ABSTRACT
Mutations in the presenilin 1 (PS1) gene are the most common genetic factor underlying the development of early onset familial Alzheimer's disease (FAD). Accumulating evidence has shown that FAD-linked mutations of PS1 enhance the generation of amyloid-beta (1-42) protein. Recently, beta-catenin has been shown to interact with PS1. beta-catenin is essential for the Wnt signalling pathway. However, the biological significance of the interaction between beta-catenin and PS1 in this signalling pathway remains to be clarified. In this study, we investigated the effect of FAD-linked PS1 (M146L) mutation in the Wnt signalling pathway using the conditioned medium containing Wnt-3A. The expression of mutated PS1 inhibited the Wnt-3A-induced accumulation of beta-catenin. Chase analysis of beta-catenin in Wnt-3A-stimulated cells following cycloheximide treatment revealed that PS1 mutation enhanced the generation of the higher molecular mass form of beta-catenin, most likely, ubiquitinated beta-catenin. In addition, the expression of mutated PS1 elevated the level of phosphorylated beta-catenin, which is targeted to the ubiquitin/proteasome pathway. Thus, it appears that PS1 (M146L) mutation down-regulates the Wnt-3A-induced accumulation of beta-catenin due to an increase in the level of phosphorylated beta-catenin.
Subject(s)
Acetylcysteine/analogs & derivatives , Cytoskeletal Proteins/metabolism , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mutation , Proto-Oncogene Proteins/metabolism , Signal Transduction , Trans-Activators , Zebrafish Proteins , Acetylcysteine/pharmacology , Animals , Culture Media, Conditioned/pharmacology , Cycloheximide/pharmacology , Down-Regulation , Electrophoresis, Polyacrylamide Gel , Mice , Phosphorylation , Presenilin-1 , Protein Synthesis Inhibitors/pharmacology , Proteins/metabolism , Time Factors , Transfection , Tumor Cells, Cultured , Wnt Proteins , Wnt3 Protein , Wnt3A Protein , beta CateninABSTRACT
Thymocyte negative selection eliminates self-reactive clones and involves both a T-cell receptor (TCR)/CD3-mediated signal and a costimulatory signal, which can be delivered via CD28. Anti-CD3/anti-CD28-triggered apoptosis in isolated CD4+CD8+ thymocytes in vitro provides a basic model for negative selection. Effects of isoform-selective and non-isoform-selective inhibitors of protein kinase C (PKC) on this apoptotic process suggest that activation of Ca2+-independent PKC isoforms during the first 2-3 hr of culture is essential for inducing apoptosis, and that Ca2+-dependent PKC isoforms may be influential, but not essential, for apoptosis. To assess the CD3/CD28-mediated activation of PKC in the apoptotic process, we prepared CD4+CD8+ thymocytes (without contamination with cells that had received negative or positive selection signals in vivo) by establishing TCR-transgenic mice with RAG-2-deficient and non-selecting major histocompatibility complex (MHC) backgrounds, in addition to a CD4+CD8+ thymocyte-enriched population from normal mice. Translocation of Ca2+-independent PKC from the cytosolic fraction to the particulate fraction of CD4+CD8+ thymocytes was induced by CD3/CD28-mediated stimulation, but not by CD3- or CD28-mediated stimulation alone, and peaked 2 hr after the start of culture. The kinase activity of the translocated Ca2+-independent PKC was dependent on cofactors in vitro, indicating that novel (n)PKC, but not atypical (a)PKC or a proteolytic PKC fragment, was responsible for the activity. Immunoblotting analysis indicated that the nPKC-theta isoform was the major contributor among nPKC isoforms, and that the classical (c)PKC-alpha isoform was the major contributor among cPKC isoforms. These results suggest that activation of nPKC (especially the theta isoform) in CD4+CD8+ thymocytes is involved in a pathway for negative selection.
Subject(s)
Apoptosis/immunology , Protein Kinase C/immunology , T-Lymphocyte Subsets/immunology , Animals , CD28 Antigens/immunology , CD3 Complex/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Calcium/physiology , Cell Culture Techniques , Clonal Anergy/immunology , Isoenzymes/metabolism , Lymphocyte Activation/immunology , Mice , Mice, Inbred BALB C , Mice, Transgenic , Protein Kinase C/metabolism , T-Lymphocyte Subsets/enzymologySubject(s)
Alzheimer Disease/genetics , Membrane Proteins/genetics , Mutation, Missense , Animals , Humans , Presenilin-1ABSTRACT
In the very early stages of target cell apoptosis induced by CTL, we found that fluorescence of labeling probes of the target plasma membrane, such as N-(3-triethylammoniumpropyl)-4-(p-dibutylaminostyryl)pyridin ium dibromide (FM1-43), was translocated into intracellular membrane structures including nuclear envelope and mitochondria. This translocation was associated with the execution of CTL-mediated killing, because neither the CTL-target conjugation alone nor the binding of noncytotoxic Th2 clone with target cell was sufficient to provoke the process. Although FM1-43 translocation was observed in perforin-mediated cytotoxicity, examinations with several other dyes failed to detect the evidence for membrane damages that may cause influx of the dye. Moreover, the translocation was also observed in Fas-dependent apoptosis. These data indicate that the translocation precedes the damage of plasma membrane and intracellular organella in the course of apoptotic cell death and may represent the existence of a membrane trafficking that mediates the translocation of plasma membrane components in the early onset of apoptotic cell death.
Subject(s)
Apoptosis/immunology , Cell Membrane Permeability/immunology , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/metabolism , Animals , Apoptosis/genetics , Caspase 3 , Caspases/deficiency , Caspases/genetics , Caspases/metabolism , Cell Line , Cell Membrane/immunology , Cell Membrane/metabolism , Cell Membrane Permeability/genetics , Cytoplasmic Granules/immunology , Cytoplasmic Granules/metabolism , Cytotoxicity, Immunologic , Fluorescent Dyes/metabolism , Intracellular Membranes/immunology , Intracellular Membranes/metabolism , Mice , Mitochondria/immunology , Mitochondria/metabolism , Permeability , Pyridinium Compounds/metabolism , Quaternary Ammonium Compounds/metabolism , T-Lymphocytes, Cytotoxic/enzymology , Tumor Cells, Cultured , fas Receptor/genetics , fas Receptor/physiologyABSTRACT
Previously, we reported that mutations in presenilin 1 (PS1) increased the intracellular levels of amyloid beta-protein (Abeta)42. However, it is still not known at which cellular site or how PS1 mutations exert their effect of enhancing Abeta42-gamma-secretase cleavage. In this study, to clarify the molecular mechanisms underlying this enhancement of Abeta42-gamma-secretase cleavage, we focused on determining the intracellular site of the cleavage. To address this issue, we used APP-C100 encoding the C-terminal beta-amyloid precursor protein (APP) fragment truncated at the N terminus of Abeta (C100); C100 requires only gamma-secretase cleavage to yield Abeta. Mutated PS1 (M146L)-induced Neuro 2a cells showed enhanced Abeta1-42 generation from transiently expressed C100 as well as from full-length APP, whereas the generation of Abeta1-40 was not increased. The intracellular generation of Abeta1-42 from transiently expressed C100 in both mutated PS1-induced and wild-type Neuro 2a cells was inhibited by brefeldin A. Moreover, the generation of Abeta1-42 and Abeta1-40 from a C100 mutant containing a di-lysine endoplasmic reticulum retention signal was greatly decreased, indicating that the major intracellular site of gamma-secretase cleavage is not the endoplasmic reticulum. The intracellular generation of Abeta1-42/40 from C100 was not influenced by monensin treatment, and the level of Abeta1-42/40 generated from C100 carrying a sorting signal for the trans-Golgi network was higher than that generated from wild-type C100. These results using PS1-mutation-harbouring and wild-type Neuro 2a cells suggest that Abeta42/40-gamma-secretase cleavages occur in the Golgi compartment and the trans-Golgi network, and that the PS1 mutation does not alter the intracelluar site of Abeta42-gamma-secretase cleavage in the normal APP proteolytic processing pathway.
Subject(s)
Amyloid beta-Peptides/biosynthesis , Endopeptidases/metabolism , Membrane Proteins/genetics , Mutation , Peptide Fragments/biosynthesis , Amyloid Precursor Protein Secretases , Animals , Aspartic Acid Endopeptidases , Base Sequence , Brefeldin A/pharmacology , DNA Primers , Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , Hydrolysis , Mice , Monensin/pharmacology , Neuroblastoma/genetics , Neuroblastoma/metabolism , Presenilin-1 , Protein Sorting Signals/metabolism , Tumor Cells, CulturedABSTRACT
The Saccharomyces cerevisiae YPS2 (formerly MKC7) gene product is a glycosylphosphatidylinositol-linked aspartyl protease that functions as a yeast secretase. Here, the glycosylphosphatidylinositol-linked form of yapsin 2 (Mkc7p) was purified to homogeneity from the membrane fraction of an overexpressing yeast strain. Purified yapsin 2 migrated diffusely in SDS-polyacrylamide gel electrophoresis (molecular mass approximately 200 kDa), suggesting extensive, heterogeneous glycosylation. Studies using internally quenched fluorogenic peptide substrates revealed cleavage by the enzyme carboxyl to Lys or Arg. No cleavage was seen when both Lys and Arg were absent. No significant enhancement was seen with multiple basic residues. However, cleavage always occurred carboxyl to the most COOH-terminal basic residue. V(max)/K(m) was insensitive to P(2) and P(3) residues except that Pro at P(2) blocked cleavage entirely. These results suggest that yapsin 2 is a monobasic amino acid-specific protease that requires a basic residue at P(1) and excludes basic residues from P(1)'. The pH dependence of V(max)/K(m) for a substrate containing a pro-alpha factor cleavage site was bell-shaped, with a maximum near pH 4.0. However, V(max)/K(m) for a substrate mimicking the alpha-secretase site in human beta amyloid precursor protein was optimal near pH 6.0, consistent with cleavage of beta amyloid precursor protein by yapsin 2 when expressed in yeast.
Subject(s)
Aspartic Acid Endopeptidases/isolation & purification , Glycosylphosphatidylinositols/physiology , Proprotein Convertases , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/enzymology , Amino Acid Sequence , Animals , Aspartic Acid Endopeptidases/analysis , Aspartic Acid Endopeptidases/chemistry , Humans , Hydrogen-Ion Concentration , Molecular Sequence Data , Pepstatins/pharmacology , Substrate Specificity , Subtilisins/physiologyABSTRACT
Mutations in presenilin 1 (PS1) and presenilin 2 (PS2) are the most common genetic factors underlying the development of early-onset familial Alzheimer's disease (FAD). To investigate the pathogenic mechanism of PS1 mutations linked to FAD, we established inducible mouse neuroblastoma (Neuro 2a) cell lines expressing the human wild-type (wt) or mutated PS1(M146L or deltaexon 10) under the control of the Lac repressor. Using this inducible PS1 system, the influence of PS1 mutations on the generation of endogenous murine Abeta species was assessed using a highly sensitive immunoblotting technique. The induction of mutated PS1 resulted in an increase in the extra- and intracellular levels of two distinct Abeta species ending at residue 42, Abeta1-42 and its N-terminally truncated variant(s), Abetax-42. In addition, the intracellular generation of these Abeta42 species was completely blocked by brefeldin A. In contrast, it exhibited differential sensitivities to monensin such that there was an increased accumulation of intracellular Abetax-42 but an inhibition of intracellular Abeta1-42 generation. These data strongly suggest that Abetax-42 is generated in a proximal Golgi compartment, whereas Abeta1-42 is generated in a distal Golgi and/or a post-Golgi compartment. Thus, it appears that PS1 mutations enhance the degree of 42-specific gamma-secretase cleavage which occurs (i) in the ER or the early Golgi apparatus prior to gamma-secretase cleavage, or (ii) in the distinct sites where Abetax-42 and Abeta1-42 are generated. To date, the site of Abeta42 generation has not been firmly established. Our data provide new information regarding the site of Abeta42 generation mediated by the FAD-linked mutant PS1.
Subject(s)
Alzheimer Disease/genetics , Membrane Proteins/genetics , Mutation , Alzheimer Disease/metabolism , Animals , Brefeldin A/pharmacology , Extracellular Space/metabolism , Gene Expression , Humans , Intracellular Fluid , Membrane Proteins/biosynthesis , Mice , Monensin/pharmacology , Neuroblastoma , Presenilin-1 , Protein Isoforms , Protein Synthesis Inhibitors/pharmacology , Tumor Cells, CulturedABSTRACT
Transgenic lpr/lpr mice expressing functional Fas selectively on B cells were produced in an attempt to elucidate the role of Fas on B cells in the regulation of autoantibody production. The homozygous lpr/lpr mice carrying the transgene did not produce anti-double-stranded DNA antibodies throughout their lives, whereas the development of abnormal lpr T cells (double negative, B220(+)) was not suppressed. Further analyses, however, revealed that the expression of the transgenic Fas on B cells of lpr/lpr homozygous mice resulted in severe impairment of the B cell function. The defect was characterized by a decrease in the number of mature peripheral B cells, a reduction in the serum Ig level and the total failure of B cells to mount antibody responses to stimulations of T-dependent as well as T-independent antigens. Such a defect was prominent only when the transgene was expressed on the lpr/lpr homozygous background. On the contrary, B cells of the transgenic lpr/lpr mice were shown to be capable of producing Ig when stimulated with anti-CD40 in the presence of IL-4 and IL-5. Furthermore, lpr/lpr T cells showed enhanced non-specific cytolytic activity. These observations suggested that the observed B cell defect was probably attributable to the destruction of activated B cells expressing transgenic Fas by aggressive lpr/lpr T cells.
Subject(s)
Autoimmune Diseases/genetics , B-Lymphocytes/immunology , fas Receptor/physiology , Animals , Autoantibodies/biosynthesis , Autoimmune Diseases/metabolism , B-Lymphocytes/metabolism , B-Lymphocytes/pathology , DNA, Complementary/genetics , DNA, Complementary/immunology , DNA, Complementary/metabolism , Female , Gene Expression , Immunoglobulins/biosynthesis , Immunoglobulins/blood , Lymphocyte Count , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Inbred MRL lpr , Mice, Transgenic , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Transgenes/immunology , fas Receptor/biosynthesis , fas Receptor/genetics , fas Receptor/immunologyABSTRACT
Recently, a novel membrane-associated metalloprotease, designated Ste24p, has been identified in Saccharomyces cerevisiae [K. Fujimura-Kamada, F.J. Nouvet, S. Michaelis, J. Cell Biol. 27 (1997) 271-285]. We cloned a human brain cDNA encoding a protein homologous to Ste24p (designated Hs Ste24p). The predicted 475-amino acid product of its open reading frame exhibited 62% similarity to Ste24p, and contained a zinc metalloprotease motif (HEXXH) and multiple predicted membrane spans. Northern blot analysis showed that this gene was expressed in most tissues. Immunofluorescence analysis of epitope-tagged Hs Ste24p constructs suggested that it is localized in the ER and possibly also in the Golgi compartment. A search of the expression sequence tag database identified a fragment of DNA encoding a segment homologous to the segment of Hs Ste24p containing the HEXXH motif in insects and nematodes. Thus, Hs Ste24p could be a member of a new family of Ste24p-like membrane-associated metalloproteases which are widely conserved in eukaryotes.
Subject(s)
DNA, Complementary/analysis , Membrane Proteins/genetics , Metalloendopeptidases/genetics , Saccharomyces cerevisiae Proteins , Amino Acid Sequence , Cell Line , Cloning, Molecular , Fungal Proteins/chemistry , Humans , Membrane Proteins/biosynthesis , Membrane Proteins/chemistry , Metalloendopeptidases/biosynthesis , Metalloendopeptidases/chemistry , Molecular Sequence Data , Sequence Alignment , Sequence HomologyABSTRACT
Neural plakophilin-related arm-repeat protein (NPRAP) is a mammalian brain protein of the armadillo family. Here, in an attempt to elucidate its function, we determined the mouse brain regions and cell types expressing the mRNAs for two NPRAP isoforms (the longer and the shorter isoforms), and examined the regulation of expression of the NPRAP mRNAs during the differentiation of P19 cells into neurons. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that both variants were expressed in various mouse brain regions, but the mRNA for the short isoform was predominant in most regions. Primary cultures of both neurons and glial cells exhibited high expression levels of both the mRNAs, indicating that NPRAP mRNA expression is not neuron-specific. The expression of the two NPRAP mRNA variants was dramatically induced even prior to the terminal neuronal and glial differentiation of P19 cells after retinoic acid treatment. These data suggest that the two NPRAP isoforms function not only in neurons and glial cells in the brain, but also play a role in the differentiation of precursor cells into neurons and glial cells.
Subject(s)
Nerve Tissue Proteins/genetics , Neuroglia/metabolism , Neurons/metabolism , RNA, Messenger/metabolism , Animals , Armadillo Domain Proteins , Catenins , Cell Adhesion Molecules , Cells, Cultured , Cytoskeletal Proteins , Mice , Neuroglia/drug effects , Neurons/drug effects , Phosphoproteins , Protein Isoforms/genetics , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Tretinoin/pharmacology , Delta CateninABSTRACT
Human beta-amyloid precursor protein (APP) introduced into yeast undergoes alpha-secretase-type cleavage, suggesting that yeast have alpha-secretase-like protease(s). Here we report that two structurally and functionally related glycosyl-phosphatidylinositol-linked yeast aspartyl proteases, Mkc7p and Yap3p (collectively termed yapsin), are responsible for alpha-secretase-type cleavage of APP expressed in yeast, resulting in release of soluble APP into the extracellular space. Disruption of MKC7 and YAP3 in a vacuolar protease-deficient strain abolished this APP cleavage/release, and APP cleavage/release could be restored by introduction of MKC7 or YAP3 on a single copy plasmid. Purified Mkc7p cleaved an internally quenched fluorogenic APP peptide substrate at the alpha-secretase cleavage site. Measurement of proteolytic activity either in yeast homogenates or on the yeast cell surface revealed that most Mkc7p and Yap3p activities were localized at the cell surface. These results establish a molecular basis for alpha-secretase-type cleavage in yeast and support the generally held concept that alpha-secretase cleavage of APP occurs at the cell surface.
Subject(s)
Amyloid beta-Protein Precursor/metabolism , Aspartic Acid Endopeptidases/metabolism , Endopeptidases/metabolism , Protein Processing, Post-Translational , Saccharomyces cerevisiae/enzymology , Amyloid Precursor Protein Secretases , Amyloid beta-Protein Precursor/genetics , Aspartic Acid Endopeptidases/genetics , Cell Membrane/enzymology , Cloning, Molecular , Endopeptidases/genetics , Genotype , Humans , Mutagenesis, Site-Directed , Plasmids , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/geneticsABSTRACT
Mutations in the presenilin genes PS1 and PS2 cause the most common form of early-onset familial Alzheimer's disease. The influence of PS1 mutations on the generation of endogenous intracellular amyloid beta-protein (A beta) species was assessed using a highly sensitive immunoblotting technique with inducible mouse neuroblastoma (Neuro 2a) cell lines expressing the human wild-type (wt) or mutated PS1 (M146L or delta exon 10). The induction of mutated PS1 increased the intracellular levels of two distinct A beta species ending at residue 42 that were likely to be A beta1-42 and its N-terminally truncated variant(s) A beta x-42. The induction of mutated PS1 resulted in a higher level of intracellular A beta1-42 than of intracellular A beta x-42, whereas extracellular levels of A beta1-42 and A beta x-42 were increased proportionally. In addition, the intracellular generation of these A beta42 species in wt and mutated PS1-induced cells was completely blocked by brefeldin A, whereas it exhibited differential sensitivities to monensin: the increased accumulation of intracellular A beta x-42 versus inhibition of intracellular A beta1-42 generation. These data strongly suggest that A beta x-42 is generated in a proximal Golgi, whereas A beta1-42 is generated in a distal Golgi and/or a post-Golgi compartment. Thus, it appears that PS1 mutations enhance the degree of 42-specific gamma-secretase cleavage that occurs in the normal beta-amyloid precursor protein processing pathway (a) in the endoplasmic reticulum or the early Golgi apparatus prior to beta-secretase cleavage or (b) in the distinct sites where A beta x-42 and A beta1-42 are generated.
Subject(s)
Alzheimer Disease/genetics , Amyloid beta-Peptides/biosynthesis , Genetic Linkage , Intracellular Fluid/metabolism , Membrane Proteins/genetics , Membrane Proteins/pharmacology , Mutation/genetics , Peptide Fragments/biosynthesis , Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Animals , Anti-Bacterial Agents/pharmacology , Brefeldin A , Cyclopentanes/pharmacology , Extracellular Space/metabolism , Gene Expression Regulation/drug effects , Humans , Macrolides , Mice , Monensin/pharmacology , Peptide Fragments/metabolism , Presenilin-1 , Time Factors , Tumor Cells, CulturedSubject(s)
Autoantibodies/biosynthesis , B-Lymphocytes/immunology , fas Receptor/metabolism , Animals , Antibodies, Antinuclear/metabolism , Apoptosis , Fas Ligand Protein , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Inbred MRL lpr , Mice, Transgenic , fas Receptor/geneticsABSTRACT
The potentials of the two major histological types of gastric carcinoma to invade through extracellular matrices were studied with cell lines. We found that the invasive potential of intestinal-type carcinoma cells (MKN-28 and MKN-74) were higher than those of diffuse-type carcinoma cells (MKN-45 and KATO-III). To investigate whether the alpha 2 and alpha 6 integrin adhesion molecules are responsible for, or involved in carcinoma invasion. We further studied alpha 2 and alpha 6 expression patterns in these two types of cell line. Although fluorescence-activated cell sorting analysis revealed that all cells examined invariably expressed these integrin molecules, their expressional patterns were different among different cell lines. The intestinal-type carcinoma cells expressed integrins mainly along the cell-cell contact region, whereas the diffuse-type carcinoma cells showed a diffuse cytoplasmic pattern of integrin expression. Invasion by MKN-28, MKN-74 and MKN-45 cells through reconstituted basement membrane or type I collagen gel was significantly inhibited (P < 0.05) by 50 micrograms/ml anti-(alpha 2 integrin) or anti-(alpha 6 integrin) monoclonal antibodies. Our results suggest that active invasiveness is stronger in the intestinal-type than in the diffuse-type carcinoma cells and that alpha 2 and alpha 6 integrins play important roles in invasion of both types of gastric carcinoma cell lines.
Subject(s)
Antigens, CD/physiology , Carcinoma/pathology , Neoplasm Invasiveness/pathology , Stomach Neoplasms/pathology , Antigens, CD/analysis , Cell Separation , Humans , Integrin alpha2 , Integrin alpha6 , Tumor Cells, CulturedABSTRACT
The MKC7 gene was isolated as a multicopy suppressor of the cold-sensitive growth phenotype of a yeast kex2 mutant, which lacks the protease that cleaves pro-alpha-factor and other secretory proproteins at pairs of basic residues in a late Golgi compartment in yeast. MKC7 encodes an aspartyl protease most closely related to product of the YAP3 gene, a previously isolated multicopy suppressor of the pro-alpha-factor processing defect of a kex2 null. Multicopy MKC7 suppressed the alpha-specific mating defect of a kex2 null as well as multicopy YAP3 did, but multicopy YAP3 was a relatively weak suppressor of kex2 cold sensitivity. Overexpression of MKC7 resulted in production of a membrane-associated proteolytic activity that cleaved an internally quenched fluorogenic peptide substrate on the carboxyl side of a Lys-Arg site. Treatment with phosphatidylinositol-specific phospholipase C shifted Mkc7 activity from the detergent to the aqueous phase in a Triton X-114 phase separation, indicating that membrane attachment of Mkc7 is mediated by a glycosyl-phosphatidylinositol anchor. Although disruption of MKC7 or YAP3 alone resulted in no observable phenotype, mkc7 yap3 double disruptants exhibited impaired growth at 37 degrees C. Disruption of MKC7 and YAP3 in a kex2 null mutant resulted in profound temperature sensitivity and more generalized cold sensitivity. The synergism of mkc7, yap3, and kex2 null mutations argues that Mkc7 and Yap3 are authentic processing enzymes whose functions overlap those of Kex2 in vivo.
Subject(s)
Aspartic Acid Endopeptidases/metabolism , Glycosylphosphatidylinositols , Membrane Proteins/metabolism , Proprotein Convertases , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/enzymology , Subtilisins/metabolism , Amino Acid Sequence , Aspartic Acid Endopeptidases/genetics , Cloning, Molecular , Crosses, Genetic , DNA-Binding Proteins/genetics , Genes, Fungal , Kruppel-Like Transcription Factors , Molecular Sequence Data , Mutagenesis , Phosphatidylinositol Diacylglycerol-Lyase , Phosphoinositide Phospholipase C , Phosphoric Diester Hydrolases/analysis , Protein Processing, Post-Translational , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Sequence Analysis, DNA , Sequence Homology, Amino AcidABSTRACT
Although T cells bearing gamma delta T-cell receptors have long been known to be present in the epithelial lining of many organs, their specificity and function remain elusive. In the present study, we examined the intestinal epithelia of T-cell-receptor mutant mice, which were deficient in either gamma delta T cells or alpha beta T cells, and of normal littermates. The absence of gamma delta T cells was associated with a reduction in epithelial cell turnover and a downregulation of the expression of major histocompatibility complex class II molecules. No such effects were observed in alpha beta T-cell-deficient mice. These findings indicate that intraepithelial gamma delta T cells regulate the generation and differentiation of intestinal epithelial cells.
Subject(s)
Intestinal Mucosa/immunology , Intestine, Small/immunology , Receptors, Antigen, T-Cell, gamma-delta/physiology , T-Lymphocytes/physiology , Animals , Base Sequence , Crosses, Genetic , DNA Primers , Epithelium/physiology , Female , Flow Cytometry , Gene Expression Regulation , Histocompatibility Antigens Class II/biosynthesis , Homeostasis , Immunohistochemistry , Lymphocyte Activation , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred Strains , Mice, Mutant Strains , Molecular Sequence Data , Polymerase Chain Reaction , Receptors, Antigen, T-Cell, gamma-delta/genetics , Species Specificity , T-Lymphocytes/immunologyABSTRACT
Human beta-amyloid precursor protein (APP), the transmembrane precursor of the Alzheimer's disease beta-amyloid peptide, was expressed in the yeast Saccharomyces cerevisiae by fusion to prepro-alpha-factor. From analysis of protease-deficient yeast strains, the fusion protein underwent partial processing by Kex2 protease to generate full-length APP and a fraction of the molecules were degraded in the vacuole. A soluble APP ectodomain fragment bearing lumenal but not cytosolic epitopes was released into the media, indicating cleavage by a "membrane protein-solubilizing proteinase" or "secretase" activity. Yeast cells contained a C-terminal APP fragment that co-migrated with authentic C-terminal fragment derived from alpha-secretase cleavage of full-length APP in human cells. The N-terminal sequence of immunoaffinity purified C-terminal APP fragment from yeast was identical to that reported in mammalian and insect cells. These results demonstrate the existence of a secretase activity in yeast. Furthermore, this yeast secretase activity may be related to an APP processing activity present in metazoan cells.
Subject(s)
Amyloid beta-Protein Precursor/metabolism , Cloning, Molecular/methods , Endopeptidases/metabolism , Protein Processing, Post-Translational , Saccharomyces cerevisiae/enzymology , Alzheimer Disease/metabolism , Amino Acid Sequence , Amyloid Precursor Protein Secretases , Amyloid beta-Protein Precursor/chemistry , Amyloid beta-Protein Precursor/isolation & purification , Aspartic Acid Endopeptidases , Chromatography, Affinity , Humans , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/isolation & purification , Plasmids , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolismABSTRACT
A prolyl endopeptidase that hydrolyses Suc-Gly-Pro-MCA (Suc, succinyl; MCA, methyl-coumaryl-7-amide) was purified to near homogeneity from NIH-Sape-4 cells derived from the flesh fly (Sarcophaga peregrina). The molecular mass of the purified enzyme was 84 kDa, and its activity was inhibited almost completely by 1 mM diisopropyl fluorophosphate (DFP). Immunoblotting and DFP-labeling experiments revealed that the leg imaginal discs of Sarcophaga contained this enzyme as a major serine proteinase. This prolyl endopeptidase is suggested to be involved in the differentiation of imaginal discs, because 2 mM DFP and 0.1 mM N-benzyloxycarbonyl-thioprolyl-thioprolynal-dimethylaceta l (ZTTA), a specific inhibitor for prolyl endopeptidase, inhibited differentiation of the imaginal discs from the eversion to the elongation stage.
Subject(s)
Diptera/enzymology , Serine Endopeptidases/metabolism , Amino Acid Sequence , Animals , Cell Differentiation/drug effects , Cell Line , Diptera/cytology , Diptera/growth & development , Hydrolysis , Larva/cytology , Larva/enzymology , Larva/growth & development , Molecular Sequence Data , Molecular Weight , Oligopeptides/metabolism , Prolyl Oligopeptidases , Serine Endopeptidases/chemistry , Serine Endopeptidases/isolation & purification , Serine Proteinase Inhibitors/pharmacology , Substrate SpecificityABSTRACT
By expression cloning using FACS, we have isolated cDNA clones encoding human CD34 from a megakaryoblastoid cell line. The predicted amino acid sequence of CD34 revealed the type I transmembrane protein consisted of a leader peptide (31 residues), an extracellular domain (258 residues), a transmembrane domain (23 residues) and a cytoplasmic domain of 73 residues. In addition, a second form of cDNA that has 194 bp insertion in the cytoplasmic region was isolated. The analysis of genomic DNA showed that this sequence is inserted between the predicted transmembrane and cytoplasmic exons due to an alternative usage of an imperfect 5' splice acceptor site in the 5' flanking region of the cytoplasmic exon. The insertion brings a stop codon so that the protein encoded by this type of mRNA has only 16 residues in the cytoplasmic domain. This truncated form of CD34 molecule can be expressed on the cell surface, and its expression seems to change in association with cell differentiation. A search of the National Biomedical Research Foundation Protein Sequence Database (NBRF) with the Predicted amino acid sequence of CD34 did not reveal homology to any known protein. Thus, the CD34 molecule represents a novel type of cell surface molecule that may have a role in early differentiation process of hematopoietic stem cells.
Subject(s)
Antigens, CD/genetics , DNA/genetics , Amino Acid Sequence , Antigens, CD34 , Base Sequence , Cells, Cultured , DNA/analysis , Exons , Flow Cytometry , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/immunology , Hematopoietic Stem Cells/physiology , Humans , Megakaryocytes/cytology , Megakaryocytes/immunology , Molecular Sequence Data , Polymerase Chain Reaction , RNA/analysis , RNA/genetics , RNA, Messenger/analysis , RNA, Messenger/geneticsABSTRACT
The nucleotide sequence of sapecin cDNA suggested that this antibacterial peptide of the flesh fly (Sarcophaga peregrina) is produced from preprosapecin by post-translational processing. We examined the production of sapecin and its prosegment by radioimmunoassay under two different physiological conditions in which its gene is activated, assuming that the prosegment has some biological role. Results suggested that the prosegment is degraded selectively during production of sapecin. We also found that imaginal discs synthesize sapecin when cultured in the presence of 20-hydroxyecdysone.