RESUMO
Presenilin 1-null mice die at birth from brain and skeletal developmental deformities due to disrupted Notch signaling. Presenilin 1-null mice also have severely reduced gamma-secretase cleavage of betaAPP. The assumption has been that facilitation of Notch signaling and betaAPP processing by presenilin 1 are analogous functions. Here we describe a presenilin 1-targetted mouse model that expresses extremely low levels ( approximately 1% of normal) of mutant PS1-M146L. Homozygous mice have significantly reduced viability due to a Notch-like phenotype. The animals that survive have severe axial skeletal deformities and markedly diminished gamma-secretase activity and accumulation of betaAPP-C100, but no obvious abnormalities in brain development. These results suggest that, in mice, a marked reduction of PS1-facilitated gamma-secretase activity is not detrimental to normal brain development.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/crescimento & desenvolvimento , Endopeptidases/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Secretases da Proteína Precursora do Amiloide , Precursor de Proteína beta-Amiloide/genética , Animais , Especificidade de Anticorpos , Ácido Aspártico Endopeptidases , Western Blotting , Encéfalo/anatomia & histologia , Encéfalo/patologia , Ensaio de Imunoadsorção Enzimática , Marcação de Genes , Genótipo , Humanos , Camundongos , Camundongos Transgênicos , Mutação/genética , Fenótipo , Presenilina-1 , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Medula Espinal/patologiaRESUMO
The purpose of this study was to ascertain the amount of stalking taking place on the Worcester Polytechnic Institute (WPI) campus in which computers play such a huge role in the lives of the students. Our results were then compared with those reported in a stalking study done at West Virginia University (WVU). Surprisingly (to us), a smaller percentage of both females and males were stalked at WPI. The use of the Internet did not play a major role in stalking as we had expected. Results reported in a TV news report and a newspaper article indicate, however, that much less stalking occurs among the general population than does at WVU and WPI.
Assuntos
Crime/estatística & dados numéricos , Internet , Adulto , Coleta de Dados , Medo , Feminino , Humanos , Incidência , Relações Interpessoais , Masculino , EstudantesRESUMO
A nested polymerase chain reaction assay was used to determine the presence of Ehrlichia chaffeensis, E. canis, and E. ewingii DNA in blood samples of free-ranging coyotes from central and northcentral Oklahoma. Of the 21 coyotes examined, 15 (71%) were positive for E. chaffeensis DNA; none was positive for E. canis or E. ewingii. Results suggest that E. chaffeensis infections are common in free-ranging coyotes in Oklahoma and that these wild canids could play a role in the epidemiology of human monocytotropic ehrlichiosis.
Assuntos
Carnívoros , DNA Bacteriano/isolamento & purificação , Ehrlichia chaffeensis/isolamento & purificação , Ehrlichiose/veterinária , Animais , Ehrlichiose/sangue , Ehrlichiose/epidemiologia , Eletroforese em Gel de Ágar , Humanos , Oklahoma/epidemiologia , Reação em Cadeia da PolimeraseRESUMO
Absence of functional presenilin 1 (PS1) protein leads to loss of gamma-secretase cleavage of the amyloid precursor protein (betaAPP), resulting in a dramatic reduction in amyloid beta peptide (Abeta) production and accumulation of alpha- or beta-secretase-cleaved COOH-terminal fragments of betaAPP (alpha- or beta-CTFs). The major COOH-terminal fragment (CTF) in brain was identified as betaAPP-CTF-(11-98), which is consistent with the observation that cultured neurons generate primarily Abeta-(11-40). In PS1(-/-) murine neurons and fibroblasts expressing the loss-of-function PS1(D385A) mutant, CTFs accumulated in the endoplasmic reticulum, Golgi, and lysosomes, but not late endosomes. There were some subtle differences in the subcellular distribution of CTFs in PS1(-/-) neurons as compared with PS1(D385A) mutant fibroblasts. However, there was no obvious redistribution of full-length betaAPP or of markers of other organelles in either mutant. Blockade of endoplasmic reticulum-to-Golgi trafficking indicated that in PS1(-/-) neurons (as in normal cells) trafficking of betaAPP to the Golgi compartment is necessary before alpha- and beta-secretase cleavages occur. Thus, although we cannot exclude a specific role for PS1 in trafficking of CTFs, these data argue against a major role in general protein trafficking. These results are more compatible with a role for PS1 either as the actual gamma-secretase catalytic activity or in other functions indirectly related to gamma-secretase catalysis (e.g. an activator of gamma-secretase, a substrate adaptor for gamma-secretase, or delivery of gamma-secretase to betaAPP-containing compartments).
Assuntos
Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Proteínas de Membrana/fisiologia , Fragmentos de Peptídeos/metabolismo , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide , Animais , Ácido Aspártico Endopeptidases , Biomarcadores , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Compartimento Celular , Eletroforese em Gel de Poliacrilamida , Endopeptidases/metabolismo , Proteínas de Membrana/deficiência , Camundongos , Camundongos Knockout , Microscopia Eletrônica , Presenilina-1RESUMO
Several thioacylating reagents have been tested toward hydrolysis under conditions suitable for protein modifications: 20-35 degrees C and buffered solutions at pH 7.5-8.5. Aliphatic dithioesters are sufficiently stable in aqueous media at room temperature (or below) if protein modification reaction time does not exceed 24 h, whereas at 35 degrees C reaction times must be limited to a few hours. Kinetic data obtained in gelatin thioacylation at room temperature using aliphatic dithioesters and dithio acid are consistent with a second-order reaction rate with respect to amine concentration. The pH dependence of the second-order reaction rate constants indicate that dithioester reacts exclusively with the free amine form of lysine residue, whereas dithiocarboxylate ion reacts with both amine and ammonium ion, probably through a more complex mechanism. Interestingly thioacylation using dithio acids may be obtained in pH near neutrality or in slightly acidic media, thus offering protein modification possibilities at pH 5-9. Thioacylation reaction rates may be expressed as R = -(dAt/dt) = k[H3O+](-b)At2[thioacylating agent] in which At is the amine concentration at time t, constants k and b depending on the reagent nature.
Assuntos
Proteínas/química , Acilação , Concentração de Íons de Hidrogênio , Hidrólise , Indicadores e Reagentes , Cinética , Lisina/química , Espectrofotometria Ultravioleta , Compostos de Sulfidrila/química , Temperatura , ÁguaRESUMO
Thioacylation is a new way for protein chemical modification. Carboxylic dithioesters and -acids react selectively and rapidly at room temperature with aliphatic amines such as lysine epsilon-amino groups leading to thioamide formation, without any other reagent or catalyst. Various thioacylating reagents were synthesized: monofunctional dithioesters bearing on the acylating end various chemical groups such as: aliphatic chains, phenyl group, mono- and dicarboxylic acids, dialkylphosphonic ester, phosphonic acid, thiol, phenol, or quaternary ammonium group. Bifunctional dithioesters containing either a polymethylene chain or an ethylene oxide oligomer as spacer group as well as some mono- and bis(dithio acids) are described. Applications of thioacylation may be involved either in enzyme chemical modifications or in the obtention of new materials from proteins. Bifunctional reagents might be used as cross-linking or coupling reagents.
Assuntos
Proteínas/química , Acilação , Alquilação , Ácidos Carboxílicos/química , Reagentes de Ligações Cruzadas , Hidrólise , Indicadores e Reagentes , Lisina/química , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Espectrofotometria Infravermelho , Espectrofotometria Ultravioleta , Compostos de Sulfidrila/químicaRESUMO
The presenilin proteins are components of high-molecular-weight protein complexes in the endoplasmic reticulum and Golgi apparatus that also contain beta-catenin. We report here that presenilin mutations associated with familial Alzheimer disease (but not the non-pathogenic Glu318Gly polymorphism) alter the intracellular trafficking of beta-catenin after activation of the Wnt/beta-catenin signal transduction pathway. As with their effect on betaAPP processing, the effect of PS1 mutations on trafficking of beta-catenin arises from a dominant 'gain of aberrant function' activity. These results indicate that mistrafficking of selected presenilin ligands is a candidate mechanism for the genesis of Alzheimer disease associated with presenilin mutations, and that dysfunction in the presenilin-beta-catenin protein complexes is central to this process.
Assuntos
Doença de Alzheimer/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas de Membrana/genética , Mutação , Transativadores , Doença de Alzheimer/metabolismo , Transporte Biológico/genética , Linhagem Celular , Núcleo Celular/metabolismo , Humanos , Proteínas de Membrana/metabolismo , NF-kappa B/metabolismo , Presenilina-1 , Presenilina-2 , Ligação Proteica , Transdução de Sinais/genética , beta CateninaRESUMO
Missense substitutions in the presenilin 1 (PS1) and presenilin 2 (PS2) proteins are associated with early-onset familial Alzheimer's disease. We have used yeast-two-hybrid and coimmunoprecipitation methods to show that the large cytoplasmic loop domains of PS1 and PS2 interact specifically with three members of the armadillo protein family, including beta-catenin, p0071, and a novel neuronal-specific armadillo protein--neural plakophilin-related armadillo protein (NPRAP). The PS1:NPRAP interaction occurs between the arm repeats of NPRAP and residues 372-399 at the C-terminal end of the large cytoplasmic loop of PS1. The latter residues contain a single arm-like domain and are highly conserved in the presenilins, suggesting that they form a functional armadillo protein binding site for the presenilins.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transativadores , Doença de Alzheimer/genética , Sequência de Aminoácidos , Animais , Proteínas do Domínio Armadillo , Cateninas , Moléculas de Adesão Celular , Células Cultivadas , Cromatografia de Afinidade , Humanos , Imuno-Histoquímica , Proteínas de Membrana/genética , Camundongos , Microscopia Confocal , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Fosfoproteínas , Placofilinas , Testes de Precipitina , Presenilina-1 , Presenilina-2 , Ligação Proteica , Transfecção , beta Catenina , delta CateninaRESUMO
Humans harboring missense mutations in the presenilin 1 (PS1) gene undergo progressive cerebral deposition of the 42-residue amyloid beta-protein (A beta 42) at an early age and develop severe Alzheimer's disease. A beta 42 is selectively elevated in the conditioned media of cells expressing mutant but not wild-type PS1, indicating that presenilin mutations alter APP processing. Here we analyze the effects of various PS1 mutant constructs on the cellular production of A beta 42. A construct expressing only the PS1 N-terminal endoproteolytic fragment with the mutation Y115H causes no significant increase in A beta 42, whereas a full-length PS1 construct with the same mutation does. This result suggests that the pathogenic effect of mutant presenilins is produced by the full-length molecule even though only a minor proportion of total PS1 occurs as holoprotein in tissues and cell lines. We demonstrate that the effects of two different PS1 mutations are additive when engineered into the same PS1 molecule. Therefore, two mutations alter gamma-secretase processing of APP more than one and the PS1 mutations described to date do not cause the maximum possible PS1-mediated rise in A beta 42. When a PS1 mutation was expressed in cells carrying the APPV717I mutation, A beta 42 rose dramatically to become the predominant secreted A beta species, an observation of interest for transgenic modeling of AD. Our results are consistent with the hypothesis that presenilin is a major regulator of the proteolytic processing of APP by gamma-secretases.
Assuntos
Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Regulação da Expressão Gênica , Proteínas de Membrana/genética , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Idade de Início , Doença de Alzheimer/genética , Doença de Alzheimer/fisiopatologia , Substituição de Aminoácidos , Peptídeos beta-Amiloides/biossíntese , Linhagem Celular , Humanos , Leucina/genética , Proteínas de Membrana/biossíntese , Metionina/genética , Fragmentos de Peptídeos/biossíntese , Presenilina-1 , Valina/genéticaRESUMO
CONTEXT: Alzheimer disease (AD) susceptibility genes have been identified on chromosomes 1, 14, 19, and 21, and a recent study has suggested a locus on chromosome 12. OBJECTIVE: To confirm or refute the existence of a familial AD susceptibility locus on chromosome 12 in an independent sample of familial AD cases. DESIGN: Retrospective cohort study. DNA data for 6 chromosome 12 genetic markers were evaluated using parametric lod score and nonparametric linkage methods and linkage heterogeneity tests. The latter include the admixture test of homogeneity in the total group of families and the predivided sample test in families stratified by the presence or absence of an apolipoprotein E (APOE) epsilon4 allele among affected members. Parametric analyses were repeated assuming autosomal dominant inheritance of AD and either age- and sex-dependent penetrance or zero penetrance for the analysis of unaffected relatives. SETTING: Clinical populations in the continental United States, Canada, Argentina, and Italy. PATIENTS: Fifty-three white families composed of multiple members affected with AD, from whom DNA samples were obtained from 173 patients with AD whose conditions were diagnosed using established criteria and from 146 nondemented relatives. MAIN OUTCOME MEASURE: Presence of an APOE epsilon4 allele among affected family members. RESULTS: Using parametric methods, no evidence for linkage to the region spanned by the chromosome 12 markers could be detected if familial AD is assumed to arise from the same genetic locus in all 53 families. However, significant evidence for linkage was detected in the presence of locus heterogeneity using the admixture test (odds ratio, 15, 135:1). The estimated proportion of linked families within the 53 families examined varied between 0.40 and 0.65, depending on the genetic model assumed and APOE status. The precise location of the AD gene could not be determined, but includes the entire region suggested previously. Nonparametric linkage analysis confirmed linkage to chromosome 12 with the strongest evidence at D12S96 (P<.001). CONCLUSIONS: Our data provide independent confirmation of the existence of an AD susceptibility locus on chromosome 12 and suggest the existence of AD susceptibility genes on other chromosomes. Screening a larger set of families with additional chromosome markers will be necessary for identifying the chromosome 12 AD gene.
Assuntos
Doença de Alzheimer/genética , Cromossomos Humanos Par 12/genética , Ligação Genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Apolipoproteínas E/genética , Mapeamento Cromossômico , DNA/análise , Feminino , Genótipo , Humanos , Escore Lod , Masculino , Pessoa de Meia-Idade , Modelos Estatísticos , Linhagem , Estudos RetrospectivosRESUMO
A novel polymorphism (-491 A/T) within the regulatory region on the apolipoprotein E gene has recently been reported to be associated with risk for Alzheimer disease (AD). To test this association in an independent data set, we have examined this polymorphism in a sample of 88 well-characterized AD cases and compared the allele frequency and genotype frequencies for this polymorphism with those observed in 112 cognitively normal subjects drawn from the same ethnic group. These results suggest that in the current data set at least, the -491 A/T polymorphism is not associated with risk for AD, but may be in partial linkage disequilibrium with the APOE epsilon2/epsilon3/epsilon4 polymorphism.
Assuntos
Doença de Alzheimer/genética , Apolipoproteínas E/genética , Polimorfismo Genético/genética , Sequências Reguladoras de Ácido Nucleico/genética , Idoso , Frequência do Gene , HumanosRESUMO
The presenilin (PS) genes associated with Alzheimer disease encode polytopic transmembrane proteins which undergo physiologic endoproteolytic cleavage to generate stable NH2- and COOH-terminal fragments (NTF or CTF) which co-localize in intracellular membranes, but are tightly regulated in their stoichiometry and abundance. We have used linear glycerol velocity and discontinuous sucrose gradient analysis to investigate the distribution and native conformation of PS1 and PS2 during this regulated processing in cultured cells and in brain. The PS1 NTF and CTF co-localize in the endoplasmic reticulum (ER) and in the Golgi apparatus, where they are components of a approximately 250-kDa complex. This complex also contains beta-catenin but not beta-amyloid precursor protein (APP). In contrast, the PS1 holoprotein precursor is predominantly localized to the rough ER and smooth ER, where it is a component of a approximately 180-kDa native complex. PS2 forms similar but independent complexes. Restricted incorporation of the presenilin NTF and CTF along with a potentially functional ligand (beta-catenin) into a multimeric complex in the ER and Golgi apparatus may provide an explanation for the regulated accumulation of the NTF and CTF.
Assuntos
Doença de Alzheimer/metabolismo , Caderinas/química , Proteínas do Citoesqueleto/química , Proteínas de Membrana/química , Transativadores , Química Encefálica , Linhagem Celular , Centrifugação com Gradiente de Concentração , Retículo Endoplasmático/química , Humanos , Peso Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Mapeamento de Peptídeos , Presenilina-1 , beta CateninaRESUMO
The K-variant of butyrylcholinesterase (BCHE-K) recently has been reported to be associated with Alzheimer disease (AD) in carriers of the epsilon4 allele of the apolipoprotein E (APOE) gene. We have re-examined the frequency of the BCHE-K allele in a large data set of both sporadic and familial cases of AD disease, and we have also examined the segregation of three genetic markers on chromosome 3 near BCHE . Our data neither support an association of BCHE-K with sporadic or familial AD, nor do they suggest the existence of another gene nearby on chromosome 3 as a common cause of familial AD.
Assuntos
Doença de Alzheimer/genética , Butirilcolinesterase/genética , Cromossomos Humanos Par 3/genética , Idoso , Alelos , Doença de Alzheimer/enzimologia , Apolipoproteína E4 , Apolipoproteínas E/genética , Feminino , Frequência do Gene , Marcadores Genéticos , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
To determine whether similar abnormalities of various soluble full-length and N-terminal truncated Abeta peptides occur in postmortem cerebral cortex of affected PS1 mutation carriers, we examined the amounts of two amyloid species ending at residue 40 or at residues 42(43) using sandwich ELISA systems. Our results indicate that PS1 mutations effect a dramatic accumulation in brain of the highly insoluble potentially neurotoxic long-tailed isoforms of the Abeta peptide such as Abeta1-42(43) and Abetax-42(43). This enhancing effect of PS1 mutation on Abetax-42(43) deposition was highly similar to that of a betaAPP mutation (Val717Ile) but the effects on Abetax-40 production were significantly different between these two causal genes. In contrast to previous studies of soluble Abeta in plasma and in supernatants from cultured fibroblasts of subjects with PS1 mutations, our studies also show that there is an increase in insoluble Abetax-40 peptides in brain of subjects with PS1 mutations.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/genética , Química Encefálica , Proteínas de Membrana/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/análise , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/análise , Precursor de Proteína beta-Amiloide/metabolismo , Ligação Genética , Humanos , Proteínas de Membrana/análise , Pessoa de Meia-Idade , Mutação Puntual , Presenilina-1 , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , SolubilidadeRESUMO
The metabolic pathways governing the turnover of presenilin 1 (PS1) have been incompletely worked out. The PS1 holoprotein has low abundance in many cells and appears to undergo endoproteolytic cleavage near residue 298. We provide evidence that one mechanism by which the PS1 holoprotein is degraded is through the action of the 26S proteasome. We also show that the proteasome does not participate in the endoproteolytic cleavage.
Assuntos
Doença de Alzheimer/metabolismo , Proteínas de Membrana/metabolismo , Peptídeo Hidrolases/metabolismo , Complexo de Endopeptidases do Proteassoma , Animais , Western Blotting , Células CACO-2 , Hipocampo/metabolismo , Humanos , Técnicas In Vitro , Camundongos , NF-kappa B/metabolismo , Presenilina-1 , Ratos , Ratos WistarAssuntos
Hiperlipidemias/genética , Lipase Lipoproteica/genética , Adulto , Idoso , Apolipoproteínas/sangue , Canadá/epidemiologia , Estudos de Coortes , DNA/genética , Análise Mutacional de DNA , Saúde da Família , Feminino , França/etnologia , Heterozigoto , Humanos , Hiperlipidemias/sangue , Hiperlipidemias/epidemiologia , Lipídeos/sangue , Lipase Lipoproteica/metabolismo , Masculino , Pessoa de Meia-Idade , MutaçãoRESUMO
We have reported three missense mutations (G188E, P207L, and D250N) in the lipoprotein lipase (LPL) gene among French-Canadians, resulting in the absence of measurable postheparin plasma LPL activity in homozygotes. Presence of triglyceride- and cholesterol-rich VLDL, as well as cholesterol-poor HDL particles, has been shown in heterozygotes affected by partial reduction in postheparin LPL activity. However, significant heterogeneity in their plasma triglyceride levels has been found, even among individuals carrying the same LPL gene mutation, indicating that factors other than LPL deficiency could affect the phenotypic expression of hypertriglyceridemia in the heterozygous state. The aim of the present study was to examine the combined effects of abdominal fat accumulation and hyperinsulinemia on plasma triglyceride levels among heterozygous patients for familial LPL deficiency. Based on sex and BMI, 43 heterozygotes (25 women and 18 men) were matched with noncarrier control subjects. Our data indicate that heterozygotes with higher abdominal fat deposition, as defined as waist girth values above the 50th percentile, had higher plasma triglyceride levels than nonobese heterozygotes. However, an important proportion of male heterozygote subjects were hypertriglyceridemic, even in absence of abdominal obesity, suggesting that another factor(s) was involved in the modulation of hypertriglyceridemia in these subjects. Indeed, multivariate analyses revealed that fasting hyperinsulinemia was a significant correlate of hypertriglyceridemia among these heterozygotes. Results of the present study indicate that abdominal obesity and hyperinsulinemia both have deleterious effects on plasma triglyceride levels in familial LPL deficiency. It is suggested that heterozygotes with moderate obesity and/or insulin resistance may be at higher risk of coronary artery disease because of the expression of an atherogenic lipoprotein phenotype among these patients.
Assuntos
Heterozigoto , Hiperinsulinismo/complicações , Hipertrigliceridemia/enzimologia , Lipase Lipoproteica/deficiência , Lipase Lipoproteica/genética , Obesidade/complicações , Abdome , Constituição Corporal , Feminino , Humanos , Masculino , Análise Multivariada , Mutação , Triglicerídeos/sangueRESUMO
Glial fibrillary acidic protein (GFAP), an astroglial marker, has been detected in the peripheral nervous system (PNS) in a shorter version and its mRNA in a longer form (beta-type) than the brain alpha-type. To determine the characteristics of the GFAP gene expression in nonneural cells, we have investigated its in vivo transcription and translation products in human lymphocytes. Using RT-PCR, we demonstrate that the GFAP gene is transcribed in these cells. Most or all of the mRNA resulting from this transcription was longer than the brain-type at its 5' end and thus may correspond to the beta-type. In addition, immunoblotting of lymphocyte extracts with a monoclonal antibody revealed a 41 KDa fragment instead of the 50 KDa expected from brain GFAP. These results suggest that GFAP expression in lymphocytes is preferentially of the PNS beta-type giving rise to longer mRNA and shorter protein. However, compared to two other astroglial mRNAs (S-100beta and aldolase C) which were synthesized in significant amounts in lymphocytes, GFAP mRNA was detected in minute amounts representing 0.03% of the brain level. This low expression may subserve a special role in lymphocytes since it is translated.
Assuntos
Proteína Glial Fibrilar Ácida/análise , Proteína Glial Fibrilar Ácida/genética , Linfócitos/química , Sistema Nervoso Periférico/química , Anticorpos Monoclonais , Astrócitos/química , Astrócitos/enzimologia , Western Blotting , Química Encefálica/fisiologia , Proteínas de Ligação ao Cálcio/genética , Frutose-Bifosfato Aldolase/análise , Frutose-Bifosfato Aldolase/genética , Expressão Gênica/fisiologia , Proteína Glial Fibrilar Ácida/imunologia , Humanos , Linfócitos/enzimologia , Fatores de Crescimento Neural , Sistema Nervoso Periférico/enzimologia , RNA Mensageiro/análise , Subunidade beta da Proteína Ligante de Cálcio S100 , Proteínas S100/análise , Proteínas S100/genética , Transcrição Gênica/fisiologiaRESUMO
Mutations in the human presenilin genes (PSEN1 and PSEN2) are associated with early onset familial Alzheimer disease. The presenilin genes encode integral membrane proteins with similar structures, which suggests that they may have closely related, but as yet unknown functions. Analysis of the 5' upstream sequence and the structure of the PSEN1 gene reveals that the 5' sequence contains multiple putative transcription regulatory elements including clusters of STAT elements involved in transcriptional activation in response to signal transduction. The first four exons contain untranslated sequences, with Exons 1 and 2 representing alternate initial transcription sites. The function of these alternate initial exons is unclear. Exon 4 bears the first ATG sequence. The last 12 bp of Exon 4 is used as an alternative splice donor site. Exon 9 is alternately spliced in leukocytes, but not in most other tissues. Splicing of Exon 9 is predicted to cause significant structural changes to the protein. The majority of transcripts expressed in most tissues are polyadenylated 1127 bp from the TAG stop codon in Exon 13. A small proportion of transcripts contain the same 5'UTR and ORF but are polyadenylated 4435 bp from the stop codon. The longer polyadenylated transcripts contain three additional palindromes and at least one additional stem-loop structure with stabilities greater than -16 kcal/mol.
Assuntos
Doença de Alzheimer/genética , Genoma Humano , Proteínas de Membrana/genética , Processamento Alternativo , Sequência de Aminoácidos , Sequência de Bases , Humanos , Dados de Sequência Molecular , Presenilina-1 , Análise de Sequência de DNARESUMO
The mechanism by which mutations in the presenilin (PS) genes cause the most aggressive form of early-onset Alzheimer's disease (AD) is unknown, but fibroblasts from mutation carriers secrete increased levels of the amyloidogenic A beta 42 peptide, the main component of AD plaques. We established transfected cell and transgenic mouse models that coexpress human PS and amyloid beta-protein precursor (APP) genes and analyzed quantitatively the effects of PS expression on APP processing. In both models, expression of wild-type PS genes did not alter APP levels, alpha- and beta-secretase activity and A beta production. In the transfected cells, PS1 and PS2 mutations caused a highly significant increase in A beta 42 secretion in all mutant clones. Likewise, mutant but not wildtype PS1 transgenic mice showed significant overproduction of A beta 42 in the brain, and this effect was detectable as early as 2-4 months of age. Different PS mutations had differential effects on A beta generation. The extent of A beta 42 increase did not correlate with presenilin expression levels. Our data demonstrate that the presenilin mutations cause a dominant gain of function and may induce AD by enhancing A beta 42 production, thus promoting cerebral beta-amyloidosis.