aggregates. I also explain why the aggregation of alpha-synuclein may lead to distinct synucleinopathies.


Subject(s)
Parkinson Disease/etiology , Parkinson Disease/metabolism , Prion Diseases/complications , alpha-Synuclein/genetics , Humans , Parkinson Disease/genetics , Prion Diseases/genetics
4.
Cell Tissue Res ; 373(1): 195-212, 2018 Jul.
Article in English | MEDLINE | ID: mdl-29704213

ABSTRACT

Several age-related neurodegenerative disorders are characterized by the deposition of aberrantly folded endogenous proteins. These proteins have prion-like propagation and amplification properties but so far appear nontransmissible between individuals. Because of the features they share with the prion protein, PrP, the characteristics of pathogenic protein aggregates in several progressive brain disorders, including different types of Lewy body diseases (LBDs), such as Parkinson's disease (PD), multiple system atrophy (MSA) and dementia with Lewy bodies (DLB), have been actively investigated. Even though the pleomorphic nature of these syndromes might suggest different underlying causes, ɑ-synuclein (ɑSyn) appears to play an important role in this heterogeneous group of diseases (the synucleinopathies). An attractive hypothesis is that different types of ɑSyn protein assemblies have a unique and causative role in distinct synucleinopathies. We will discuss the recent research progress on ɑSyn assemblies involved in PD, MSA and DLB; their behavior as strains; current spreading hypotheses; their ability to seed centrally and peripherally; and their implication for disease pathogenesis.


Subject(s)
Lewy Body Disease/metabolism , Multiple System Atrophy/metabolism , Parkinson Disease/metabolism , alpha-Synuclein/metabolism , Animals , Humans , Lewy Body Disease/pathology , Multiple System Atrophy/pathology , Parkinson Disease/pathology , Prions/metabolism , Protein Aggregates , alpha-Synuclein/chemistry
5.
Neuropathol Appl Neurobiol ; 42(2): 137-52, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26111612

ABSTRACT

AIMS: The aggregation of Huntingtin (HTT) protein and of its moiety encoded by its Exon1 (HTTExon1) into fibrillar structures inside neurons is the molecular hallmark of Huntington's disease. Prion-like transmission of these aggregates between cells has been demonstrated. The cell-to-cell transmission mechanisms of these protein aggregates and the susceptibility of different kinds of neuronal cells to these toxic assemblies still need assessment. METHODS: Here, we documented the binding to and internalization by differentiated and undifferentiated neuroblastoma cells of exogenous fibrillar HTTExon1 and polyglutamine (polyQ) polypeptides containing the same number of glutamines. We assessed the contribution of endocytosis to fibrillar HTTExon1 uptake, their intracellular localization and fate. RESULTS: We observed that undifferentiated neuroblastoma cells were more susceptible to fibrillar HTTExon1 and polyQ than their differentiated counterparts. Furthermore, we demonstrated that exogenous HTTExon1 aggregates are mainly taken up by endocytosis and directed to lysosomal compartments in both mitotic and quiescent cells. CONCLUSIONS: These data suggest that the rates of endocytic processes that differ in mitotic and quiescent cells strongly impact the uptake of exogenous HTTExon1 and polyQ fibrils. This may be either the consequence of distinct metabolisms or distributions of specific protein partners for amyloid-like assemblies at the surface of highly dividing versus quiescent cells. Our results highlight the importance of endocytic processes in the internalization of exogenous HTTExon1 fibrils and suggest that a proportion of those assemblies reach the cytosol where they can amplify by recruiting the endogenous protein after escaping, by yet an unknown process, from the endo-lysosomal compartments.


Subject(s)
Huntingtin Protein/metabolism , Huntington Disease/metabolism , Neurons/cytology , Neurons/metabolism , Protein Aggregation, Pathological/metabolism , Cell Differentiation , Cell Line, Tumor , Endocytosis/physiology , Exons , Humans , Huntington Disease/pathology , Microscopy, Confocal , Microscopy, Electron, Transmission , Mitosis , Neuroblastoma , Peptide Fragments/metabolism , Peptides/metabolism , Protein Aggregation, Pathological/pathology , Transfection
6.
Nature ; 522(7556): 340-4, 2015 Jun 18.
Article in English | MEDLINE | ID: mdl-26061766

ABSTRACT

Misfolded protein aggregates represent a continuum with overlapping features in neurodegenerative diseases, but differences in protein components and affected brain regions. The molecular hallmark of synucleinopathies such as Parkinson's disease, dementia with Lewy bodies and multiple system atrophy are megadalton α-synuclein-rich deposits suggestive of one molecular event causing distinct disease phenotypes. Glial α-synuclein (α-SYN) filamentous deposits are prominent in multiple system atrophy and neuronal α-SYN inclusions are found in Parkinson's disease and dementia with Lewy bodies. The discovery of α-SYN assemblies with different structural characteristics or 'strains' has led to the hypothesis that strains could account for the different clinico-pathological traits within synucleinopathies. In this study we show that α-SYN strain conformation and seeding propensity lead to distinct histopathological and behavioural phenotypes. We assess the properties of structurally well-defined α-SYN assemblies (oligomers, ribbons and fibrils) after injection in rat brain. We prove that α-SYN strains amplify in vivo. Fibrils seem to be the major toxic strain, resulting in progressive motor impairment and cell death, whereas ribbons cause a distinct histopathological phenotype displaying Parkinson's disease and multiple system atrophy traits. Additionally, we show that α-SYN assemblies cross the blood-brain barrier and distribute to the central nervous system after intravenous injection. Our results demonstrate that distinct α-SYN strains display differential seeding capacities, inducing strain-specific pathology and neurotoxic phenotypes.


Subject(s)
Lewy Body Disease/chemically induced , Multiple System Atrophy/chemically induced , Parkinson Disease/pathology , alpha-Synuclein/administration & dosage , alpha-Synuclein/toxicity , Animals , Blood-Brain Barrier , Brain/drug effects , Brain/metabolism , Female , Humans , Lewy Body Disease/metabolism , Lewy Body Disease/pathology , Multiple System Atrophy/metabolism , Multiple System Atrophy/pathology , Parkinson Disease/metabolism , Phenotype , Rats , Rats, Wistar , Substantia Nigra/drug effects , Substantia Nigra/metabolism , Substantia Nigra/pathology , Synapses/metabolism , Synapses/pathology , alpha-Synuclein/chemistry , alpha-Synuclein/classification
7.
Pathol Biol (Paris) ; 62(3): 162-8, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24698014

ABSTRACT

Prion protein and prion-like proteins share a number of characteristics. From the molecular point of view, they are constitutive proteins that aggregate following conformational changes into insoluble particles. These particles escape the cellular clearance machinery and amplify by recruiting the soluble for of their constituting proteins. The resulting protein aggregates are responsible for a number of neurodegenerative diseases such as Creutzfeldt-Jacob, Alzheimer, Parkinson and Huntington diseases. In addition, there are increasing evidences supporting the inter-cellular trafficking of these aggregates, meaning that they are "transmissible" between cells. There are also evidences that brain homogenates from individuals developing Alzheimer and Parkinson diseases propagate the disease in recipient model animals in a manner similar to brain extracts of patients developing Creutzfeldt-Jacob's disease. Thus, the propagation of protein aggregates from cell to cell may be a generic phenomenon that contributes to the evolution of neurodegenerative diseases, which has important consequences on human health issues. Moreover, although the distribution of protein aggregates is characteristic for each disease, new evidences indicate the possibility of overlaps and crosstalk between the different disorders. Despite the increasing evidences that support prion or prion-like propagation of protein aggregates, there are many unanswered questions regarding the mechanisms of toxicity and this is a field of intensive research nowadays.


Subject(s)
Nerve Tissue Proteins/chemistry , Neurodegenerative Diseases/metabolism , Prion Diseases/metabolism , Protein Aggregation, Pathological/metabolism , Aging , Alzheimer Disease/prevention & control , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/pathology , Animals , Autophagy , Biopolymers , Clinical Trials, Phase II as Topic , Disease Models, Animal , Drug Evaluation, Preclinical , Endocytosis , Humans , Inclusion Bodies/chemistry , Inclusion Bodies/pathology , Mice , Neurodegenerative Diseases/pathology , Neurofibrillary Tangles/chemistry , Neurofibrillary Tangles/pathology , Plaque, Amyloid/chemistry , Plaque, Amyloid/pathology , Polysaccharides/therapeutic use , Prion Diseases/pathology , Prion Diseases/veterinary , Prions/chemistry , Protein Aggregation, Pathological/pathology , Protein Conformation , Solubility
8.
J Mol Biol ; 357(4): 1306-21, 2006 Apr 07.
Article in English | MEDLINE | ID: mdl-16483608

ABSTRACT

A novel computational approach to the structural analysis of ordered beta-aggregation is presented and validated on three known amyloidogenic polypeptides. The strategy is based on the decomposition of the sequence into overlapping stretches and equilibrium implicit solvent molecular dynamics (MD) simulations of an oligomeric system for each stretch. The structural stability of the in-register parallel aggregates sampled in the implicit solvent runs is further evaluated using explicit water simulations for a subset of the stretches. The beta-aggregation propensity along the sequence of the Alzheimer's amyloid-beta peptide (Abeta(42)) is found to be highly heterogeneous with a maximum in the segment V(12)HHQKLVFFAE(22) and minima at S(8)G(9), G(25)S(26), G(29)A(30), and G(38)V(39), which are turn-like segments. The simulation results suggest that these sites may play a crucial role in determining the aggregation tendency and the fibrillar structure of Abeta(42). Similar findings are obtained for the human amylin, a 37-residue peptide that displays a maximal beta-aggregation propensity at Q(10)RLANFLVHSSNN(22) and two turn-like sites at G(24)A(25) and G(33)S(34). In the third application, the MD approach is used to identify beta-aggregation "hot-spots" within the N-terminal domain of the yeast prion Ure2p (Ure2p(1-94)) and to design a double-point mutant (Ure2p-N4748S(1-94)) with lower beta-aggregation propensity. The change in the aggregation propensity of Ure2p-N4748S(1-94) is verified in vitro using the thioflavin T binding assay.


Subject(s)
Amyloid beta-Peptides/chemistry , Amyloid/chemistry , Computer Simulation , Peptide Fragments/chemistry , Prions/chemistry , Protein Structure, Secondary , Saccharomyces cerevisiae Proteins/chemistry , Amino Acid Sequence , Amyloid/genetics , Amyloid/metabolism , Amyloid beta-Peptides/genetics , Amyloid beta-Peptides/metabolism , Fungal Proteins/chemistry , Fungal Proteins/genetics , Fungal Proteins/metabolism , Glutathione Peroxidase , Humans , Islet Amyloid Polypeptide , Models, Molecular , Molecular Conformation , Molecular Sequence Data , Peptide Fragments/genetics , Peptide Fragments/metabolism , Point Mutation , Prions/genetics , Prions/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Sequence Alignment
9.
J Med Genet ; 41(9): 647-51, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15342693

ABSTRACT

INTRODUCTION: Primary open-angle glaucoma (POAG) is a leading cause of visual impairment worldwide and a complex genetic disorder that affects mostly adults. Mutations in the MYOCILIN (MYOC) and OPTINEURIN genes account for rare forms with a Mendelian inheritance and for <5% of all POAG cases. The CYP1B1 gene, a member of the cytochrome P450 gene family, is a major cause of primary congenital glaucoma (PCG), a rare and severely blinding disease with recessive inheritance. However, CYP1B1 mutations have also been associated with cases of juvenile-onset glaucoma in some PCG families or shown to modify the age of onset of glaucoma linked to a MYOC mutation in a large family. OBJECTIVE: To investigate the role of CYP1B1 mutations in POAG predisposition, irrespective of the presence of a MYOC mutation. METHODS AND SUBJECTS: CYP1B1 coding region variation was characterised by denaturing high performance liquid chromatography (DHPLC) and sequencing in 236 unrelated French Caucasian POAG patients and 47 population-matched controls. RESULTS: Eleven (4.6%) patients carried one or two mutated CYP1B1 gene(s) and no MYOC mutation. They showed juvenile or middle-age onset of disease (median age at diagnosis, 40 years, range 13-52), significantly earlier than in non-carrier patients. Apart from one, all mutations detected in POAG patients were previously associated with PCG. CONCLUSION: CYP1B1 mutations might pose a significant risk for early-onset POAG and might also modify glaucoma phenotype in patients who do not carry a MYOC mutation.


Subject(s)
Aryl Hydrocarbon Hydroxylases/genetics , Glaucoma, Open-Angle/genetics , Mutation/genetics , Adolescent , Adult , Age of Onset , Aged , Aged, 80 and over , Child , Cytochrome P-450 CYP1B1 , DNA Mutational Analysis , Female , France , Genetic Testing , Genetic Variation/genetics , Glaucoma, Open-Angle/physiopathology , Humans , Male , Middle Aged , Pedigree
11.
J Musculoskelet Neuronal Interact ; 3(3): 232-9, 2003 Sep.
Article in English | MEDLINE | ID: mdl-15758346

ABSTRACT

OBJECTIVE: This study was aimed to assess age changes in quantitative ultrasonometry (QUS) in a large sample of Lebanese women to determine a Lebanese reference population. DESIGN: Cross-sectional study. SUBJECTS AND METHODS: Broadband ultrasound attenuation (BUA) and speed of sound (SOS) and the stiffness index (SI) of the os calcaneus was measured in 4,320 women with a mean age of 52.5 years (age range 20 to 79 years) using three identical Achilles Express (GE/Lunar) and one Achilles Plus (GE/Lunar) ultrasonometry devices. Women were randomly selected and asked to participate in a nationwide screening program using the media, conferences, telephone calls etc. Measurements were performed at Red Cross centers located all over the country. No inclusion or exclusion criteria were used. RESULTS: There was an overall decline of 19.2% for BUA, 3.1% for SOS and 30.3% for SI between late adolescence and old age. In premenopausal women, BUA decreased only slightly by 3%, while postmenopausal women showed a significant decline of 16.2%. In contrast, SOS continuously decreased from the age of 42; there was a decline of 0.8% from adolescence to the menopause; postmenopausal women showed a larger decline of 2.4%. The SI of premenopausal women decreased by 6%, while postmenopausal women showed a significantly larger decline of 24.3%. SI value for the female Lebanese young adult reference is 8% lower than that of the American and European women (92 SI units compared to 100). At the age of 42, SI value for the Lebanese women is 10.4% lower than the American women and 7.5% lower than the European women (86 SI units compared to 96 and 93, respectively). At the age of 75, SI values for the Lebanese women is 4.4% lower than the American women and the European women (65 SI units compared to 68). The decline in stiffness index for the Lebanese women between age 20 and 75 years is about 30.3% compared to 32% for the American or European reference curves. The rate of decrease for the Lebanese women was 0.2 SI units per year for the premenopausal period, and 0.7 SI units per year for the postmenopausal period. CONCLUSION: The age-related female, Lebanese reference curve was significantly different from the American and the European reference curves used by the manufacturer. Therefore, the use of our standardized reference data instead of the proposed US or European database reduces the risk of overestimating osteoporosis in the Lebanese population. The impact of our results on the prevalence of osteoporotic fracture in Lebanon has to be evaluated later on.

12.
Clin Genet ; 62(4): 334-9, 2002 Oct.
Article in English | MEDLINE | ID: mdl-12372064

ABSTRACT

Primary congenital glaucoma (PCG) is a heterogeneous autosomal recessive disorder caused by unknown developmental defect(s) of the anterior chamber of the eye. A member of the cytochrome P450 gene family, CYP1B1, was found to be mutated in PCG patients in different populations, albeit to a variable extent. In this study, CYP1B1 mutations were searched for in 32 unrelated PCG patients from Morocco. Two mutations were detected in 11 (34%) patients. One, 4339delG, is novel and causes a frameshift at residue 179. The other, G61E, was previously found in patients from Turkey and Saudi Arabia. Seven patients were homozygous for 4339delG and two other patients for G61E, whereas the two remaining patients were compound heterozygotes. The close association of 4339delG with a rare allele of D2S177, a microsatellite marker located 270 kb upstream of CYP1B1, strongly suggested a founder effect for 4339delG. The occurrence of this mutation was tentatively dated at between 900 and 1700 years ago. Typing 4339delG and G61E mutations should help to prevent blindness resulting from a delayed diagnosis of PCG in Morocco.


Subject(s)
Aryl Hydrocarbon Hydroxylases/genetics , Frameshift Mutation , Glaucoma/genetics , Cytochrome P-450 CYP1B1 , DNA Mutational Analysis , Exons , Founder Effect , Genotype , Glaucoma/congenital , Humans , Microsatellite Repeats , Morocco , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length
13.
Biochemistry ; 40(45): 13564-73, 2001 Nov 13.
Article in English | MEDLINE | ID: mdl-11695904

ABSTRACT

The [URE3] phenotype in yeast Saccharomyces cerevisiae is due to an altered prion form of Ure2p, a protein involved in nitrogen catabolism. To understand possible conformational changes at the origin of prion propagation, we previously solved the crystal structure of the Ure2p functional region [Bousset et al. (2001) Structure 9, 39-46]. We showed the protein to have a fold similar to that of the beta class of glutathione S-transferases (GSTs). Here we report crystal structures of the Ure2p functional region (extending from residues 95-354) in complex with glutathione (GSH), the substrate of all GSTs, and two widely used GST inhibitors, namely, S-hexylglutathione and S-p-nitrobenzylglutathione. In a manner similar to what is observed in many GSTs, ligand binding is not accompanied by a significant change in the conformation of the protein. We identify one GSH and one hydrophobic electrophile binding site per monomer as observed in all other GSTs. The sulfur group of GSH, that conjugates electrophiles, is located near the amide group of Asn124, allowing a hydrogen bond to be formed. Biochemical data indicate that GSH binds to Ure2p with high affinity. Its binding affects Ure2p oligomerization but has no effect on the assembly of the protein into amyloid fibrils. Despite results indicating that Ure2p lacks GST activity, we propose that Ure2p is a member of the GST superfamily that may describe a novel GST class. Our data bring new insights into the function of the Ure2p active region.


Subject(s)
Fungal Proteins/chemistry , Glutathione/analogs & derivatives , Glutathione/chemistry , Prions , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/chemistry , Amino Acid Sequence , Binding Sites , Crystallization , Crystallography, X-Ray , Fungal Proteins/metabolism , Glutathione Peroxidase , Models, Molecular , Molecular Sequence Data , Sequence Homology, Amino Acid
14.
J Struct Biol ; 135(2): 170-5, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11580266

ABSTRACT

Current biochemical and structural studies on the conformational changes induced by the nature of nucleotide bound to the chaperonin containing testis complex polypeptide 1 (CCT) are examined to see how consistent the data are. This exercise suggests that the biochemical and structural data are in good agreement. CCT clearly appears as a folding nano-machine fueled by ATP. A careful comparison of the biochemical and structural data, however, highlights a number of points that remain to be carefully documented in order to better understand the nature of the conformational changes in CCT that yield folded target proteins. Special effort should be made to clearly answer the points listed at the end of this review in order to obtain the dynamic sequence of events yielding folded proteins in the eukaryotic cytoplasm similar to what has been obtained for prokaryotes.


Subject(s)
Adenosine Triphosphate/pharmacology , Chaperonins/chemistry , Animals , Chaperonin Containing TCP-1 , Chaperonins/drug effects , Eukaryotic Cells/chemistry , Humans , Protein Conformation/drug effects , Protein Folding
15.
Biochemistry ; 40(6): 1764-73, 2001 Feb 13.
Article in English | MEDLINE | ID: mdl-11327838

ABSTRACT

The [URE3] factor of Saccharomyces cerevisiae propagates by a prion-like mechanism and corresponds to the loss of the function of the cellular protein Ure2. The molecular basis of the propagation of this phenotype is unknown. We recently expressed Ure2p in Escherichia coli and demonstrated that the N-terminal region of the protein is flexible and unstructured, while its C-terminal region is compactly folded. Ure2p oligomerizes in solution to form mainly dimers that assemble into fibrils [Thual et al. (1999) J. Biol. Chem. 274, 13666-13674]. To determine the role played by each domain of Ure2p in the overall properties of the protein, specifically, its stability, conformation, and capacity to assemble into fibrils, we have further analyzed the properties of Ure2p N- and C-terminal regions. We show here that Ure2p dimerizes through its C-terminal region. We also show that the N-terminal region is essential for directing the assembly of the protein into a particular pathway that yields amyloid fibrils. A full-length Ure2p variant that possesses an additional tryptophan residue in its N-terminal moiety was generated to follow conformational changes affecting this domain. Comparison of the overall conformation, folding, and unfolding properties, and the behavior upon proteolytic treatments of full-length Ure2p, Ure2pW37 variant, and Ure2p C-terminal fragment reveals that Ure2p N-terminal domain confers no additional stability to the protein. This study reveals the existence of a stable unfolding intermediate of Ure2p under conditions where the protein assembles into amyloid fibrils. Our results contradict the intramolecular interaction between the N- and C-terminal moieties of Ure2p and the single unfolding transitions reported in a number of previous studies.


Subject(s)
Fungal Proteins/metabolism , Prions/metabolism , Protein Folding , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/metabolism , Amyloid/metabolism , Circular Dichroism , Dimerization , Fungal Proteins/chemistry , Fungal Proteins/genetics , Fungal Proteins/ultrastructure , Glutathione Peroxidase , Guanidine , Kinetics , Molecular Weight , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Peptide Fragments/ultrastructure , Prions/chemistry , Prions/genetics , Prions/ultrastructure , Protein Denaturation , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructure , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/ultrastructure , Solubility , Spectrometry, Fluorescence
16.
Structure ; 9(1): 39-46, 2001 Jan 10.
Article in English | MEDLINE | ID: mdl-11342133

ABSTRACT

BACKGROUND: The [URE3] non-Mendelian element of the yeast S. cerevisiae is due to the propagation of a transmissible form of the protein Ure2. The infectivity of Ure2p is thought to originate from a conformational change of the normal form of the prion protein. This conformational change generates a form of Ure2p that assembles into amyloid fibrils. Hence, knowledge of the three-dimensional structure of prion proteins such as Ure2p should help in understanding the mechanism of amyloid formation associated with a number of neurodegenerative diseases. RESULTS: Here we report the three-dimensional crystal structure of the globular region of Ure2p (residues 95--354), also called the functional region, solved at 2.5 A resolution by the MAD method. The structure of Ure2p 95--354 shows a two-domain protein forming a globular dimer. The N-terminal domain is composed of a central 4 strand beta sheet flanked by four alpha helices, two on each side. In contrast, the C-terminal domain is entirely alpha-helical. The fold of Ure2p 95--354 resembles that of the beta class glutathione S-transferases (GST), in line with a weak similarity in the amino acid sequence that exists between these proteins. Ure2p dimerizes as GST does and possesses a potential ligand binding site, although it lacks GST activity. CONCLUSIONS: The structure of the functional region of Ure2p is the first crystal structure of a prion protein. Structure comparisons between Ure2p 95--354 and GST identified a 32 amino acid residues cap region in Ure2p exposed to the solvent. The cap region is highly flexible and may interact with the N-terminal region of the partner subunit in the dimer. The implication of this interaction in the assembly of Ure2p into amyloid fibrils is discussed.


Subject(s)
Fungal Proteins/chemistry , Prions/chemistry , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/chemistry , Amino Acid Sequence , Amyloid/chemistry , Binding Sites , Crystallography, X-Ray , Dimerization , Glutathione Peroxidase , Glutathione Transferase/chemistry , Ligands , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Homology, Amino Acid
17.
J Biol Chem ; 275(29): 21975-80, 2000 Jul 21.
Article in English | MEDLINE | ID: mdl-10764751

ABSTRACT

gamma-Tubulin is required for nucleation and polarized organization of microtubules in vivo. The mechanism of microtubule nucleation by gamma-tubulin and the role of associated proteins is not understood. Here we show that in vitro translated monomeric gamma-tubulin nucleates microtubules by lowering the size of the nucleus from seven to three tubulin subunits. In capping the minus end with high affinity (10(10) m(-1)) and a binding stoichiometry of one molecule of gamma-tubulin/microtubule, gamma-tubulin establishes the critical concentration of the plus end in the medium and prevents minus end growth. gamma-Tubulin interacts strongly with beta-tubulin. A structural model accounts for these results.


Subject(s)
Microtubules/chemistry , Tubulin/chemistry , Dimerization , Humans , Microtubules/metabolism , Tubulin/metabolism
18.
J Cell Sci ; 113 ( Pt 5): 877-86, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10671377

ABSTRACT

Actin related protein of vertebrate, Arp1, is a major component of the dynactin complex. To characterise and localise Arp1 during mammalian spermatogenesis, polyclonal antibodies were raised against a human recombinant Arp1. Anti-Arp1 antibodies were used for western-immunoblotting, indirect immunofluorescence and immunoelectron microscopy. In round spermatids, Arp1 was detected at the centrosome and at the Golgi apparatus. In elongated spermatids, Arp1 was predominantly found along microtubules of the manchette and at their site of attachment to the nuclear envelope. In maturing spermatids, Arp1 was still present in the pericentriolar material, but in testicular spermatozoa it was not detectable. These various localisations of Arp1 and their changes during spermatid differentiation suggest that the dynactin complex in association with dynein might contribute to several activities: the functional organisation of the centrosome and of the Golgi apparatus and the shaping of the nucleus by manchette microtubules.


Subject(s)
Cell Nucleus/physiology , DNA-Binding Proteins/physiology , Golgi Apparatus/metabolism , Microtubules/physiology , Receptors, Steroid , Spermatogenesis/physiology , Transcription Factors/physiology , Animals , Antibody Specificity , COUP Transcription Factor II , COUP Transcription Factors , Cell Nucleus/chemistry , Cell Nucleus/ultrastructure , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/ultrastructure , Fluorescent Antibody Technique, Indirect , Golgi Apparatus/chemistry , Golgi Apparatus/ultrastructure , Humans , Immune Sera/chemistry , Immunohistochemistry , Macaca fascicularis , Male , Mice , Microscopy, Immunoelectron , Microtubules/chemistry , Microtubules/ultrastructure , Nuclear Envelope/immunology , Nuclear Envelope/metabolism , Nuclear Envelope/ultrastructure , Rabbits , Rats , Rats, Sprague-Dawley , Swine , Transcription Factors/chemistry , Transcription Factors/metabolism , Transcription Factors/ultrastructure
19.
J Biol Chem ; 274(19): 13666-74, 1999 May 07.
Article in English | MEDLINE | ID: mdl-10224139

ABSTRACT

Sacchromyces cerevisiae prion-like protein Ure2 was expressed in Escherichia coli and was purified to homogeneity. We show here that Ure2p is a soluble protein that can assemble into fibers that are similar to the fibers observed in the case of PrP in its scrapie prion filaments form or that form on Sup35 self-assembly. Ure2p self-assembly is a cooperative process where one can distinguish a lag phase followed by an elongation phase preceding a plateau. A combination of size exclusion chromatography, sedimentation velocity, and electron microscopy demonstrates that the soluble form of Ure2p consists at least of three forms of the protein as follows: a monomeric, dimeric, and tetrameric form whose abundance is concentration-dependent. By the use of limited proteolysis, intrinsic fluorescence, and circular dichroism measurements, we bring strong evidence for the existence of at least two structural domains in Ure2p molecules. Indeed, Ure2p NH2-terminal region is found poorly structured, whereas its COOH-terminal domain appears to be compactly folded. Finally, we show that only slight conformational changes accompany Ure2p assembly into insoluble high molecular weight oligomers. These changes essentially affect the COOH-terminal part of the molecule. The properties of Ure2p are compared in the discussion to that of other prion-like proteins such as Sup35 and mammalian prion protein PrP.


Subject(s)
Fungal Proteins/chemistry , Prions , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/chemistry , Base Sequence , Biopolymers , Cloning, Molecular , DNA Primers , Escherichia coli/genetics , Fungal Proteins/genetics , Glutathione Peroxidase , Microscopy, Electron , Molecular Weight , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Saccharomyces cerevisiae/genetics , Solubility
20.
Biochemistry ; 38(11): 3246-57, 1999 Mar 16.
Article in English | MEDLINE | ID: mdl-10079067

ABSTRACT

The nonhomologous proteins actin and alpha- and beta-tubulin need the assistance of the cytosolic chaperonin containing TCP-1 (CCT) to reach their correct native state, and their folding requires a transient binary complex formation with CCT. We show that separate or combined deletion of three delineated hydrophobic sequences in actin disturbs the interaction with CCT. These sites are situated between residues 125-179, 244-285, and 340-375. Also, alpha- and beta-tubulin contain at least one recognition region, and intriguingly, it has a similar distribution of hydrophobic residues as region 244-285 in actin. Internal deletion of the sites in actin favor a model for cooperative binding of target proteins to CCT. Peptide mimetics, representing the binding regions, inhibit target polypeptide binding to CCT, suggesting that actin and tubulin contact similar CCT subunits. In addition, we show that actin recognition by class II chaperonins is different from that by class I.


Subject(s)
Chaperonins/metabolism , Cytosol/metabolism , Actins/genetics , Actins/metabolism , Amino Acid Sequence , Animals , Binding Sites/genetics , Binding, Competitive/genetics , Cell-Free System , Chaperonin 60/metabolism , Chaperonin Containing TCP-1 , Chaperonins/genetics , Mice , Models, Molecular , Molecular Mimicry , Molecular Sequence Data , Mutagenesis, Site-Directed , Peptide Fragments/genetics , Peptide Fragments/metabolism , Rabbits , Reticulocytes , Sequence Deletion , Tubulin/metabolism
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