RESUMEN
The risk of developing Alzheimer's disease (AD) significantly increases in individuals carrying the APOEε4 allele. Elderly cognitively healthy individuals with APOEε4 also exist, suggesting the presence of cellular mechanisms that counteract the pathological effects of APOEε4; however, these mechanisms are unknown. We hypothesized that APOEε4 carriers without dementia might carry genetic variations that could protect them from developing APOEε4-mediated AD pathology. To test this, we leveraged whole-genome sequencing (WGS) data in the National Institute on Aging Alzheimer's Disease Family Based Study (NIA-AD FBS), Washington Heights/Inwood Columbia Aging Project (WHICAP), and Estudio Familiar de Influencia Genetica en Alzheimer (EFIGA) cohorts and identified potentially protective variants segregating exclusively among unaffected APOEε4 carriers. In homozygous unaffected carriers above 70 years old, we identified 510 rare coding variants. Pathway analysis of the genes harboring these variants showed significant enrichment in extracellular matrix (ECM)-related processes, suggesting protective effects of functional modifications in ECM proteins. We prioritized two genes that were highly represented in the ECM-related gene ontology terms, (FN1) and collagen type VI alpha 2 chain (COL6A2) and are known to be expressed at the blood-brain barrier (BBB), for postmortem validation and in vivo functional studies. An independent analysis in a large cohort of 7185 APOEε4 homozygous carriers found that rs140926439 variant in FN1 was protective of AD (OR = 0.29; 95% CI [0.11, 0.78], P = 0.014) and delayed age at onset of disease by 3.37 years (95% CI [0.42, 6.32], P = 0.025). The FN1 and COL6A2 protein levels were increased at the BBB in APOEε4 carriers with AD. Brain expression of cognitively unaffected homozygous APOEε4 carriers had significantly lower FN1 deposition and less reactive gliosis compared to homozygous APOEε4 carriers with AD, suggesting that FN1 might be a downstream driver of APOEε4-mediated AD-related pathology and cognitive decline. To validate our findings, we used zebrafish models with loss-of-function (LOF) mutations in fn1b-the ortholog for human FN1. We found that fibronectin LOF reduced gliosis, enhanced gliovascular remodeling, and potentiated the microglial response, suggesting that pathological accumulation of FN1 could impair toxic protein clearance, which is ameliorated with FN1 LOF. Our study suggests that vascular deposition of FN1 is related to the pathogenicity of APOEε4, and LOF variants in FN1 may reduce APOEε4-related AD risk, providing novel clues to potential therapeutic interventions targeting the ECM to mitigate AD risk.
Asunto(s)
Enfermedad de Alzheimer , Fibronectinas , Anciano , Animales , Humanos , Enfermedad de Alzheimer/genética , Fibronectinas/genética , Variación Genética/genética , Gliosis , Pez CebraRESUMEN
Serine palmitoyltransferase (SPT), an endoplasmic reticulum-localized membrane enzymecomposed of acatalytic LCB1/LCB2 heterodimer and a small activating subunit (Tsc3 in yeast; ssSPTs in mammals), is negatively regulated by the evolutionarily conserved family of proteins known as the ORMs. In yeast, SPT, the ORMs, and the PI4P phosphatase Sac1, copurify in the "SPOTs" complex. However, neither the mechanism of ORM inhibition of SPT nor details of the interactions of the ORMs and Sac1 with SPT are known. Here we report that the first transmembrane domain (TMD1) of Lcb1 is required for ORM binding to SPT. Loss of binding is not due to altered membrane topology of Lcb1 since replacing TMD1 with a heterologous TMD restores membrane topology but not ORM binding. TMD1 deletion also eliminates ORM-dependent formation of SPT oligomers as assessed by co-immunoprecipitation assays and in vivo imaging. Expression of ORMs lacking derepressive phosphorylation sites results in constitutive SPT oligomerization, while phosphomimetic ORMs fail to induce oligomerization under any conditions. Significantly, when LCB1-RFP and LCB1ΔTMD1-GFP were coexpressed, more LCB1ΔTMD1-GFP was in the peripheral ER, suggesting ORM regulation is partially accomplished by SPT redistribution. Tsc3 deletion does not abolish ORM inhibition of SPT, indicating the ORMs do not simply prevent activation by Tsc3. Binding of Sac1 to SPT requires Tsc3, but not the ORMs, and Sac1 does not influence ORM-mediated oligomerization of SPT. Finally, yeast mutants lacking ORM regulation of SPT require the LCB-P lyase Dpl1 to maintain long-chain bases at sublethal levels.
Asunto(s)
Proteínas de Saccharomyces cerevisiae/metabolismo , Serina C-Palmitoiltransferasa/metabolismo , Aciltransferasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/fisiología , Secuencia de Aminoácidos , Animales , Células CHO , Cricetulus , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Monoéster Fosfórico Hidrolasas/metabolismo , Unión Proteica , Proteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Serina C-Palmitoiltransferasa/fisiología , Esfingolípidos/metabolismoRESUMEN
The topological and functional organization of the two isoforms of the small subunits of human serine palmitoyltransferase (hssSPTs) that activate the catalytic hLCB1/hLCB2 heterodimer was investigated. A variety of experimental approaches placed the N termini of the ssSPTs in the cytosol, their C termini in the lumen, and showed that they contain a single transmembrane domain. Deletion analysis revealed that the ability to activate the heterodimer is contained in a conserved 33-amino acid core domain that has the same membrane topology as the full-length protein. In combination with analysis of isoform chimera and site-directed mutagenesis, a single amino acid residue in this core (Met(25) in ssSPTa and Val(25) in ssSPTb) was identified which confers specificity for palmitoyl- or stearoyl-CoA, respectively, in both yeast and mammalian cells. This same residue also determines which isoform is a better activator of a mutant heterodimer, hLCB1(S331F)/hLCB2a, which has increased basal SPT activity and decreased amino acid substrate selectivity. This suggests that the role of the ssSPTs is to increase SPT activity without compromising substrate specificity. In addition, the observation that the C-terminal domains of ssSPTa and ssSPTb, which are highly conserved within each subfamily but are the most divergent regions between isoform subfamilies, are not required for activation of the heterodimer or for acyl-CoA selectivity suggests that the ssSPTs have additional roles that remain to be discovered.
Asunto(s)
Serina C-Palmitoiltransferasa/fisiología , Secuencia de Aminoácidos , Aminoácidos/química , Animales , Membrana Celular/metabolismo , Dimerización , Activación Enzimática , Genes Fúngicos , Glicosilación , Humanos , Lípidos/química , Microsomas/metabolismo , Datos de Secuencia Molecular , Mutación , Plásmidos/metabolismo , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Serina C-Palmitoiltransferasa/química , Esfingolípidos/química , Especificidad por SustratoRESUMEN
Deletion of the sla1(+) gene, which encodes a homologue of the human RNA-binding protein La in Schizosaccharomyces pombe, causes irregularities in tRNA processing, with altered distribution of pre-tRNA intermediates. We show, using mRNA profiling, that cells lacking sla1(+) have increased mRNAs from amino acid metabolism (AAM) genes and, furthermore, exhibit slow growth in Edinburgh minimal medium. A subset of these AAM genes is under control of the AP-1-like, stress-responsive transcription factors Atf1p and Pcr1p. Although S. pombe growth is resistant to rapamycin, sla1-Δ cells are sensitive, consistent with deficiency of leucine uptake, hypersensitivity to NH4, and genetic links to the target of rapamycin (TOR) pathway. Considering that perturbed intranuclear pre-tRNA metabolism and apparent deficiency in tRNA nuclear export in sla1-Δ cells may trigger the AAM response, we show that modest overexpression of S. pombe los1(+) (also known as Xpo-t), encoding the nuclear exportin for tRNA, suppresses the reduction in pre-tRNA levels, AAM gene up-regulation, and slow growth of sla1-Δ cells. The conclusion that emerges is that sla1(+) regulates AAM mRNA production in S. pombe through its effects on nuclear tRNA processing and probably nuclear export. Finally, the results are discussed in the context of stress response programs in Saccharomyces cerevisiae.
Asunto(s)
Factor de Transcripción Activador 1/metabolismo , Factores de Transcripción Activadores/metabolismo , ARN de Hongos/metabolismo , ARN de Transferencia/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Factor de Transcripción Activador 1/genética , Factores de Transcripción Activadores/genética , Núcleo Celular/metabolismo , Regulación Fúngica de la Expresión Génica , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/metabolismo , ARN de Hongos/análisis , ARN Mensajero/análisis , Proteínas de Unión al ARN/metabolismo , Schizosaccharomyces/genética , Estrés FisiológicoRESUMEN
We used a genetic screen based on tRNA-mediated suppression (TMS) in a Schizosaccharomyces pombe La protein (Sla1p) mutant. Suppressor pre-tRNA(Ser)UCA-C47:6U with a debilitating substitution in its variable arm fails to produce tRNA in a sla1-rrm mutant deficient for RNA chaperone-like activity. The parent strain and spontaneous mutant were analyzed using Solexa sequencing. One synonymous single-nucleotide polymorphism (SNP), unrelated to the phenotype, was identified. Further sequence analyses found a duplication of the tRNA(Ser)UCA-C47:6U gene, which was shown to cause the phenotype. Ninety percent of 28 isolated mutants contain duplicated tRNA(Ser)UCA-C47:6U genes. The tRNA gene duplication led to a disproportionately large increase in tRNA(Ser)UCA-C47:6U levels in sla1-rrm but not sla1-null cells, consistent with non-specific low-affinity interactions contributing to the RNA chaperone-like activity of La, similar to other RNA chaperones. Our analysis also identified 24 SNPs between ours and S. pombe 972h- strain yFS101 that was recently sequenced using Solexa. By including mitochondrial (mt) DNA in our analysis, overall coverage increased from 52% to 96%. mtDNA from our strain and yFS101 shared 14 mtSNPs relative to a 'reference' mtDNA, providing the first identification of these S. pombe mtDNA discrepancies. Thus, strain-specific and spontaneous phenotypic mutations can be mapped in S. pombe by Solexa sequencing.
Asunto(s)
Duplicación de Gen , Polimorfismo de Nucleótido Simple , ARN de Transferencia de Serina/genética , Proteínas de Unión al ARN/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Alelos , Secuencia de Bases , Núcleo Celular/genética , ADN Mitocondrial/química , Genes Fúngicos , Genoma Fúngico , Datos de Secuencia Molecular , Mutación , Fenotipo , ARN de Transferencia de Serina/química , ARN de Transferencia de Serina/metabolismo , Análisis de Secuencia de ADN , Supresión GenéticaRESUMEN
After the second transesterification step of pre-mRNA splicing, the Prp22 helicase catalyzes release of spliced mRNA by disrupting contacts in the spliceosome that likely involve Prp8. Mutations at Arg1753 in Prp8, which suppress helicase-defective prp22 mutants, elicit temperature-sensitive growth phenotypes, indicating that interactions in the spliceosome involving Prp8-R1753 might be broken prematurely at 37 degrees C. Here we report that mutations in loop I of the U5 snRNA or in Prp18 can suppress the temperature-sensitive prp8-R1753 mutants. The same gain-of-function PRP18 alleles can also alleviate the growth phenotypes of multiple slu7-ts mutants, indicating a functional link between Prp8 and the second step splicing factors Prp18 and Slu7. These findings, together with the demonstration that changes at Arg1753 in Prp8 impair step 2 of pre-mRNA splicing in vitro, are consistent with a model in which (1) Arg1753 plays a role in stabilizing U5/exon interactions prior to exon joining and (2) these contacts persist until they are broken by the helicase Prp22.
Asunto(s)
Proteínas Nucleares/genética , Precursores del ARN/metabolismo , Empalme del ARN/genética , ARN de Hongos/metabolismo , ARN Nuclear Pequeño/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Células Cultivadas , Esterificación , Exones/genética , Humanos , Proteínas Nucleares/metabolismo , Precursores del ARN/genética , Factores de Empalme de ARN , ARN de Hongos/química , ARN Nuclear Pequeño/genética , Ribonucleoproteína Nuclear Pequeña U4-U6 , Ribonucleoproteína Nuclear Pequeña U5 , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Interaction of the ends of the exons with loop 1 of the U5 snRNA aligns the exons for ligation in the second step of pre-mRNA splicing. To study the effect of Prp18 on the exons' interactions, we analyzed the splicing of pre-mRNAs with random sequences in the exon bases at the splice junctions. The exon mutations had large effects on splicing in yeast with a Prp18 protein lacking its most conserved region, but not in wild-type yeast. Analysis of splicing kinetics demonstrated that only the second step was affected in vivo and in vitro, showing that Prp18 - and specifically its conserved region - plays a key role in stabilizing the interaction of the exons with the spliceosome at the time of exon joining. Superior exon sequences defined by the prp18 results accelerated the second step of splicing by wild-type spliceosomes with inefficient AT-AC pre-mRNAs, implying that normal exon interactions follow the rules we discerned for prp18 splicing. Our results show that As are preferred at the ends of both exons and support a revised model of the interactions of the exons with U5 in which the exons are arranged in a continuous double helix that facilitates the second reaction.
Asunto(s)
Exones/genética , Proteínas Nucleares/genética , Precursores del ARN/genética , Empalme del ARN/genética , ARN Nuclear Pequeño/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Empalmosomas/metabolismo , Emparejamiento Base , Secuencia de Bases , Cartilla de ADN , Datos de Secuencia Molecular , Mutación/genética , ARN Nuclear Pequeño/genética , Ribonucleoproteína Nuclear Pequeña U5 , Empalmosomas/genética , LevadurasRESUMEN
Both the Prp18 protein and the U5 snRNA function in the second step of pre-mRNA splicing. We identified suppressors of mutant prp18 alleles in the gene for the U5 snRNA (SNR7). The suppressors' U5 snRNAs have either a U4-to-A or an A8-to-C mutation in the evolutionarily invariant loop 1 of U5. Suppression is specific for prp18 alleles that encode proteins with mutations in a highly conserved region of Prp18 which forms an unstructured loop in crystals of Prp18. The snr7 suppressors partly restored the pre-mRNA splicing activity that was lost in the prp18 mutants. The close functional relationship of Prp18 and U5 is emphasized by the finding that two snr7 alleles, U5A and U6A, are dominant synthetic lethal with prp18 alleles. Our results support the idea that Prp18 and the U5 snRNA act in concert during the second step of pre-mRNA splicing and suggest a model in which the conserved loop of Prp18 acts to stabilize the interaction of loop 1 of the U5 snRNA with the splicing intermediates.
Asunto(s)
Proteínas Nucleares/fisiología , Empalme del ARN/fisiología , ARN Nuclear Pequeño/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/genética , Supresión Genética/genética , Alelos , Secuencia de Aminoácidos , Secuencia Conservada/genética , Secuencia Conservada/fisiología , Evolución Molecular , Genes Fúngicos/genética , Genes Fúngicos/fisiología , Datos de Secuencia Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Conformación de Ácido Nucleico , Mutación Puntual/genética , Empalme del ARN/genética , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Mensajero/análisis , ARN Mensajero/metabolismo , ARN Nuclear Pequeño/química , ARN Nuclear Pequeño/genética , Ribonucleoproteína Nuclear Pequeña U5 , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Supresión Genética/fisiologíaRESUMEN
Prp18 functions in the second step of pre-mRNA splicing, joining the spliceosome just prior to the transesterification reaction that creates the mature mRNA. Prp18 interacts with Slu7, and the functions of the two proteins are intertwined. Using the X-ray structure of Prp18, we have designed mutants in Prp18 that imply that Prp18 has two distinct roles in splicing. Deletion mutations were used to delineate the surface of Prp18 that interacts with Slu7, and point mutations in Prp18 were used to define amino acids that contact Slu7. Experiments in which Slu7 and mutant Prp18 proteins were expressed at different levels support a model in which interaction between the proteins is needed for stable binding of both proteins to the spliceosome. Mutations in an evolutionarily conserved region show that it is critical for Prp18 function but is not involved in binding Slu7. Alleles with mutations in the conserved region are dominant negative, suggesting that the resulting mutant prp18 proteins make proper contacts with the spliceosome, but fail to carry out a Prp18-specific function. Prp18 thus appears to have two separable roles in splicing, one in stabilizing interaction of Slu7 with the spliceosome, and a second that requires the conserved loop.
Asunto(s)
Proteínas Nucleares , Empalme del ARN , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Secuencia Conservada , Análisis Mutacional de ADN , Regulación Fúngica de la Expresión Génica , Datos de Secuencia Molecular , Conformación Proteica , Factores de Empalme de ARN , Ribonucleoproteína Nuclear Pequeña U5 , Ribonucleoproteínas Nucleares Pequeñas/genética , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Eliminación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
It was recently demonstrated that mutations in the human SPTLC1 gene, encoding the Lcb1p subunit of serine palmitoyltransferase (SPT), cause hereditary sensory neuropathy type I . As a member of the subfamily of pyridoxal 5'-phosphate enzymes known as the alpha-oxoamine synthases, serine palmitoyltransferase catalyzes the committed step of sphingolipid synthesis. The residues that are mutated to cause hereditary sensory neuropathy type I reside in a highly conserved region of Lcb1p that is predicted to be a catalytic domain of Lcb1p on the basis of alignments with other members of the alpha-oxoamine synthase family. We found that the corresponding mutations in the LCB1 gene of Saccharomyces cerevisiae reduce serine palmitoyltransferase activity. These mutations are dominant and decrease serine palmitoyltransferase activity by 50% when the wild-type and mutant LCB1 alleles are coexpressed. We also show that serine palmitoyltransferase is an Lcb1p small middle dotLcb2p heterodimer and that the mutated Lcb1p proteins retain their ability to interact with Lcb2p. Modeling studies suggest that serine palmitoyltransferase is likely to have a single active site that lies at the Lcb1p small middle dotLcb2p interface and that the mutations in Lcb1p reside near the lysine in Lcb2p that is expected to form the Schiff's base with the pyridoxal 5'-phosphate cofactor. Furthermore, mutations in this lysine and in a histidine residue that is also predicted to be important for pyridoxal 5'-phosphate binding to Lcb2p also dominantly inactivate SPT similar to the hereditary sensory neuropathy type 1-like mutations in Lcb1p.