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1.
Zh Nevrol Psikhiatr Im S S Korsakova ; 121(11. Vyp. 2): 31-37, 2021.
Article in Russian | MEDLINE | ID: mdl-35038844

ABSTRACT

OBJECTIVE: To evaluate effectiveness and safety of umbilical cord blood cells (UCBC) in children with ASD. MATERIAL AND METHODS: The study comprised 13 boys and 2 girls, mean age 7.0±0.5 years (test group), and 9 boys and 1 girl, mean age 6.0±1.3 years (control group) diagnosed with autism or autistic syndrome. UCBC were infused intravenously in a single dose of 250±20 million cells, four times at 14±3-day intervals. Dynamics of cognitive functions were assessed with WISC subscales, questionnaire Skvortsov Developmental Profile Survey and brainstem auditory evoked potentials (BAEPs) conducted before therapy and six months after the first injection. The dynamic of autistic symptoms was explored with CASD and ATEC questionnaires, fulfilled at the start of the study, 3 and 6 months later (test group) or at the start of the study, and 6 months later (control group). RESULTS: UCBC was well tolerated and caused no appreciable adverse effects. Observation revealed improvement in cognitive functioning and alleviation of autistic symptoms in patients of the test group six months after the first UCBC injection. Positive dynamics were more noticeable in the test group than in the control group receiving standard therapy. CONCLUSION: The use of UCBC is safe and might be effective in the complex therapy of autism.


Subject(s)
Autism Spectrum Disorder , Autistic Disorder , Hematopoietic Stem Cell Transplantation , Autistic Disorder/therapy , Cell- and Tissue-Based Therapy , Child , Child, Preschool , Female , Fetal Blood , Humans , Male
3.
Ross Fiziol Zh Im I M Sechenova ; 90(5): 522-36, 2004 May.
Article in Russian | MEDLINE | ID: mdl-15341079

ABSTRACT

We used double immunofluorescence and electron microscopy to study the spatial relationships between Weibel--Palade bodies (WPBs) and cytoskeletal elements in endothelial cells treated with thrombin or cytoskeleton-damaging agents. We have found that some WPBs undergo translocation towards the centrosome in 5 min in the cells treated with thrombin, cytochalasin B or calyculin A. The cells treated with thrombin or cytochalasin exhibit depletion of WPBs, whereas WPBs found at the cell periphery were colocalized with intermediate filaments. There was a precise colocalization observed between the WPBs and microtubules in the calyculin-treated cells in which all WPBs undergo centrosome-directed translocation within 15 min after the agent addition. When vimentin filaments were induced to collapse by demecolcine, intermediate filaments and WPBs both translocated to the perinuclear region. The data provide the first direct evidence that secretory granules utilize microtubules to move in retrograde direction, i.e., away from the plasma membrane, towards the centrosome. We suggest that anterograde movement of WPBs is dependent on their interaction with vimentin filaments.


Subject(s)
Aorta/metabolism , Endothelial Cells/metabolism , Microtubules/metabolism , Weibel-Palade Bodies/metabolism , von Willebrand Factor/metabolism , Aorta/cytology , Aorta/ultrastructure , Cells, Cultured , Endothelial Cells/ultrastructure , Humans , Intermediate Filaments/metabolism , Microscopy, Electron, Transmission , Microtubules/ultrastructure , Protein Transport/physiology , Vimentin/metabolism , Weibel-Palade Bodies/ultrastructure
4.
Mol Microbiol ; 53(2): 687-96, 2004 Jul.
Article in English | MEDLINE | ID: mdl-15228544

ABSTRACT

The translation termination factor eRF1 recognizes stop codons at the A site of the ribosome and induces peptidyl-tRNA hydrolysis at the peptidyl transferase centre. Recent data show that, besides translation, yeast eRF1 is also involved in cell cycle regulation. To clarify the mechanisms of non-translational functions of eRF1, we performed a genetic screen for its novel partner proteins. This screen revealed the gene for myosin light chain, Mlc1p, acting as a dosage suppressor of a temperature-sensitive mutation in the SUP45 gene encoding eRF1. eRF1 and Mlc1p are able to interact with each other and, similarly to depletion of Mlc1p, mutations in the SUP45 gene may affect cytokinesis. Immunofluorescent staining performed to determine localization of Mlc1p has shown that the sup45 mutation, which arrests cytokinesis, redistributed Mlc1p, causing its disappearance from the bud tip and the bud neck. The data obtained demonstrate that yeast eRF1 has an important non-translational function effecting cytokinesis via interaction with Mlc1p.


Subject(s)
Cytokinesis , Myosin Light Chains/physiology , Peptide Termination Factors/physiology , Saccharomyces cerevisiae Proteins/isolation & purification , Saccharomyces cerevisiae Proteins/physiology , Saccharomyces cerevisiae/physiology , Cytoplasm/chemistry , Genes, Fungal , Genes, Suppressor , Mutation , Myosin Light Chains/genetics , Peptide Chain Termination, Translational , Peptide Termination Factors/genetics , Precipitin Tests , Protein Binding , Protein Biosynthesis , Protein Interaction Mapping , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/genetics , Suppression, Genetic
5.
Ross Fiziol Zh Im I M Sechenova ; 90(10): 1203-14, 2004 Oct.
Article in Russian | MEDLINE | ID: mdl-15628176

ABSTRACT

The intima hyperplasia is a major morphological feature of various arterial pathologies such as atherosclerosis, postangioplasty restenosis and transplantation arteriopathy. It is commonly assumed that smooth muscle cells (SMC) comprising loci of the intima hyperplasia originate from arterial media. However, recent studies suggest that the bone marrow could also supply circulating vascular progenitor of SMCs and endothelial cells (EC). Such bone marrow progenitors participate in the formation of a cellular mass of neointima after experimental allotransplantation, mechanical vessel injury or hyperlipidemia induced experimental atherosclerosis. Circulating SMC and EC progenitors are also likely to be involved in the transplantation arteriopathy development in humans but their roles in the atherosclerosis and restenosis remain to be determined. Stages of the mobilization, defferentiation and proliferation of SMC progenitors could provide point of attack for new therapeutic strategies for the treatment of proliferative vascular diseases. The precise understanding of the neointima cells origin could provide a key for development of the optimal therapeutic strategy of treatnent of such disorders. This review is focused on the pathological significance of circulating progenitors of the bone marrow origin, particularly on the SMC progenitors, for development of vascular wall disorders.


Subject(s)
Arteries/physiopathology , Tunica Intima/pathology , Tunica Intima/physiopathology , Animals , Arteries/pathology , Bone Marrow Cells/metabolism , Bone Marrow Cells/pathology , Endothelial Cells/metabolism , Endothelial Cells/pathology , Humans , Hyperplasia/pathology , Hyperplasia/physiopathology , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/pathology
6.
Prikl Biokhim Mikrobiol ; 38(5): 523-8, 2002.
Article in Russian | MEDLINE | ID: mdl-12391753

ABSTRACT

Results of industrial exploitation of a biofiltration plant tailored for purifying gaseous discharges of hazardous organic components such as toluene, cyclohexane, and xylene, are examined. Both numerical and compositional variations were monitored for a long-term (more than 1.5 years) utilization process in an association of microorganisms decomposing organic pollutants. A population of microbial association composed by one yeast and two bacterial strains in the biofilm on the surface of filtering sheets was abundant (10(8)-10(9) yeast cells/cm2 and 10(10)-10(11) bacterial cells/cm2) and stable during the whole period of monitoring. A microbial association in the culture medium averaging 10(6) yeast cells/l and 10(8) bacterial cells/l is more susceptible to technogenic impacts and seasonal fluctuations. Overall, the biofilter as an open and autonomic system maintained its microbial association, thereby providing a high-degree (93-98%) purification of industrial gaseous discharges from organic pollutants.


Subject(s)
Filtration/methods , Industrial Waste , Microbiology , Colony Count, Microbial , Membranes, Artificial
7.
BMC Mol Biol ; 2: 9, 2001.
Article in English | MEDLINE | ID: mdl-11570975

ABSTRACT

BACKGROUND: Termination of translation in eukaryotes is controlled by two interacting polypeptide chain release factors, eRFl and eRF3. eRFl recognizes nonsense codons UAA, UAG and UGA, while eRF3 stimulates polypeptide release from the ribosome in a GTP- and eRFl - dependent manner. Recent studies has shown that proteins interacting with these release factors can modulate the efficiency of nonsense codon readthrough. RESULTS: We have isolated a nonessential yeast gene, which causes suppression of nonsense mutations, being in a multicopy state. This gene encodes a protein designated Itt1p, possessing a zinc finger domain characteristic of the TRIAD proteins of higher eukaryotes. Overexpression of Itt1p decreases the efficiency of translation termination, resulting in the readthrough of all three types of nonsense codons. Itt1p interacts in vitro with both eRFl and eRF3. Overexpression of eRFl, but not of eRF3, abolishes the nonsense suppressor effect of overexpressed Itt1p. CONCLUSIONS: The data obtained demonstrate that Itt1p can modulate the efficiency of translation termination in yeast. This protein possesses a zinc finger domain characteristic of the TRIAD proteins of higher eukaryotes, and this is a first observation of such protein being involved in translation.

8.
Yeast ; 18(6): 489-97, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11284005

ABSTRACT

The yeast cytoplasmically-inherited nonsense suppressor [PSI(+)] determinant is presumed to be a manifestation of the aggregated prion-like state of the Sup35 protein. Overexpression of the Sup35 protein induces generation of [PSI(+)] determinants with various suppressor efficiency and mitotic stabilities. Here, we demonstrate that the relative frequency of appearance of [PSI(+)] with different properties depends on the SUP35 allele used to induce their generation. The difference in properties of [PSI(+)] determinants was preserved after their transmission from one yeast strain to another. This difference correlated with variation in properties of the Sup35 protein. A novel type of prion instability was observed: some [PSI(+)] with weak suppressor efficiency could convert spontaneously into strong suppressor determinants.


Subject(s)
Fungal Proteins/genetics , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Suppression, Genetic/genetics , Alleles , Blotting, Western , Cell Aggregation/genetics , Cell Cycle , Fungal Proteins/biosynthesis , Gene Expression Regulation, Fungal/genetics , Peptide Termination Factors , Phenotype , Plasmids , Prions/genetics , Prions/metabolism , Saccharomyces cerevisiae/metabolism , Species Specificity
9.
Curr Biol ; 10(22): 1443-6, 2000 Nov 16.
Article in English | MEDLINE | ID: mdl-11102806

ABSTRACT

The [PSI(+)] nonsense-suppressor determinant of Saccharomyces cerevisiae results from the ability of Sup35 (eRF3) translation termination factor to undergo prion-like aggregation [1]. Although this process is autocatalytic, in vivo it depends on the chaperone Hsp104, whose lack or overexpression can cure [PSI(+)] [2]. Overproduction of the chaperone protein Ssb1 increased the [PSI(+)] curing by excess Hsp104, although it had no effect on its own, and excess chaperone protein Ssa1 protected [PSI(+)] against Hsp104 [3,4]. We used an artificial [PSI(+)(PS)] based on the Sup35 prion-forming domain from yeast Pichia methanolica [5] to find other prion-curing factors. Both [PSI(+)(PS)] and [PSI(+)] have prion 'strains', differing in their suppressor efficiency and mitotic stability. We show that [PSI(+)(PS)] and a 'weak' strain of [PSI(+)] can be cured by overexpression of chaperones Ssa1, Ssb1 and Ydj1. The ability of different chaperones to cure [PSI(+)(PS)] showed significant prion strain specificity, which could be related to variation in Sup35 prion structure. Our results imply that homologs of these chaperones may be active against mammalian prion and amyloid diseases.


Subject(s)
Fungal Proteins/metabolism , HSP70 Heat-Shock Proteins/metabolism , Heat-Shock Proteins/metabolism , Prions/metabolism , Saccharomyces cerevisiae Proteins , Adenosine Triphosphatases , Fungal Proteins/genetics , HSP40 Heat-Shock Proteins , HSP70 Heat-Shock Proteins/genetics , Heat-Shock Proteins/genetics , Molecular Chaperones , Peptide Termination Factors , Pichia/genetics , Pichia/metabolism
10.
Cell Motil Cytoskeleton ; 47(2): 141-53, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11013394

ABSTRACT

To examine the possible role of the cytoskeleton in exocytosis of Weibel-Palade bodies (WPBs), we used double immunofluorescence and electron microscopy to study the spatial relationships between WPBs and main cytoskeletal elements in endothelial cells treated with secretagogue, such as thrombin, or cytoskeleton-damaging agents. Unexpectedly, we have found that WPBs undergo rapid translocation towards the centrosome both in cells treated with thrombin and in those treated with cytochalasin B or calyculin A. Typically, 3 or 5 min after agent addition compact cluster of WPBs became visible near the microtubule-organizing center (MTOC) in most endothelial cells in which a fivefold increase in WPBs localized in close proximity to the mother centriole had been detected. In both thrombin- and cytochalasin-treated cells that exhibit a noticeable depletion in WPBs compared to control cells, WPBs located at the cell periphery were found to colocalize with vimentin intermediate filaments, but not with microtubules. In contrast, there was precise colocalization observed between WPBs and microtubules in calyculin-treated cells in which all WPBs undergo centrosome-directed translocation within 15 min after the agent addition. When vimentin filaments were induced to collapse to a perinuclear location by the microtubule-disrupting agent demecolcine, WPBs also translocated to the perinuclear region, where numerous WPBs were found to be localized within the bundles of intermediate-sized filaments. The data provide the first direct evidence that secretory granules utilize microtubule-based transport system to move in retrograde direction, i.e., away from the plasma membrane, towards the centrosome. We suggest that anterograde movement of WPBs is primarily dependent on their interaction with vimentin intermediate filaments.


Subject(s)
Centrosome/metabolism , Cytochalasin B/pharmacology , Demecolcine/pharmacology , Endothelium, Vascular/metabolism , Microtubules/physiology , Oxazoles/pharmacology , Thrombin/pharmacology , Weibel-Palade Bodies/metabolism , Adult , Aorta/drug effects , Aorta/metabolism , Cells, Cultured , Centrosome/ultrastructure , Endothelium, Vascular/drug effects , Endothelium, Vascular/ultrastructure , Exocytosis , Female , Fluorescent Antibody Technique , Humans , Marine Toxins , Microscopy, Electron , Microtubules/ultrastructure , Middle Aged , Protein Transport , Vimentin/metabolism , Vimentin/ultrastructure , Weibel-Palade Bodies/ultrastructure
11.
Virchows Arch ; 436(6): 539-52, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10917167

ABSTRACT

A number of data suggest that reactivation of cytomegalovirus (CMV) latent in arterial wall cells may contribute to atherogenesis; however, there is no direct evidence available. To address this issue, we have examined, using in situ hybridization or immunohistochemical staining, the frequency of occurrence of cells containing viral genome and of those expressing the IE 70 viral antigen in the endothelial layer and in deeper layers of human aortas with or without visible atherosclerotic lesions. Using endothelial cell cultures or tissue endothelial preparations, we found CMV-hybridizing endothelial cells in 6 of 8 grossly normal aortas and in 16 of 18 lesioned aortas. Antigen-positive endothelial cells were detected in 1 of 5 grossly normal vessels and in 6 of 7 lesioned vessels. Infected endothelial cells were abundant in areas adjacent to orifices of intercostal arteries of grossly normal aortas and in fatty spots of lesioned aortas, but no infected endothelial cells were observed in most plaques examined. In paraffin sections of grossly normal vessels, we detected CMV genome in cells adjacent to lumen and in cells randomly scattered through subendothelial intima and the media; however, no immunoreactive viral protein was found in the same tissue samples. In sections of lesioned vessels, clusters of CMV-hybridizing cells were found in the media in addition to infected cells randomly scattered through the intima and the media. In these samples of lesioned vessels, viral antigen was detected in cells adjacent to lumen and in cells clustered at the intima/media border. We found antigen-positive cells in grossly normal areas of lesioned aortas and in fatty lesions, but not in plaques of the same vessels. The data suggest that accumulation of the immediate-early CMV antigen in cells of endothelial layer and development of antigen-positive cell clusters in deeper layers of vascular wall accompany early atherogenic events in human aorta.


Subject(s)
Antigens, Viral/analysis , Aorta, Thoracic/metabolism , Cytomegalovirus/genetics , Genome, Viral , Immediate-Early Proteins/analysis , Adolescent , Adult , Aged , Aorta, Thoracic/cytology , Arteriosclerosis/etiology , Arteriosclerosis/virology , DNA, Viral/analysis , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Female , Humans , Immunohistochemistry , In Situ Hybridization , Male , Middle Aged , Polymerase Chain Reaction , Tunica Intima/cytology , Tunica Intima/metabolism
12.
Yeast ; 15(14): 1485-501, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10514566

ABSTRACT

Using a screening procedure for obtaining yeast strains with enhanced ability to secrete heterologous protein, we have isolated a mutant with alteration of the cell wall structure. This mutant displayed strong decrease in cell wall mannoprotein content, which was not accompanied by decreased glycosylation of secreted proteins. The mutation defines a gene, designated SSU21(identical to previously characterized MCD4), which encodes a novel vacuolar protein. SSU21 is probably connected to the cell integrity protein kinase C-mediated pathway, since ssu21 and pkc1Delta double mutant is synthetic lethal. To our knowledge, this is the first example of a yeast vacuolar protein whose alteration results in a cell wall defect.


Subject(s)
Fungal Proteins/physiology , Membrane Proteins/physiology , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/chemistry , Vacuoles/chemistry , Cell Wall/chemistry , Genes, Fungal , Membrane Proteins/analysis , Membrane Proteins/genetics , Mutation , Protein Kinase C/physiology , Saccharomyces cerevisiae/genetics , Urokinase-Type Plasminogen Activator/metabolism
13.
Ross Fiziol Zh Im I M Sechenova ; 85(7): 984-96, 1999 Jul.
Article in Russian | MEDLINE | ID: mdl-10643621

ABSTRACT

The data obtained suggest that the fatal changes in brain tissue associated with the prion diseases, are initiated by a conformational rearrangement of constitutively expressed cellular protein PrP. Possible mechanisms of such a conversion of this protein are discussed. Existence of the proteins with the prion properties in low eukaryotes may determine the unusual mechanisms of the "protein" inheritance. A new experimental model for studying the proteins with the prion properties in the yeast Saccharomyces cerevisiae, is described.


Subject(s)
Prion Diseases/genetics , Prions/genetics , Animals , Fungal Proteins/genetics , Humans , Prions/chemistry , Prions/pathogenicity , Protein Conformation
14.
Curr Genet ; 34(2): 146-51, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9724418

ABSTRACT

The yeast non-Mendelian [PSI+] determinant is presumed to be the manifestation of the aggregated prion-like state of the Sup35 protein. Plasmid-mediated amplification of the SUP35 gene greatly increases the frequency of Sup35p transition to this prion-like state. Here we show that the 3'-deletions of plasmid SUP35, leading to the C-terminal truncation of Sup35p, further increase the frequency of [PSI+] induction despite a marked decrease in Sup35p expression levels. The data suggest that the presence of Sup35p N-terminal proteolytic fragments can cause [PSI+] appearance in wild-type yeast cells.


Subject(s)
Fungal Proteins/genetics , Prions/genetics , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Alleles , Gene Amplification , Gene Expression , Genes, Fungal , Peptide Termination Factors , Plasmids/genetics , Sequence Deletion , Transformation, Genetic
15.
J Struct Biol ; 120(1): 52-60, 1997 Oct.
Article in English | MEDLINE | ID: mdl-9356291

ABSTRACT

The organization of the mitotic apparatus was studied in human embryo lung fibroblasts (HEL) and Vero cells at 4 days postinfection with human cytomegalovirus (HCMV) strain AD 169. The bipolar spindle was detected by immunofluorescence in p72-positive mitotic cells exhibiting a regular or C-metaphase-like chromosome configuration. Electron-microscopic study of C-metaphase-like cells revealed alteration of the centrosome structure which is characterized by the following features: (1) breakdown of the diplosome, (2) separation of the fibrillar material from centrioles, and (3) disruption of the centriolar cylinder. The spindle pole in the aberrant mitotic cells consisted of one or several foci of microtubules converging on the fibrillar aggregates. There are not any signs of the nuclear envelope reconstruction found in mitotic cells with highly condensed scattered chromosomes. Unlike in HEL cells, viral particles were not detected in Vero cells. A question arises as to whether centrosome injury is an integral part of the events leading to cell death unrelated to the reproduction of HCMV.


Subject(s)
Cell Cycle , Centrosome/ultrastructure , Cytomegalovirus/physiology , Spindle Apparatus/ultrastructure , Anaphase , Animals , Antigens, Viral/analysis , Cell Line , Cell Polarity , Centrioles/ultrastructure , Centrioles/virology , Centrosome/virology , Chlorocebus aethiops , Humans , Lung , Microscopy, Electron , Microtubules/ultrastructure , Microtubules/virology , Mitosis , Mitotic Index , Spindle Apparatus/virology , Time Factors , Vero Cells
16.
Vopr Virusol ; 42(4): 157-61, 1997.
Article in Russian | MEDLINE | ID: mdl-9304294

ABSTRACT

The incidence of markers of hepatitis C virus (HCV in the blood of 4216 normal subjects living in the European Russia (Northern, North-Western, Central, Central Chernozem, Volga-Vyatka, Volga, and North Caucasian regions), in the Urals, in Siberia (Eastern Siberian region), in the Far East, and in Monogolia is assessed. The incidence of antibodies to HCV varied from 0.7% in the Central region to 3.8% in the Central Chernozem and 10.7% in Mongolia. HCV genotyping (identification of 1a, 1b, 2a, 2b, and 3a genotypes) was performed using 469 RNA of HCV-positive sera of donors and patients collected in Russia, Moldova, Turkmenistan, and Mongolia. The 1b genotype predominated everywhere (68.9%), its incidence being the highest in Moldova (96%). Unclassifiable variants of HCV were found in 28 (6%) of sera. The regularities of HCV genotypes circulation in the European Russia were the same as in other European countries, whereas their prevalence in Eastern Russia was rather like that in China or Japan. The prevalence of genotypes did not depend on the clinical manifestations of diseases caused by HCV.


Subject(s)
Hepacivirus/genetics , Hepatitis C/epidemiology , Adolescent , Adult , Commonwealth of Independent States/epidemiology , Female , Genotype , Hepacivirus/classification , Hepatitis C/virology , Humans , Prevalence , Species Specificity
17.
Science ; 277(5324): 381-3, 1997 Jul 18.
Article in English | MEDLINE | ID: mdl-9219697

ABSTRACT

The yeast cytoplasmically inherited genetic determinant [PSI+] is presumed to be a manifestation of the prion-like properties of the Sup35 protein (Sup35p). Here, cell-free conversion of Sup35p from [psi-] cells (Sup35ppsi-) to the prion-like [PSI+]-specific form (Sup35pPSI+) was observed. The conversion reaction could be repeated for several consecutive cycles, thus modeling in vitro continuous [PSI+] propagation. Size fractionation of lysates of [PSI+] cells demonstrated that the converting activity was associated solely with Sup35pPSI+ aggregates, which agrees with the nucleation model for [PSI+] propagation. Sup35pPSI+ was purified and showed high conversion activity, thus confirming the prion hypothesis for Sup35p.


Subject(s)
Fungal Proteins/chemistry , Prions/chemistry , Protein Conformation , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/chemistry , Endopeptidases/metabolism , Fungal Proteins/genetics , Fungal Proteins/isolation & purification , Models, Chemical , Peptide Termination Factors , Phenotype , PrPC Proteins/chemistry , PrPSc Proteins/chemistry , Protein Biosynthesis , Protein Folding , Saccharomyces cerevisiae/genetics , Solubility , Transformation, Genetic
18.
Mol Cell Biol ; 17(5): 2798-805, 1997 May.
Article in English | MEDLINE | ID: mdl-9111351

ABSTRACT

The SUP45 and SUP35 genes of Saccharomyces cerevisiae encode polypeptide chain release factors eRF1 and eRF3, respectively. It has been suggested that the Sup35 protein (Sup35p) is subject to a heritable conformational switch, similar to mammalian prions, thus giving rise to the non-Mendelian [PSI+] nonsense suppressor determinant. In a [PSI+] state, Sup35p forms high-molecular-weight aggregates which may inhibit Sup35p activity, leading to the [PSI+] phenotype. Sup35p is composed of the N-terminal domain (N) required for [PSI+] maintenance, the presumably nonfunctional middle region (M), and the C-terminal domain (C) essential for translation termination. In this study, we observed that the N domain, alone or as a part of larger fragments, can form aggregates in [PSI+] cells. Two sites for Sup45p binding were found within Sup35p: one is formed by the N and M domains, and the other is located within the C domain. Similarly to Sup35p, in [PSI+] cells Sup45p was found in aggregates. The aggregation of Sup45p is caused by its binding to Sup35p and was not observed when the aggregated Sup35p fragments did not contain sites for Sup45p binding. The incorporation of Sup45p into the aggregates should inhibit its activity. The N domain of Sup35p, responsible for its aggregation in [PSI+] cells, may thus act as a repressor of another polypeptide chain release factor, Sup45p. This phenomenon represents a novel mechanism of regulation of gene expression at the posttranslational level.


Subject(s)
Fungal Proteins/metabolism , Peptide Termination Factors , Prions/metabolism , Protein Biosynthesis , Saccharomyces cerevisiae Proteins , Electrophoresis, Polyacrylamide Gel , Endopeptidases/metabolism , Heat-Shock Proteins/metabolism , Molecular Weight , Phenotype , Saccharomyces cerevisiae
19.
EMBO J ; 15(12): 3127-34, 1996 Jun 17.
Article in English | MEDLINE | ID: mdl-8670813

ABSTRACT

The Sup35p protein of yeast Saccharomyces cerevisiae is a homologue of the polypeptide chain release factor 3 (eRF3) of higher eukaryotes. It has been suggested that this protein may adopt a specific self-propagating conformation, similar to mammalian prions, giving rise to the [psi+] nonsense suppressor determinant, inherited in a non-Mendelian fashion. Here we present data confirming the prion-like nature of [psi+]. We show that Sup35p molecules interact with each other through their N-terminal domains in [psi+], but not [psi-] cells. This interaction is critical for [psi+] propagation, since its disruption leads to a loss of [psi+]. Similarly to mammalian prions, in [psi+] cells Sup35p forms high molecular weight aggregates, accumulating most of this protein. The aggregation inhibits Sup35p activity leading to a [psi+] nonsense-suppressor phenotype. N-terminally altered Sup35p molecules are unable to interact with the [psi+] Sup35p isoform, remain soluble and improve the translation termination in [psi+] strains, thus causing an antisuppressor phenotype. The overexpression of Hsp104p chaperone protein partially solubilizes Sup35P aggregates in the [psi+] strain, also causing an antisuppressor phenotype. We propose that Hsp104p plays a role in establishing stable [psi+] inheritance by splitting up Sup35p aggregates and thus ensuring equidistribution of the prion-like Sup35p isoform to daughter cells at cell divisions.


Subject(s)
Fungal Proteins/genetics , Prions/genetics , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Alleles , Base Sequence , Biopolymers , Endopeptidases/metabolism , Molecular Sequence Data , Oligodeoxyribonucleotides , Peptide Termination Factors , Prions/metabolism , Protein Binding , Saccharomyces cerevisiae/metabolism
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