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1.
Brain ; 144(9): 2759-2770, 2021 10 22.
Article in English | MEDLINE | ID: mdl-34428276

ABSTRACT

The molecular link between amyloid-ß plaques and neurofibrillary tangles, the two pathological hallmarks of Alzheimer's disease, is still unclear. Increasing evidence suggests that amyloid-ß peptide activates multiple regulators of cell cycle pathways, including transcription factors CDKs and E2F1, leading to hyperphosphorylation of tau protein. However, the exact pathways downstream of amyloid-ß-induced cell cycle imbalance are unknown. Here, we show that PAX6, a transcription factor essential for eye and brain development which is quiescent in adults, is increased in the brains of patients with Alzheimer's disease and in APP transgenic mice, and plays a key role between amyloid-ß and tau hyperphosphorylation. Downregulation of PAX6 protects against amyloid-ß peptide-induced neuronal death, suggesting that PAX6 is a key executor of the amyloid-ß toxicity pathway. Mechanistically, amyloid-ß upregulates E2F1, followed by the induction of PAX6 and c-Myb, while Pax6 is a direct target for both E2F1 and its downstream target c-Myb. Furthermore, PAX6 directly regulates transcription of GSK-3ß, a kinase involved in tau hyperphosphorylation and neurofibrillary tangles formation, and its phosphorylation of tau at Ser356, Ser396 and Ser404. In conclusion, we show that signalling pathways that include CDK/pRB/E2F1 modulate neuronal death signals by activating downstream transcription factors c-Myb and PAX6, leading to GSK-3ß activation and tau pathology, providing novel potential targets for pharmaceutical intervention.


Subject(s)
Alzheimer Disease/metabolism , Amyloid beta-Peptides/toxicity , PAX6 Transcription Factor/metabolism , Peptide Fragments/toxicity , tau Proteins/metabolism , Alzheimer Disease/pathology , Animals , Brain/drug effects , Brain/metabolism , Brain/pathology , Cells, Cultured , HEK293 Cells , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Phosphorylation/drug effects , Phosphorylation/physiology , Signal Transduction/drug effects , Signal Transduction/physiology
2.
Neurol Genet ; 2(6): e116, 2016 Dec.
Article in English | MEDLINE | ID: mdl-27822510

ABSTRACT

OBJECTIVE: To characterize the clinical and molecular effect of mutations in the sortilin-related receptor (SORL1) gene. METHODS: We performed whole-exome sequencing in early-onset Alzheimer disease (EOAD) and late-onset Alzheimer disease (LOAD) families followed by functional studies of select variants. The phenotypic consequences associated with SORL1 mutations were characterized based on clinical reviews of medical records. Functional studies were completed to evaluate ß-amyloid (Aß) production and amyloid precursor protein (APP) trafficking associated with SORL1 mutations. RESULTS: SORL1 alterations were present in 2 EOAD families. In one, a SORL1 T588I change was identified in 4 individuals with AD, 2 of whom had parkinsonian features. In the second, an SORL1 T2134 alteration was found in 3 of 4 AD cases, one of whom had postmortem Lewy bodies. Among LOAD cases, 4 individuals with either SORL1 A528T or T947M alterations had parkinsonian features. Functionally, the variants weaken the interaction of the SORL1 protein with full-length APP, altering levels of Aß and interfering with APP trafficking. CONCLUSIONS: The findings from this study support an important role for SORL1 mutations in AD pathogenesis by way of altering Aß levels and interfering with APP trafficking. In addition, the presence of parkinsonian features among select individuals with AD and SORL1 mutations merits further investigation.

3.
Ann Neurol ; 77(2): 215-27, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25382023

ABSTRACT

OBJECTIVE: Common single nucleotide polymorphisms in the SORL1 gene have been associated with late onset Alzheimer disease (LOAD), but causal variants have not been fully characterized nor has the mechanism been established. The study was undertaken to identify functional SORL1 mutations in patients with LOAD. METHODS: This was a family- and cohort-based genetic association study. Caribbean Hispanics with familial and sporadic LOAD and similarly aged controls were recruited from the United States and the Dominican Republic, and patients with sporadic disease of Northern European origin were recruited from Canada. Prioritized coding variants in SORL1 were detected by targeted resequencing and validated by genotyping in additional family members and unrelated healthy controls. Variants transfected into human embryonic kidney 293 cell lines were tested for Aß40 and Aß42 secretion, and the amount of the amyloid precursor protein (APP) secreted at the cell surface was determined. RESULTS: Seventeen coding exonic variants were significantly associated with disease. Two rare variants (rs117260922-E270K and rs143571823-T947M) with minor allele frequency (MAF) < 1% and 1 common variant (rs2298813-A528T) with MAF = 14.9% segregated within families and were deemed deleterious to the coding protein. Transfected cell lines showed increased Aß40 and Aß42 secretion for the rare variants (E270K and T947M) and increased Aß42 secretion for the common variant (A528T). All mutants increased the amount of APP at the cell surface, although in slightly different ways, thereby failing to direct full-length APP into the retromer-recycling endosome pathway. INTERPRETATION: Common and rare variants in SORL1 elevate the risk of LOAD by directly affecting APP processing, which in turn can result in increased Aß40 and Aß42 secretion.


Subject(s)
Alzheimer Disease/diagnosis , Alzheimer Disease/genetics , Genetic Association Studies/methods , LDL-Receptor Related Proteins/genetics , Membrane Transport Proteins/genetics , Mutation/genetics , Open Reading Frames/genetics , Aged , Aged, 80 and over , Cohort Studies , Female , HEK293 Cells , Humans , Male , Middle Aged , Polymorphism, Single Nucleotide/genetics
4.
Mol Neurodegener ; 9: 59, 2014 Dec 18.
Article in English | MEDLINE | ID: mdl-25523933

ABSTRACT

The presenilin genes were first identified as the site of missense mutations causing early onset autosomal dominant familial Alzheimer's disease. Subsequent work has shown that the presenilin proteins are the catalytic subunits of a hetero-tetrameric complex containing APH1, nicastrin and PEN-2. This complex (variously termed presenilin complex or gamma-secretase complex) performs an unusual type of proteolysis in which the transmembrane domains of Type I proteins are cleaved within the hydrophobic compartment of the membrane. This review describes some of the molecular and structural biology of this unusual enzyme complex. The presenilin complex is a bilobed structure. The head domain contains the ectodomain of nicastrin. The base domain contains a central cavity with a lateral cleft that likely provides the route for access of the substrate to the catalytic cavity within the centre of the base domain. There are reciprocal allosteric interactions between various sites in the complex that affect its function. For instance, binding of Compound E, a peptidomimetic inhibitor to the PS1 N-terminus, induces significant conformational changes that reduces substrate binding at the initial substrate docking site, and thus inhibits substrate cleavage. However, there is a reciprocal allosteric interaction between these sites such that prior binding of the substrate to the initial docking site paradoxically increases the binding of the Compound E peptidomimetic inhibitor. Such reciprocal interactions are likely to form the basis of a gating mechanism that underlies access of substrate to the catalytic site. An increasingly detailed understanding of the structural biology of the presenilin complex is an essential step towards rational design of substrate- and/or cleavage site-specific modulators of presenilin complex function.


Subject(s)
Alzheimer Disease/metabolism , Amyloid Precursor Protein Secretases/metabolism , Cell Membrane/metabolism , Presenilin-1/metabolism , Proteolysis , Animals , Humans , Molecular Docking Simulation/methods , Presenilin-1/chemistry , Protein Binding
5.
Structure ; 22(1): 125-35, 2014 Jan 07.
Article in English | MEDLINE | ID: mdl-24210759

ABSTRACT

Presenilin-mediated endoproteolysis of transmembrane proteins plays a key role in physiological signaling and in the pathogenesis of Alzheimer disease and some cancers. Numerous inhibitors have been found via library screens, but their structural mechanisms remain unknown. We used several biophysical techniques to investigate the structure of human presenilin complexes and the effects of peptidomimetic γ-secretase inhibitors. The complexes are bilobed. The head contains nicastrin ectodomain. The membrane-embedded base has a central channel and a lateral cleft, which may represent the initial substrate docking site. Inhibitor binding induces widespread structural changes, including rotation of the head and closure of the lateral cleft. These changes block substrate access to the catalytic pocket and inhibit the enzyme. Intriguingly, peptide substrate docking has reciprocal effects on the inhibitor binding site. Similar reciprocal shifts may underlie the mechanisms of other inhibitors and of the "lateral gate" through which substrates access to the catalytic site.


Subject(s)
Benzodiazepinones/chemistry , Enzyme Inhibitors/chemistry , Peptidomimetics/chemistry , Presenilin-1/chemistry , Recombinant Fusion Proteins/chemistry , Allosteric Regulation , Amyloid Precursor Protein Secretases/chemistry , Catalytic Domain , HEK293 Cells , Humans , Ligands , Membrane Glycoproteins/chemistry , Molecular Docking Simulation , Presenilin-1/antagonists & inhibitors , Presenilin-1/genetics , Protein Structure, Tertiary , Proteolysis , Recombinant Fusion Proteins/genetics , Structure-Activity Relationship
6.
J Biol Chem ; 288(21): 15352-66, 2013 May 24.
Article in English | MEDLINE | ID: mdl-23589300

ABSTRACT

γ-Secretase plays a pivotal role in the production of neurotoxic amyloid ß-peptides (Aß) in Alzheimer disease (AD) and consists of a heterotetrameric core complex that includes the aspartyl intramembrane protease presenilin (PS). The human genome codes for two presenilin paralogs. To understand the causes for distinct phenotypes of PS paralog-deficient mice and elucidate whether PS mutations associated with early-onset AD affect the molecular environment of mature γ-secretase complexes, quantitative interactome comparisons were undertaken. Brains of mice engineered to express wild-type or mutant PS1, or HEK293 cells stably expressing PS paralogs with N-terminal tandem-affinity purification tags served as biological source materials. The analyses revealed novel interactions of the γ-secretase core complex with a molecular machinery that targets and fuses synaptic vesicles to cellular membranes and with the H(+)-transporting lysosomal ATPase macrocomplex but uncovered no differences in the interactomes of wild-type and mutant PS1. The catenin/cadherin network was almost exclusively found associated with PS1. Another intramembrane protease, signal peptide peptidase, predominantly co-purified with PS2-containing γ-secretase complexes and was observed to influence Aß production.


Subject(s)
Alzheimer Disease/enzymology , Amyloid Precursor Protein Secretases/immunology , Membrane Proteins/metabolism , Presenilin-2/metabolism , Serine Endopeptidases/metabolism , Alzheimer Disease/genetics , Alzheimer Disease/pathology , Amyloid Precursor Protein Secretases/genetics , Amyloid beta-Peptides/genetics , Amyloid beta-Peptides/metabolism , Animals , Cadherins/genetics , Cadherins/metabolism , Catenins/genetics , Catenins/metabolism , HEK293 Cells , Humans , Membrane Proteins/genetics , Mice , Mice, Transgenic , Mutation , Presenilin-2/genetics , Protein Binding/genetics , Serine Endopeptidases/genetics , Vacuolar Proton-Translocating ATPases/genetics , Vacuolar Proton-Translocating ATPases/metabolism
7.
Proteome Sci ; 10(1): 33, 2012 May 18.
Article in English | MEDLINE | ID: mdl-22607704

ABSTRACT

BACKGROUND: Signal peptide peptidase (SPP), a member of the presenilin-like intra-membrane cleaving aspartyl protease family, migrates on Blue Native (BN) gels as 100 kDa, 200 kDa and 450 kDa species. SPP has recently been implicated in other non-proteolytic functions such as retro-translocation of MHC Class I molecules and binding of misfolded proteins in the endoplasmic reticulum (ER). These high molecular weight SPP complexes might contain additional proteins that regulate the proteolytic activity of SPP or support its non-catalytic functions. RESULTS: In this study, an unbiased iTRAQ-labeling mass spectrometry approach was used to identify SPP-interacting proteins. We found that vigilin, a ubiquitous multi-KH domain containing cytoplasmic protein involved in RNA binding and protein translation control, selectively enriched with SPP. Vigilin interacted with SPP and both proteins co-localized in restricted intracellular domains near the ER, biochemically co-fractionated and were part of the same 450 kDa complex on BN gels. However, vigilin does not alter the protease activity of SPP, suggesting that the SPP-vigilin interaction might be involved in the non-proteolytic functions of SPP. CONCLUSIONS: We have identified and validated vigilin as a novel interacting partner of SPP that could play an important role in the non-proteolytic functions of SPP. This data adds further weight to the idea that intramembrane-cleaving aspartyl proteases, such as presenilin and SPPs, could have other functions besides the proteolysis of short membrane stubs.

8.
Hum Mol Genet ; 21(1): 1-9, 2012 Jan 01.
Article in English | MEDLINE | ID: mdl-21949354

ABSTRACT

It is unclear whether mutations in fused in sarcoma (FUS) cause familial amyotrophic lateral sclerosis via a loss-of-function effect due to titrating FUS from the nucleus or a gain-of-function effect from cytoplasmic overabundance. To investigate this question, we generated a series of independent Caenorhabditis elegans lines expressing mutant or wild-type (WT) human FUS. We show that mutant FUS, but not WT-FUS, causes cytoplasmic mislocalization associated with progressive motor dysfunction and reduced lifespan. The severity of the mutant phenotype in C. elegans was directly correlated with the severity of the illness caused by the same mutation in humans, arguing that this model closely replicates key features of the human illness. Importantly, the mutant phenotype could not be rescued by overexpression of WT-FUS, even though WT-FUS had physiological intracellular localization, and was not recruited to the cytoplasmic mutant FUS aggregates. Our data suggest that FUS mutants cause neuronal dysfunction by a dominant gain-of-function effect related either to neurotoxic aggregates of mutant FUS in the cytoplasm or to dysfunction in its RNA-binding functions.


Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , Caenorhabditis elegans/metabolism , Motor Neurons/cytology , Mutation , RNA-Binding Protein FUS/genetics , RNA-Binding Protein FUS/metabolism , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/physiopathology , Animals , Animals, Genetically Modified , Caenorhabditis elegans/cytology , Caenorhabditis elegans/genetics , Cell Death , Disease Models, Animal , Female , Humans , Male , Motor Neurons/metabolism
9.
J Proteome Res ; 10(10): 4388-404, 2011 Oct 07.
Article in English | MEDLINE | ID: mdl-21819105

ABSTRACT

DJ-1 is a small but relatively abundant protein of unknown function that may undergo stress-dependent cellular translocation and has been implicated in both neurodegenerative diseases and cancer. As such, DJ-1 may be an excellent study object to elucidate the relative influence of the cellular context on its interactome and for exploring whether acute exposure to oxidative stressors alters its molecular environment. Using quantitative mass spectrometry, we conducted comparative DJ-1 interactome analyses from in vivo cross-linked brains or livers and from hydrogen peroxide-treated or naïve embryonic stem cells. The analysis identified a subset of glycolytic enzymes, heat shock proteins 70 and 90, and peroxiredoxins as interactors of DJ-1. Consistent with a role of DJ-1 in Hsp90 chaperone biology, we document destabilization of Hsp90 clients in DJ-1 knockout cells. We further demonstrate the existence of a C106 sulfinic acid modification within DJ-1 and thereby establish that this previously inferred modification also exists in vivo. Our data suggest that caution has to be exerted in interpreting interactome data obtained from a single biological source material and identify a role of DJ-1 as an oxidative stress sensor and partner of a molecular machinery notorious for its involvement in cell fate decisions.


Subject(s)
Gene Expression Regulation, Neoplastic , HSP90 Heat-Shock Proteins/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Oncogene Proteins/metabolism , Oxidative Stress , Proteomics/methods , Animals , Cysteine/chemistry , HSC70 Heat-Shock Proteins/metabolism , Humans , Mass Spectrometry/methods , Mice , Mice, Knockout , Peroxiredoxins/chemistry , Protein Deglycase DJ-1 , Proteome , Sulfinic Acids/chemistry
10.
J Biol Chem ; 284(42): 28634-41, 2009 Oct 16.
Article in English | MEDLINE | ID: mdl-19710022

ABSTRACT

TMP21 has been shown to be associated with the gamma-secretase complex and can specifically regulate gamma-cleavage without affecting epsilon-mediated proteolysis. To explore the basis of this activity, TMP21 modulation of gamma-secretase activity was investigated independent of epsilon-cleavage using an amyloid-beta precursor proteinepsilon (APPepsilon) construct which lacks the amyloid intracellular domain domain. The APPepsilon construct behaves similarly to the full-length precursor protein with respect to alpha- and beta-cleavages and is able to undergo normal gamma-processing. Co-expression of APPepsilon and TMP21 resulted in the accumulation of membrane-embedded higher molecular weight Abeta-positive fragments, consistent with an inhibition of gamma-secretase cleavage. The APPepsilon system was used to examine the functional domains of TMP21 through the investigation of a series of TMP21-p24a chimera proteins. It was found that chimeras containing the transmembrane domain bound to the gamma-secretase complex and could decrease gamma-secretase proteolytic processing. This was confirmed though investigation of a synthetic peptide corresponding to the TMP21 transmembrane helix. The isolated TMP21 TM peptide but not the homologous p24a domain was able to reduce Abeta production in a dose-dependent fashion. These observations suggest that the TMP21 transmembrane domain promotes its association with the presenilin complex that results in decreased gamma-cleavage activity.


Subject(s)
Amyloid Precursor Protein Secretases/metabolism , Membrane Proteins/metabolism , Peptides/chemistry , Amyloid beta-Peptides/chemistry , Cell Line , Cell Membrane/metabolism , Cell-Free System , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Genes, Reporter , Humans , Lysine/chemistry , Mutagenesis , Nucleocytoplasmic Transport Proteins , Protein Structure, Tertiary , Recombinant Fusion Proteins/chemistry
11.
J Biol Chem ; 284(24): 16298-16307, 2009 Jun 12.
Article in English | MEDLINE | ID: mdl-19369254

ABSTRACT

Complexes involved in the gamma/epsilon-secretase-regulated intramembranous proteolysis of substrates such as the amyloid-beta precursor protein are composed primarily of presenilin (PS1 or PS2), nicastrin, anterior pharynx defective-1 (APH1), and PEN2. The presenilin aspartyl residues form the catalytic site, and similar potentially functional polar transmembrane residues in APH1 have been identified. Substitution of charged (E84A, R87A) or polar (Q83A) residues in TM3 had no effect on complex assembly or activity. In contrast, changes to either of two highly conserved histidines (H171A, H197A) located in TM5 and TM6 negatively affected PS1 cleavage and altered binding to other secretase components, resulting in decreased amyloid generating activity. Charge replacement with His-to-Lys substitutions rescued nicastrin maturation and PS1 endoproteolysis leading to assembly of the formation of structurally normal but proteolytically inactive gamma-secretase complexes. Substitution with a negatively charged side chain (His-to-Asp) or altering the structural location of the histidines also disrupted gamma-secretase binding and abolished functionality of APH1. These results suggest that the conserved transmembrane histidine residues contribute to APH1 function and can affect presenilin catalytic activity.


Subject(s)
Membrane Proteins/genetics , Membrane Proteins/metabolism , Multiprotein Complexes/metabolism , Peptide Hydrolases/genetics , Peptide Hydrolases/metabolism , Presenilins/metabolism , Amino Acid Sequence , Amino Acid Substitution , Amyloid Precursor Protein Secretases/metabolism , Animals , Aspartic Acid/genetics , Catalysis , Cells, Cultured , Conserved Sequence , Endopeptidases , Fibroblasts/cytology , Fibroblasts/physiology , Histidine/genetics , Humans , Membrane Proteins/chemistry , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed , Peptide Hydrolases/chemistry , Protein Structure, Tertiary , Transfection
12.
Nat Genet ; 39(2): 168-77, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17220890

ABSTRACT

The recycling of the amyloid precursor protein (APP) from the cell surface via the endocytic pathways plays a key role in the generation of amyloid beta peptide (Abeta) in Alzheimer disease. We report here that inherited variants in the SORL1 neuronal sorting receptor are associated with late-onset Alzheimer disease. These variants, which occur in at least two different clusters of intronic sequences within the SORL1 gene (also known as LR11 or SORLA) may regulate tissue-specific expression of SORL1. We also show that SORL1 directs trafficking of APP into recycling pathways and that when SORL1 is underexpressed, APP is sorted into Abeta-generating compartments. These data suggest that inherited or acquired changes in SORL1 expression or function are mechanistically involved in causing Alzheimer disease.


Subject(s)
Alzheimer Disease/genetics , LDL-Receptor Related Proteins/genetics , Membrane Transport Proteins/genetics , Age of Onset , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/metabolism , Cell Line , Endosomes/metabolism , Genetic Variation , Haplotypes , Humans , Introns , Models, Genetic , Organ Specificity , Polymorphism, Single Nucleotide , Protease Nexins , Receptors, Cell Surface/metabolism , Vesicular Transport Proteins/metabolism
13.
Nature ; 440(7088): 1208-12, 2006 Apr 27.
Article in English | MEDLINE | ID: mdl-16641999

ABSTRACT

The presenilin proteins (PS1 and PS2) and their interacting partners nicastrin, aph-1 (refs 4, 5) and pen-2 (ref. 5) form a series of high-molecular-mass, membrane-bound protein complexes that are necessary for gamma-secretase and epsilon-secretase cleavage of selected type 1 transmembrane proteins, including the amyloid precursor protein, Notch and cadherins. Modest cleavage activity can be generated by reconstituting these four proteins in yeast and Spodoptera frugiperda (sf9) cells. However, a critical but unanswered question about the biology of the presenilin complexes is how their activity is modulated in terms of substrate specificity and/or relative activities at the gamma and epsilon sites. A corollary to this question is whether additional proteins in the presenilin complexes might subsume these putative regulatory functions. The hypothesis that additional proteins might exist in the presenilin complexes is supported by the fact that enzymatically active complexes have a mass that is much greater than predicted for a 1:1:1:1 stoichiometric complex (at least 650 kDa observed, compared with about 220 kDa predicted). To address these questions we undertook a search for presenilin-interacting proteins that differentially affected gamma- and epsilon-site cleavage events. Here we report that TMP21, a member of the p24 cargo protein family, is a component of presenilin complexes and differentially regulates gamma-secretase cleavage without affecting epsilon-secretase activity.


Subject(s)
Endopeptidases/metabolism , Membrane Proteins/metabolism , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Amyloid Precursor Protein Secretases , Amyloid beta-Peptides/biosynthesis , Amyloid beta-Peptides/genetics , Animals , Aspartic Acid Endopeptidases , Cell Line , Endopeptidases/chemistry , Humans , Membrane Proteins/chemistry , Membrane Proteins/deficiency , Membrane Proteins/genetics , Mice , Models, Biological , Nucleocytoplasmic Transport Proteins , Presenilin-1 , Presenilin-2 , Protein Binding , Substrate Specificity
14.
J Biol Chem ; 281(21): 14547-53, 2006 May 26.
Article in English | MEDLINE | ID: mdl-16531402

ABSTRACT

The single-transmembrane receptor SorLA/LR11 contains binding domains typical for lipoprotein receptors and a VPS10 domain, which binds the neuropeptide head-activator. This undecapeptide enhances proliferation of neuronal precursor cells in a SorLA-dependent manner. Using specific inhibitors we found previously that head activator activates shedding of SorLA by the metalloprotease TACE close to the transmembrane domain releasing the large extra-cellular part of the receptor. Here we show that the remaining COOH-terminal membrane fragment of SorLA is processed by gamma-secretase. Inhibition of gamma-secretase by specific inhibitors or overexpression of dominant negative presenilin mutants and knock out of the presenilin genes led to accumulation of the SorLA membrane fragment and also of full-length SorLA in the membrane. In an in vitro assay we observed the gamma-secretase-dependent release of the two soluble cleavage products, the SorLA cytoplasmic domain and the SorLA beta-peptide. These processing steps are reminiscent of a novel signaling pathway that has been described for the notch receptor. Here, the notch cytoplasmic domain is released into the cytoplasm by the gamma-secretase and migrates to the nucleus where it acts as a transcriptional regulator. In parallel we found that a fusion protein of the released cytoplasmic tail of SorLA with EGFP located to the nucleus only if the nuclear localization signal of SorLA was intact. In a reporter gene assay the cytoplasmic domain of SorLA acted as a transcriptional activator indicating that SorLA might directly regulate transcription after activation by gamma-secretase.


Subject(s)
Membrane Transport Proteins/physiology , Receptors, LDL/physiology , Amino Acid Sequence , Amyloid Precursor Protein Secretases , Aspartic Acid Endopeptidases , Cell Nucleus/metabolism , Cytoplasm/metabolism , Endopeptidases/metabolism , Gene Expression Regulation , Green Fluorescent Proteins/metabolism , Humans , LDL-Receptor Related Proteins , Membrane Proteins/metabolism , Membrane Transport Proteins/metabolism , Molecular Sequence Data , Nuclear Localization Signals , Peptides/chemistry , Presenilin-1 , Protein Sorting Signals , Receptors, LDL/metabolism , Signal Transduction
15.
FEBS J ; 272(7): 1718-26, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15794758

ABSTRACT

Hu-K4 is a human protein homologous to the K4L protein of vaccinia virus. Due to the presence of two HKD motifs, Hu-K4 was assigned to the family of Phospholipase D proteins although so far no catalytic activity has been shown. The Hu-K4 mRNA is found in many human organs with highest expression levels in the central nervous system. We extended the ORF of Hu-K4 to the 5' direction. As a consequence the protein is 53 amino acids larger than originally predicted, now harbouring a putative transmembrane domain. The exon/intron structure of the Hu-K4 gene reveals extensive alternative splicing in the 5' untranslated region. Due to the absence of G/C-rich regions and upstream ATG codons, the mRNA isoform in brain may be translated with higher efficacy leading to a high Hu-K4 protein concentration in this tissue. Using a specific antiserum produced against Hu-K4 we found that Hu-K4 is a membrane-bound protein colocalizing with protein disulfide isomerase, a marker of the endoplasmic reticulum. Glycosylation of Hu-K4 as shown by treatment with peptide N-glycosidase F or tunicamycin indicates that Hu-K4 has a type 2 transmembrane topology.


Subject(s)
Endoplasmic Reticulum/metabolism , Membrane Proteins/chemistry , Amino Acid Sequence , Base Sequence , Endoplasmic Reticulum/chemistry , Gene Expression Profiling , Humans , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , Molecular Sequence Data , RNA, Messenger/metabolism , Sequence Analysis, DNA
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