The vacuolar protein sorting genes in insects: A comparative genome view.

PubMed

Li, Zhaofei; Blissard, Gary

2015-07-01

In eukaryotic cells, regulated vesicular trafficking is critical for directing protein transport and for recycling and degradation of membrane lipids and proteins. Through carefully regulated transport vesicles, the endomembrane system performs a large and important array of dynamic cellular functions while maintaining the integrity of the cellular membrane system. Genetic studies in yeast Saccharomyces cerevisiae have identified approximately 50 vacuolar protein sorting (VPS) genes involved in vesicle trafficking, and most of these genes are also characterized in mammals. The VPS proteins form distinct functional complexes, which include complexes known as ESCRT, retromer, CORVET, HOPS, GARP, and PI3K-III. Little is known about the orthologs of VPS proteins in insects. Here, with the newly annotated Manduca sexta genome, we carried out genomic comparative analysis of VPS proteins in yeast, humans, and 13 sequenced insect genomes representing the Orders Hymenoptera, Diptera, Hemiptera, Phthiraptera, Lepidoptera, and Coleoptera. Amino acid sequence alignments and domain/motif structure analyses reveal that most of the components of ESCRT, retromer, CORVET, HOPS, GARP, and PI3K-III are evolutionarily conserved across yeast, insects, and humans. However, in contrast to the VPS gene expansions observed in the human genome, only four VPS genes (VPS13, VPS16, VPS33, and VPS37) were expanded in the six insect Orders. Additionally, VPS2 was expanded only in species from Phthiraptera, Lepidoptera, and Coleoptera. These studies provide a baseline for understanding the evolution of vesicular trafficking across yeast, insect, and human genomes, and also provide a basis for further addressing specific functional roles of VPS proteins in insects. Copyright © 2014 Elsevier Ltd. All rights reserved.

Avt5p is required for vacuolar uptake of amino acids in the fission yeast Schizosaccharomyces pombe.

PubMed

Chardwiriyapreecha, Soracom; Mukaiyama, Hiroyuki; Sekito, Takayuki; Iwaki, Tomoko; Takegawa, Kaoru; Kakinuma, Yoshimi

2010-06-03

We identified SPBC1685.07c of Schizosaccharomyces pombe as a novel vacuolar protein, Avt5p, with similarity to vacuolar amino acid transporters Avt5p from Saccharomyces cerevisiae. Avt5p localizes to the vacuolar membrane and upon disruption of avt5, uptake of histidine, glutamate, tyrosine, arginine, lysine or serine was impaired. During nitrogen starvation, the transient increase of vacuolar lysine transport observed for wild-type cells still occurred in the mutant cells, however, uptake of glutamate did not significantly increase in response to nitrogen starvation. Our results show that under diverse growth conditions Avt5p is involved in vacuolar transport of a selective set of amino acids. Copyright 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Identification of the fnx1+ and fnx2+ genes for vacuolar amino acid transporters in Schizosaccharomyces pombe.

PubMed

Chardwiriyapreecha, Soracom; Shimazu, Masamitsu; Morita, Tomotake; Sekito, Takayuki; Akiyama, Koichi; Takegawa, Kaoru; Kakinuma, Yoshimi

2008-06-25

We have identified the Schizosaccharomyces pombe SPBC3E7.06c gene (fnx2(+)) from a homology search with the fnx1(+) gene involving in G(0) arrest upon nitrogen starvation. Green fluorescent protein-fused Fnx1p and Fnx2p localized exclusively to the vacuolar membrane. Uptake of histidine or isoleucine by S. pombe cells was inhibited by concanamycin A, a specific inhibitor of the vacuolar H(+)-ATPase. Amino acid uptake was also defective in the vacuolar ATPase mutant, suggesting that vacuolar compartmentalization is critical for amino acid uptake by whole cells. In both Deltafnx1 and Deltafnx2 mutant cells, uptake of lysine, isoleucine or asparagine was impaired. These results suggest that fnx1(+) and fnx2(+) are involved in vacuolar amino acid uptake in S. pombe.

Vacuolar morphology of Saccharomyces cerevisiae during the process of wine making and Japanese sake brewing.

PubMed

Izawa, Shingo; Ikeda, Kayo; Miki, Takeo; Wakai, Yoshinori; Inoue, Yoshiharu

2010-09-01

Although ethanol and osmotic stress affect the vacuolar morphology of Saccharomyces cerevisiae, little information is available about changes in vacuolar morphology during the processes of wine making and Japanese sake (rice wine) brewing. Here, we elucidated changes in the morphology of yeast vacuoles using Zrc1p-GFP, a vacuolar membrane protein, so as to better understand yeast physiology during the brewing process. Wine yeast cells (OC-2 and EC1118) contained highly fragmented vacuoles in the sake mash (moromi) as well as in the grape must. Although sake yeast cells (Kyokai no. 9 and no. 10) also contained highly fragmented vacuoles during the wine-making process, they showed quite a distinct vacuolar morphology during sake brewing. Since the environment surrounding sake yeast cells in the sake mash did not differ much from that surrounding wine yeast cells, the difference in vacuolar morphology during sake brewing between wine yeast and sake yeast was likely caused by innate characters.

Loss of ATP-dependent lysine uptake in the vacuolar membrane vesicles of Saccharomyces cerevisiae ypq1∆ mutant.

PubMed

Sekito, Takayuki; Nakamura, Kyosuke; Manabe, Kunio; Tone, Junichi; Sato, Yumika; Murao, Nami; Kawano-Kawada, Miyuki; Kakinuma, Yoshimi

2014-01-01

Saccharomyces cerevisiae Ypq1p is a vacuolar membrane protein of the PQ-loop protein family. We found that ATP-dependent uptake activities of amino acids by vacuolar membrane vesicles were impaired by ypq1∆ mutation. Loss of lysine uptake was most remarkable, and the uptake was recovered by overproduction of Ypq1p. Ypq1p is thus involved in transport of amino acids into vacuoles.

A mobile precursor determines protein resistance on nanostructured surfaces.

PubMed

Wang, Kang; Chen, Ye; Gong, Xiangjun; Xia, Jianlong; Zhao, Junpeng; Shen, Lei

2018-05-09

Biomaterials are often engineered with nanostructured surfaces to control interactions with proteins and thus regulate their biofunctions. However, the mechanism of how nanostructured surfaces resist or attract proteins together with the underlying design rules remains poorly understood at a molecular level, greatly limiting attempts to develop high-performance biomaterials and devices through the rational design of nanostructures. Here, we study the dynamics of nonspecific protein adsorption on block copolymer nanostructures of varying adhesive domain areas in a resistant matrix. Using surface plasmon resonance and single molecule tracking techniques, we show that weakly adsorbed proteins with two-dimensional diffusivity are critical precursors to protein resistance on nanostructured surfaces. The adhesive domain areas must be more than tens or hundreds of times those of the protein footprints to slow down the 2D-mobility of the precursor proteins for their irreversible adsorption. This precursor model can be used to quantitatively analyze the kinetics of nonspecific protein adsorption on nanostructured surfaces. Our method is applicable to precisely manipulate protein adsorption and resistance on various nanostructured surfaces, e.g., amphiphilic, low-surface-energy, and charged nanostructures, for the design of protein-compatible materials.

Vacuolar processing enzyme in plant programmed cell death

PubMed Central

Hatsugai, Noriyuki; Yamada, Kenji; Goto-Yamada, Shino; Hara-Nishimura, Ikuko

2015-01-01

Vacuolar processing enzyme (VPE) is a cysteine proteinase originally identified as the proteinase responsible for the maturation and activation of vacuolar proteins in plants, and it is known to be an ortholog of animal asparaginyl endopeptidase (AEP/VPE/legumain). VPE has been shown to exhibit enzymatic properties similar to that of caspase 1, which is a cysteine protease that mediates the programmed cell death (PCD) pathway in animals. Although there is limited sequence identity between VPE and caspase 1, their predicted three-dimensional structures revealed that the essential amino-acid residues for these enzymes form similar pockets for the substrate peptide YVAD. In contrast to the cytosolic localization of caspases, VPE is localized in vacuoles. VPE provokes vacuolar rupture, initiating the proteolytic cascade leading to PCD in the plant immune response. It has become apparent that the VPE-dependent PCD pathway is involved not only in the immune response, but also in the responses to a variety of stress inducers and in the development of various tissues. This review summarizes the current knowledge on the contribution of VPE to plant PCD and its role in vacuole-mediated cell death, and it also compares VPE with the animal cell death executor caspase 1. PMID:25914711

Amyloid precursor protein modulates macrophage phenotype and diet-dependent weight gain

PubMed Central

Puig, Kendra L.; Brose, Stephen A.; Zhou, Xudong; Sens, Mary A.; Combs, Gerald F.; Jensen, Michael D.; Golovko, Mikhail Y.; Combs, Colin K.

2017-01-01

It is well known that mutations in the gene coding for amyloid precursor protein are responsible for autosomal dominant forms of Alzheimer’s disease. Proteolytic processing of the protein leads to a number of metabolites including the amyloid beta peptide. Although brain amyloid precursor protein expression and amyloid beta production are associated with the pathophysiology of Alzheimer’s disease, it is clear that amyloid precursor protein is expressed in numerous cell types and tissues. Here we demonstrate that amyloid precursor protein is involved in regulating the phenotype of both adipocytes and peripheral macrophages and is required for high fat diet-dependent weight gain in mice. These data suggest that functions of this protein include modulation of the peripheral immune system and lipid metabolism. This biology may have relevance not only to the pathophysiology of Alzheimer’s disease but also diet-associated obesity. PMID:28262782

Increased KPI containing amyloid precursor protein in experimental autoimmune encephalomyelitis brains.

PubMed

Beilin, Orit; Karussis, Dimitrios M; Korczyn, Amos D; Gurwitz, David; Aronovich, Ramona; Mizrachi-Kol, Rachel; Chapman, Joab

2007-04-16

Amyloid precursor protein can be translated from three alternatively spliced mRNAs. We measured levels of amyloid precursor protein isoforms containing the Kunitz protease inhibitor domain (KPIAPP), and amyloid precursor protein without the Kunitz protease inhibitor domain (KPIAPP) in brain homogenates of acute experimental autoimmune encephalomyelitis mice. At the preclinical phase of the disease, both KPIAPP and KPIAPP levels were significantly higher in homogenates from brains of autoimmune encephalomyelitis mice, whereas at the acute phase of the disease only KPIAPP remained significantly elevated compared with controls. At the recovery phase, no differences were observed between the groups. The early and isoform-specific elevation of KPIAPP in autoimmune encephalomyelitis mice suggests a possible role for amyloid precursor protein in the immune response mediating the disease.

Thermoinactivation analysis of vacuolar H(+)-pyrophosphatase.

PubMed

Yang, Su J; Jiang, Shih S; Hsiao, Yi Y; Van, Ru C; Pan, Yih J; Pan, Rong L

2004-06-07

Vacuolar H(+)-translocating pyrophosphatase (H(+)-PPase; EC 3.6.1.1) catalyzes both the hydrolysis of PP(i) and the electrogenic translocation of proton from the cytosol to the lumen of the vacuole. Vacuolar H(+)-PPase, purified from etiolated hypocotyls of mung bean (Vigna radiata L.), is a homodimer with a molecular mass of 145 kDa. To investigate the relationship between structure and function of this H(+)-translocating enzyme, thermoinactivation analysis was employed. Thermoinactivation studies suggested that vacuolar H(+)-PPase consists of two distinct states upon heat treatment and exhibited different transition temperatures in the presence and absence of ligands (substrate and inhibitors). Substrate protection of H(+)-PPase stabilizes enzyme structure by increasing activation energy from 54.9 to 70.2 kJ/mol. We believe that the conformation of this enzyme was altered in the presence of substrate to protect against the thermoinactivation. In contrast, the modification of H(+)-PPase by inhibitor (fluorescein 5'-isothiocyanate; FITC) augmented the inactivation by heat treatment. The native, substrate-bound, and FITC-labeled vacuolar H(+)-PPases possess probably distinct conformation and show different modes of susceptibility to thermoinactivation. Our results also indicate that the structure of one subunit of this homodimer exerts long distance effect on the other, suggesting a specific subunit-subunit interaction in vacuolar H(+)-PPase. A working model was proposed to interpret the relationship of the structure and function of vacuolar H(+)-PPase.

Nuclear localization of foamy virus Gag precursor protein.

PubMed Central

Schliephake, A W; Rethwilm, A

1994-01-01

All foamy viruses give rise to a strong nuclear staining when infected cells are reacted with sera from infected hosts. This nuclear fluorescence distinguishes foamy viruses from all other retroviruses. The experiments reported here indicate that the foamy virus Gag precursor protein is transiently located in the nuclei of infected cells and this is the likely reason for the typical foamy virus nuclear fluorescence. By using the vaccinia virus expression system, a conserved basic sequence motif in the nucleocapsid domain of foamy virus Gag proteins was identified to be responsible for the nuclear transport of the gag precursor molecule. This motif was also found to be able to direct a heterologous protein, the Gag protein of human immunodeficiency virus, into the nucleus. Images PMID:8035493

Functional expression of Schizosaccharomyces pombe Vba2p in the vacuolar membrane of Saccharomyces cerevisiae.

PubMed

Pongcharoen, Pongsanat; Kawano-Kawada, Miyuki; Iwaki, Tomoko; Sugimoto, Naoko; Sekito, Takayuki; Akiyama, Koichi; Takegawa, Kaoru; Kakinuma, Yoshimi

2013-01-01

A vacuolar membrane protein, Vba2p of Schizosaccharomyces pombe, is involved in basic amino acid uptake by intact cells. Here we found evidence that Vba2p mediated ATP-dependent lysine uptake by vacuolar membrane vesicles of Saccharomyces cerevisiae. Vba2p was also responsible for quinidine sensitivity, and the addition of lysine improved cell growth on quinidine-containing media. These findings should be useful for further characterization of Vba2p.

Genes Required for Vacuolar Acidity in Saccharomyces Cerevisiae

PubMed Central

Preston, R. A.; Reinagel, P. S.; Jones, E. W.

1992-01-01

Mutations that cause loss of acidity in the vacuole (lysosome) of Saccharomyces cerevisiae were identified by screening colonies labeled with the fluorescent, pH-sensitive, vacuolar labeling agent, 6-carboxyfluorescein. Thirty nine vacuolar pH (Vph(-)) mutants were identified. Four of these contained mutant alleles of the previously described PEP3, PEP5, PEP6 and PEP7 genes. The remaining mutants defined eight complementation groups of vph mutations. No alleles of the VAT2 or TFP1 genes (known to encode subunits of the vacuolar H(+)-ATPase) were identified in the Vph(-) screen. Strains bearing mutations in any of six of the VPH genes failed to grow on medium buffered at neutral pH; otherwise, none of the vph mutations caused notable growth inhibition on standard yeast media. Expression of the vacuolar protease, carboxypeptidase Y, was defective in strains bearing vph4 mutations but was apparently normal in strains bearing any of the other vph mutations. Defects in vacuolar morphology at the light microscope level were evident in all Vph(-) mutants. Strains that contained representative mutant alleles of the 17 previously described PEP genes were assayed for vacuolar pH; mutations in seven of the PEP genes (including PEP3, PEP5, PEP6 and PEP7) caused loss of vacuolar acidity. PMID:1628805

Evolution of tonoplast P-ATPase transporters involved in vacuolar acidification.

PubMed

Li, Yanbang; Provenzano, Sofia; Bliek, Mattijs; Spelt, Cornelis; Appelhagen, Ingo; Machado de Faria, Laura; Verweij, Walter; Schubert, Andrea; Sagasser, Martin; Seidel, Thorsten; Weisshaar, Bernd; Koes, Ronald; Quattrocchio, Francesca

2016-08-01

Petunia mutants (Petunia hybrida) with blue flowers defined a novel vacuolar proton pump consisting of two interacting P-ATPases, PH1 and PH5, that hyper-acidify the vacuoles of petal cells. PH5 is similar to plasma membrane H(+) P3A -ATPase, whereas PH1 is the only known eukaryoticP3B -ATPase. As there were no indications that this tonoplast pump is widespread in plants, we investigated the distribution and evolution of PH1 and PH5. We combined database mining and phylogenetic and synteny analyses of PH1- and PH5-like proteins from all kingdoms with functional analyses (mutant complementation and intracellular localization) of homologs from diverse angiosperms. We identified functional PH1 and PH5 homologs in divergent angiosperms. PH5 homologs evolved from plasma membrane P3A -ATPases, acquiring an N-terminal tonoplast-sorting sequence and new cellular function before angiosperms appeared. PH1 is widespread among seed plants and related proteins are found in some groups of bacteria and fungi and in one moss, but is absent in most algae, suggesting that its evolution involved several cases of gene loss and possibly horizontal transfer events. The distribution of PH1 and PH5 in the plant kingdom suggests that vacuolar acidification by P-ATPases appeared in gymnosperms before flowers. This implies that, next to flower color determination, vacuolar hyper-acidification is required for yet unknown processes. © 2016 European Union. New Phytologist © 2016 New Phytologist Trust.

Subcellular localization and vacuolar targeting of sorbitol dehydrogenase in apple seed.

PubMed

Wang, Xiu-Ling; Hu, Zi-Ying; You, Chun-Xiang; Kong, Xiu-Zhen; Shi, Xiao-Pu

2013-09-01

Sorbitol is the primary photosynthate and translocated carbohydrate in fruit trees of the Rosaceae family. NAD(+)-dependent sorbitol dehydrogenase (NAD-SDH, EC 1.1.1.14), which mainly catalyzes the oxidation of sorbitol to fructose, plays a key role in regulating sink strength in apple. In this study, we found that apple NAD-SDH was ubiquitously distributed in epidermis, parenchyma, and vascular bundle in developing cotyledon. NAD-SDH was localized in the cytosol, the membranes of endoplasmic reticulum and vesicles, and the vacuolar lumen in the cotyledon at the middle stage of seed development. In contrast, NAD-SDH was mainly distributed in the protein storage vacuoles in cotyledon at the late stage of seed development. Sequence analysis revealed there is a putative signal peptide (SP), also being predicated to be a transmembrane domain, in the middle of proteins of apple NAD-SDH isoforms. To investigate whether the putative internal SP functions in the vacuolar targeting of NAD-SDH, we analyzed the localization of the SP-deletion mutants of MdSDH5 and MdSDH6 (two NAD-SDH isoforms in apple) by the transient expression system in Arabidopsis protoplasts. MdSDH5 and MdSDH6 were not localized in the vacuoles after their SPs were deleted, suggesting the internal SP functions in the vacuolar targeting of apple NAD-SDH. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Mutational Analysis of the Rift Valley Fever Virus Glycoprotein Precursor Proteins for Gn Protein Expression

PubMed Central

Phoenix, Inaia; Lokugamage, Nandadeva; Nishiyama, Shoko; Ikegami, Tetsuro

2016-01-01

The Rift Valley fever virus (RVFV) M-segment encodes the 78 kD, NSm, Gn, and Gc proteins. The 1st AUG generates the 78 kD-Gc precursor, the 2nd AUG generates the NSm-Gn-Gc precursor, and the 3rd AUG makes the NSm’-Gn-Gc precursor. To understand biological changes due to abolishment of the precursors, we quantitatively measured Gn secretion using a reporter assay, in which a Gaussia luciferase (gLuc) protein is fused to the RVFV M-segment pre-Gn region. Using the reporter assay, the relative expression of Gn/gLuc fusion proteins was analyzed among various AUG mutants. The reporter assay showed efficient secretion of Gn/gLuc protein from the precursor made from the 2nd AUG, while the removal of the untranslated region upstream of the 2nd AUG (AUG2-M) increased the secretion of the Gn/gLuc protein. Subsequently, recombinant MP-12 strains encoding mutations in the pre-Gn region were rescued, and virological phenotypes were characterized. Recombinant MP-12 encoding the AUG2-M mutation replicated slightly less efficiently than the control, indicating that viral replication is further influenced by the biological processes occurring after Gn expression, rather than the Gn abundance. This study showed that, not only the abolishment of AUG, but also the truncation of viral UTR, affects the expression of Gn protein by the RVFV M-segment. PMID:27231931

Mutational Analysis of the Rift Valley Fever Virus Glycoprotein Precursor Proteins for Gn Protein Expression.

PubMed

Phoenix, Inaia; Lokugamage, Nandadeva; Nishiyama, Shoko; Ikegami, Tetsuro

2016-05-24

The Rift Valley fever virus (RVFV) M-segment encodes the 78 kD, NSm, Gn, and Gc proteins. The 1st AUG generates the 78 kD-Gc precursor, the 2nd AUG generates the NSm-Gn-Gc precursor, and the 3rd AUG makes the NSm'-Gn-Gc precursor. To understand biological changes due to abolishment of the precursors, we quantitatively measured Gn secretion using a reporter assay, in which a Gaussia luciferase (gLuc) protein is fused to the RVFV M-segment pre-Gn region. Using the reporter assay, the relative expression of Gn/gLuc fusion proteins was analyzed among various AUG mutants. The reporter assay showed efficient secretion of Gn/gLuc protein from the precursor made from the 2nd AUG, while the removal of the untranslated region upstream of the 2nd AUG (AUG2-M) increased the secretion of the Gn/gLuc protein. Subsequently, recombinant MP-12 strains encoding mutations in the pre-Gn region were rescued, and virological phenotypes were characterized. Recombinant MP-12 encoding the AUG2-M mutation replicated slightly less efficiently than the control, indicating that viral replication is further influenced by the biological processes occurring after Gn expression, rather than the Gn abundance. This study showed that, not only the abolishment of AUG, but also the truncation of viral UTR, affects the expression of Gn protein by the RVFV M-segment.

Chloroplast precursor proteins compete to form early import intermediates in isolated pea chloroplasts.

PubMed

Row, P E; Gray, J C

2001-01-01

In order to ascertain whether there is one site for the import of precursor proteins into chloroplasts or whether different precursor proteins are imported via different import machineries, chloroplasts were incubated with large quantities of the precursor of the 33 kDa subunit of the oxygen-evolving complex (pOE33) or the precursor of the light-harvesting chlorophyll a/b-binding protein (pLHCP) and tested for their ability to import a wide range of other chloroplast precursor proteins. Both pOE33 and pLHCP competed for import into chloroplasts with precursors of the stromally-targeted small subunit of Rubisco (pSSu), ferredoxin NADP(+) reductase (pFNR) and porphobilinogen deaminase; the thylakoid membrane proteins LHCP and the Rieske iron-sulphur protein (pRieske protein); ferrochelatase and the gamma subunit of the ATP synthase (which are both associated with the thylakoid membrane); the thylakoid lumenal protein plastocyanin and the phosphate translocator, an integral membrane protein of the inner envelope. The concentrations of pOE33 or pLHCP required to cause half-maximal inhibition of import ranged between 0.2 and 4.9 microM. These results indicate that all of these proteins are imported into the chloroplast by a common import machinery. Incubation of chloroplasts with pOE33 inhibited the formation of early import intermediates of pSSu, pFNR and pRieske protein.

Choline but not its derivative betaine blocks slow vacuolar channels in the halophyte Chenopodium quinoa: implications for salinity stress responses.

PubMed

Pottosin, Igor; Bonales-Alatorre, Edgar; Shabala, Sergey

2014-11-03

Activity of tonoplast slow vacuolar (SV, or TPC1) channels has to be under a tight control, to avoid undesirable leak of cations stored in the vacuole. This is particularly important for salt-grown plants, to ensure efficient vacuolar Na(+) sequestration. In this study we show that choline, a cationic precursor of glycine betaine, efficiently blocks SV channels in leaf and root vacuoles of the two chenopods, Chenopodium quinoa (halophyte) and Beta vulgaris (glycophyte). At the same time, betaine and proline, two major cytosolic organic osmolytes, have no significant effect on SV channel activity. Physiological implications of these findings are discussed. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Functionally Similar WRKY Proteins Regulate Vacuolar Acidification in Petunia and Hair Development in Arabidopsis

PubMed Central

de Vries, Michel

2016-01-01

The WD40 proteins ANTHOCYANIN11 (AN11) from petunia (Petunia hybrida) and TRANSPARENT TESTA GLABRA1 (TTG1) from Arabidopsis thaliana and associated basic helix-loop-helix (bHLH) and MYB transcription factors activate a variety of differentiation processes. In petunia petals, AN11 and the bHLH protein AN1 activate, together with the MYB protein AN2, anthocyanin biosynthesis and, together with the MYB protein PH4, distinct genes, such as PH1 and PH5, that acidify the vacuole. To understand how AN1 and AN11 activate anthocyanin biosynthetic and PH genes independently, we isolated PH3. We found that PH3 is a target gene of the AN11-AN1-PH4 complex and encodes a WRKY protein that can bind to AN11 and is required, in a feed-forward loop, together with AN11-AN1-PH4 for transcription of PH5. PH3 is highly similar to TTG2, which regulates hair development, tannin accumulation, and mucilage production in Arabidopsis. Like PH3, TTG2 can bind to petunia AN11 and the Arabidopsis homolog TTG1, complement ph3 in petunia, and reactivate the PH3 target gene PH5. Our findings show that the specificity of WD40-bHLH-MYB complexes is in part determined by interacting proteins, such as PH3 and TTG2, and reveal an unanticipated similarity in the regulatory circuitry that controls petunia vacuolar acidification and Arabidopsis hair development. PMID:26977085

Functionally Similar WRKY Proteins Regulate Vacuolar Acidification in Petunia and Hair Development in Arabidopsis.

PubMed

Verweij, Walter; Spelt, Cornelis E; Bliek, Mattijs; de Vries, Michel; Wit, Niek; Faraco, Marianna; Koes, Ronald; Quattrocchio, Francesca M

2016-03-01

The WD40 proteins ANTHOCYANIN11 (AN11) from petunia (Petunia hybrida) and TRANSPARENT TESTA GLABRA1 (TTG1) from Arabidopsis thaliana and associated basic helix-loop-helix (bHLH) and MYB transcription factors activate a variety of differentiation processes. In petunia petals, AN11 and the bHLH protein AN1 activate, together with the MYB protein AN2, anthocyanin biosynthesis and, together with the MYB protein PH4, distinct genes, such as PH1 and PH5, that acidify the vacuole. To understand how AN1 and AN11 activate anthocyanin biosynthetic and PH genes independently, we isolated PH3. We found that PH3 is a target gene of the AN11-AN1-PH4 complex and encodes a WRKY protein that can bind to AN11 and is required, in a feed-forward loop, together with AN11-AN1-PH4 for transcription of PH5. PH3 is highly similar to TTG2, which regulates hair development, tannin accumulation, and mucilage production in Arabidopsis. Like PH3, TTG2 can bind to petunia AN11 and the Arabidopsis homolog TTG1, complement ph3 in petunia, and reactivate the PH3 target gene PH5. Our findings show that the specificity of WD40-bHLH-MYB complexes is in part determined by interacting proteins, such as PH3 and TTG2, and reveal an unanticipated similarity in the regulatory circuitry that controls petunia vacuolar acidification and Arabidopsis hair development. © 2016 American Society of Plant Biologists. All rights reserved.

Plasmodium vivax: a monoclonal antibody recognizes a circumsporozoite protein precursor on the sporozoite surface.

PubMed

Gonzalez-Ceron, L; Rodriguez, M H; Wirtz, R A; Sina, B J; Palomeque, O L; Nettel, J A; Tsutsumi, V

1998-11-01

The major surface circumsporozoite (CS) proteins are known to play a role in malaria sporozoite development and invasion of invertebrate and vertebrate host cells. Plasmodium vivax CS protein processing during mosquito midgut oocyst and salivary gland sporozoite development was studied using monoclonal antibodies which recognize different CS protein epitopes. Monoclonal antibodies which react with the CS amino acid repeat sequences by ELISA recognized a 50-kDa precursor protein in immature oocyst and additional 47- and 42-kDa proteins in older oocysts. A 42-kDa CS protein was detected after initial sporozoite invasion of mosquito salivary glands and an additional 50-kDa precursor CS protein observed later in infected salivary glands. These data confirm previous results with other Plasmodium species, in which more CS protein precursors were detected in oocysts than in salivary gland sporozoites. A monoclonal antibody (PvPCS) was characterized which reacts with an epitope found only in the 50-kDa precursor CS protein. PvPCS reacted with all P. vivax sporozoite strains tested by indirect immunofluorescent assay, homogeneously staining the sporozoite periphery with much lower intensity than that produced by anti-CS repeat antibodies. Immunoelectron microscopy using PvPCS showed that the CS protein precursor was associated with peripheral cytoplasmic vacuoles and membranes of sporoblast and budding sporozoites in development oocysts. In salivary gland sporozoites, the CS protein precursor was primarily associated with micronemes and sporozoite membranes. Our results suggest that the 50-kDa CS protein precursor is synthesized intracellularly and secreted on the membrane surface, where it is proteolytically processed to form the 42-kDa mature CS protein. These data indicate that differences in CS protein processing in oocyst and salivary gland sporozoites development may occur. Copyright 1998 Academic Press.

Atg22p, a vacuolar membrane protein involved in the amino acid compartmentalization of Schizosaccharomyces pombe.

PubMed

Sugimoto, Naoko; Iwaki, Tomoko; Chardwiriyapreecha, Soracom; Shimazu, Masamitsu; Kawano, Miyuki; Sekito, Takayuki; Takegawa, Kaoru; Kakinuma, Yoshimi

2011-01-01

The fission yeast Schizosaccharomyces pombe has a homolog of the budding yeast Atg22p, which is involved in spore formation (Mukaiyama H. et al., Microbiology, 155, 3816-3826 (2009)). GFP-tagged Atg22p in the fission yeast was localized to the vacuolar membrane. Upon disruption of atg22, the amino acid levels of the cellular fraction as well as the vacuolar fraction decreased. The uptake of several amino acids, such as lysine, histidine, and arginine, was impaired in atg22Δ cells. S. pombe Atg22p plays an important role in the compartmentalization of amino acids.

Vba2p, a vacuolar membrane protein involved in basic amino acid transport in Schizosaccharomyces pombe.

PubMed

Sugimoto, Naoko; Iwaki, Tomoko; Chardwiriyapreecha, Soracom; Shimazu, Masamitsu; Sekito, Takayuki; Takegawa, Kaoru; Kakinuma, Yoshimi

2010-01-01

A recent study filling the gap in the genome sequence in the left arm of chromosome 2 of Schizosaccharomyces pombe revealed a homolog of budding yeast Vba2p, a vacuolar transporter of basic amino acids. GFP-tagged Vba2p in fission yeast was localized to the vacuolar membrane. Upon disruption of vba2, the uptake of several amino acids, including lysine, histidine, and arginine, was impaired. A transient increase in lysine uptake under nitrogen starvation was lowered by this mutation. These findings suggest that Vba2p is involved in basic amino acid transport in S. pombe under diverse conditions.

Protein Mobilization in Germinating Mung Bean Seeds Involves Vacuolar Sorting Receptors and Multivesicular Bodies1[W][OA

PubMed Central

Wang, Junqi; Li, Yubing; Lo, Sze Wan; Hillmer, Stefan; Sun, Samuel S.M.; Robinson, David G.; Jiang, Liwen

2007-01-01

Plants accumulate and store proteins in protein storage vacuoles (PSVs) during seed development and maturation. Upon seed germination, these storage proteins are mobilized to provide nutrients for seedling growth. However, little is known about the molecular mechanisms of protein degradation during seed germination. Here we test the hypothesis that vacuolar sorting receptor (VSR) proteins play a role in mediating protein degradation in germinating seeds. We demonstrate that both VSR proteins and hydrolytic enzymes are synthesized de novo during mung bean (Vigna radiata) seed germination. Immunogold electron microscopy with VSR antibodies demonstrate that VSRs mainly locate to the peripheral membrane of multivesicular bodies (MVBs), presumably as recycling receptors in day 1 germinating seeds, but become internalized to the MVB lumen, presumably for degradation at day 3 germination. Chemical cross-linking and immunoprecipitation with VSR antibodies have identified the cysteine protease aleurain as a specific VSR-interacting protein in germinating seeds. Further confocal immunofluorescence and immunogold electron microscopy studies demonstrate that VSR and aleurain colocalize to MVBs as well as PSVs in germinating seeds. Thus, MVBs in germinating seeds exercise dual functions: as a storage compartment for proteases that are physically separated from PSVs in the mature seed and as an intermediate compartment for VSR-mediated delivery of proteases from the Golgi apparatus to the PSV for protein degradation during seed germination. PMID:17322331

A novel chlorophyll a/b binding (Cab) protein gene from petunia which encodes the lower molecular weight Cab precursor protein.

PubMed

Stayton, M M; Black, M; Bedbrook, J; Dunsmuir, P

1986-12-22

The 16 petunia Cab genes which have been characterized are all closely related at the nucleotide sequence level and they encode Cab precursor polypeptides which are similar in sequence and length. Here we describe a novel petunia Cab gene which encodes a unique Cab precursor protein. This protein is a member of the smallest class of Cab precursor proteins for which no gene has previously been assigned in petunia or any other species. The features of this Cab precursor protein are that it is shorter by 2-3 amino acids than the formerly characterized Cab precursors, its transit peptide sequence is unrelated, and the mature polypeptide is significantly diverged at the functionally important N terminus from other petunia Cab proteins. Gene structure also discriminates this gene which is the only intron containing Cab gene in petunia genomic DNA.

Vacuolar Transporters – Companions on a Longtime Journey[OPEN

PubMed Central

2018-01-01

Biochemical and electrophysiological studies on plant vacuolar transporters became feasible in the late 1970s and early 1980s, when methods to isolate large quantities of intact vacuoles and purified vacuolar membrane vesicles were established. However, with the exception of the H+-ATPase and H+-PPase, which could be followed due to their hydrolytic activities, attempts to purify tonoplast transporters were for a long time not successful. Heterologous complementation, T-DNA insertion mutants, and later proteomic studies allowed the next steps, starting from the 1990s. Nowadays, our knowledge about vacuolar transporters has increased greatly. Nevertheless, there are several transporters of central importance that have still to be identified at the molecular level or have even not been characterized biochemically. Furthermore, our knowledge about regulation of the vacuolar transporters is very limited, and much work is needed to get a holistic view about the interplay of the vacuolar transportome. The huge amount of information generated during the last 35 years cannot be summarized in such a review. Therefore, I decided to concentrate on some aspects where we were involved during my research on vacuolar transporters, for some our laboratories contributed more, while others contributed less. PMID:29295940

The plant homolog to the human sodium/dicarboxylic cotransporter is the vacuolar malate carrier

PubMed Central

Emmerlich, Vera; Linka, Nicole; Reinhold, Thomas; Hurth, Marco A.; Traub, Michaela; Martinoia, Enrico; Neuhaus, H. Ekkehard

2003-01-01

Malate plays a central role in plant metabolism. It is an intermediate in the Krebs and glyoxylate cycles, it is the store for CO2 in C4 and crassulacean acid metabolism plants, it protects plants from aluminum toxicity, it is essential for maintaining the osmotic pressure and charge balance, and it is therefore involved in regulation of stomatal aperture. To fulfil many of these roles, malate has to be accumulated within the large central vacuole. Many unsuccessful efforts have been made in the past to identify the vacuolar malate transporter; here, we describe the identification of the vacuolar malate transporter [A. thaliana tonoplast dicarboxylate transporter (AttDT)]. This transporter exhibits highest sequence similarity to the human sodium/dicarboxylate cotransporter. Independent T-DNA [portion of the Ti (tumor-inducing) plasmid that is transferred to plant cells] Arabidopsis mutants exhibit substantially reduced levels of leaf malate, but respire exogenously applied [14C]malate faster than the WT. An AttDT-GFP fusion protein was localized to vacuole. Vacuoles isolated from Arabidopsis WT leaves exhibited carbonylcyanide m-chlorophenylhydrazone and citrate inhibitable malate transport, which was not stimulated by sodium. Vacuoles isolated from mutant plants import [14C]-malate at strongly reduced rates, confirming that this protein is the vacuolar malate transporter. PMID:12947042

The plant homolog to the human sodium/dicarboxylic cotransporter is the vacuolar malate carrier.

PubMed

Emmerlich, Vera; Linka, Nicole; Reinhold, Thomas; Hurth, Marco A; Traub, Michaela; Martinoia, Enrico; Neuhaus, H Ekkehard

2003-09-16

Malate plays a central role in plant metabolism. It is an intermediate in the Krebs and glyoxylate cycles, it is the store for CO2 in C4 and crassulacean acid metabolism plants, it protects plants from aluminum toxicity, it is essential for maintaining the osmotic pressure and charge balance, and it is therefore involved in regulation of stomatal aperture. To fulfil many of these roles, malate has to be accumulated within the large central vacuole. Many unsuccessful efforts have been made in the past to identify the vacuolar malate transporter; here, we describe the identification of the vacuolar malate transporter [A. thaliana tonoplast dicarboxylate transporter (AttDT)]. This transporter exhibits highest sequence similarity to the human sodium/dicarboxylate cotransporter. Independent T-DNA [portion of the Ti (tumor-inducing) plasmid that is transferred to plant cells] Arabidopsis mutants exhibit substantially reduced levels of leaf malate, but respire exogenously applied [14C]malate faster than the WT. An AttDT-GFP fusion protein was localized to vacuole. Vacuoles isolated from Arabidopsis WT leaves exhibited carbonylcyanide m-chlorophenylhydrazone and citrate inhibitable malate transport, which was not stimulated by sodium. Vacuoles isolated from mutant plants import [14C]-malate at strongly reduced rates, confirming that this protein is the vacuolar malate transporter.

The R2R3-MYB transcription factor MdMYB73 is involved in malate accumulation and vacuolar acidification in apple.

PubMed

Hu, Da-Gang; Li, Yuan-Yuan; Zhang, Quan-Yan; Li, Ming; Sun, Cui-Hui; Yu, Jian-Qiang; Hao, Yu-Jin

2017-08-01

Malate, the predominant organic acid in many fruits, is a crucial component of the organoleptic quality of fruit, including taste and flavor. The genetic and environmental mechanisms affecting malate metabolism in fruit cells have been studied extensively. However, the transcriptional regulation of malate-metabolizing enzymes and vacuolar transporters remains poorly understood. Our previous studies demonstrated that MdMYB1 modulates anthocyanin accumulation and vacuolar acidification by directly activating vacuolar transporters, including MdVHA-B1, MdVHA-E, MdVHP1 and MdtDT. Interestingly, we isolated and identified a MYB transcription factor, MdMYB73, a distant relative of MdMYB1 in this study. It was subsequently found that MdMYB73 protein bound directly to the promoters of MdALMT9 (aluminum-activated malate transporter 9), MdVHA-A (vacuolar ATPase subunit A) and MdVHP1 (vacuolar pyrophosphatase 1), transcriptionally activating their expression and thereby enhancing their activities. Analyses of transgenic apple calli demonstrated that MdMYB73 influenced malate accumulation and vacuolar pH. Furthermore, MdCIbHLH1 interacted with MdMYB73 and enhanced its activity upon downstream target genes. These findings help to elucidate how MdMYB73 directly modulates the vacuolar transport system to affect malate accumulation and vacuolar pH in apple. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Genes encoding the vacuolar Na+/H+ exchanger and flower coloration.

PubMed

Yamaguchi, T; Fukada-Tanaka, S; Inagaki, Y; Saito, N; Yonekura-Sakakibara, K; Tanaka, Y; Kusumi, T; Iida, S

2001-05-01

Vacuolar pH plays an important role in flower coloration: an increase in the vacuolar pH causes blueing of flower color. In the Japanese morning glory (Ipomoea nil or Pharbitis nil), a shift from reddish-purple buds to blue open flowers correlates with an increase in the vacuolar pH. We describe details of the characterization of a mutant that carries a recessive mutation in the Purple (Pr) gene encoding a vacuolar Na+/H+ exchanger termed InNHX1. The genome of I. nil carries one copy of the Pr (or InNHX1) gene and its pseudogene, and it showed functional complementation to the yeast nhx1 mutation. The mutant of I. nil, called purple (pr), showed a partial increase in the vacuolar pH during flower-opening and its reddish-purple buds change into purple open flowers. The vacuolar pH in the purple open flowers of the mutant was significantly lower than that in the blue open flowers. The InNHX1 gene is most abundantly expressed in the petals at around 12 h before flower-opening, accompanying the increase in the vacuolar pH for the blue flower coloration. No such massive expression was observed in the petunia flowers. Since the NHX1 genes that promote the transport of Na+ into the vacuoles have been regarded to be involved in salt tolerance by accumulating Na+ in the vacuoles, we can add a new biological role for blue flower coloration in the Japanese morning glory by the vacuolar alkalization.

The vacuolar ATPase from Entamoeba histolytica: molecular cloning of the gene encoding for the B subunit and subcellular localization of the protein.

PubMed

Meléndez-Hernández, Mayra Gisela; Barrios, María Luisa Labra; Orozco, Esther; Luna-Arias, Juan Pedro

2008-12-23

Entamoeba histolytica is a professional phagocytic cell where the vacuolar ATPase plays a key role. This enzyme is a multisubunit complex that regulates pH in many subcellular compartments, even in those that are not measurably acidic. It participates in a wide variety of cellular processes such as endocytosis, intracellular transport and membrane fusion. The presence of a vacuolar type H+-ATPase in E. histolytica trophozoites has been inferred previously from inhibition assays of its activity, the isolation of the Ehvma1 and Ehvma3 genes, and by proteomic analysis of purified phagosomes. We report the isolation and characterization of the Ehvma2 gene, which encodes for the subunit B of the vacuolar ATPase. This polypeptide is a 55.3 kDa highly conserved protein with 34 to 80% identity to orthologous proteins from other species. Particularly, in silico studies showed that EhV-ATPase subunit B displays 78% identity and 90% similarity to its Dictyostelium ortholog. A 462 bp DNA fragment of the Ehvma2 gene was expressed in bacteria and recombinant polypeptide was used to raise mouse polyclonal antibodies. EhV-ATPase subunit B antibodies detected a 55 kDa band in whole cell extracts and in an enriched fraction of DNA-containing organelles named EhkOs. The V-ATPase subunit B was located by immunofluorescence and confocal microscopy in many vesicles, in phagosomes, plasma membrane and in EhkOs. We also identified the genes encoding for the majority of the V-ATPase subunits in the E. histolytica genome, and proposed a putative model for this proton pump. We have isolated the Ehvma2 gene which encodes for the V-ATPase subunit B from the E. histolytica clone A. This gene has a 154 bp intron and encodes for a highly conserved polypeptide. Specific antibodies localized EhV-ATPase subunit B in many vesicles, phagosomes, plasma membrane and in EhkOs. Most of the orthologous genes encoding for the EhV-ATPase subunits were found in the E. histolytica genome, indicating the

The VPH1 gene encodes a 95-kDa integral membrane polypeptide required for in vivo assembly and activity of the yeast vacuolar H(+)-ATPase.

PubMed

Manolson, M F; Proteau, D; Preston, R A; Stenbit, A; Roberts, B T; Hoyt, M A; Preuss, D; Mulholland, J; Botstein, D; Jones, E W

1992-07-15

Yeast vacuolar acidification-defective (vph) mutants were identified using the pH-sensitive fluorescence of 6-carboxyfluorescein diacetate (Preston, R. A., Murphy, R. F., and Jones, E. W. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7027-7031). Vacuoles purified from yeast bearing the vph1-1 mutation had no detectable bafilomycin-sensitive ATPase activity or ATP-dependent proton pumping. The peripherally bound nucleotide-binding subunits of the vacuolar H(+)-ATPase (60 and 69 kDa) were no longer associated with vacuolar membranes yet were present in wild type levels in yeast whole cell extracts. The VPH1 gene was cloned by complementation of the vph1-1 mutation and independently cloned by screening a lambda gt11 expression library with antibodies directed against a 95-kDa vacuolar integral membrane protein. Deletion disruption of the VPH1 gene revealed that the VPH1 gene is not essential for viability but is required for vacuolar H(+)-ATPase assembly and vacuolar acidification. VPH1 encodes a predicted polypeptide of 840 amino acid residues (molecular mass 95.6 kDa) and contains six putative membrane-spanning regions. Cell fractionation and immunodetection demonstrate that Vph1p is a vacuolar integral membrane protein that co-purifies with vacuolar H(+)-ATPase activity. Multiple sequence alignments show extensive homology over the entire lengths of the following four polypeptides: Vph1p, the 116-kDa polypeptide of the rat clathrin-coated vesicles/synaptic vesicle proton pump, the predicted polypeptide encoded by the yeast gene STV1 (Similar To VPH1, identified as an open reading frame next to the BUB2 gene), and the TJ6 mouse immune suppressor factor.

Cloning, 3D modeling and expression analysis of three vacuolar invertase genes from cassava (Manihot Esculenta Crantz).

PubMed

Yao, Yuan; Wu, Xiao-Hui; Geng, Meng-Ting; Li, Rui-Mei; Liu, Jiao; Hu, Xin-Wen; Guo, Jian-Chun

2014-05-15

Vacuolar invertase is one of the key enzymes in sucrose metabolism that irreversibly catalyzes the hydrolysis of sucrose to glucose and fructose in plants. In this research, three vacuolar invertase genes, named MeVINV1-3, and with 653, 660 and 639 amino acids, respectively, were cloned from cassava. The motifs of NDPNG (β-fructosidase motif), RDP and WECVD, which are conserved and essential for catalytic activity in the vacuolar invertase family, were found in MeVINV1 and MeVINV2. Meanwhile, in MeVINV3, instead of NDPNG we found the motif NGPDG, in which the three amino acids GPD are different from those in other vacuolar invertases (DPN) that might result in MeVINV3 being an inactivated protein. The N-terminal leader sequence of MeVINVs contains a signal anchor, which is associated with the sorting of vacuolar invertase to vacuole. The overall predicted 3D structure of the MeVINVs consists of a five bladed β-propeller module at N-terminus domain, and forms a β-sandwich module at the C-terminus domain. The active site of the protein is situated in the β-propeller module. MeVINVs are classified in two subfamilies, α and β groups, in which α group members of MeVINV1 and 2 are highly expressed in reproductive organs and tuber roots (considered as sink organs), while β group members of MeVINV3 are highly expressed in leaves (source organs). All MeVINVs are highly expressed in leaves, while only MeVINV1 and 2 are highly expressed in tubers at cassava tuber maturity stage. Thus, MeVINV1 and 2 play an important role in sucrose unloading and starch accumulation, as well in buffering the pools of sucrose, hexoses and sugar phosphates in leaves, specifically at later stages of plant development.

Diethylpyrocarbonate inhibition of vacuolar H+-pyrophosphatase possibly involves a histidine residue.

PubMed

Hsiao, Yi Yuong; Van, Ru Chuan; Hung, Hsiao Hui; Pan, Rong Long

2002-01-01

Vacuolar proton pumping pyrophosphatase (H+-PPase; EC 3.6.1.1) plays a pivotal role in electrogenic translocation of protons from cytosol to the vacuolar lumen at the expense of PPi hydrolysis. A histidine-specific modifier, diethylpyrocarbonate (DEPC), could substantially inhibit enzymic activity and H+-translocation of vacuolar H+-PPase in a concentration-dependent manner. Absorbance of vacuolar H+-PPase at 240 nm was increased upon incubation with DEPC, demonstrating that an N-carbethoxyhistidine moiety was probably formed. On the other hand, hydroxylamine, a reagent that can deacylate N-carbethoxyhistidine, could reverse the absorption change at 240 nm and partially restore PPi hydrolysis activity as well. The pKa of modified residues of the enzyme was determined to be 6.4, a value close to that of histidine. Thus, we speculate that inhibition of vacuolar H+-PPase by DEPC possibly could be attributed to the modification of histidyl residues on the enzyme. Furthermore, inhibition of vacuolar H+-PPase by DEPC follows pseudo-first-order rate kinetics. A reaction order of 0.85 was calculated from a double logarithmic plot of the apparent reaction constant against DEPC concentration, suggesting that the modification of one single histidine residue on the enzyme suffices to inhibit vacuolar H+-PPase. Inhibition of vacuolar H+-PPase by DEPC changes Vmax but not Km values. Moreover, DEPC inhibition of vacuolar H+-PPase could be substantially protected against by its physiological substrate, Mg2+-PPi. These results indicated that DEPC specifically competes with the substrate at the active site and the DEPC-labeled histidine residue might locate in or near the catalytic domain of the enzyme. Besides, pretreatment of the enzyme with N-ethylmaleimide decreased the degree of subsequent labeling of H+-PPase by DEPC. Taken together, we suggest that vacuolar H+-PPase likely contains a substrate-protectable histidine residue contributing to the inhibition of its activity by

Generation of amyloid-β is reduced by the interaction of calreticulin with amyloid precursor protein, presenilin and nicastrin.

PubMed

Stemmer, Nina; Strekalova, Elena; Djogo, Nevena; Plöger, Frank; Loers, Gabriele; Lutz, David; Buck, Friedrich; Michalak, Marek; Schachner, Melitta; Kleene, Ralf

2013-01-01

Dysregulation of the proteolytic processing of amyloid precursor protein by γ-secretase and the ensuing generation of amyloid-β is associated with the pathogenesis of Alzheimer's disease. Thus, the identification of amyloid precursor protein binding proteins involved in regulating processing of amyloid precursor protein by the γ-secretase complex is essential for understanding the mechanisms underlying the molecular pathology of the disease. We identified calreticulin as novel amyloid precursor protein interaction partner that binds to the γ-secretase cleavage site within amyloid precursor protein and showed that this Ca(2+)- and N-glycan-independent interaction is mediated by amino acids 330-344 in the C-terminal C-domain of calreticulin. Co-immunoprecipitation confirmed that calreticulin is not only associated with amyloid precursor protein but also with the γ-secretase complex members presenilin and nicastrin. Calreticulin was detected at the cell surface by surface biotinylation of cells overexpressing amyloid precursor protein and was co-localized by immunostaining with amyloid precursor protein and presenilin at the cell surface of hippocampal neurons. The P-domain of calreticulin located between the N-terminal N-domain and the C-domain interacts with presenilin, the catalytic subunit of the γ-secretase complex. The P- and C-domains also interact with nicastrin, another functionally important subunit of this complex. Transfection of amyloid precursor protein overexpressing cells with full-length calreticulin leads to a decrease in amyloid-β42 levels in culture supernatants, while transfection with the P-domain increases amyloid-β40 levels. Similarly, application of the recombinant P- or C-domains and of a synthetic calreticulin peptide comprising amino acid 330-344 to amyloid precursor protein overexpressing cells result in elevated amyloid-β40 and amyloid-β42 levels, respectively. These findings indicate that the interaction of calreticulin with

A Novel Arabidopsis Vacuolar Glucose Exporter Is Involved in Cellular Sugar Homeostasis and Affects the Composition of Seed Storage Compounds1[W][OA

PubMed Central

Poschet, Gernot; Hannich, Barbara; Raab, Sabine; Jungkunz, Isabel; Klemens, Patrick A.W.; Krueger, Stephan; Wic, Stefan; Neuhaus, H. Ekkehard; Büttner, Michael

2011-01-01

Subcellular sugar partitioning in plants is strongly regulated in response to developmental cues and changes in external conditions. Besides transitory starch, the vacuolar sugars represent a highly dynamic pool of instantly accessible metabolites that serve as energy source and osmoprotectant. Here, we present the molecular identification and functional characterization of the vacuolar glucose (Glc) exporter Arabidopsis (Arabidopsis thaliana) Early Responsive to Dehydration-Like6 (AtERDL6). We demonstrate tonoplast localization of AtERDL6 in plants. In Arabidopsis, AtERDL6 expression is induced in response to factors that activate vacuolar Glc pools, like darkness, heat stress, and wounding. On the other hand, AtERDL6 transcript levels drop during conditions that trigger Glc accumulation in the vacuole, like cold stress and external sugar supply. Accordingly, sugar analyses revealed that Aterdl6 mutants have elevated vacuolar Glc levels and that Glc flux across the tonoplast is impaired under stress conditions. Interestingly, overexpressor lines indicated a very similar function for the ERDL6 ortholog Integral Membrane Protein from sugar beet (Beta vulgaris). Aterdl6 mutant plants display increased sensitivity against external Glc, and mutant seeds exhibit a 10% increase in seed weight due to enhanced levels of seed sugars, proteins, and lipids. Our findings underline the importance of vacuolar Glc export during the regulation of cellular Glc homeostasis and the composition of seed reserves. PMID:21984725

Appropriate vacuolar acidification in Saccharomyces cerevisiae is associated with efficient high sugar fermentation.

PubMed

Nguyen, Trung D; Walker, Michelle E; Gardner, Jennifer M; Jiranek, Vladimir

2018-04-01

Vacuolar acidification serves as a homeostatic mechanism to regulate intracellular pH, ion and chemical balance, as well as trafficking and recycling of proteins and nutrients, critical for normal cellular function. This study reports on the importance of vacuole acidification during wine-like fermentation. Ninety-three mutants (homozygous deletions in lab yeast strain, BY4743), which result in protracted fermentation when grown in a chemically defined grape juice with 200 g L -1 sugar (pH 3.5), were examined to determine whether fermentation protraction was in part due to a dysfunction in vacuolar acidification (VA) during the early stages of fermentation, and whether VA was responsive to the initial sugar concentration in the medium. Cells after 24 h growth were dual-labelled with propidium iodide and vacuolar specific probe 6-carboxyfluorescein diacetate (6-CFDA) and examined with a FACS analyser for viability and impaired VA, respectively. Twenty mutants showed a greater than two-fold increase in fluorescence intensity; the experimental indicator for vacuolar dysfunction; 10 of which have not been previously annotated to this process. With the exception of Δhog1, Δpbs2 and Δvph1 mutants, where dysfunction was directly related to osmolality; the remainder exhibited increased CF-fluorescence, independent of sugar concentration at 20 g L -1 or 200 g L -1 . These findings offer insight to the importance of VA to cell growth in high sugar media. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cytosolic Nucleotides Block and Regulate the Arabidopsis Vacuolar Anion Channel AtALMT9*

PubMed Central

Zhang, Jingbo; Martinoia, Enrico; De Angeli, Alexis

2014-01-01

The aluminum-activated malate transporters (ALMTs) form a membrane protein family exhibiting different physiological roles in plants, varying from conferring tolerance to environmental Al3+ to the regulation of stomatal movement. The regulation of the anion channels of the ALMT family is largely unknown. Identifying intracellular modulators of the activity of anion channels is fundamental to understanding their physiological functions. In this study we investigated the role of cytosolic nucleotides in regulating the activity of the vacuolar anion channel AtALMT9. We found that cytosolic nucleotides modulate the transport activity of AtALMT9. This modulation was based on a direct block of the pore of the channel at negative membrane potentials (open channel block) by the nucleotide and not by a phosphorylation mechanism. The block by nucleotides of AtALMT9-mediated currents was voltage dependent. The blocking efficiency of intracellular nucleotides increased with the number of phosphate groups and ATP was the most effective cellular blocker. Interestingly, the ATP block induced a marked modification of the current-voltage characteristic of AtALMT9. In addition, increased concentrations of vacuolar anions were able to shift the ATP block threshold to a more negative membrane potential. The block of AtALMT9-mediated anion currents by ATP at negative membrane potentials acts as a gate of the channel and vacuolar anion tune this gating mechanism. Our results suggest that anion transport across the vacuolar membrane in plant cells is controlled by cytosolic nucleotides and the energetic status of the cell. PMID:25028514

Cytosolic nucleotides block and regulate the Arabidopsis vacuolar anion channel AtALMT9.

PubMed

Zhang, Jingbo; Martinoia, Enrico; De Angeli, Alexis

2014-09-12

The aluminum-activated malate transporters (ALMTs) form a membrane protein family exhibiting different physiological roles in plants, varying from conferring tolerance to environmental Al(3+) to the regulation of stomatal movement. The regulation of the anion channels of the ALMT family is largely unknown. Identifying intracellular modulators of the activity of anion channels is fundamental to understanding their physiological functions. In this study we investigated the role of cytosolic nucleotides in regulating the activity of the vacuolar anion channel AtALMT9. We found that cytosolic nucleotides modulate the transport activity of AtALMT9. This modulation was based on a direct block of the pore of the channel at negative membrane potentials (open channel block) by the nucleotide and not by a phosphorylation mechanism. The block by nucleotides of AtALMT9-mediated currents was voltage dependent. The blocking efficiency of intracellular nucleotides increased with the number of phosphate groups and ATP was the most effective cellular blocker. Interestingly, the ATP block induced a marked modification of the current-voltage characteristic of AtALMT9. In addition, increased concentrations of vacuolar anions were able to shift the ATP block threshold to a more negative membrane potential. The block of AtALMT9-mediated anion currents by ATP at negative membrane potentials acts as a gate of the channel and vacuolar anion tune this gating mechanism. Our results suggest that anion transport across the vacuolar membrane in plant cells is controlled by cytosolic nucleotides and the energetic status of the cell. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

A vacuolar iron transporter in tulip, TgVit1, is responsible for blue coloration in petal cells through iron accumulation.

PubMed

Momonoi, Kazumi; Yoshida, Kumi; Mano, Shoji; Takahashi, Hideyuki; Nakamori, Chihiro; Shoji, Kazuaki; Nitta, Akira; Nishimura, Mikio

2009-08-01

Blue color in flowers is due mainly to anthocyanins, and a considerable part of blue coloration can be attributed to metal-complexed anthocyanins. However, the mechanism of metal ion transport into vacuoles and subsequent flower color development has yet to be fully explored. Previously, we studied the mechanism of blue color development specifically at the bottom of the inner perianth in purple tulip petals of Tulipa gesneriana cv. Murasakizuisho. We found that differences in iron content were associated with the development of blue- and purple-colored cells. Here, we identify a vacuolar iron transporter in T. gesneriana (TgVit1), and characterize the localization and function of this transporter protein in tulip petals. The amino acid sequence of TgVit1 is 85% similar that of the Arabidopsis thaliana vacuolar iron transporter AtVIT1, and also showed similarity to the AtVIT1 homolog in yeast, Ca(2+)-sensitive cross-complementer 1 (CCC1). The gene TgVit1 was expressed exclusively in blue-colored epidermal cells, and protein levels increased with increasing mRNA expression and blue coloration. Transient expression experiments revealed that TgVit1 localizes to the vacuolar membrane, and is responsible for the development of the blue color in purple cells. Expression of TgVit1 in yeast rescued the growth defect of ccc1 mutant cells in the presence of high concentrations of FeSO(4). Our results indicate that TgVit1 plays an essential role in blue coloration as a vacuolar iron transporter in tulip petals. These results suggest a new role for involvement of a vacuolar iron transporter in blue flower color development.

Supplementation with tocotrienol-rich fraction alters the plasma levels of Apolipoprotein A-I precursor, Apolipoprotein E precursor, and C-reactive protein precursor from young and old individuals.

PubMed

Heng, Eng Chee; Karsani, Saiful Anuar; Abdul Rahman, Mariati; Abdul Hamid, Noor Aini; Hamid, Zalina; Wan Ngah, Wan Zurinah

2013-10-01

Tocotrienol possess beneficial effects not exhibited by tocopherol. In vitro studies using animal models have suggested that these effects are caused via modulation of gene and protein expression. However, human supplementation studies using tocotrienol-rich isomers are limited. This study aims to identify plasma proteins that changed in expression following tocotrienol-rich fraction (TRF) supplementation within two different age groups. Subjects were divided into two age groups-32 ± 2 (young) and 52 ± 2 (old) years old. Four subjects from each group were assigned with TRF (78% tocotrienol and 22% tocopherol, 150 mg/day) or placebo capsules for 6 months. Fasting plasma were obtained at 0, 3, and 6 months. Plasma tocopherol and tocotrienol levels were determined. Plasma proteome was resolved by 2DE, and differentially expressed proteins identified by MS. The expressions of three proteins were validated by Western blotting. Six months of TRF supplementation significantly increased plasma levels of tocopherols and tocotrienols. Proteins identified as being differentially expressed were related to cholesterol homeostasis, acute-phase response, protease inhibitor, and immune response. The expressions of Apolipoprotein A-I precursor, Apolipoprotein E precursor, and C-reactive protein precursor were validated. The old groups showed more proteins changing in expression. TRF appears to not only affect plasma levels of tocopherols and tocotrienols, but also the levels of plasma proteins. The identity of these proteins may provide insights into how TRF exerts its beneficial effects. They may also be potentially developed into biomarkers for the study of the effects and effectiveness of TRF supplementation.

Fe deficiency differentially affects the vacuolar proton pumps in cucumber and soybean roots

PubMed Central

Dell’Orto, Marta; Nisi, Patrizia De; Vigani, Gianpiero; Zocchi, Graziano

2013-01-01

Iron uptake in dicots depends on their ability to induce a set of responses in root cells including rhizosphere acidification through H+ extrusion and apoplastic Fe(III) reduction by Fe(III)-chelate reductase. These responses must be sustained by metabolic rearrangements aimed at providing the required NAD(P)H, ATP and H+. Previous results in Fe-deficient cucumber roots showed that high H+ extrusion is accompanied by increased phosphoenolpyruvate carboxylase (PEPC) activity, involved in the cytosol pH-stat; moreover 31P-NMR analysis revealed increased vacuolar pH and decreased vacuolar [inorganic phosphate (Pi)]. The opposite was found in soybean: low rhizosphere acidification, decreased PEPC activity, vacuole acidification, and increased vacuolar [Pi]. These findings, highlighting a different impact of the Fe deficiency responses on cytosolic pH in the two species, lead to hypothesize different roles for H+ and Pi movements across the tonoplast in pH homeostasis. The role of vacuole in cytosolic pH-stat involves the vacuolar H+-ATPase (V-ATPase) and vacuolar H+-pyrophosphatase (V-PPase) activities, which generating the ΔpH and ΔΨ, mediate the transport of solutes, among which Pi, across the tonoplast. Fluxes of Pi itself in its two ionic forms, H2PO4- predominating in the vacuole and HPO42- in the cytosol, may be involved in pH homeostasis owing to its pH-dependent protonation/deprotonation reactions. Tonoplast enriched fractions were obtained from cucumber and soybean roots grown with or without Fe. Both V-ATPase and V-PPase activities were analyzed and the enrichment and localization of the corresponding proteins in root tissues were determined by Western blot and immunolocalization. V-ATPase did not change its activity and expression level in response to Fe starvation in both species. V-PPase showed a different behavior: in cucumber roots its activity and abundance were decreased, while in Fe-deficient soybean roots they were increased. The distinct role of

ATP-dependent export of neutral amino acids by vacuolar membrane vesicles of Saccharomyces cerevisiae.

PubMed

Ishimoto, Masaya; Sugimoto, Naoko; Sekito, Takayuki; Kawano-Kawada, Miyuki; Kakinuma, Yoshimi

2012-01-01

Amino acid analysis of Saccharomyces cerevisiae cells indicated that neutral amino acids such as glycine and alanine were probably excluded from the vacuoles, and that vacuolar H(+)-ATPase (V-ATPase) was involved in the vacuolar compartmentalization of these amino acids. We found that vacuolar membrane vesicles export neutral amino acids in an ATP-dependent manner. This is important in identifying vacuolar transporters for neutral amino acids.

Redundancy and divergence in the amyloid precursor protein family.

PubMed

Shariati, S Ali M; De Strooper, Bart

2013-06-27

Gene duplication provides genetic material required for functional diversification. An interesting example is the amyloid precursor protein (APP) protein family. The APP gene family has experienced both expansion and contraction during evolution. The three mammalian members have been studied quite extensively in combined knock out models. The underlying assumption is that APP, amyloid precursor like protein 1 and 2 (APLP1, APLP2) are functionally redundant. This assumption is primarily supported by the similarities in biochemical processing of APP and APLPs and on the fact that the different APP genes appear to genetically interact at the level of the phenotype in combined knockout mice. However, unique features in each member of the APP family possibly contribute to specification of their function. In the current review, we discuss the evolution and the biology of the APP protein family with special attention to the distinct properties of each homologue. We propose that the functions of APP, APLP1 and APLP2 have diverged after duplication to contribute distinctly to different neuronal events. Our analysis reveals that APLP2 is significantly diverged from APP and APLP1. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Identification and functional expression of the Arabidopsis thaliana vacuolar glucose transporter 1 and its role in seed germination and flowering.

PubMed

Aluri, Sirisha; Büttner, Michael

2007-02-13

Sugar compartmentation into vacuoles of higher plants is a very important physiological process, providing extra space for transient and long-term sugar storage and contributing to the osmoregulation of cell turgor and shape. Despite the long-standing knowledge of this subcellular sugar partitioning, the proteins responsible for these transport steps have remained unknown. We have identified a gene family in Arabidopsis consisting of three members homologous to known sugar transporters. One member of this family, Arabidopsis thaliana vacuolar glucose transporter 1 (AtVGT1), was localized to the vacuolar membrane. Moreover, we provide evidence for transport activity of a tonoplast sugar transporter based on its functional expression in bakers' yeast and uptake studies in isolated yeast vacuoles. Analyses of Atvgt1 mutant lines indicate an important function of this vacuolar glucose transporter during developmental processes like seed germination and flowering.

Gc protein (vitamin D-binding protein): Gc genotyping and GcMAF precursor activity.

PubMed

Nagasawa, Hideko; Uto, Yoshihiro; Sasaki, Hideyuki; Okamura, Natsuko; Murakami, Aya; Kubo, Shinichi; Kirk, Kenneth L; Hori, Hitoshi

2005-01-01

The Gc protein (human group-specific component (Gc), a vitamin D-binding protein or Gc globulin), has important physiological functions that include involvement in vitamin D transport and storage, scavenging of extracellular G-actin, enhancement of the chemotactic activity of C5a for neutrophils in inflammation and macrophage activation (mediated by a GalNAc-modified Gc protein (GcMAF)). In this review, the structure and function of the Gc protein is focused on especially with regard to Gc genotyping and GcMAF precursor activity. A discussion of the research strategy "GcMAF as a target for drug discovery" is included, based on our own research.

Novel families of vacuolar amino acid transporters.

PubMed

Sekito, Takayuki; Fujiki, Yuki; Ohsumi, Yoshinori; Kakinuma, Yoshimi

2008-08-01

Amino acids are compartmentalized in the vacuoles of microorganisms and plants. In Saccharomyces cerevisiae, basic amino acids accumulate preferentially into vacuoles but acidic amino acids are almost excluded from them. This indicates that selective machineries operate at the vacuolar membrane. The members of the amino acid/auxin permease family and the major facilitator superfamily involved in the vacuolar compartmentalization of amino acids have been recently identified in studies using S. cerevisiae. Homologous genes for these transporters are also found in plant and mammalian genomes. The physiological significance in response to nitrogen starvation can now be discussed. (c) 2008 IUBMB

Association of the macrophage activating factor (MAF) precursor activity with polymorphism in vitamin D-binding protein.

PubMed

Nagasawa, Hideko; Sasaki, Hideyuki; Uto, Yoshihiro; Kubo, Shinichi; Hori, Hitoshi

2004-01-01

Serum vitamin D-binding protein (Gc protein or DBP) is a highly expressed polymorphic protein, which is a precursor of the inflammation-primed macrophage activating factor, GcMAF, by a cascade of carbohydrate processing reactions. In order to elucidate the relationship between Gc polymorphism and GcMAF precursor activity, we estimated the phagocytic ability of three homotypes of Gc protein, Gc1F-1F, Gc1S-1S and Gc2-2, through processing of their carbohydrate moiety. We performed Gc typing of human serum samples by isoelectric focusing (IEF). Gc protein from human serum was purified by affinity chromatography with 25-hydroxyvitamin D3-sepharose. A phagocytosis assay of Gc proteins, modified using beta-glycosidase and sialidase, was carried out. The Gc1F-1F phenotype was revealed to possess Galbeta1-4GalNAc linkage by the analysis of GcMAF precursor activity using beta1-4 linkage-specific galactosidase from jack bean. The GcMAF precursor activity of the Gc1F-1F phenotype was highest among three Gc homotypes. The Gc polymorphism and carbohydrate diversity of Gc protein are significant for its pleiotropic effects.

RhVI1 is a membrane-anchored vacuolar invertase highly expressed in Rosa hybrida L. petals

PubMed Central

Farci, Domenica; Collu, Gabriella; Kirkpatrick, Joanna; Esposito, Francesca; Piano, Dario

2016-01-01

Invertases are a widespread group of enzymes that catalyse the conversion of sucrose into fructose and glucose. Plants invertases and their substrates are essential factors that play an active role in primary metabolism and in cellular differentiation and by these activities they sustain development and growth. Being naturally present in multiple isoforms, invertases are known to be highly differentiated and tissue specific in such a way that every isoform is characteristic of a specific part of the plant. In this work, we report the identification of the invertase RhVI1 that was found to be highly expressed in rose petals. A characterization of this protein revealed that RhVI1 is a glycosylated membrane-anchored protein associated with the cytosolic side of the vacuolar membrane which occurs in vivo in a monomeric form. Purification yields have shown that the levels of expression decreased during the passage of petals from buds to mature and pre-senescent flowers. Moreover, the activity assay indicates RhVI1 to be an acidic vacuolar invertase. The physiological implications of these findings are discussed, suggesting a possible role of this protein during anthesis. PMID:27083698

Salinity Tolerance of Two Potato Cultivars (Solanum tuberosum) Correlates With Differences in Vacuolar Transport Activity

PubMed Central

Jaarsma, Rinse; de Boer, Albertus H.

2018-01-01

Potato is an important cultivated crop species and since it is moderately salt sensitive there is a need to develop more salt tolerant cultivars. A high activity of Na+ transport across the tonoplast in exchange for H+ is essential to reduce Na+ toxicity. The proton motive force (PMF) generated by the V-H+-ATPase and the V-H+-PPase energizes the Na+(K+)/H+ antiport. We compared the activity, gene expression, and protein levels of the vacuolar proton pumps and the Na+/H+ antiporters in two potato cultivars (Solanum tuberosum) contrasting in their salt tolerance (cv. Desiree; tolerant and Mozart; sensitive) grown at 0 and 60 mM NaCl. Tonoplast-enriched vesicles were used to study the pump activity and protein levels of the V-H+-ATPase and the V-H+-PPase and the activity of the Na+/H+ antiporter. Although salt stress reduced the V-H+-ATPase and the V-H+-PPase activity in both cultivars, the decline in H+ pump activity was more severe in the salt-sensitive cultivar Mozart. After salt treatment, protein amounts of the vacuolar H+ pumps decreased in Mozart but remained unchanged in the cultivar Desiree. Decreased protein amounts of the V-H+-PPase found in Mozart may explain the reduced V-H+-PPase activity found for Mozart after salt stress. Under non-stress conditions, protein amounts of V-H+-PPase were equal in both cultivars while the V-H+-PPase activity was already twice as high and remained higher after salt treatment in the cultivar Desiree as compared to Mozart. This cultivar-dependent V-H+-PPase activity may explain the higher salt tolerance of Desiree. Moreover, combined with reduced vacuolar H+ pump activity, Mozart showed a lower Na+/H+ exchange activity and the Km for Na+ is at least twofold lower in tonoplast vesicles from Desiree, what suggests that NHXs from Desiree have a higher affinity for Na+ as compared to Mozart. From these results, we conclude that the higher capacity in combination with the higher affinity for Na+ uptake can be an important factor

Salinity Tolerance of Two Potato Cultivars (Solanum tuberosum) Correlates With Differences in Vacuolar Transport Activity.

PubMed

Jaarsma, Rinse; de Boer, Albertus H

2018-01-01

Potato is an important cultivated crop species and since it is moderately salt sensitive there is a need to develop more salt tolerant cultivars. A high activity of Na + transport across the tonoplast in exchange for H + is essential to reduce Na + toxicity. The proton motive force (PMF) generated by the V-H + -ATPase and the V-H + -PPase energizes the Na + (K + )/H + antiport. We compared the activity, gene expression, and protein levels of the vacuolar proton pumps and the Na + /H + antiporters in two potato cultivars ( Solanum tuberosum ) contrasting in their salt tolerance (cv. Desiree; tolerant and Mozart; sensitive) grown at 0 and 60 mM NaCl. Tonoplast-enriched vesicles were used to study the pump activity and protein levels of the V-H + -ATPase and the V-H + -PPase and the activity of the Na + /H + antiporter. Although salt stress reduced the V-H + -ATPase and the V-H + -PPase activity in both cultivars, the decline in H + pump activity was more severe in the salt-sensitive cultivar Mozart. After salt treatment, protein amounts of the vacuolar H + pumps decreased in Mozart but remained unchanged in the cultivar Desiree. Decreased protein amounts of the V-H + -PPase found in Mozart may explain the reduced V-H + -PPase activity found for Mozart after salt stress. Under non-stress conditions, protein amounts of V-H + -PPase were equal in both cultivars while the V-H + -PPase activity was already twice as high and remained higher after salt treatment in the cultivar Desiree as compared to Mozart. This cultivar-dependent V-H + -PPase activity may explain the higher salt tolerance of Desiree. Moreover, combined with reduced vacuolar H + pump activity, Mozart showed a lower Na + /H + exchange activity and the K m for Na + is at least twofold lower in tonoplast vesicles from Desiree, what suggests that NHXs from Desiree have a higher affinity for Na + as compared to Mozart. From these results, we conclude that the higher capacity in combination with the higher

Increased neuronal beta-amyloid precursor protein expression in human temporal lobe epilepsy: association with interleukin-1 alpha immunoreactivity.

PubMed

Sheng, J G; Boop, F A; Mrak, R E; Griffin, W S

1994-11-01

Levels of immunoreactive beta-amyloid precursor protein and interleukin-1 alpha were found to be elevated in surgically resected human temporal lobe tissue from patients with intractable epilepsy compared with postmortem tissue from neurologically unaffected patients (controls). In tissue from epileptics, the levels of the 135-kDa beta-amyloid precursor protein isoform were elevated to fourfold (p < 0.05) those of controls and those of the 130-kDa isoform to threefold (p < 0.05), whereas those of the 120-kDa isoform (p > 0.05) were not different from control values. beta-Amyloid precursor protein-immunoreactive neurons were 16 times more numerous, and their cytoplasm and proximal processes were more intensely immunoreactive in tissue sections from epileptics than controls (133 +/- 12 vs. 8 +/- 3/mm2; p < 0.001). However, neither beta-amyloid precursor protein-immunoreactive dystrophic neurites nor beta-amyloid deposits were found in this tissue. Interleukin-1 alpha-immunoreactive cells (microglia) were three times more numerous in epileptics than in controls (80 +/- 8 vs. 25 +/- 5/mm2; p < 0.001), and these cells were often found adjacent to beta-amyloid precursor protein-immunoreactive neuronal cell bodies. Our findings, together with functions established in vitro for interleukin-1, suggest that increased expression of this protein contributes to the increased levels of beta-amyloid precursor protein in epileptics, thus indicating a potential role for both of these proteins in the neuronal dysfunctions, e.g., hyperexcitability, characteristic of epilepsy.

RhVI1 is a membrane-anchored vacuolar invertase highly expressed in Rosa hybrida L. petals.

PubMed

Farci, Domenica; Collu, Gabriella; Kirkpatrick, Joanna; Esposito, Francesca; Piano, Dario

2016-05-01

Invertases are a widespread group of enzymes that catalyse the conversion of sucrose into fructose and glucose. Plants invertases and their substrates are essential factors that play an active role in primary metabolism and in cellular differentiation and by these activities they sustain development and growth. Being naturally present in multiple isoforms, invertases are known to be highly differentiated and tissue specific in such a way that every isoform is characteristic of a specific part of the plant. In this work, we report the identification of the invertase RhVI1 that was found to be highly expressed in rose petals. A characterization of this protein revealed that RhVI1 is a glycosylated membrane-anchored protein associated with the cytosolic side of the vacuolar membrane which occurs in vivo in a monomeric form. Purification yields have shown that the levels of expression decreased during the passage of petals from buds to mature and pre-senescent flowers. Moreover, the activity assay indicates RhVI1 to be an acidic vacuolar invertase. The physiological implications of these findings are discussed, suggesting a possible role of this protein during anthesis. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Reflections on protein splicing: structures, functions and mechanisms

PubMed Central

Anraku, Yasuhiro; Satow, Yoshinori

2009-01-01

Twenty years ago, evidence that one gene produces two enzymes via protein splicing emerged from structural and expression studies of the VMA1 gene in Saccharomyces cerevisiae. VMA1 consists of a single open reading frame and contains two independent genetic information for Vma1p (a catalytic 70-kDa subunit of the vacuolar H+-ATPase) and VDE (a 50-kDa DNA endonuclease) as an in-frame spliced insert in the gene. Protein splicing is a posttranslational cellular process, in which an intervening polypeptide termed as the VMA1 intein is self-catalytically excised out from a nascent 120-kDa VMA1 precursor and two flanking polypeptides of the N- and C-exteins are ligated to produce the mature Vma1p. Subsequent studies have demonstrated that protein splicing is not unique to the VMA1 precursor and there are many operons in nature, which implement genetic information editing at protein level. To elucidate its structure-directed chemical mechanisms, a series of biochemical and crystal structural studies has been carried out with the use of various VMA1 recombinants. This article summarizes a VDE-mediated self-catalytic mechanism for protein splicing that is triggered and terminated solely via thiazolidine intermediates with tetrahedral configurations formed within the splicing sites where proton ingress and egress are driven by balanced protonation and deprotonation. PMID:19907126

In-depth glycoproteomic characterisation of grape berry vacuolar invertase using a combination of mass spectrometry-based approaches.

PubMed

Hovasse, Agnès; Alayi, Tchilabalo Dilezitoko; Van Dorsselaer, Alain; Marchal, Richard; Jégou, Sandrine; Schaeffer-Reiss, Christine

2016-06-01

Vacuolar invertase is a key enzyme of sugar metabolism in grape berries. A full characterisation of this highly N-glycosylated protein is required to help understand its biological and biochemical significance in grapes. We have developed a mass spectrometry (MS)-based glycoproteomic approach wherein deglycosylated peptides are analysed by LC-MS/MS, while intact glycopeptides are characterised using a dedicated MS method to determine the attachment sites and micro-heterogeneity. For grape invertase, in parallel with deglycosylated peptides analysis, different enzymatic digestions were performed and glycopeptide detection was improved by enrichment method, nanoLC-MS and oxonium glycan ions. This MS-based glycoproteomic approach demonstrates that vacuolar invertase is glycosylated at all twelve potential N-glycosylation sites. Glycosylation is heterogeneous, with twelve glycoforms identified at six of the sites. The identification of several types of N-glycans is a major result to correlate with the surface and foaming properties of wine, the solubility, allergenicity, and protease resistance of wine proteins. Copyright © 2016 Elsevier Ltd. All rights reserved.

Evidence for Avt6 as a vacuolar exporter of acidic amino acids in Saccharomyces cerevisiae cells.

PubMed

Chahomchuen, Thippayarat; Hondo, Kana; Ohsaki, Mariko; Sekito, Takayuki; Kakinuma, Yoshimi

2009-12-01

Here we examined the significance of Avt6, a vacuolar exporter of glutamate and aspartate suggested by the in vitro membrane vesicle experiment, in vacuolar compartmentalization of amino acids in Saccharomyces cerevisiae cells. Fluorescent microscopic observation of GFP-fused Avt6 revealed it to be exclusively localized to the vacuolar membrane, with the amount of Myc-tagged Avt6 significantly increased under nitrogen starvation. Glutamate uptake by cells was enhanced by deletion of the AVT6 gene, indicating indirect involvement of Avt6 in cellular glutamate accumulation. Differences in acidic amino acid content of both total and vacuolar fractions were insignificant between the parent and avt6Delta cells when cultured in nutrient-rich conditions. However, in nitrogen-starved conditions, the amount of glutamate and aspartate in the vacuolar fraction was notably increased in the avt6Delta cells. Avt6 is thus involved in vacuolar amino acid compartmentalization in S. cerevisiae cells, especially under conditions of nitrogen starvation.

The Arabidopsis vacuolar malate channel is a member of the ALMT family.

PubMed

Kovermann, Peter; Meyer, Stefan; Hörtensteiner, Stefan; Picco, Cristiana; Scholz-Starke, Joachim; Ravera, Silvia; Lee, Youngsook; Martinoia, Enrico

2007-12-01

In plants, malate is a central metabolite and fulfills a large number of functions. Vacuolar malate may reach very high concentrations and fluctuate rapidly, whereas cytosolic malate is kept at a constant level allowing optimal metabolism. Recently, a vacuolar malate transporter (Arabidopsis thaliana tonoplast dicarboxylate transporter, AttDT) was identified that did not correspond to the well-characterized vacuolar malate channel. We therefore hypothesized that a member of the aluminum-activated malate transporter (ALMT) gene family could code for a vacuolar malate channel. Using GFP fusion constructs, we could show that AtALMT9 (A. thaliana ALMT9) is targeted to the vacuole. Promoter-GUS fusion constructs demonstrated that this gene is expressed in all organs, but is cell-type specific as GUS activity in leaves was detected nearly exclusively in mesophyll cells. Patch-clamp analysis of an Atalmt9 T-DNA insertion mutant exhibited strongly reduced vacuolar malate channel activity. In order to functionally characterize AtALMT9 as a malate channel, we heterologously expressed this gene in tobacco and in oocytes. Overexpression of AtALMT9-GFP in Nicotiana benthamiana leaves strongly enhanced the malate current densities across the mesophyll tonoplasts. Functional expression of AtALMT9 in Xenopus oocytes induced anion currents, which were clearly distinguishable from endogenous oocyte currents. Our results demonstrate that AtALMT9 is a vacuolar malate channel. Deletion mutants for AtALMT9 exhibit only slightly reduced malate content in mesophyll protoplasts and no visible phenotype, indicating that AttDT and the residual malate channel activity are sufficient to sustain the transport activity necessary to regulate the cytosolic malate homeostasis.

Cleavage of precursors by the mitochondrial processing peptidase requires a compatible mature protein or an intermediate octapeptide

PubMed Central

1991-01-01

Many precursors of mitochondrial proteins are processed in two successive steps by independent matrix peptidases (MPP and MIP), whereas others are cleaved in a single step by MPP alone. To explain this dichotomy, we have constructed deletions of all or part of the octapeptide characteristic of a twice cleaved precursor (human ornithine transcarbamylase [pOTC]), have exchanged leader peptide sequences between once-cleaved (human methylmalonyl-CoA mutase [pMUT]; yeast F1ATPase beta-subunit [pF1 beta]) and twice-cleaved (pOTC; rat malate dehydrogenase (pMDH); Neurospora ubiquinol-cytochrome c reductase iron-sulfur subunit [pFe/S]) precursors, and have incubated these proteins with purified MPP and MIP. When the octapeptide of pOTC was deleted, or when the entire leader peptide of a once-cleaved precursor (pMUT or pF1 beta) was joined to the mature amino terminus of a twice-cleaved precursor (pOTC or pFe/S), no cleavage was produced by either protease. Cleavage of these constructs by MPP was restored by re- inserting as few as two amino-terminal residues of the octapeptide or of the mature amino terminus of a once-cleaved precursor. We conclude that the mature amino terminus of a twice-cleaved precursor is structurally incompatible with cleavage by MPP; such proteins have evolved octapeptides cleaved by MIP to overcome this incompatibility. PMID:1672532

In silico modeling and experimental evidence of coagulant protein interaction with precursors for nanoparticle functionalization.

PubMed

Okoli, Chuka; Sengottaiyan, Selvaraj; Arul Murugan, N; Pavankumar, Asalapuram R; Agren, Hans; Kuttuva Rajarao, Gunaratna

2013-10-01

The design of novel protein-nanoparticle hybrid systems has applications in many fields of science ranging from biomedicine, catalysis, water treatment, etc. The main barrier in devising such tool is lack of adequate information or poor understanding of protein-ligand chemistry. Here, we establish a new strategy based on computational modeling for protein and precursor linkers that can decorate the nanoparticles. Moringa oleifera (MO2.1) seed protein that has coagulation and antimicrobial properties was used. Superparamagnetic nanoparticles (SPION) with precursor ligands were used for the protein-ligand interaction studies. The molecular docking studies reveal that there are two binding sites, one is located at the core binding site; tetraethoxysilane (TEOS) or 3-aminopropyl trimethoxysilane (APTES) binds to this site while the other one is located at the side chain residues where trisodium citrate (TSC) or Si60 binds to this site. The protein-ligand distance profile analysis explains the differences in functional activity of the decorated SPION. Experimentally, TSC-coated nanoparticles showed higher coagulation activity as compared to TEOS- and APTES-coated SPION. To our knowledge, this is the first report on in vitro experimental data, which endorses the computational modeling studies as a powerful tool to design novel precursors for functionalization of nanomaterials; and develop interface hybrid systems for various applications.

Defects of Vps15 in skeletal muscles lead to autophagic vacuolar myopathy and lysosomal disease

PubMed Central

Nemazanyy, Ivan; Blaauw, Bert; Paolini, Cecilia; Caillaud, Catherine; Protasi, Feliciano; Mueller, Amelie; Proikas-Cezanne, Tassula; Russell, Ryan C; Guan, Kun-Liang; Nishino, Ichizo; Sandri, Marco; Pende, Mario; Panasyuk, Ganna

2013-01-01

The complex of Vacuolar Protein Sorting 34 and 15 (Vps34 and Vps15) has Class III phosphatidylinositol 3-kinase activity and putative roles in nutrient sensing, mammalian Target Of Rapamycin (mTOR) activation by amino acids, cell growth, vesicular trafficking and autophagy. Contrary to expectations, here we show that Vps15-deficient mouse tissues are competent for LC3-positive autophagosome formation and maintain mTOR activation. However, an impaired lysosomal function in mutant cells is traced by accumulation of adaptor protein p62, LC3 and Lamp2 positive vesicles, which can be reverted to normal levels after ectopic overexpression of Vps15. Mice lacking Vps15 in skeletal muscles, develop a severe myopathy. Distinct from the autophagy deficient Atg7−/− mutants, pathognomonic morphological hallmarks of autophagic vacuolar myopathy (AVM) are observed in Vps15−/− mutants, including elevated creatine kinase plasma levels, accumulation of autophagosomes, glycogen and sarcolemmal features within the fibres. Importantly, Vps34/Vps15 overexpression in myoblasts of Danon AVM disease patients alleviates the glycogen accumulation. Thus, the activity of the Vps34/Vps15 complex is critical in disease conditions such as AVMs, and possibly a variety of other lysosomal storage diseases. PMID:23630012

The amino-terminal hydrophilic region of the vacuolar transporter Avt3p is dispensable for the vacuolar amino acid compartmentalization of Schizosaccharomyces pombe.

PubMed

Kawano-Kawada, Miyuki; Chardwiriyapreecha, Soracom; Manabe, Kunio; Sekito, Takayuki; Akiyama, Koichi; Takegawa, Kaoru; Kakinuma, Yoshimi

2016-12-01

Avt3p, a vacuolar amino acid exporter (656 amino acid residues) that is important for vacuolar amino acid compartmentalization as well as spore formation in Schizosaccharomyces pombe, has an extremely long hydrophilic region (approximately 290 amino acid residues) at its N-terminus. Because known functional domains have not been found in this region, its functional role was examined with a deletion mutant avt3 (∆1-270) expressed in S. pombe avt3∆ cells. The deletion of this region did not affect its intracellular localization or vacuolar contents of basic amino acids as well as neutral ones. The defect of avt3Δ cells in spore formation was rescued by the expression of avt3 + but was not completely rescued by the expression of avt3 (∆1-270) . The N-terminal region is thus dispensable for the function of Avt3p as an amino acid exporter, but it is likely to be involved in the role of Avt3p under nutritional starvation conditions.

Spore coat protein of Bacillus subtilis. Structure and precursor synthesis.

PubMed

Munoz, L; Sadaie, Y; Doi, R H

1978-10-10

The coat protein of Bacillus subtilis spores comprises about 10% of the total dry weight of spores and 25% of the total spore protein. One protein with a molecular weight of 13,000 to 15,000 comprises a major portion of the spore coat. This mature spore coat protein has histidine at its NH2 terminus and is relatively rich in hydrophobic amino acids. Netropsin, and antibiotic which binds to A-T-rich regions of DNA and inhibits sporulation, but not growth, decreased the synthesis of this spore coat protein by 75%. A precursor spore coat protein with a molecular weight of 25,000 is made initially at t1 of sporulation and is converted to the mature spore coat protein with a molecular weight of 13,500 at t2 - t3. These data indicate that the spore coat protein gene is expressed very early in sporulation prior to the modifications of RNA polymerase which have been noted.

Pollination in Nicotiana alata stimulates synthesis and transfer to the stigmatic surface of NaStEP, a vacuolar Kunitz proteinase inhibitor homologue

PubMed Central

Busot, Grethel Yanet; McClure, Bruce; Ibarra-Sánchez, Claudia Patricia; Jiménez-Durán, Karina; Vázquez-Santana, Sonia; Cruz-García, Felipe

2008-01-01

After landing on a wet stigma, pollen grains hydrate and germination generally occurs. However, there is no certainty of the pollen tube growth through the style to reach the ovary. The pistil is a gatekeeper that evolved in many species to recognize and reject the self-pollen, avoiding endogamy and encouraging cross-pollination. However, recognition is a complex process, and specific factors are needed. Here the isolation and characterization of a stigma-specific protein from N. alata, NaStEP (N. alata Stigma Expressed Protein), that is homologous to Kunitz-type proteinase inhibitors, are reported. Activity gel assays showed that NaStEP is not a functional serine proteinase inhibitor. Immunohistochemical and protein blot analyses revealed that NaStEP is detectable in stigmas of self-incompatible (SI) species N. alata, N. forgetiana, and N. bonariensis, but not in self-compatible (SC) species N. tabacum, N. plumbaginifolia, N. benthamiana, N. longiflora, and N. glauca. NaStEP contains the vacuolar targeting sequence NPIVL, and immunocytochemistry experiments showed vacuolar localization in unpollinated stigmas. After self-pollination or pollination with pollen from the SC species N. tabacum or N. plumbaginifolia, NaStEP was also found in the stigmatic exudate. The synthesis and presence in the stigmatic exudate of this protein was strongly induced in N. alata following incompatible pollination with N. tabacum pollen. The transfer of NaStEP to the stigmatic exudate was accompanied by perforation of the stigmatic cell wall, which appeared to release the vacuolar contents to the apoplastic space. The increase in NaStEP synthesis after pollination and its presence in the stigmatic exudates suggest that this protein may play a role in the early pollen–stigma interactions that regulate pollen tube growth in Nicotiana. PMID:18689443

Pollination in Nicotiana alata stimulates synthesis and transfer to the stigmatic surface of NaStEP, a vacuolar Kunitz proteinase inhibitor homologue.

PubMed

Busot, Grethel Yanet; McClure, Bruce; Ibarra-Sánchez, Claudia Patricia; Jiménez-Durán, Karina; Vázquez-Santana, Sonia; Cruz-García, Felipe

2008-01-01

After landing on a wet stigma, pollen grains hydrate and germination generally occurs. However, there is no certainty of the pollen tube growth through the style to reach the ovary. The pistil is a gatekeeper that evolved in many species to recognize and reject the self-pollen, avoiding endogamy and encouraging cross-pollination. However, recognition is a complex process, and specific factors are needed. Here the isolation and characterization of a stigma-specific protein from N. alata, NaStEP (N. alata Stigma Expressed Protein), that is homologous to Kunitz-type proteinase inhibitors, are reported. Activity gel assays showed that NaStEP is not a functional serine proteinase inhibitor. Immunohistochemical and protein blot analyses revealed that NaStEP is detectable in stigmas of self-incompatible (SI) species N. alata, N. forgetiana, and N. bonariensis, but not in self-compatible (SC) species N. tabacum, N. plumbaginifolia, N. benthamiana, N. longiflora, and N. glauca. NaStEP contains the vacuolar targeting sequence NPIVL, and immunocytochemistry experiments showed vacuolar localization in unpollinated stigmas. After self-pollination or pollination with pollen from the SC species N. tabacum or N. plumbaginifolia, NaStEP was also found in the stigmatic exudate. The synthesis and presence in the stigmatic exudate of this protein was strongly induced in N. alata following incompatible pollination with N. tabacum pollen. The transfer of NaStEP to the stigmatic exudate was accompanied by perforation of the stigmatic cell wall, which appeared to release the vacuolar contents to the apoplastic space. The increase in NaStEP synthesis after pollination and its presence in the stigmatic exudates suggest that this protein may play a role in the early pollen-stigma interactions that regulate pollen tube growth in Nicotiana.

Characterization of Avt1p as a vacuolar proton/amino acid antiporter in Saccharomyces cerevisiae.

PubMed

Tone, Junichi; Yoshimura, Ayumi; Manabe, Kunio; Murao, Nami; Sekito, Takayuki; Kawano-Kawada, Miyuki; Kakinuma, Yoshimi

2015-01-01

Several genes for vacuolar amino acid transport were reported in Saccharomyces cerevisiae, but have not well been investigated. We characterized AVT1, a member of the AVT vacuolar transporter family, which is reported to be involved in lifespan of yeast. ATP-dependent uptake of isoleucine and histidine by the vacuolar vesicles of an AVT exporter mutant was lost by introducing avt1∆ mutation. Uptake activity was inhibited by the V-ATPase inhibitor: concanamycin A and a protonophore. Isoleucine uptake was inhibited by various neutral amino acids and histidine, but not by γ-aminobutyric acid, glutamate, and aspartate. V-ATPase-dependent acidification of the vesicles was declined by the addition of isoleucine or histidine, depending upon Avt1p. Taken together with the data of the amino acid contents of vacuolar fractions in cells, the results suggested that Avt1p is a proton/amino acid antiporter important for vacuolar compartmentalization of various amino acids.

Amyloid Precursor Protein Processing and Alzheimer’s Disease

PubMed Central

O’Brien, Richard J.; Wong, Philip C.

2011-01-01

Alzheimer’s disease (AD), the leading cause of dementia worldwide, is characterized by the accumulation of the β-amyloid peptide (Aβ) within the brain along with hyperphosphorylated and cleaved forms of the microtubule-associated protein tau. Genetic, biochemical, and behavioral research suggest that physiologic generation of the neurotoxic Aβ peptide from sequential amyloid precursor protein (APP) proteolysis is the crucial step in the development of AD. APP is a single-pass transmembrane protein expressed at high levels in the brain and metabolized in a rapid and highly complex fashion by a series of sequential proteases, including the intramembranous γ-secretase complex, which also process other key regulatory molecules. Why Aβ accumulates in the brains of elderly individuals is unclear but could relate to changes in APP metabolism or Aβ elimination. Lessons learned from biochemical and genetic studies of APP processing will be crucial to the development of therapeutic targets to treat AD. PMID:21456963

Manduca Contactin Regulates Amyloid Precursor Protein-Dependent Neuronal Migration

PubMed Central

Ramaker, Jenna M.; Swanson, Tracy L.

2016-01-01

Amyloid precursor protein (APP) was originally identified as the source of β-amyloid peptides that accumulate in Alzheimer's disease (AD), but it also has been implicated in the control of multiple aspects of neuronal motility. APP belongs to an evolutionarily conserved family of transmembrane proteins that can interact with a variety of adapter and signaling molecules. Recently, we showed that both APP and its insect ortholog [APPL (APP-Like)] directly bind the heterotrimeric G-protein Goα, supporting the model that APP can function as an unconventional Goα-coupled receptor. We also adapted a well characterized assay of neuronal migration in the hawkmoth, Manduca sexta, to show that APPL–Goα signaling restricts ectopic growth within the developing nervous system, analogous to the role postulated for APP family proteins in controlling migration within the mammalian cortex. Using this assay, we have now identified Manduca Contactin (MsContactin) as an endogenous ligand for APPL, consistent with previous work showing that Contactins interact with APP family proteins in other systems. Using antisense-based knockdown protocols and fusion proteins targeting both proteins, we have shown that MsContactin is selectively expressed by glial cells that ensheath the migratory neurons (expressing APPL), and that MsContactin–APPL interactions normally prevent inappropriate migration and outgrowth. These results provide new evidence that Contactins can function as authentic ligands for APP family proteins that regulate APP-dependent responses in the developing nervous system. They also support the model that misregulated Contactin–APP interactions might provoke aberrant activation of Goα and its effectors, thereby contributing to the neurodegenerative sequelae that typify AD. SIGNIFICANCE STATEMENT Members of the amyloid precursor protein (APP) family participate in many aspects of neuronal development, but the ligands that normally activate APP signaling have remained

A Grapevine TTG2-Like WRKY Transcription Factor Is Involved in Regulating Vacuolar Transport and Flavonoid Biosynthesis.

PubMed

Amato, Alessandra; Cavallini, Erika; Zenoni, Sara; Finezzo, Laura; Begheldo, Maura; Ruperti, Benedetto; Tornielli, Giovanni Battista

2016-01-01

A small set of TTG2-like homolog proteins from different species belonging to the WRKY family of transcription factors were shown to share a similar mechanism of action and to control partially conserved biochemical/developmental processes in their native species. In particular, by activating P-ATPases residing on the tonoplast, PH3 from Petunia hybrida promotes vacuolar acidification in petal epidermal cells whereas TTG2 from Arabidopsis thaliana enables the accumulation of proanthocyanidins in the seed coat. In this work we functionally characterized VvWRKY26 identified as the closest grapevine homolog of PhPH3 and AtTTG2 . When constitutively expressed in petunia ph3 mutant, VvWRKY26 can fulfill the PH3 function in the regulation of vacuolar pH and restores the wild type pigmentation phenotype. By a global correlation analysis of gene expression and by transient over-expression in Vitis vinifera , we showed transcriptomic relationships of VvWRKY26 with many genes related to vacuolar acidification and transport in grapevine. Moreover, our results indicate an involvement in flavonoid pathway possibly restricted to the control of proanthocyanidin biosynthesis that is consistent with its expression pattern in grape berry tissues. Overall, the results show that, in addition to regulative mechanisms and biological roles shared with TTG2-like orthologs, VvWRKY26 can play roles in fleshy fruit development that have not been previously reported in studies from dry fruit species. This study paves the way toward the comprehension of the regulatory network controlling vacuolar acidification and flavonoid accumulation mechanisms that contribute to the final berry quality traits in grapevine.

A Grapevine TTG2-Like WRKY Transcription Factor Is Involved in Regulating Vacuolar Transport and Flavonoid Biosynthesis

PubMed Central

Amato, Alessandra; Cavallini, Erika; Zenoni, Sara; Finezzo, Laura; Begheldo, Maura; Ruperti, Benedetto; Tornielli, Giovanni Battista

2017-01-01

A small set of TTG2-like homolog proteins from different species belonging to the WRKY family of transcription factors were shown to share a similar mechanism of action and to control partially conserved biochemical/developmental processes in their native species. In particular, by activating P-ATPases residing on the tonoplast, PH3 from Petunia hybrida promotes vacuolar acidification in petal epidermal cells whereas TTG2 from Arabidopsis thaliana enables the accumulation of proanthocyanidins in the seed coat. In this work we functionally characterized VvWRKY26 identified as the closest grapevine homolog of PhPH3 and AtTTG2. When constitutively expressed in petunia ph3 mutant, VvWRKY26 can fulfill the PH3 function in the regulation of vacuolar pH and restores the wild type pigmentation phenotype. By a global correlation analysis of gene expression and by transient over-expression in Vitis vinifera, we showed transcriptomic relationships of VvWRKY26 with many genes related to vacuolar acidification and transport in grapevine. Moreover, our results indicate an involvement in flavonoid pathway possibly restricted to the control of proanthocyanidin biosynthesis that is consistent with its expression pattern in grape berry tissues. Overall, the results show that, in addition to regulative mechanisms and biological roles shared with TTG2-like orthologs, VvWRKY26 can play roles in fleshy fruit development that have not been previously reported in studies from dry fruit species. This study paves the way toward the comprehension of the regulatory network controlling vacuolar acidification and flavonoid accumulation mechanisms that contribute to the final berry quality traits in grapevine. PMID:28105033

Listeria monocytogenes switches from dissemination to persistence by adopting a vacuolar lifestyle in epithelial cells.

PubMed

Kortebi, Mounia; Milohanic, Eliane; Mitchell, Gabriel; Péchoux, Christine; Prevost, Marie-Christine; Cossart, Pascale; Bierne, Hélène

2017-11-01

Listeria monocytogenes causes listeriosis, a foodborne disease that poses serious risks to fetuses, newborns and immunocompromised adults. This intracellular bacterial pathogen proliferates in the host cytosol and exploits the host actin polymerization machinery to spread from cell-to-cell and disseminate in the host. Here, we report that during several days of infection in human hepatocytes or trophoblast cells, L. monocytogenes switches from this active motile lifestyle to a stage of persistence in vacuoles. Upon intercellular spread, bacteria gradually stopped producing the actin-nucleating protein ActA and became trapped in lysosome-like vacuoles termed Listeria-Containing Vacuoles (LisCVs). Subpopulations of bacteria resisted degradation in LisCVs and entered a slow/non-replicative state. During the subculture of host cells harboring LisCVs, bacteria showed a capacity to cycle between the vacuolar and the actin-based motility stages. When ActA was absent, such as in ΔactA mutants, vacuolar bacteria parasitized host cells in the so-called "viable but non-culturable" state (VBNC), preventing their detection by conventional colony counting methods. The exposure of infected cells to high doses of gentamicin did not trigger the formation of LisCVs, but selected for vacuolar and VBNC bacteria. Together, these results reveal the ability of L. monocytogenes to enter a persistent state in a subset of epithelial cells, which may favor the asymptomatic carriage of this pathogen, lengthen the incubation period of listeriosis, and promote bacterial survival during antibiotic therapy.

Listeria monocytogenes switches from dissemination to persistence by adopting a vacuolar lifestyle in epithelial cells

PubMed Central

Mitchell, Gabriel

2017-01-01

Listeria monocytogenes causes listeriosis, a foodborne disease that poses serious risks to fetuses, newborns and immunocompromised adults. This intracellular bacterial pathogen proliferates in the host cytosol and exploits the host actin polymerization machinery to spread from cell-to-cell and disseminate in the host. Here, we report that during several days of infection in human hepatocytes or trophoblast cells, L. monocytogenes switches from this active motile lifestyle to a stage of persistence in vacuoles. Upon intercellular spread, bacteria gradually stopped producing the actin-nucleating protein ActA and became trapped in lysosome-like vacuoles termed Listeria-Containing Vacuoles (LisCVs). Subpopulations of bacteria resisted degradation in LisCVs and entered a slow/non-replicative state. During the subculture of host cells harboring LisCVs, bacteria showed a capacity to cycle between the vacuolar and the actin-based motility stages. When ActA was absent, such as in ΔactA mutants, vacuolar bacteria parasitized host cells in the so-called “viable but non-culturable” state (VBNC), preventing their detection by conventional colony counting methods. The exposure of infected cells to high doses of gentamicin did not trigger the formation of LisCVs, but selected for vacuolar and VBNC bacteria. Together, these results reveal the ability of L. monocytogenes to enter a persistent state in a subset of epithelial cells, which may favor the asymptomatic carriage of this pathogen, lengthen the incubation period of listeriosis, and promote bacterial survival during antibiotic therapy. PMID:29190284

The V-ATPase subunit A is essential for salt tolerance through participating in vacuolar Na+ compartmentalization in Salicornia europaea.

PubMed

Lv, Sulian; Jiang, Ping; Tai, Fang; Wang, Duoliya; Feng, Juanjuan; Fan, Pengxiang; Bao, Hexigeduleng; Li, Yinxin

2017-12-01

The V-ATPase subunit A participates in vacuolar Na + compartmentalization in Salicornia europaea regulating V-ATPase and V-PPase activities. Na + sequestration into the vacuole is an efficient strategy in response to salinity in many halophytes. However, it is not yet fully understood how this process is achieved. Particularly, the role of vacuolar H + -ATPase (V-ATPase) in this process is controversial. Our previous proteomic investigation in the euhalophyte Salicornia europaea L. found a significant increase of the abundance of V-ATPase subunit A under salinity. Here, the gene encoding this subunit named SeVHA-A was characterized, and its role in salt tolerance was demonstrated by RNAi directed downregulation in suspension-cultured cells of S. europaea. The transcripts of genes encoding vacuolar H + -PPase (V-PPase) and vacuolar Na + /H + antiporter (SeNHX1) also decreased significantly in the RNAi cells. Knockdown of SeVHA-A resulted in a reduction in both V-ATPase and vacuolar H + -PPase (V-PPase) activities. Accordingly, the SeVHA-A-RNAi cells showed increased vacuolar pH and decreased cell viability under different NaCl concentrations. Further Na + staining showed the reduced vacuolar Na + sequestration in RNAi cells. Taken together, our results evidenced that SeVHA-A participates in vacuolar Na + sequestration regulating V-ATPase and V-PPase activities and thereby vacuolar pH in S. europaea. The possible mechanisms underlying the reduction of vacuolar V-PPase activity in SeVHA-A-RNAi cells were also discussed.

The Citrus transcription factor, CitERF13, regulates citric acid accumulation via a protein-protein interaction with the vacuolar proton pump, CitVHA-c4

PubMed Central

Li, Shao-jia; Yin, Xue-ren; Xie, Xiu-lan; Allan, Andrew C.; Ge, Hang; Shen, Shu-ling; Chen, Kun-song

2016-01-01

Organic acids are essential to fruit flavor. The vacuolar H+ transporting adenosine triphosphatase (V-ATPase) plays an important role in organic acid transport and accumulation. However, less is known of V-ATPase interacting proteins and their relationship with organic acid accumulation. The relationship between V-ATPase and citric acid was investigated, using the citrus tangerine varieties ‘Ordinary Ponkan (OPK)’ and an early maturing mutant ‘Zaoshu Ponkan (ZPK)’. Five V-ATPase genes (CitVHA) were predicted as important to citric acid accumulation. Among the genes, CitVHA-c4 was observed, using a yeast two-hybrid screen, to interact at the protein level with an ethylene response factor, CitERF13. This was verified using bimolecular fluorescence complementation assays. A similar interaction was also observed between Arabidopsis AtERF017 (a CitERF13 homolog) and AtVHA-c4 (a CitVHA-c4 homolog). A synergistic effect on citric acid levels was observed between V-ATPase proteins and interacting ERFs when analyzed using transient over-expression in tobacco and Arabidopsis mutants. Furthermore, the transcript abundance of CitERF13 was concomitant with CitVHA-c4. CitERF13 or AtERF017 over-expression leads to significant citric acid accumulation. This accumulation was abolished in an AtVHA-c4 mutant background. ERF-VHA interactions appear to be involved in citric acid accumulation, which was observed in both citrus and Arabidopsis. PMID:26837571

The Citrus transcription factor, CitERF13, regulates citric acid accumulation via a protein-protein interaction with the vacuolar proton pump, CitVHA-c4.

PubMed

Li, Shao-jia; Yin, Xue-ren; Xie, Xiu-lan; Allan, Andrew C; Ge, Hang; Shen, Shu-ling; Chen, Kun-song

2016-02-03

Organic acids are essential to fruit flavor. The vacuolar H(+) transporting adenosine triphosphatase (V-ATPase) plays an important role in organic acid transport and accumulation. However, less is known of V-ATPase interacting proteins and their relationship with organic acid accumulation. The relationship between V-ATPase and citric acid was investigated, using the citrus tangerine varieties 'Ordinary Ponkan (OPK)' and an early maturing mutant 'Zaoshu Ponkan (ZPK)'. Five V-ATPase genes (CitVHA) were predicted as important to citric acid accumulation. Among the genes, CitVHA-c4 was observed, using a yeast two-hybrid screen, to interact at the protein level with an ethylene response factor, CitERF13. This was verified using bimolecular fluorescence complementation assays. A similar interaction was also observed between Arabidopsis AtERF017 (a CitERF13 homolog) and AtVHA-c4 (a CitVHA-c4 homolog). A synergistic effect on citric acid levels was observed between V-ATPase proteins and interacting ERFs when analyzed using transient over-expression in tobacco and Arabidopsis mutants. Furthermore, the transcript abundance of CitERF13 was concomitant with CitVHA-c4. CitERF13 or AtERF017 over-expression leads to significant citric acid accumulation. This accumulation was abolished in an AtVHA-c4 mutant background. ERF-VHA interactions appear to be involved in citric acid accumulation, which was observed in both citrus and Arabidopsis.

Vacuolar Protein Sorting Genes in Parkinson's Disease: A Re-appraisal of Mutations Detection Rate and Neurobiology of Disease.

PubMed

Gambardella, Stefano; Biagioni, Francesca; Ferese, Rosangela; Busceti, Carla L; Frati, Alessandro; Novelli, Giuseppe; Ruggieri, Stefano; Fornai, Francesco

2016-01-01

Mammalian retromers play a critical role in protein trans-membrane sorting from endosome to the trans-Golgi network (TGN). Recently, retromer alterations have been related to the onset of Parkinson's Disease (PD) since the variant p.Asp620Asn in VPS35 (Vacuolar Protein Sorting 35) was identified as a cause of late onset PD. This variant causes a primary defect in endosomal trafficking and retromers formation. Other mutations in VPS genes have been reported in both sporadic and familial PD. These mutations are less defined. Understanding the specific prevalence of all VPS gene mutations is key to understand the relevance of retromers impairment in the onset of PD. A number of PD-related mutations despite affecting different biochemical systems (autophagy, mitophagy, proteasome, endosomes, protein folding), all converge in producing an impairment in cell clearance. This may explain how genetic predispositions to PD may derive from slightly deleterious VPS mutations when combined with environmental agents overwhelming the clearance of the cell. This manuscript reviews genetic data produced in the last 5 years to re-define the actual prevalence of VPS gene mutations in the onset of PD. The prevalence of p.Asp620Asn mutation in VPS35 is 0.286 of familial PD. This increases up to 0.548 when considering mutations affecting all VPS genes. This configures mutations in VPS genes as the second most frequent autosomal dominant PD genotype. This high prevalence, joined with increased awareness of the role played by retromers in the neurobiology of PD, suggests environmentally-induced VPS alterations as crucial in the genesis of PD.

Trafficking modulator TENin1 inhibits endocytosis, causes endomembrane protein accumulation at the pre-vacuolar compartment and impairs gravitropic response in Arabidopsis thaliana.

PubMed

Paudyal, Rupesh; Jamaluddin, Adam; Warren, James P; Doyle, Siamsa M; Robert, Stéphanie; Warriner, Stuart L; Baker, Alison

2014-06-01

Auxin gradients are established and maintained by polarized distribution of auxin transporters that undergo constitutive endocytic recycling from the PM (plasma membrane) and are essential for the gravitropic response in plants. The present study characterizes an inhibitor of endomembrane protein trafficking, TE1 (trafficking and endocytosis inhibitor 1/TENin1) that reduces gravitropic root bending in Arabidopsis thaliana seedlings. Short-term TE1 treatment causes accumulation of PM proteins, including the BR (brassinosteroid) receptor BRI1 (BR insensitive 1), PIP2a (PM intrinsic protein 2a) and the auxin transporter PIN2 (PIN-FORMED 2) in a PVC (pre-vacuolar related compartment), which is sensitive to BFA (Brefeldin A). This compound inhibits endocytosis from the PM and promotes trafficking to the vacuole, consistent with inhibition of retrieval of proteins to the TGN (trans-Golgi network) from the PVC and the PM. However, trafficking of newly synthesized proteins to the PM is unaffected. The short-term protein trafficking inhibition and long-term effect on plant growth and survival caused by TE1 were fully reversible upon drug washout. Structure-activity relationship studies revealed that only minor modifications were possible without loss of biological activity. Diversity in Arabidopsis ecotypes was also exploited to identify two Arabidopsis accessions that display reduced sensitivity to TE1. This compound and the resistant Arabidopsis accessions may be used as a resource in future studies to better understand endomembrane trafficking in plants.

Effects of N-glycan precursor length diversity on quality control of protein folding and on protein glycosylation

PubMed Central

Samuelson, John; Robbins, Phillips W.

2014-01-01

Asparagine-linked glycans (N-glycans) of medically important protists have much to tell us about the evolution of N-glycosylation and of N-glycan-dependent quality control (N-glycan QC) of protein folding in the endoplasmic reticulum. While host N-glycans are built upon a dolichol-pyrophosphate-linked precursor with 14 sugars (Glc3Man9GlcNAc2), protist N-glycan precursors vary from Glc3Man9GlcNAc2 (Acanthamoeba) to Man9GlcNAc2 (Trypanosoma) to Glc3Man5GlcNAc2 (Toxoplasma) to Man5GlcNAc2 (Entamoeba, Trichomonas, and Eimeria) to GlcNAc2 (Plasmodium and Giardia) to zero (Theileria). As related organisms have differing N-glycan lengths (e.g. Toxoplasma, Eimeria, Plasmodium, and Theileria), the present N-glycan variation is based upon secondary loss of Alg genes, which encode enzymes that add sugars to the N-glycan precursor. An N-glycan precursor with Man5GlcNAc2 is necessary but not sufficient for N-glycan QC, which is predicted by the presence of the UDP-glucose:glucosyltransferase (UGGT) plus calreticulin and/or calnexin. As many parasites lack glucose in their N-glycan precursor, UGGT product may be identified by inhibition of glucosidase II. The presence of an armless calnexin in Toxoplasma suggests secondary loss of N-glycan QC from coccidia. Positive selection for N-glycan sites occurs in secreted proteins of organisms with NG-QC and is based upon an increased likelihood of threonine but not serine in the second position versus asparagine. In contrast, there appears to be selection against N-glycan length in Plasmodium and N-glycan site density in Toxoplasma. Finally, there is suggestive evidence for N-glycan-dependent ERAD in Trichomonas, which glycosylates and degrades the exogenous reporter mutant carboxypeptidase Y (CPY*). PMID:25475176

Structural basis for precursor protein-directed ribosomal peptide macrocyclization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Kunhua; Condurso, Heather L.; Li, Gengnan

Macrocyclization is a common feature of natural product biosynthetic pathways including the diverse family of ribosomal peptides. Microviridins are architecturally complex cyanobacterial ribosomal peptides that target proteases with potent reversible inhibition. The product structure is constructed via three macrocyclizations catalyzed sequentially by two members of the ATP-grasp family, a unique strategy for ribosomal peptide macrocyclization. Here we describe in detail the structural basis for the enzyme-catalyzed macrocyclizations in the microviridin J pathway of Microcystis aeruginosa. The macrocyclases MdnC and MdnB interact with a conserved α-helix of the precursor peptide using a novel precursor-peptide recognition mechanism. The results provide insight intomore » the unique protein–protein interactions that are key to the chemistry, suggest an origin for the natural combinatorial synthesis of microviridin peptides, and provide a framework for future engineering efforts to generate designed compounds.« less

A mutation in the melon Vacuolar Protein Sorting 41prevents systemic infection of Cucumber mosaic virus.

PubMed

Giner, Ana; Pascual, Laura; Bourgeois, Michael; Gyetvai, Gabor; Rios, Pablo; Picó, Belén; Troadec, Christelle; Bendahmane, Abdel; Garcia-Mas, Jordi; Martín-Hernández, Ana Montserrat

2017-09-05

In the melon exotic accession PI 161375, the gene cmv1, confers recessive resistance to Cucumber mosaic virus (CMV) strains of subgroup II. cmv1 prevents the systemic infection by restricting the virus to the bundle sheath cells and impeding viral loading to the phloem. Here we report the fine mapping and cloning of cmv1. Screening of an F2 population reduced the cmv1 region to a 132 Kb interval that includes a Vacuolar Protein Sorting 41 gene. CmVPS41 is conserved among plants, animals and yeast and is required for post-Golgi vesicle trafficking towards the vacuole. We have validated CmVPS41 as the gene responsible for the resistance, both by generating CMV susceptible transgenic melon plants, expressing the susceptible allele in the resistant cultivar and by characterizing CmVPS41 TILLING mutants with reduced susceptibility to CMV. Finally, a core collection of 52 melon accessions allowed us to identify a single amino acid substitution (L348R) as the only polymorphism associated with the resistant phenotype. CmVPS41 is the first natural recessive resistance gene found to be involved in viral transport and its cellular function suggests that CMV might use CmVPS41 for its own transport towards the phloem.

Vacuolar transporter Avt4 is involved in excretion of basic amino acids from the vacuoles of Saccharomyces cerevisiae.

PubMed

Sekito, Takayuki; Chardwiriyapreecha, Soracom; Sugimoto, Naoko; Ishimoto, Masaya; Kawano-Kawada, Miyuki; Kakinuma, Yoshimi

2014-01-01

Basic amino acids (lysine, histidine and arginine) accumulated in Saccharomyces cerevisiae vacuoles should be mobilized to cytosolic nitrogen metabolism under starvation. We found that the decrease of vacuolar basic amino acids in response to nitrogen starvation was impaired by the deletion of AVT4 gene encoding a vacuolar transporter. In addition, overexpression of AVT4 reduced the accumulation of basic amino acids in vacuoles under nutrient-rich condition. In contrast to AVT4, the deletion and overexpression of AVT3, which encodes the closest homologue of Avt4p, did not affect the contents of vacuolar basic amino acids. Consistent with these, arginine uptake into vacuolar membrane vesicles was decreased by Avt4p-, but not by Avt3p-overproduction, whereas various neutral amino acids were excreted from vacuolar membrane vesicles in a manner dependent on either Avt4p or Avt3p. These results suggest that Avt4p is a vacuolar amino acid exporter involving in the recycling of basic amino acids.

Phylogenetic appearance of Neuropeptide S precursor proteins in tetrapods

PubMed Central

Reinscheid, Rainer K.

2007-01-01

Sleep and emotional behavior are two hallmarks of vertebrate animal behavior, implying that specialized neuronal circuits and dedicated neurochemical messengers may have been developed during evolution to regulate such complex behaviors. Neuropeptide S (NPS) is a newly identified peptide transmitter that activates a typical G protein-coupled receptor. Central administration of NPS produces profound arousal, enhances wakefulness and suppresses all stages of sleep. In addition, NPS can alleviate behavioral responses to stress by producing anxiolytic-like effects. A bioinformatic analysis of current genome databases revealed that the NPS peptide precursor gene is present in all vertebrates with the exception of fish. A high level of sequence conservation, especially of aminoterminal structures was detected, indicating stringent requirements for agonist-induced receptor activation. Duplication of the NPS precursor gene was only found in one out of two marsupial species with sufficient genome coverage (Monodelphis domestica; opossum), indicating that the duplicated opossum NPS sequence might have arisen as an isolated event. Pharmacological analysis of both Monodelphis NPS peptides revealed that only the closely related NPS peptide retained agonistic activity at NPS receptors. The duplicated precursor might be either a pseudogene or could have evolved different receptor selectivity. Together, these data show that NPS is a relatively recent gene in vertebrate evolution whose appearance might coincide with its specialized physiological functions in terrestrial vertebrates. PMID:17293003

Viral precursor protein P3 and its processed products perform discrete and essential functions in the poliovirus RNA replication complex

USDA-ARS?s Scientific Manuscript database

The differential use of protein precursors and their products is a key strategy used during poliovirus replication. To characterize the role of protein precursors during replication, we examined the complementation profiles of mutants that inhibited 3D polymerase or 3C-RNA binding activity. We showe...

Four-way regulation of mosquito yolk protein precursor genes by juvenile hormone-, ecdysone-, nutrient-, and insulin-like peptide signaling pathways.

PubMed

Hansen, Immo A; Attardo, Geoffrey M; Rodriguez, Stacy D; Drake, Lisa L

2014-01-01

Anautogenous mosquito females require a meal of vertebrate blood in order to initiate the production of yolk protein precursors by the fat body. Yolk protein precursor gene expression is tightly repressed in a state-of-arrest before blood meal-related signals activate it and expression levels rise rapidly. The best understood example of yolk protein precursor gene regulation is the vitellogenin-A gene (vg) of the yellow fever mosquito Aedes aegypti. Vg-A is regulated by (1) juvenile hormone signaling, (2) the ecdysone-signaling cascade, (3) the nutrient sensitive target-of-rapamycin signaling pathway, and (4) the insulin-like peptide (ILP) signaling pathway. A plethora of new studies have refined our understanding of the regulation of yolk protein precursor genes since the last review on this topic in 2005 (Attardo et al., 2005). This review summarizes the role of these four signaling pathways in the regulation of vg-A and focuses upon new findings regarding the interplay between them on an organismal level.

Four-way regulation of mosquito yolk protein precursor genes by juvenile hormone-, ecdysone-, nutrient-, and insulin-like peptide signaling pathways

PubMed Central

Hansen, Immo A.; Attardo, Geoffrey M.; Rodriguez, Stacy D.; Drake, Lisa L.

2014-01-01

Anautogenous mosquito females require a meal of vertebrate blood in order to initiate the production of yolk protein precursors by the fat body. Yolk protein precursor gene expression is tightly repressed in a state-of-arrest before blood meal-related signals activate it and expression levels rise rapidly. The best understood example of yolk protein precursor gene regulation is the vitellogenin-A gene (vg) of the yellow fever mosquito Aedes aegypti. Vg-A is regulated by (1) juvenile hormone signaling, (2) the ecdysone-signaling cascade, (3) the nutrient sensitive target-of-rapamycin signaling pathway, and (4) the insulin-like peptide (ILP) signaling pathway. A plethora of new studies have refined our understanding of the regulation of yolk protein precursor genes since the last review on this topic in 2005 (Attardo et al., 2005). This review summarizes the role of these four signaling pathways in the regulation of vg-A and focuses upon new findings regarding the interplay between them on an organismal level. PMID:24688471

Isolation, characterization, and structure analysis of a vacuolar processing enzyme gene (MhVPEγ) from Malus hupehensis (Pamp) Rehd.

PubMed

Ran, Kun; Yang, Hongqiang; Sun, Xiaoli; Li, Qiang; Jiang, Qianqian; Zhang, Weiwei; Shen, Wei

2014-05-01

Vacuolar processing enzymes (VPEs) have received considerable attention recently, as they exhibit caspase-1-like cleavage activity and regulate the process of PCD. However, knowledge about their detailed characteristics and structures is relatively limited. In this study, a gamma vacuolar processing enzyme gene, MhVPEγ, has been isolated from the leaves of Malus hupehensis (Ramp) Rehd. var pinyiensis Jiang. MhVPEγ coded-translated protein sequence comprised of 494 amino acids with a signal peptide and a transmembrane helix structure at N-terminal, peptidase_C13 domain, and vacuolar sorting signal at C-terminal. Consequently, genomic walking approach was performed for the isolation of its upstream sequence. Computational analysis demonstrated several motifs of the promoter exhibiting hypothetic MeJA, ABA, and light-induced characteristics, as well as some typical domains universally discovered in promoter, such as TATA-box and CAAT-box. MhVPEγ transcript level was enhanced during wounding treatment, and WUN-motif, as one of the cis-acting regulatory elements existing in the upstream sequence perhaps regulates its expression. In silico-constructed 3D models revealed that MhCPYL successively interacts with MhVPEγ like that of "Induced Fit-Lock and Key" model, providing molecular conformation evidence that CPY is a direct substrate of VPEγ. This study is the first stride to understand the molecular mechanism of VPEγ and CPYL interactions.

Involvement of MoVMA11, a Putative Vacuolar ATPase c’ Subunit, in Vacuolar Acidification and Infection-Related Morphogenesis of Magnaporthe oryzae

PubMed Central

Chen, Guoqing; Liu, Xiaohong; Zhang, Lilin; Cao, Huijuan; Lu, Jianping; Lin, Fucheng

2013-01-01

Many functions of vacuole depend on the activity of vacuolar ATPase which is essential to maintain an acidic lumen and create the driving forces for massive fluxes of ions and metabolites through vacuolar membrane. In filamentous fungus Magnaporthe oryzae , subcellular colocalization and quinacrine staining suggested that the V1V0 domains of V-ATPase were fully assembled and the vacuoles were kept acidic during infection-related developments. Targeted gene disruption of MoVMA11 gene, encoding the putative c’ subunit of V-ATPase, impaired vacuolar acidification and mimicked the phenotypes of yeast V-ATPase mutants in the poor colony morphology, abolished asexual and sexual reproductions, selective carbon source utilization, and increased calcium and heavy metals sensitivities, however, not in the typical pH conditional lethality. Strikingly, aerial hyphae of the MoVMA11 null mutant intertwined with each other to form extremely thick filamentous structures. The results also implicated that MoVMA11 was involved in cell wall integrity and appressorium formation. Abundant non-melanized swollen structures and rare, small appressoria without penetration ability were produced at the hyphal tips of the ΔMovma11 mutant on onion epidermal cells. Finally, the MoVMA11 null mutant lost pathogenicity on both intact and wounded host leaves. Overall, our data indicated that MoVMA11, like other fungal VMA genes, is associated with numerous cellular functions and highlighted that V-ATPase is essential for infection-related morphogenesis and pathogenesis in M . oryzae . PMID:23826342

Clinical Utility of LC3 and p62 Immunohistochemistry in Diagnosis of Drug-Induced Autophagic Vacuolar Myopathies: A Case-Control Study

PubMed Central

Lee, Han S.; Daniels, Brianne H.; Salas, Eduardo; Bollen, Andrew W.; Debnath, Jayanta; Margeta, Marta

2012-01-01

Background Some patients treated with chloroquine, hydroxychloroquine, or colchicine develop autophagic vacuolar myopathy, the diagnosis of which currently requires electron microscopy. The goal of the current study was to develop an immunohistochemical diagnostic marker for this pathologic entity. Methodology Microtubule-associated protein light chain 3 (LC3) has emerged as a robust marker of autophagosomes. LC3 binds p62/SQSTM1, an adapter protein that is selectively degraded via autophagy. In this study, we evaluated the utility of immunohistochemical stains for LC3 and p62 as diagnostic markers of drug-induced autophagic vacuolar myopathy. The staining was performed on archival muscle biopsy material, with subject assignment to normal control, drug-treated control, and autophagic myopathy groups based on history of drug use and morphologic criteria. Principal Findings In all drug-treated subjects, but not in normal controls, LC3 and p62 showed punctate staining characteristic of autophagosome buildup. In the autophagic myopathy subjects, puncta were coarser and tended to coalesce into linear structures aligned with the longitudinal axis of the fiber, often in the vicinity of vacuoles. The percentage of LC3- and p62-positive fibers was significantly higher in the autophagic myopathy group compared to either the normal control (p<0.001) or the drug-treated control group (p<0.05). With the diagnostic threshold set between 8% and 15% positive fibers (depending on the desired level of sensitivity and specificity), immunohistochemical staining for either LC3 or p62 could be used to identify subjects with autophagic vacuolar myopathy within the drug-treated subject group (p≤0.001). Significance Immunohistochemistry for LC3 and p62 can facilitate tissue-based diagnosis of drug-induced autophagic vacuolar myopathies. By limiting the need for electron microscopy (a time consuming and costly technique with high specificity, but low sensitivity), clinical use of these

Clinical utility of LC3 and p62 immunohistochemistry in diagnosis of drug-induced autophagic vacuolar myopathies: a case-control study.

PubMed

Lee, Han S; Daniels, Brianne H; Salas, Eduardo; Bollen, Andrew W; Debnath, Jayanta; Margeta, Marta

2012-01-01

Some patients treated with chloroquine, hydroxychloroquine, or colchicine develop autophagic vacuolar myopathy, the diagnosis of which currently requires electron microscopy. The goal of the current study was to develop an immunohistochemical diagnostic marker for this pathologic entity. Microtubule-associated protein light chain 3 (LC3) has emerged as a robust marker of autophagosomes. LC3 binds p62/SQSTM1, an adapter protein that is selectively degraded via autophagy. In this study, we evaluated the utility of immunohistochemical stains for LC3 and p62 as diagnostic markers of drug-induced autophagic vacuolar myopathy. The staining was performed on archival muscle biopsy material, with subject assignment to normal control, drug-treated control, and autophagic myopathy groups based on history of drug use and morphologic criteria. In all drug-treated subjects, but not in normal controls, LC3 and p62 showed punctate staining characteristic of autophagosome buildup. In the autophagic myopathy subjects, puncta were coarser and tended to coalesce into linear structures aligned with the longitudinal axis of the fiber, often in the vicinity of vacuoles. The percentage of LC3- and p62-positive fibers was significantly higher in the autophagic myopathy group compared to either the normal control (p<0.001) or the drug-treated control group (p<0.05). With the diagnostic threshold set between 8% and 15% positive fibers (depending on the desired level of sensitivity and specificity), immunohistochemical staining for either LC3 or p62 could be used to identify subjects with autophagic vacuolar myopathy within the drug-treated subject group (p ≤ 0.001). Immunohistochemistry for LC3 and p62 can facilitate tissue-based diagnosis of drug-induced autophagic vacuolar myopathies. By limiting the need for electron microscopy (a time consuming and costly technique with high specificity, but low sensitivity), clinical use of these markers will improve the speed and accuracy of

Vacuolar Chloride Fluxes Impact Ion Content and Distribution during Early Salinity Stress1

PubMed Central

Baetz, Ulrike; Tohge, Takayuki; Martinoia, Enrico; De Angeli, Alexis

2016-01-01

The ability to control the cytoplasmic environment is a prerequisite for plants to cope with changing environmental conditions. During salt stress, for instance, Na+ and Cl− are sequestered into the vacuole to help maintain cytosolic ion homeostasis and avoid cellular damage. It has been observed that vacuolar ion uptake is tied to fluxes across the plasma membrane. The coordination of both transport processes and relative contribution to plant adaptation, however, is still poorly understood. To investigate the link between vacuolar anion uptake and whole-plant ion distribution during salinity, we used mutants of the only vacuolar Cl− channel described to date: the Arabidopsis (Arabidopsis thaliana) ALMT9. After 24-h NaCl treatment, almt9 knock-out mutants had reduced shoot accumulation of both Cl− and Na+. In contrast, almt9 plants complemented with a mutant variant of ALMT9 that exhibits enhanced channel activity showed higher Cl− and Na+ accumulation. The altered shoot ion contents were not based on differences in transpiration, pointing to a vacuolar function in regulating xylem loading during salinity. In line with this finding, GUS staining demonstrated that ALMT9 is highly expressed in the vasculature of shoots and roots. RNA-seq analysis of almt9 mutants under salinity revealed specific expression profiles of transporters involved in long-distance ion translocation. Taken together, our study uncovers that the capacity of vacuolar Cl− loading in vascular cells plays a crucial role in controlling whole-plant ion movement rapidly after onset of salinity. PMID:27503602

Activity of the C-terminal-dependent vacuolar sorting signal of horseradish peroxidase C1a is enhanced by its secondary structure.

PubMed

Matsui, Takeshi; Tabayashi, Ayako; Iwano, Megumi; Shinmyo, Atsuhiko; Kato, Ko; Nakayama, Hideki

2011-02-01

Plant class III peroxidase (PRX) catalyzes the oxidation and oxidative polymerization of a variety of phenolic compounds while reducing hydrogen peroxide. PRX proteins are classified into apoplast type and vacuole type based on the absence or the presence of C-terminal propeptides, which probably function as vacuolar sorting signals (VSSs). In this study, in order to improve our understanding of vacuole-type PRX, we analyzed regulatory mechanisms of vacuolar sorting of a model vacuole-type PRX, the C1a isozyme of horseradish (Armoracia rusticana) (HRP C1a). Using cultured transgenic tobacco cells and protoplasts derived from horseradish leaves, we characterized HRP C1a's VSS, which is a 15 amino acid C-terminal propeptide (C15). We found that the C-terminal hexapeptide of C15 (C6), which is well conserved among vacuole-type PRX proteins, forms the core of the C-terminal-dependent VSS. We also found that the function of C6 is enhanced by the remaining N-terminal part of C15 which probably folds into an amphiphilic α-helix.

Precursor binding to an 880-kDa Toc complex as an early step during active import of protein into chloroplasts.

PubMed

Chen, Kuan-Yu; Li, Hsou-min

2007-01-01

The import of protein into chloroplasts is mediated by translocon components located in the chloroplast outer (the Toc proteins) and inner (the Tic proteins) envelope membranes. To identify intermediate steps during active import, we used sucrose density gradient centrifugation and blue-native polyacrylamide gel electrophoresis (BN-PAGE) to identify complexes of translocon components associated with precursor proteins under active import conditions instead of arrested binding conditions. Importing precursor proteins in solubilized chloroplast membranes formed a two-peak distribution in the sucrose density gradient. The heavier peak was in a similar position as the previously reported Tic/Toc supercomplex and was too large to be analyzed by BN-PAGE. The BN-PAGE analyses of the lighter peak revealed that precursors accumulated in at least two complexes. The first complex migrated at a position close to the ferritin dimer (approximately 880 kDa) and contained only the Toc components. Kinetic analyses suggested that this Toc complex represented an earlier step in the import process than the Tic/Toc supercomplex. The second complex in the lighter peak migrated at the position of the ferritin trimer (approximately 1320 kDa). It contained, in addition to the Toc components, Tic110, Hsp93, and an hsp70 homolog, but not Tic40. Two different precursor proteins were shown to associate with the same complexes. Processed mature proteins first appeared in the membranes at the same fractions as the Tic/Toc supercomplex, suggesting that processing of transit peptides occurs while precursors are still associated with the supercomplex.

Precursor binding to an 880-kDa Toc complex as an early step during active import of protein into chloroplasts

PubMed Central

Chen, Kuan-Yu; Li, Hsou-min

2007-01-01

The import of protein into chloroplasts is mediated by translocon components located in the chloroplast outer (the Toc proteins) and inner (the Tic proteins) envelope membranes. To identify intermediate steps during active import, we used sucrose density gradient centrifugation and blue-native polyacrylamide gel electrophoresis (BN-PAGE) to identify complexes of translocon components associated with precursor proteins under active import conditions instead of arrested binding conditions. Importing precursor proteins in solubilized chloroplast membranes formed a two-peak distribution in the sucrose density gradient. The heavier peak was in a similar position as the previously reported Tic/Toc supercomplex and was too large to be analyzed by BN-PAGE. The BN-PAGE analyses of the lighter peak revealed that precursors accumulated in at least two complexes. The first complex migrated at a position close to the ferritin dimer (approximately 880 kDa) and contained only the Toc components. Kinetic analyses suggested that this Toc complex represented an earlier step in the import process than the Tic/Toc supercomplex. The second complex in the lighter peak migrated at the position of the ferritin trimer (approximately 1320 kDa). It contained, in addition to the Toc components, Tic110, Hsp93, and an hsp70 homolog, but not Tic40. Two different precursor proteins were shown to associate with the same complexes. Processed mature proteins first appeared in the membranes at the same fractions as the Tic/Toc supercomplex, suggesting that processing of transit peptides occurs while precursors are still associated with the supercomplex. PMID:17144891

Molecular cloning and functional characterization of DkMATE1 involved in proanthocyanidin precursor transport in persimmon (Diospyros kaki Thunb.) fruit.

PubMed

Yang, Sichao; Jiang, Yun; Xu, Liqing; Shiratake, Katsuhiro; Luo, Zhengrong; Zhang, Qinglin

2016-11-01

Persimmon fruits accumulate a large amount of proanthocyanidins (PAs) in "tannin cells" during development that cause the sensation of astringency due to coagulation of oral proteins. Pollination-constant non-astringent (PCNA) is a spontaneous mutant persimmon phenotype that loses its astringency naturally on the tree at maturity; while the more common non-PCNA fruits remain rich in PAs until they are fully ripened. Here, we isolated a DkMATE1 gene encoding a Multidrug And Toxic Compound Extrusion (MATE) family protein from the Chinese PCNA (C-PCNA) 'Eshi 1'. Expression patterns of DkMATE1 were positively correlated with the accumulation of PAs in different types of persimmons fruits during fruit development. An analysis of the inferred amino acid sequences and phylogenetic relationships indicated that DkMATE1 is a putative PA precursor transporter, and subcellular localization assays revealed that DkMATE1 is localized in the vacuolar membrane. Ectopic expression of the DkMATE1 in Arabidopsis tt12 mutant supported that DkMATE1 could complement its biological function in transporting epicatechin 3'-O-glucoside as a PAs precursor from the cytoplasm to vacuole. Furthermore, the transient over-expression and silencing of DkMATE1 in 'Mopanshi' persimmon leaves resulted in a significant increase and a decrease in PA content, respectively. The analysis of cis-elements in DkMATE1 promoter regions indicated that DkMATE1 might be regulated by DkMYB4, another well-known structural gene in persimmon. Overall, our results show that DkMATE1 may be an essential PA precursor membrane transporter that plays an important role in PA biosynthesis in persimmon. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Expression analysis of Arabidopsis vacuolar sorting receptor 3 reveals a putative function in guard cells.

PubMed

Avila, Emily L; Brown, Michelle; Pan, Songqin; Desikan, Radhika; Neill, Steven J; Girke, Thomas; Surpin, Marci; Raikhel, Natasha V

2008-01-01

Vacuolar sorting receptors (VSRs) are responsible for the proper targeting of soluble cargo proteins to their destination compartments. The Arabidopsis genome encodes seven VSRs. In this work, the spatio-temporal expression of one of the members of this gene family, AtVSR3, was determined by RT-PCR and promoter::reporter gene fusions. AtVSR3 was expressed specifically in guard cells. Consequently, a reverse genetics approach was taken to determine the function of AtVSR3 by using RNA interference (RNAi) technology. Plants expressing little or no AtVSR3 transcript had a compressed life cycle, bolting approximately 1 week earlier and senescing up to 2 weeks earlier than the wild-type parent line. While the development and distribution of stomata in AtVSR3 RNAi plants appeared normal, stomatal function was altered. The guard cells of mutant plants did not close in response to abscisic acid treatment, and the mean leaf temperatures of the RNAi plants were on average 0.8 degrees C lower than both wild type and another vacuolar sorting receptor mutant, atvsr1-1. Furthermore, the loss of AtVSR3 protein caused the accumulation of nitric oxide and hydrogen peroxide, signalling molecules implicated in the regulation of stomatal opening and closing. Finally, proteomics and western blot analyses of cellular proteins isolated from wild-type and AtVSR3 RNAi leaves showed that phospholipase Dgamma, which may play a role in abscisic acid signalling, accumulated to higher levels in AtVSR3 RNAi guard cells. Thus, AtVSR3 may play an important role in responses to plant stress.

Functional Expression and Characterization of Schizosaccharomyces pombe Avt3p as a Vacuolar Amino Acid Exporter in Saccharomyces cerevisiae.

PubMed

Chardwiriyapreecha, Soracom; Manabe, Kunio; Iwaki, Tomoko; Kawano-Kawada, Miyuki; Sekito, Takayuki; Lunprom, Siriporn; Akiyama, Koichi; Takegawa, Kaoru; Kakinuma, Yoshimi

2015-01-01

In Saccharomyces cerevisiae, Avt3p and Avt4p mediate the extrusion of several amino acids from the vacuolar lumen into the cytosol. SpAvt3p of Schizosaccharomyces pombe, a homologue of these vacuolar amino acid transporters, has been indicated to be involved in spore formation. In this study, we confirmed that GFP-SpAvt3p localized to the vacuolar membrane in S. pombe. The amounts of various amino acids increased significantly in the vacuolar pool of avt3Δ cells, but decreased in that of avt3+-overexpressing avt3Δ cells. These results suggest that SpAvt3p participates in the vacuolar compartmentalization of amino acids in S. pombe. To examine the export activity of SpAvt3p, we expressed the avt3+ gene in S. cerevisiae cells. We found that the heterologously overproduced GFP-SpAvt3p localized to the vacuolar membrane in S. cerevisiae. Using the vacuolar membrane vesicles isolated from avt3+-overexpressing S. cerevisiae cells, we detected the export activities of alanine and tyrosine in an ATP-dependent manner. These activities were inhibited by the addition of a V-ATPase inhibitor, concanamycin A, thereby suggesting that the activity of SpAvt3p is dependent on a proton electrochemical gradient generated by the action of V-ATPase. In addition, the amounts of various amino acids in the vacuolar pools of S. cerevisiae cells were decreased by the overproduction of SpAvt3p, which indicated that SpAvt3p was functional in S. cerevisiae cells. Thus, SpAvt3p is a vacuolar transporter that is involved in the export of amino acids from S. pombe vacuoles.

Identification of two structurally related proteins involved in proteolytic processing of precursors targeted to the chloroplast.

PubMed Central

Oblong, J E; Lamppa, G K

1992-01-01

Two proteins of 145 and 143 kDa were identified in pea which co-purify with a chloroplast processing activity that cleaves the precursor for the major light-harvesting chlorophyll binding protein (preLHCP). Antiserum generated against the 145/143 kDa doublet recognizes only these two polypeptides in a chloroplast soluble extract. In immunodepletion experiments the antiserum removed the doublet, and there was a concomitant loss of cleavage of preLHCP as well as of precursors for the small subunit of Rubisco and the acyl carrier protein. The 145 and 143 kDa proteins co-eluted in parallel with the peak of processing activity during all fractionation procedures, but they were not detectable as a homo- or heterodimeric complex. The 145 and 143 kDa proteins were used separately to affinity purify immunoglobulins; each preparation recognized both polypeptides, indicating that they are antigenically related. Wheat chloroplasts contain a soluble species similar in size to the 145/143 kDa doublet. Images PMID:1385116

Regulation of Vacuolar pH in Citrus limon

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lincoln Taiz

The primary objective of this grant was to characterize the vacuolar V-ATPase of lemon fruits. Lemon fruit vacuoles have an internal pH of about 2.5. Since a typical plant vacuole has a luminal pH of around 5.5, the lemon fruit V-APTase must have special properties which allow it to acidify the lumen to such a low pH: (1) it might have a different structure; (2) it might have a different H{sup +}/ATP stoichiometry; and (3) it might be regulated differently. During the course of the investigations (which began in 1996) they characterized these aspects of the V-ATPases of both lemonmore » fruits and lime fruits. They examined lime fruits because of the availability of both acidic limes with a low vacuolar pH and sweet limes, which have a much higher vacuolar pH. The existence of two types of lime fruits allowed a comparison of the V-ATPases of the two varieties. In this report they are including two publications from 1996 and 1997 as background for the later publications. A review article with Heven Sze on V-ATPase nomenclature was also generated during the funding period. In addition to the studies on citrus fruit vacuoles, they also initiated studies in two new areas: polar auxin transport and the regulation of stomatal opening by UV-B irradiation. These studies were intended to serve as a basis of future separate grants, but the proposals they submitted on these topics were not funded.« less

vph6 mutants of Saccharomyces cerevisiae require calcineurin for growth and are defective in vacuolar H(+)-ATPase assembly.

PubMed

Hemenway, C S; Dolinski, K; Cardenas, M E; Hiller, M A; Jones, E W; Heitman, J

1995-11-01

We have characterized a Saccharomyces cerevisiae mutant strain that is hypersensitive to cyclosporin A (CsA) and FK506, immunosuppressants that inhibit calcineurin, a serine-threonine-specific phosphatase (PP2B). A single nuclear mutation, designated cev1 for calcineurin essential for viability, is responsible for the CsA-FK506-sensitive phenotype. The peptidyl-prolyl cis-trans isomerases cyclophilin A and FKBP12, respectively, mediate CsA and FK506 toxicity in the cev1 mutant strain. We demonstrate that cev1 is an allele of the VPH6 gene and that vph6 mutant strains fail to assemble the vacuolar H(+)-ATPase (V-ATPase). The VPH6 gene was mapped on chromosome VIII and is predicted to encode a 181-amino acid (21 kD) protein with no identity to other known proteins. We find that calcineurin is essential for viability in many mutant strains with defects in V-ATPase function or vacuolar acidification. In addition, we find that calcineurin modulates extracellular acidification in response to glucose, which we propose occurs via calcineurin regulation of the plasma membrane H(+)-ATPase PMA1. Taken together, our findings suggest calcineurin plays a general role in the regulation of cation transport and homeostasis.

Adaptor protein 2–mediated endocytosis of the β-secretase BACE1 is dispensable for amyloid precursor protein processing

PubMed Central

Prabhu, Yogikala; Burgos, Patricia V.; Schindler, Christina; Farías, Ginny G.; Magadár, Javier G.; Bonifacino, Juan S.

2012-01-01

The β-site amyloid precursor protein (APP)–cleaving enzyme 1 (BACE1) is a transmembrane aspartyl protease that catalyzes the proteolytic processing of APP and other plasma membrane protein precursors. BACE1 cycles between the trans-Golgi network (TGN), the plasma membrane, and endosomes by virtue of signals contained within its cytosolic C-terminal domain. One of these signals is the DXXLL-motif sequence DISLL, which controls transport between the TGN and endosomes via interaction with GGA proteins. Here we show that the DISLL sequence is embedded within a longer [DE]XXXL[LI]-motif sequence, DDISLL, which mediates internalization from the plasma membrane by interaction with the clathrin-associated, heterotetrameric adaptor protein 2 (AP-2) complex. Mutation of this signal or knockdown of either AP-2 or clathrin decreases endosomal localization and increases plasma membrane localization of BACE1. Remarkably, internalization-defective BACE1 is able to cleave an APP mutant that itself cannot be delivered to endosomes. The drug brefeldin A reversibly prevents BACE1-catalyzed APP cleavage, ruling out that this reaction occurs in the endoplasmic reticulum (ER) or ER–Golgi intermediate compartment. Taken together, these observations support the notion that BACE1 is capable of cleaving APP in late compartments of the secretory pathway. PMID:22553349

Vacuolar ATPase in Phagosome-Lysosome Fusion

PubMed Central

Kissing, Sandra; Hermsen, Christina; Repnik, Urska; Nesset, Cecilie Kåsi; von Bargen, Kristine; Griffiths, Gareth; Ichihara, Atsuhiro; Lee, Beth S.; Schwake, Michael; De Brabander, Jef; Haas, Albert; Saftig, Paul

2015-01-01

The vacuolar H+-ATPase (v-ATPase) complex is instrumental in establishing and maintaining acidification of some cellular compartments, thereby ensuring their functionality. Recently it has been proposed that the transmembrane V0 sector of v-ATPase and its a-subunits promote membrane fusion in the endocytic and exocytic pathways independent of their acidification functions. Here, we tested if such a proton-pumping independent role of v-ATPase also applies to phagosome-lysosome fusion. Surprisingly, endo(lyso)somes in mouse embryonic fibroblasts lacking the V0 a3 subunit of the v-ATPase acidified normally, and endosome and lysosome marker proteins were recruited to phagosomes with similar kinetics in the presence or absence of the a3 subunit. Further experiments used macrophages with a knockdown of v-ATPase accessory protein 2 (ATP6AP2) expression, resulting in a strongly reduced level of the V0 sector of the v-ATPase. However, acidification appeared undisturbed, and fusion between latex bead-containing phagosomes and lysosomes, as analyzed by electron microscopy, was even slightly enhanced, as was killing of non-pathogenic bacteria by V0 mutant macrophages. Pharmacologically neutralized lysosome pH did not affect maturation of phagosomes in mouse embryonic cells or macrophages. Finally, locking the two large parts of the v-ATPase complex together by the drug saliphenylhalamide A did not inhibit in vitro and in cellulo fusion of phagosomes with lysosomes. Hence, our data do not suggest a fusion-promoting role of the v-ATPase in the formation of phagolysosomes. PMID:25903133

Vacuolar ATPase in phagosome-lysosome fusion.

PubMed

Kissing, Sandra; Hermsen, Christina; Repnik, Urska; Nesset, Cecilie Kåsi; von Bargen, Kristine; Griffiths, Gareth; Ichihara, Atsuhiro; Lee, Beth S; Schwake, Michael; De Brabander, Jef; Haas, Albert; Saftig, Paul

2015-05-29

The vacuolar H(+)-ATPase (v-ATPase) complex is instrumental in establishing and maintaining acidification of some cellular compartments, thereby ensuring their functionality. Recently it has been proposed that the transmembrane V0 sector of v-ATPase and its a-subunits promote membrane fusion in the endocytic and exocytic pathways independent of their acidification functions. Here, we tested if such a proton-pumping independent role of v-ATPase also applies to phagosome-lysosome fusion. Surprisingly, endo(lyso)somes in mouse embryonic fibroblasts lacking the V0 a3 subunit of the v-ATPase acidified normally, and endosome and lysosome marker proteins were recruited to phagosomes with similar kinetics in the presence or absence of the a3 subunit. Further experiments used macrophages with a knockdown of v-ATPase accessory protein 2 (ATP6AP2) expression, resulting in a strongly reduced level of the V0 sector of the v-ATPase. However, acidification appeared undisturbed, and fusion between latex bead-containing phagosomes and lysosomes, as analyzed by electron microscopy, was even slightly enhanced, as was killing of non-pathogenic bacteria by V0 mutant macrophages. Pharmacologically neutralized lysosome pH did not affect maturation of phagosomes in mouse embryonic cells or macrophages. Finally, locking the two large parts of the v-ATPase complex together by the drug saliphenylhalamide A did not inhibit in vitro and in cellulo fusion of phagosomes with lysosomes. Hence, our data do not suggest a fusion-promoting role of the v-ATPase in the formation of phagolysosomes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Targeting vacuolar H+-ATPases as a new strategy against cancer.

PubMed

Fais, Stefano; De Milito, Angelo; You, Haiyan; Qin, Wenxin

2007-11-15

Growing evidence suggests a key role of tumor acidic microenvironment in cancer development, progression, and metastasis. As a consequence, the need for compounds that specifically target the mechanism(s) responsible for the low pH of tumors is increasing. Among the key regulators of the tumor acidic microenvironment, vacuolar H(+)-ATPases (V-ATPases) play an important role. These proteins cover a number of functions in a variety of normal as well as tumor cells, in which they pump ions across the membranes. We discuss here some recent results showing that a molecular inhibition of V-ATPases by small interfering RNA in vivo as well as a pharmacologic inhibition through proton pump inhibitors led to tumor cytotoxicity and marked inhibition of human tumor growth in xenograft models. These results propose V-ATPases as a key target for new strategies in cancer treatment.

Amyloid precursor protein interaction network in human testis: sentinel proteins for male reproduction.

PubMed

Silva, Joana Vieira; Yoon, Sooyeon; Domingues, Sara; Guimarães, Sofia; Goltsev, Alexander V; da Cruz E Silva, Edgar Figueiredo; Mendes, José Fernando F; da Cruz E Silva, Odete Abreu Beirão; Fardilha, Margarida

2015-01-16

Amyloid precursor protein (APP) is widely recognized for playing a central role in Alzheimer's disease pathogenesis. Although APP is expressed in several tissues outside the human central nervous system, the functions of APP and its family members in other tissues are still poorly understood. APP is involved in several biological functions which might be potentially important for male fertility, such as cell adhesion, cell motility, signaling, and apoptosis. Furthermore, APP superfamily members are known to be associated with fertility. Knowledge on the protein networks of APP in human testis and spermatozoa will shed light on the function of APP in the male reproductive system. We performed a Yeast Two-Hybrid screen and a database search to study the interaction network of APP in human testis and sperm. To gain insights into the role of APP superfamily members in fertility, the study was extended to APP-like protein 2 (APLP2). We analyzed several topological properties of the APP interaction network and the biological and physiological properties of the proteins in the APP interaction network were also specified by gene ontologyand pathways analyses. We classified significant features related to the human male reproduction for the APP interacting proteins and identified modules of proteins with similar functional roles which may show cooperative behavior for male fertility. The present work provides the first report on the APP interactome in human testis. Our approach allowed the identification of novel interactions and recognition of key APP interacting proteins for male reproduction, particularly in sperm-oocyte interaction.

Regulation of amyloid precursor protein processing by its KFERQ motif.

PubMed

Park, Ji-Seon; Kim, Dong-Hou; Yoon, Seung-Yong

2016-06-01

Understanding of trafficking, processing, and degradation mechanisms of amyloid precursor protein (APP) is important because APP can be processed to produce β-amyloid (Aβ), a key pathogenic molecule in Alzheimer's disease (AD). Here, we found that APP contains KFERQ motif at its C-terminus, a consensus sequence for chaperone-mediated autophagy (CMA) or microautophagy which are another types of autophagy for degradation of pathogenic molecules in neurodegenerative diseases. Deletion of KFERQ in APP increased C-terminal fragments (CTFs) and secreted N-terminal fragments of APP and kept it away from lysosomes. KFERQ deletion did not abolish the interaction of APP or its cleaved products with heat shock cognate protein 70 (Hsc70), a protein necessary for CMA or microautophagy. These findings suggest that KFERQ motif is important for normal processing and degradation of APP to preclude the accumulation of APP-CTFs although it may not be important for CMA or microautophagy. [BMB Reports 2016; 49(6): 337-342].

Plant polycistronic precursors containing non-homologous microRNAs target transcripts encoding functionally related proteins

PubMed Central

2009-01-01

Background MicroRNAs (miRNAs) are endogenous single-stranded small RNAs that regulate the expression of specific mRNAs involved in diverse biological processes. In plants, miRNAs are generally encoded as a single species in independent transcriptional units, referred to as MIRNA genes, in contrast to animal miRNAs, which are frequently clustered. Results We performed a comparative genomic analysis in three model plants (rice, poplar and Arabidopsis) and characterized miRNA clusters containing two to eight miRNA species. These clusters usually encode miRNAs of the same family and certain share a common evolutionary origin across monocot and dicot lineages. In addition, we identified miRNA clusters harboring miRNAs with unrelated sequences that are usually not evolutionarily conserved. Strikingly, non-homologous miRNAs from the same cluster were predicted to target transcripts encoding related proteins. At least four Arabidopsis non-homologous clusters were expressed as single transcriptional units. Overexpression of one of these polycistronic precursors, producing Ath-miR859 and Ath-miR774, led to the DCL1-dependent accumulation of both miRNAs and down-regulation of their different mRNA targets encoding F-box proteins. Conclusions In addition to polycistronic precursors carrying related miRNAs, plants also contain precursors allowing coordinated expression of non-homologous miRNAs to co-regulate functionally related target transcripts. This mechanism paves the way for using polycistronic MIRNA precursors as a new molecular tool for plant biologists to simultaneously control the expression of different genes. PMID:19951405

Ablation of Prion Protein in Wild Type Human Amyloid Precursor Protein (APP) Transgenic Mice Does Not Alter The Proteolysis of APP, Levels of Amyloid-β or Pathologic Phenotype

PubMed Central

Baybutt, Herbert; Diack, Abigail B.; Kellett, Katherine A. B.; Piccardo, Pedro; Manson, Jean C.

2016-01-01

The cellular prion protein (PrPC) has been proposed to play an important role in the pathogenesis of Alzheimer’s disease. In cellular models PrPC inhibited the action of the β-secretase BACE1 on wild type amyloid precursor protein resulting in a reduction in amyloid-β (Aβ) peptides. Here we have assessed the effect of genetic ablation of PrPC in transgenic mice expressing human wild type amyloid precursor protein (line I5). Deletion of PrPC had no effect on the α- and β-secretase proteolysis of the amyloid precursor protein (APP) nor on the amount of Aβ38, Aβ40 or Aβ42 in the brains of the mice. In addition, ablation of PrPC did not alter Aβ deposition or histopathology phenotype in this transgenic model. Thus using this transgenic model we could not provide evidence to support the hypothesis that PrPC regulates Aβ production. PMID:27447728

ABG1, a Novel and Essential Candida albicans Gene Encoding a Vacuolar Protein Involved in Cytokinesis and Hyphal Branching

PubMed Central

Veses, Verónica; Casanova, Manuel; Murgui, Amelia; Domínguez, Ángel; Gow, Neil A. R.; Martínez, José P.

2005-01-01

Immunoscreening of a Candida albicans expression library resulted in the isolation of a novel gene encoding a 32.9-kDa polypeptide (288 amino acids), with 27.7% homology to the product of Saccharomyces cerevisiae YGR106c, a putative vacuolar protein. Heterozygous mutants in this gene displayed an altered budding growth pattern, characterized by the formation of chains of buds, decreasingly in size towards the apex, without separation of the daughter buds. Consequently, this gene was designated ABG1. A conditional mutant for ABG1 with the remaining allele under the control of the MET3 promoter did not grow in the presence of methionine and cysteine, demonstrating that ABG1 was essential for viability. Western analysis revealed the presence of a major 32.9-kDa band, mainly in a particulate fraction (P40) enriched in vacuoles, and tagging with green fluorescent protein confirmed that Abg1p localized to the vacuole. Vacuole inheritance has been linked to the regulation of branching frequency in C. albicans. Under repressing conditions, the conditional mutant had an increased frequency of branching under hyphal inducing conditions and an altered sensitivity to substances that interfered with cell wall assembly. Repression of ABG1 in the conditional mutant strain caused disturbance of normal size and number of vacuoles both in yeast and mycelial cells and also in the asymmetric vacuole inheritance associated with the characteristic pattern of germ tubes and branching in C. albicans. These observations indicate that ABG1 plays a key role in vacuole biogenesis, cytokinesis, and hyphal branching. PMID:15947201

ABG1, a novel and essential Candida albicans gene encoding a vacuolar protein involved in cytokinesis and hyphal branching.

PubMed

Veses, Verónica; Casanova, Manuel; Murgui, Amelia; Domínguez, Angel; Gow, Neil A R; Martínez, José P

2005-06-01

Immunoscreening of a Candida albicans expression library resulted in the isolation of a novel gene encoding a 32.9-kDa polypeptide (288 amino acids), with 27.7% homology to the product of Saccharomyces cerevisiae YGR106c, a putative vacuolar protein. Heterozygous mutants in this gene displayed an altered budding growth pattern, characterized by the formation of chains of buds, decreasingly in size towards the apex, without separation of the daughter buds. Consequently, this gene was designated ABG1. A conditional mutant for ABG1 with the remaining allele under the control of the MET3 promoter did not grow in the presence of methionine and cysteine, demonstrating that ABG1 was essential for viability. Western analysis revealed the presence of a major 32.9-kDa band, mainly in a particulate fraction (P40) enriched in vacuoles, and tagging with green fluorescent protein confirmed that Abg1p localized to the vacuole. Vacuole inheritance has been linked to the regulation of branching frequency in C. albicans. Under repressing conditions, the conditional mutant had an increased frequency of branching under hyphal inducing conditions and an altered sensitivity to substances that interfered with cell wall assembly. Repression of ABG1 in the conditional mutant strain caused disturbance of normal size and number of vacuoles both in yeast and mycelial cells and also in the asymmetric vacuole inheritance associated with the characteristic pattern of germ tubes and branching in C. albicans. These observations indicate that ABG1 plays a key role in vacuole biogenesis, cytokinesis, and hyphal branching.

Overexpression of human virus surface glycoprotein precursors induces cytosolic unfolded protein response in Saccharomyces cerevisiae

PubMed Central

2011-01-01

Background The expression of human virus surface proteins, as well as other mammalian glycoproteins, is much more efficient in cells of higher eukaryotes rather than yeasts. The limitations to high-level expression of active viral surface glycoproteins in yeast are not well understood. To identify possible bottlenecks we performed a detailed study on overexpression of recombinant mumps hemagglutinin-neuraminidase (MuHN) and measles hemagglutinin (MeH) in yeast Saccharomyces cerevisiae, combining the analysis of recombinant proteins with a proteomic approach. Results Overexpressed recombinant MuHN and MeH proteins were present in large aggregates, were inactive and totally insoluble under native conditions. Moreover, the majority of recombinant protein was found in immature form of non-glycosylated precursors. Fractionation of yeast lysates revealed that the core of viral surface protein aggregates consists of MuHN or MeH disulfide-linked multimers involving eukaryotic translation elongation factor 1A (eEF1A) and is closely associated with small heat shock proteins (sHsps) that can be removed only under denaturing conditions. Complexes of large Hsps seem to be bound to aggregate core peripherally as they can be easily removed at high salt concentrations. Proteomic analysis revealed that the accumulation of unglycosylated viral protein precursors results in specific cytosolic unfolded protein response (UPR-Cyto) in yeast cells, characterized by different action and regulation of small Hsps versus large chaperones of Hsp70, Hsp90 and Hsp110 families. In contrast to most environmental stresses, in the response to synthesis of recombinant MuHN and MeH, only the large Hsps were upregulated whereas sHsps were not. Interestingly, the amount of eEF1A was also increased during this stress response. Conclusions Inefficient translocation of MuHN and MeH precursors through ER membrane is a bottleneck for high-level expression in yeast. Overexpression of these recombinant

Calmodulin-stimulated Ca(2+)-ATPases in the vacuolar and plasma membranes in cauliflower.

PubMed

Askerlund, P

1997-07-01

The subcellular locations of Ca(2+)-ATPases in the membranes of cauliflower (Brassica oleracea L.) inflorescences were investigated. After continuous sucrose gradient centrifugation a 111-kD calmodulin (CaM)-stimulated and caM-binding Ca(2+)-ATPase (BCA1; P. Askerlund [1996] Plant Physiol 110: 913-922; S. Malmström, P. Askerlund, M.G. Plamgren [1997] FEBS Lett 400: 324-328) comigrated with vacuolar membrane markers, whereas a 116-kD caM-binding Ca(2+)-ATPase co-migrated with a marker for the plasma membrane. The 116 kD Ca(2+)-ATPase was enriched in plasma membranes obtained by aqueous two-phase partitioning, which is in agreement with a plasma membrane location of this Ca(2+)-ATPase. Countercurrent distribution of a low-density intracellular membrane fraction in an aqueous two-phase system resulted in the separation of the endoplasmic reticulum and vacuolar membranes. The 111-kD Ca(2+)-ATPase co-migrated with a vacuolar membrane marker after countercurrent distribution but not with markers for the endoplasmic reticulum. A vacuolar membrane location of the 111-kD Ca(2+)-AtPase was further supported by experiments with isolated vacuoles from cauliflower: (a) Immunoblotting with an antibody against the 111-kD Ca(2+)-ATPase showed that it was associated with the vacuoles, and (b) ATP-dependent Ca2+ uptake by the intact vacuoles was found to be CaM stimulated and partly protonophore insensitive.

LC3 and p62 as diagnostic markers of drug-induced autophagic vacuolar cardiomyopathy: a study of 3 cases.

PubMed

Daniels, Brianne H; McComb, Rodney D; Mobley, Bret C; Gultekin, Sakir Humayun; Lee, Han S; Margeta, Marta

2013-07-01

Autophagic vacuolar cardiomyopathy is an underrecognized, but potentially fatal, complication of treatment with chloroquine (CQ) and its derivative hydroxychloroquine (HCQ), which are used as therapy for malaria and common connective tissue disorders. Currently, the diagnosis of autophagic vacuolar cardiomyopathy is established through an endomyocardial biopsy and requires electron microscopy, which is not widely available and has a significant potential for sampling error. Recently, we have reported that immunohistochemistry for autophagic markers LC3 and p62 can replace electron microscopy in the diagnosis of HCQ-induced and colchicine-induced autophagic vacuolar skeletal myopathies. In the current study, we use 3 cases of CQ-induced or HCQ-induced cardiomyopathy and 1 HCQ-treated control case to show that the same two markers can be used to diagnose autophagic vacuolar cardiomyopathies by light microscopy. CQ-induced or HCQ-induced autophagic vacuolar cardiomyopathy is not universally fatal, but successful treatment requires early detection. By lowering the barriers to diagnosis, the application of these immunohistochemical markers will decrease the number of misdiagnosed patients, thus increasing the likelihood of favorable clinical outcomes.

Natural Modulators of Amyloid-Beta Precursor Protein Processing

PubMed Central

Zhang, Can; Tanzi, Rudolph E.

2013-01-01

Alzheimer’s disease (AD) is a devastating neurodegenerative disease and the primary cause of dementia, with no cure currently available. The pathogenesis of AD is believed to be primarily driven by Aβ, the principal component of senile plaques. Aβ is an ~4 kDa peptide generated from the amyloid-β precursor protein (APP) through proteolytic secretases. Natural products, particularly those utilized in traditional Chinese medicine (TCM), have a long history alleviating common clinical disorders, including dementia. However, the cell/molecular pathways mediated by these natural products are largely unknown until recently when the underlying molecular mechanisms of the disorders begin to be elucidated. Here, the mechanisms with which natural products modulate the pathogenesis of AD are discussed, in particular, by focusing on their roles in the processing of APP. PMID:22998566

Molecular cloning, functional expression and subcellular localization of two putative vacuolar voltage-gated chloride channels in rice (Oryza sativa L.).

PubMed

Nakamura, Atsuko; Fukuda, Atsunori; Sakai, Shingo; Tanaka, Yoshiyuki

2006-01-01

We isolated two cDNA clones (OsCLC-1 and OsCLC-2) homologous to tobacco CLC-Nt1, which encodes a voltage-gated chloride channel, from rice (Oryza sativa L. ssp. japonica, cv. Nipponbare). The deduced amino acid sequences were highly conserved (87.9% identity with each other). Southern blot analysis of the rice genomic DNA revealed that OsCLC-1 and OsCLC-2 were single-copy genes on chromosomes 4 and 2, respectively. OsCLC-1 was expressed in most tissues, whereas OsCLC-2 was expressed only in the roots, nodes, internodes and leaf sheaths. The level of expression of OsCLC-1, but not of OsCLC-2, was increased by treatment with NaCl. Both genes could partly substitute for GEF1, which encodes the sole chloride channel in yeast, by restoring growth under ionic stress. These results indicate that both genes are chloride channel genes. The proteins from both genes were immunochemically detected in the tonoplast fraction. Tagged synthetic green fluorescent protein which was fused to OsCLC-1 or OsCLC-2 localized in the vacuolar membranes. These results indicate that the proteins may play a role in the transport of chloride ions across the vacuolar membrane. We isolated loss-of-function mutants of both genes from a panel of rice mutants produced by the insertion of a retrotransposon, Tos17, in the exon region, and found inhibition of growth at all life stages.

Vacuolar biogenesis and aquaporin expression at early germination of broad bean seeds.

PubMed

Novikova, Galina V; Tournaire-Roux, Colette; Sinkevich, Irina A; Lityagina, Snejana V; Maurel, Christophe; Obroucheva, Natalie

2014-09-01

A key event in seed germination is water uptake-mediated growth initiation in embryonic axes. Vicia faba var. minor (broad bean) seeds were used for studying cell growth, vacuolar biogenesis, expression and function of tonoplast water channel proteins (aquaporins) in embryonic axes during seed imbibition, radicle emergence and growth. Hypocotyl and radicle basal cells showed vacuole restoration from protein storage vacuoles, whereas de novo vacuole formation from provacuoles was observed in cells newly produced by root meristem. cDNA fragments of seven novel aquaporin isoforms including five Tonoplast Intrinsic Proteins (TIP) from three sub-types were amplified by PCR. The expression was probed using q-RT-PCR and when possible with isoform-specific antibodies. Decreased expression of TIP3s was associated to the transformation of protein storage vacuoles to vacuoles, whereas enhanced expression of a TIP2 homologue was closely linked to the fast cell elongation. Water channel functioning checked by inhibitory test with mercuric chloride showed closed water channels prior to growth initiation and active water transport into elongating cells. The data point to a crucial role of tonoplast aquaporins during germination, especially during growth of embryonic axes, due to accelerated water uptake and vacuole enlargement resulting in rapid cell elongation. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

The Loss of Vacuolar Protein Sorting 11 (vps11) Causes Retinal Pathogenesis in a Vertebrate Model of Syndromic Albinism

PubMed Central

Thomas, Jennifer L.; Vihtelic, Thomas S.; denDekker, Aaron D.; Willer, Gregory; Luo, Xixia; Murphy, Taylor R.; Gregg, Ronald G.; Hyde, David R.

2011-01-01

Purpose. To establish the zebrafish platinum mutant as a model for studying vision defects caused by syndromic albinism diseases such as Chediak-Higashi syndrome, Griscelli syndrome, and Hermansky-Pudlak syndrome (HPS). Methods. Bulked segregant analysis and candidate gene sequencing revealed that the zebrafish platinum mutation is a single-nucleotide insertion in the vps11 (vacuolar protein sorting 11) gene. Expression of vps11 was determined by RT-PCR and in situ hybridization. Mutants were analyzed for pigmentation defects and retinal disease by histology, immunohistochemistry, and transmission electron microscopy. Results. Phenocopy and rescue experiments determined that a loss of Vps11 results in the platinum phenotype. Expression of vps11 appeared ubiquitous during zebrafish development, with stronger expression in the developing retina and retinal pigmented epithelium (RPE). Zebrafish platinum mutants exhibited reduced pigmentation in the body and RPE; however, melanophore development, migration, and dispersion occurred normally. RPE, photoreceptors, and inner retinal neurons formed normally in zebrafish platinum mutants. However, a gradual loss of RPE, an absence of mature melanosomes, and the subsequent degradation of RPE/photoreceptor interdigitation was observed. Conclusions. These data show that Vps11 is not necessary for normal retinal development or initiation of melanin biosynthesis, but is essential for melanosome maturation and healthy maintenance of the RPE and photoreceptors. PMID:21330665

Proton Gradient-Driven Nickel Uptake by Vacuolar Membrane Vesicles of Saccharomyces cerevisiae

PubMed Central

Nishimura, Ken; Igarashi, Kazuei; Kakinuma, Yoshimi

1998-01-01

A vacuolar H+-ATPase-negative mutant of Saccharomyces cerevisiae was highly sensitive to nickel ion. Accumulation of nickel ion in the cells of this mutant of less than 60% of the value for the parent strain arrested growth, suggesting a role for this ATPase in sequestering nickel ion into vacuoles. An artificially imposed pH gradient (interior acid) induced transient nickel ion uptake by vacuolar membrane vesicles, which was inhibited by collapse of the pH difference but not of the membrane potential. Nickel ion transport into vacuoles in a pH gradient-dependent manner is thus important for its detoxification in yeast. PMID:9537401

Expression of amyloid-β protein and amyloid-β precursor protein after primary brain-stem injury in rats.

PubMed

Yang, Shudong; Sun, Rongchao; Zhou, Zhiyi; Zhou, Jing; Liang, Jiabei; Mu, Huijun

2014-09-01

Amyloid-β (Aβ) protein and its precursor, amyloid-β precursor protein (β-APP), have traditionally been used in the diagnosis of Alzheimer disease. Their use in diagnosis of traumatic brain injury by forensic analysis is becoming more widespread. However, to date, no reliable small animal model exists to evaluate these brain injury indicators. To address this, we have studied primary brain-stem injury in rats to assess the appearance of diffuse axonal injury in brain sections and correlate these findings with appearance of Aβ and relative β-APP mRNA levels. Using an EnVision 2-step immunohistochemical staining method to measure axon diameter, we found that there was significant difference in axon diameters within the medulla oblongata and several time points after brain injury, ranging from 3 to 24 hours. In addition, mRNA expression levels of β-APP increased following brain injury, peaking 3 hours following injury and decreasing back to baseline levels by 24 hours after injury. These results suggest that using immunohistochemistry and reverse transcription-polymerase chain reaction to detect changes in Aβ-associated axonal changes and β-APP mRNA levels, respectively, can be useful for the diagnosis of diffuse axonal injury during autopsy at early time points following fatal brain injury.

Identification of snake bradykinin-potentiating peptides (BPPs)-simile sequences in rat brain--Potential BPP-like precursor protein?

PubMed

Campeiro, Joana D'Arc; Neshich, Izabella P; Sant'Anna, Osvaldo A; Lopes, Robson; Ianzer, Danielle; Assakura, Marina T; Neshich, Goran; Hayashi, Mirian A F

2015-08-01

Bradykinin-potentiating peptides (BPPs) from the South American pit viper snake venom were the first natural inhibitors of the human angiotensin I-converting enzyme (ACE) described. The pioneer characterization of the BPPs precursor from the snake venom glands by our group showed for the first time the presence of the C-type natriuretic peptide (CNP) in this same viper precursor protein. The confirmation of the BPP/CNP expression in snake brain regions correlated with neuroendocrine functions stimulated us to pursue the physiological correlates of these vasoactive peptides in mammals. Notably, several snake toxins were shown to have endogenous physiological correlates in mammals. In the present work, we expressed in bacteria the BPPs domain of the snake venom gland precursor protein, and this purified recombinant protein was used to raise specific polyclonal anti-BPPs antibodies. The correspondent single protein band immune-recognized in adult rat brain cytosol was isolated by 2D-SDS/PAGE and/or HPLC, before characterization by MS fingerprint analysis, which identified this protein as superoxide dismutase (SOD, EC 1.15.1.1), a classically known enzyme with antioxidant activity and important roles in the blood pressure modulation. In silico analysis showed the exposition of the BPP-like peptide sequences on the surface of the 3D structure of rat SOD. These peptides were chemically synthesized to show the BPP-like biological activities in ex vivo and in vivo pharmacological bioassays. Taken together, our data suggest that SOD protein have the potential to be a source for putative BPP-like bioactive peptides, which once released may contribute to the blood pressure control in mammals. Copyright © 2015 Elsevier Inc. All rights reserved.

Heat-Treatment-Responsive Proteins in Different Developmental Stages of Tomato Pollen Detected by Targeted Mass Accuracy Precursor Alignment (tMAPA).

PubMed

Chaturvedi, Palak; Doerfler, Hannes; Jegadeesan, Sridharan; Ghatak, Arindam; Pressman, Etan; Castillejo, Maria Angeles; Wienkoop, Stefanie; Egelhofer, Volker; Firon, Nurit; Weckwerth, Wolfram

2015-11-06

Recently, we have developed a quantitative shotgun proteomics strategy called mass accuracy precursor alignment (MAPA). The MAPA algorithm uses high mass accuracy to bin mass-to-charge (m/z) ratios of precursor ions from LC-MS analyses, determines their intensities, and extracts a quantitative sample versus m/z ratio data alignment matrix from a multitude of samples. Here, we introduce a novel feature of this algorithm that allows the extraction and alignment of proteotypic peptide precursor ions or any other target peptide from complex shotgun proteomics data for accurate quantification of unique proteins. This strategy circumvents the problem of confusing the quantification of proteins due to indistinguishable protein isoforms by a typical shotgun proteomics approach. We applied this strategy to a comparison of control and heat-treated tomato pollen grains at two developmental stages, post-meiotic and mature. Pollen is a temperature-sensitive tissue involved in the reproductive cycle of plants and plays a major role in fruit setting and yield. By LC-MS-based shotgun proteomics, we identified more than 2000 proteins in total for all different tissues. By applying the targeted MAPA data-processing strategy, 51 unique proteins were identified as heat-treatment-responsive protein candidates. The potential function of the identified candidates in a specific developmental stage is discussed.

Functional characterization of a vacuolar invertase from Solanum lycopersicum: post-translational regulation by N-glycosylation and a proteinaceous inhibitor.

PubMed

Tauzin, Alexandra S; Sulzenbacher, Gerlind; Lafond, Mickael; Desseaux, Véronique; Reca, Ida Barbara; Perrier, Josette; Bellincampi, Daniela; Fourquet, Patrick; Lévêque, Christian; Giardina, Thierry

2014-06-01

Plant vacuolar invertases, which belong to family 32 of glycoside hydrolases (GH32), are key enzymes in sugar metabolism. They hydrolyse sucrose into glucose and fructose. The cDNA encoding a vacuolar invertase from Solanum lycopersicum (TIV-1) was cloned and heterologously expressed in Pichia pastoris. The functional role of four N-glycosylation sites in TIV-1 has been investigated by site-directed mutagenesis. Single mutations to Asp of residues Asn52, Asn119 and Asn184, as well as the triple mutant (Asn52, Asn119 and Asn184), lead to enzymes with reduced specific invertase activity and thermostability. Expression of the N516D mutant, as well as of the quadruple mutant (N52D, N119D, N184D and N516D) could not be detected, indicating that these mutations dramatically affected the folding of the protein. Our data indicate that N-glycosylation is important for TIV-1 activity and that glycosylation of N516 is crucial for recombinant enzyme stability. Using a functional genomics approach a new vacuolar invertase inhibitor of S. lycopersicum (SolyVIF) has been identified. SolyVIF cDNA was cloned and heterologously expressed in Escherichia coli. Specific interactions between SolyVIF and TIV-1 were investigated by an enzymatic approach and surface plasmon resonance (SPR). Finally, qRT-PCR analysis of TIV-1 and SolyVIF transcript levels showed a specific tissue and developmental expression. TIV-1 was mainly expressed in flowers and both genes were expressed in senescent leaves. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

The Flavivirus Precursor Membrane-Envelope Protein Complex: Structure and Maturation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Long; Lok, Shee-Mei; Yu, I-Mei

2008-09-17

Many viruses go through a maturation step in the final stages of assembly before being transmitted to another host. The maturation process of flaviviruses is directed by the proteolytic cleavage of the precursor membrane protein (prM), turning inert virus into infectious particles. We have determined the 2.2 angstrom resolution crystal structure of a recombinant protein in which the dengue virus prM is linked to the envelope glycoprotein E. The structure represents the prM-E heterodimer and fits well into the cryo-electron microscopy density of immature virus at neutral pH. The pr peptide {beta}-barrel structure covers the fusion loop in E, preventingmore » fusion with host cell membranes. The structure provides a basis for identifying the stages of its pH-directed conformational metamorphosis during maturation, ending with release of pr when budding from the host.« less

Increased Activity of the Vacuolar Monosaccharide Transporter TMT1 Alters Cellular Sugar Partitioning, Sugar Signaling, and Seed Yield in Arabidopsis1[OA

PubMed Central

Wingenter, Karina; Schulz, Alexander; Wormit, Alexandra; Wic, Stefan; Trentmann, Oliver; Hoermiller, Imke I.; Heyer, Arnd G.; Marten, Irene; Hedrich, Rainer; Neuhaus, H. Ekkehard

2010-01-01

The extent to which vacuolar sugar transport activity affects molecular, cellular, and developmental processes in Arabidopsis (Arabidopsis thaliana) is unknown. Electrophysiological analysis revealed that overexpression of the tonoplast monosaccharide transporter TMT1 in a tmt1-2::tDNA mutant led to increased proton-coupled monosaccharide import into isolated mesophyll vacuoles in comparison with wild-type vacuoles. TMT1 overexpressor mutants grew faster than wild-type plants on soil and in high-glucose (Glc)-containing liquid medium. These effects were correlated with increased vacuolar monosaccharide compartmentation, as revealed by nonaqueous fractionation and by chlorophyllab-binding protein1 and nitrate reductase1 gene expression studies. Soil-grown TMT1 overexpressor plants respired less Glc than wild-type plants and only about half the amount of Glc respired by tmt1-2::tDNA mutants. In sum, these data show that TMT activity in wild-type plants limits vacuolar monosaccharide loading. Remarkably, TMT1 overexpressor mutants produced larger seeds and greater total seed yield, which was associated with increased lipid and protein content. These changes in seed properties were correlated with slightly decreased nocturnal CO2 release and increased sugar export rates from detached source leaves. The SUC2 gene, which codes for a sucrose transporter that may be critical for phloem loading in leaves, has been identified as Glc repressed. Thus, the observation that SUC2 mRNA increased slightly in TMT1 overexpressor leaves, characterized by lowered cytosolic Glc levels than wild-type leaves, provided further evidence of a stimulated source capacity. In summary, increased TMT activity in Arabidopsis induced modified subcellular sugar compartmentation, altered cellular sugar sensing, affected assimilate allocation, increased the biomass of Arabidopsis seeds, and accelerated early plant development. PMID:20709831

Effect of vacuolar ATPase subunit H (VmaH) on cellular pH, asexual cycle, stress tolerance and virulence in Beauveria bassiana.

PubMed

Zhu, Jing; Zhu, Xiao-Guan; Ying, Sheng-Hua; Feng, Ming-Guang

2017-01-01

Vacuolar ATPase (V-ATPase) is a conserved multi-subunit protein complex that mediates intracellular acidification in fungi. Here we show functional diversity of V-ATPase subunit H (BbVmaH) in Beauveria bassiana, a filamentous fungal insect pathogen. Deletion of BbvmaH resulted in elevated vacuolar pH, increased Ca 2+ level in cytosol but not in vacuoles, accelerated culture acidification and reduced accumulation of extracellular ammonia. Aerial conidiation and submerged blastospore production were largely delayed and reduced in the deletion mutant, respectively, accompanied with a significant delay in conidial germination, alterations of conidia and blastospores in morphology, size and/or density, and severe growth defects in minimal media with different carbon and nitrogen sources. Despite null responses to osmotic, oxidative and cell wall perturbing stresses, the deletion mutant showed increased sensitivity to Ca 2+ , Zn 2+ and Cu 2+ during growth while its conidia were less tolerant to a wet-heat stress at 45°C and UV-B irradiation. Intracellular glycerol and mannitol contents also decreased significantly. Its virulence to Galleria mellonella larvae was significantly attenuated when conidia were topically applied for normal cuticle infection or injected into haemocoel for cuticle-bypassing infection. All phenotypic changes were restored by targeted gene complementation. Our results indicate that BbVmaH plays an important role in sustaining not only vacuolar acidification but also cytosolic calcium accumulation, ambient pH homeostasis, in vitro asexual cycle and virulence in B. bassiana. Copyright © 2016 Elsevier Inc. All rights reserved.

The amyloid precursor protein and postnatal neurogenesis/neuroregeneration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen Yanan; Tang, Bor Luen

2006-03-03

The amyloid precursor protein (APP) is the source of amyloid-beta (A{beta}) peptide, produced via its sequential cleavage {beta}- and {gamma}-secretases. Various biophysical forms of A{beta} (and the mutations of APP which results in their elevated levels) have been implicated in the etiology and early onset of Alzheimer's disease. APP's evolutionary conservation and the existence of APP-like isoforms (APLP1 and APLP2) which lack the A{beta} sequence, however, suggest that these might have important physiological functions that are unrelated to A{beta} production. Soluble N-terminal fragments of APP have been known to be neuroprotective, and the interaction of its cytoplasmic C-terminus with amore » myriad of proteins associates it with diverse processes such as axonal transport and transcriptional regulation. The notion for an essential postnatal function of APP has been demonstrated genetically, as mice deficient in both APP and APLP2 or all three APP isoforms exhibit early postnatal lethality and neuroanatomical abnormalities. Recent findings have also brought to light two possible functions of the APP family in Brain-regulation of neural progenitor cell proliferation and axonal outgrowth after injury. Interestingly, these two apparently related neurogenic/neuroregenerative functions of APP involve two separate domains of the molecule.« less

Herpes simplex virus interferes with amyloid precursor protein processing.

PubMed

Shipley, Suzanne J; Parkin, Edward T; Itzhaki, Ruth F; Dobson, Curtis B

2005-08-18

The early events underlying Alzheimer's disease (AD) remain uncertain, although environmental factors may be involved. Work in this laboratory has shown that the combination of herpes simplex virus type 1 (HSV1) in brain and carriage of the APOE-epsilon4 allele of the APOE gene strongly increases the risk of developing AD. The development of AD is thought to involve abnormal aggregation or deposition of a 39-43 amino acid protein--beta amyloid (Abeta)--within the brain. This is cleaved from the much larger transmembranal protein 'amyloid precursor protein' (APP). Any agent able to interfere directly with Abeta or APP metabolism may therefore have the capacity to contribute towards AD. One recent report showed that certain HSV1 glycoprotein peptides may aggregate like Abeta; a second study described a role for APP in transport of virus in squid axons. However to date the effects of acute herpesvirus infection on metabolism of APP in human neuronal-type cells have not been investigated. In order to find if HSV1 directly affects APP and its degradation, we have examined this protein from human neuroblastoma cells (normal and transfected with APP 695) infected with the virus, using Western blotting. We have found that acute HSV1 (and also HSV2) infection rapidly reduces full length APP levels--as might be expected--yet surprisingly markedly increases levels of a novel C-terminal fragment of APP of about 55 kDa. This band was not increased in cells treated with the protein synthesis inhibitor cycloheximide Herpes virus infection leads to rapid loss of full length APP from cells, yet also causes increased levels of a novel 55 kDa C-terminal APP fragment. These data suggest that infection can directly alter the processing of a transmembranal protein intimately linked to the aetiology of AD.

Herpes simplex virus interferes with amyloid precursor protein processing

PubMed Central

Shipley, Suzanne J; Parkin, Edward T; Itzhaki, Ruth F; Dobson, Curtis B

2005-01-01

Background The early events underlying Alzheimer's disease (AD) remain uncertain, although environmental factors may be involved. Work in this laboratory has shown that the combination of herpes simplex virus type 1 (HSV1) in brain and carriage of the APOE-ε4 allele of the APOE gene strongly increases the risk of developing AD. The development of AD is thought to involve abnormal aggregation or deposition of a 39–43 amino acid protein – β amyloid (Aβ) – within the brain. This is cleaved from the much larger transmembranal protein 'amyloid precursor protein' (APP). Any agent able to interfere directly with Aβ or APP metabolism may therefore have the capacity to contribute towards AD. One recent report showed that certain HSV1 glycoprotein peptides may aggregate like Aβ; a second study described a role for APP in transport of virus in squid axons. However to date the effects of acute herpesvirus infection on metabolism of APP in human neuronal-type cells have not been investigated. In order to find if HSV1 directly affects APP and its degradation, we have examined this protein from human neuroblastoma cells (normal and transfected with APP 695) infected with the virus, using Western blotting. Results We have found that acute HSV1 (and also HSV2) infection rapidly reduces full length APP levels – as might be expected – yet surprisingly markedly increases levels of a novel C-terminal fragment of APP of about 55 kDa. This band was not increased in cells treated with the protein synthesis inhibitor cycloheximide Conclusion Herpes virus infection leads to rapid loss of full length APP from cells, yet also causes increased levels of a novel 55 kDa C-terminal APP fragment. These data suggest that infection can directly alter the processing of a transmembranal protein intimately linked to the aetiology of AD. PMID:16109164

Chemical synthesis of the precursor molecule of the Aequorea green fluorescent protein, subsequent folding, and development of fluorescence

PubMed Central

Nishiuchi, Yuji; Inui, Tatsuya; Nishio, Hideki; Bódi, József; Kimura, Terutoshi; Tsuji, Frederick I.; Sakakibara, Shumpei

1998-01-01

The present paper describes the total chemical synthesis of the precursor molecule of the Aequorea green fluorescent protein (GFP). The molecule is made up of 238 amino acid residues in a single polypeptide chain and is nonfluorescent. To carry out the synthesis, a procedure, first described in 1981 for the synthesis of complex peptides, was used. The procedure is based on performing segment condensation reactions in solution while providing maximum protection to the segment. The effectiveness of the procedure has been demonstrated by the synthesis of various biologically active peptides and small proteins, such as human angiogenin, a 123-residue protein analogue of ribonuclease A, human midkine, a 121-residue protein, and pleiotrophin, a 136-residue protein analogue of midkine. The GFP precursor molecule was synthesized from 26 fully protected segments in solution, and the final 238-residue peptide was treated with anhydrous hydrogen fluoride to obtain the precursor molecule of GFP containing two Cys(acetamidomethyl) residues. After removal of the acetamidomethyl groups, the product was dissolved in 0.1 M Tris⋅HCl buffer (pH 8.0) in the presence of DTT. After several hours at room temperature, the solution began to emit a green fluorescence (λmax = 509 nm) under near-UV light. Both fluorescence excitation and fluorescence emission spectra were measured and were found to have the same shape and maxima as those reported for native GFP. The present results demonstrate the utility of the segment condensation procedure in synthesizing large protein molecules such as GFP. The result also provides evidence that the formation of the chromophore in GFP is not dependent on any external cofactor. PMID:9811837

Job Sharing in the Endomembrane System: Vacuolar Acidification Requires the Combined Activity of V-ATPase and V-PPase.

PubMed

Kriegel, Anne; Andrés, Zaida; Medzihradszky, Anna; Krüger, Falco; Scholl, Stefan; Delang, Simon; Patir-Nebioglu, M Görkem; Gute, Gezahegn; Yang, Haibing; Murphy, Angus S; Peer, Wendy Ann; Pfeiffer, Anne; Krebs, Melanie; Lohmann, Jan U; Schumacher, Karin

2015-12-01

The presence of a large central vacuole is one of the hallmarks of a prototypical plant cell, and the multiple functions of this compartment require massive fluxes of molecules across its limiting membrane, the tonoplast. Transport is assumed to be energized by the membrane potential and the proton gradient established by the combined activity of two proton pumps, the vacuolar H(+)-pyrophosphatase (V-PPase) and the vacuolar H(+)-ATPase (V-ATPase). Exactly how labor is divided between these two enzymes has remained elusive. Here, we provide evidence using gain- and loss-of-function approaches that lack of the V-ATPase cannot be compensated for by increased V-PPase activity. Moreover, we show that increased V-ATPase activity during cold acclimation requires the presence of the V-PPase. Most importantly, we demonstrate that a mutant lacking both of these proton pumps is conditionally viable and retains significant vacuolar acidification, pointing to a so far undetected contribution of the trans-Golgi network/early endosome-localized V-ATPase to vacuolar pH. © 2015 American Society of Plant Biologists. All rights reserved.

Phosphatidylinositol 3-Kinase Promotes V-ATPase Activation and Vacuolar Acidification and Delays Methyl Jasmonate-Induced Leaf Senescence1

PubMed Central

Liu, Jian; Ji, Yingbin; Zhou, Jun; Xing, Da

2016-01-01

PI3K and its product PI3P are both involved in plant development and stress responses. In this study, the down-regulation of PI3K activity accelerated leaf senescence induced by methyl jasmonate (MeJA) and suppressed the activation of vacuolar H+-ATPase (V-ATPase). Yeast two-hybrid analyses indicated that PI3K bound to the V-ATPase B subunit (VHA-B). Analysis of bimolecular fluorescence complementation in tobacco guard cells showed that PI3K interacted with VHA-B2 in the tonoplasts. Through the use of pharmacological and genetic tools, we found that PI3K and V-ATPase promoted vacuolar acidification and stomatal closure during leaf senescence. Vacuolar acidification was suppressed by the PIKfyve inhibitor in 35S:AtVPS34-YFP Arabidopsis during MeJA-induced leaf senescence, but the decrease was lower than that in YFP-labeled Arabidopsis. These results suggest that PI3K promotes V-ATPase activation and consequently induces vacuolar acidification and stomatal closure, thereby delaying MeJA-induced leaf senescence. PMID:26739232

Genome-wide screen in Saccharomyces cerevisiae identifies vacuolar protein sorting, autophagy, biosynthetic, and tRNA methylation genes involved in life span regulation.

PubMed

Fabrizio, Paola; Hoon, Shawn; Shamalnasab, Mehrnaz; Galbani, Abdulaye; Wei, Min; Giaever, Guri; Nislow, Corey; Longo, Valter D

2010-07-15

The study of the chronological life span of Saccharomyces cerevisiae, which measures the survival of populations of non-dividing yeast, has resulted in the identification of homologous genes and pathways that promote aging in organisms ranging from yeast to mammals. Using a competitive genome-wide approach, we performed a screen of a complete set of approximately 4,800 viable deletion mutants to identify genes that either increase or decrease chronological life span. Half of the putative short-/long-lived mutants retested from the primary screen were confirmed, demonstrating the utility of our approach. Deletion of genes involved in vacuolar protein sorting, autophagy, and mitochondrial function shortened life span, confirming that respiration and degradation processes are essential for long-term survival. Among the genes whose deletion significantly extended life span are ACB1, CKA2, and TRM9, implicated in fatty acid transport and biosynthesis, cell signaling, and tRNA methylation, respectively. Deletion of these genes conferred heat-shock resistance, supporting the link between life span extension and cellular protection observed in several model organisms. The high degree of conservation of these novel yeast longevity determinants in other species raises the possibility that their role in senescence might be conserved.

Toxoplasma gondii sequesters lysosomes from mammalian hosts in the vacuolar space.

PubMed

Coppens, Isabelle; Dunn, Joe Dan; Romano, Julia D; Pypaert, Marc; Zhang, Hui; Boothroyd, John C; Joiner, Keith A

2006-04-21

The intracellular compartment harboring Toxoplasma gondii satisfies the parasite's nutritional needs for rapid growth in mammalian cells. We demonstrate that the parasitophorous vacuole (PV) of T. gondii accumulates material coming from the host mammalian cell via the exploitation of the host endo-lysosomal system. The parasite actively recruits host microtubules, resulting in selective attraction of endo-lysosomes to the PV. Microtubule-based invaginations of the PV membrane serve as conduits for the delivery of host endo-lysosomes within the PV. These tubular conduits are decorated by a parasite coat, including the tubulogenic protein GRA7, which acts like a garrote that sequesters host endocytic organelles in the vacuolar space. These data define an unanticipated process allowing the parasite intimate and concentrated access to a diverse range of low molecular weight components produced by the endo-lysosomal system. More generally, they identify a unique mechanism for unidirectional transport and sequestration of host organelles.

A vacuolar iron-transporter homologue acts as a detoxifier in Plasmodium

PubMed Central

Slavic, Ksenija; Krishna, Sanjeev; Lahree, Aparajita; Bouyer, Guillaume; Hanson, Kirsten K.; Vera, Iset; Pittman, Jon K.; Staines, Henry M.; Mota, Maria M.

2016-01-01

Iron is an essential micronutrient but is also highly toxic. In yeast and plant cells, a key detoxifying mechanism involves iron sequestration into intracellular storage compartments, mediated by members of the vacuolar iron-transporter (VIT) family of proteins. Here we study the VIT homologue from the malaria parasites Plasmodium falciparum (PfVIT) and Plasmodium berghei (PbVIT). PfVIT-mediated iron transport in a yeast heterologous expression system is saturable (Km∼14.7 μM), and selective for Fe2+ over other divalent cations. PbVIT-deficient P. berghei lines (Pbvit−) show a reduction in parasite load in both liver and blood stages of infection in mice. Moreover, Pbvit− parasites have higher levels of labile iron in blood stages and are more sensitive to increased iron levels in liver stages, when compared with wild-type parasites. Our data are consistent with Plasmodium VITs playing a major role in iron detoxification and, thus, normal development of malaria parasites in their mammalian host. PMID:26786069

A vacuolar iron-transporter homologue acts as a detoxifier in Plasmodium.

PubMed

Slavic, Ksenija; Krishna, Sanjeev; Lahree, Aparajita; Bouyer, Guillaume; Hanson, Kirsten K; Vera, Iset; Pittman, Jon K; Staines, Henry M; Mota, Maria M

2016-01-20

Iron is an essential micronutrient but is also highly toxic. In yeast and plant cells, a key detoxifying mechanism involves iron sequestration into intracellular storage compartments, mediated by members of the vacuolar iron-transporter (VIT) family of proteins. Here we study the VIT homologue from the malaria parasites Plasmodium falciparum (PfVIT) and Plasmodium berghei (PbVIT). PfVIT-mediated iron transport in a yeast heterologous expression system is saturable (Km ∼ 14.7 μM), and selective for Fe(2+) over other divalent cations. PbVIT-deficient P. berghei lines (Pbvit(-)) show a reduction in parasite load in both liver and blood stages of infection in mice. Moreover, Pbvit(-) parasites have higher levels of labile iron in blood stages and are more sensitive to increased iron levels in liver stages, when compared with wild-type parasites. Our data are consistent with Plasmodium VITs playing a major role in iron detoxification and, thus, normal development of malaria parasites in their mammalian host.

Nerve Growth Factor Increases mRNA Levels for the Prion Protein and the β -amyloid Protein Precursor in Developing Hamster Brain

NASA Astrophysics Data System (ADS)

Mobley, William C.; Neve, Rachael L.; Prusiner, Stanley B.; McKinley, Michael P.

1988-12-01

Deposition of amyloid filaments serves as a pathologic hallmark for some neurodegenerative disorders. The prion protein (PrP) is found in amyloid of animals with scrapie and humans with Creutzfeldt-Jakob disease; the β protein is present in amyloid deposits in Alzheimer disease and Down syndrome patients. These two proteins are derived from precursors that in the brain are expressed primarily in neurons and are membrane bound. We found that gene expression for PrP and the β -protein precursor (β -PP) is regulated in developing hamster brain. Specific brain regions showed distinct patterns of ontogenesis for PrP and β -PP mRNAs. The increases in PrP and β -PP mRNAs in developing basal forebrain coincided with an increase in choline acetyltransferase activity, raising the possibility that these markers might be coordinately controlled in cholinergic neurons and regulated by nerve growth factor (NGF). Injections of NGF into the brains of neonatal hamsters increased both PrP and β -PP mRNA levels. Increased PrP and β -PP mRNA levels induced by NGF were confined to regions that contain NGF-responsive cholinergic neurons and were accompanied by elevations in choline acetyltransferase. It remains to be established whether or not exogenous NGF acts to increase PrP and β -PP gene expression selectively in forebrain cholinergic neurons in the developing hamster and endogenous NGF regulates expression of these genes.

Peptide reranking with protein-peptide correspondence and precursor peak intensity information.

PubMed

Yang, Chao; He, Zengyou; Yang, Can; Yu, Weichuan

2012-01-01

Searching tandem mass spectra against a protein database has been a mainstream method for peptide identification. Improving peptide identification results by ranking true Peptide-Spectrum Matches (PSMs) over their false counterparts leads to the development of various reranking algorithms. In peptide reranking, discriminative information is essential to distinguish true PSMs from false PSMs. Generally, most peptide reranking methods obtain discriminative information directly from database search scores or by training machine learning models. Information in the protein database and MS1 spectra (i.e., single stage MS spectra) is ignored. In this paper, we propose to use information in the protein database and MS1 spectra to rerank peptide identification results. To quantitatively analyze their effects to peptide reranking results, three peptide reranking methods are proposed: PPMRanker, PPIRanker, and MIRanker. PPMRanker only uses Protein-Peptide Map (PPM) information from the protein database, PPIRanker only uses Precursor Peak Intensity (PPI) information, and MIRanker employs both PPM information and PPI information. According to our experiments on a standard protein mixture data set, a human data set and a mouse data set, PPMRanker and MIRanker achieve better peptide reranking results than PetideProphet, PeptideProphet+NSP (number of sibling peptides) and a score regularization method SRPI. The source codes of PPMRanker, PPIRanker, and MIRanker, and all supplementary documents are available at our website: http://bioinformatics.ust.hk/pepreranking/. Alternatively, these documents can also be downloaded from: http://sourceforge.net/projects/pepreranking/.

A single amino acid difference between the intracellular domains of amyloid precursor protein and amyloid-like precursor protein 2 enables induction of synaptic depression and block of long-term potentiation.

PubMed

Trillaud-Doppia, Emilie; Paradis-Isler, Nicolas; Boehm, Jannic

2016-07-01

Alzheimer disease (AD) is initially characterized as a disease of the synapse that affects synaptic transmission and synaptic plasticity. While amyloid-beta and tau have been traditionally implicated in causing AD, recent studies suggest that other factors, such as the intracellular domain of the amyloid-precursor protein (APP-ICD), can also play a role in the development of AD. Here, we show that the expression of APP-ICD induces synaptic depression, while the intracellular domain of its homolog amyloid-like precursor protein 2 (APLP2-ICD) does not. We are able to show that this effect by APP-ICD is due to a single alanine vs. proline difference between APP-ICD and APLP2-ICD. The alanine in APP-ICD and the proline in APLP2-ICD lie directly behind a conserved caspase cleavage site. Inhibition of caspase cleavage of APP-ICD prevents the induction of synaptic depression. Finally, we show that the expression of APP-ICD increases and facilitates long-term depression and blocks induction of long-term potentiation. The block in long-term potentiation can be overcome by mutating the aforementioned alanine in APP-ICD to the proline of APLP2. Based on our results, we propose the emergence of a new APP critical domain for the regulation of synaptic plasticity and in consequence for the development of AD. Copyright © 2016 Elsevier Inc. All rights reserved.

AtALMT9 is a malate-activated vacuolar chloride channel required for stomatal opening in Arabidopsis

PubMed Central

De Angeli, Alexis; Zhang, Jingbo; Meyer, Stefan; Martinoia, Enrico

2013-01-01

Water deficit strongly affects crop productivity. Plants control water loss and CO2 uptake by regulating the aperture of the stomatal pores within the leaf epidermis. Stomata aperture is regulated by the two guard cells forming the pore and changing their size in response to ion uptake and release. While our knowledge about potassium and chloride fluxes across the plasma membrane of guard cells is advanced, little is known about fluxes across the vacuolar membrane. Here we present the molecular identification of the long-sought-after vacuolar chloride channel. AtALMT9 is a chloride channel activated by physiological concentrations of cytosolic malate. Single-channel measurements demonstrate that this activation is due to a malate-dependent increase in the channel open probability. Arabidopsis thaliana atalmt9 knockout mutants exhibited impaired stomatal opening and wilt more slowly than the wild type. Our findings show that AtALMT9 is a vacuolar chloride channel having a major role in controlling stomata aperture. PMID:23653216

Dissection of autophagy in tobacco BY-2 cells under sucrose starvation conditions using the vacuolar H+-ATPase inhibitor concanamycin A and the autophagy-related protein Atg8

PubMed Central

Yano, Kanako; Yanagisawa, Takahiro; Mukae, Kyosuke; Niwa, Yasuo; Inoue, Yuko; Moriyasu, Yuji

2015-01-01

Tobacco BY-2 cells undergo autophagy in sucrose-free culture medium, which is the process mostly responsible for intracellular protein degradation under these conditions. Autophagy was inhibited by the vacuolar H+-ATPase inhibitors concanamycin A and bafilomycin A1, which caused the accumulation of autophagic bodies in the central vacuoles. Such accumulation did not occur in the presence of the autophagy inhibitor 3-methyladenine, and concanamycin in turn inhibited the accumulation of autolysosomes in the presence of the cysteine protease inhibitor E-64c. Electron microscopy revealed not only that the autophagic bodies were accumulated in the central vacuole, but also that autophagosome-like structures were more frequently observed in the cytoplasm in treatments with concanamycin, suggesting that concanamycin affects the morphology of autophagosomes in addition to raising the pH of the central vacuole. Using BY-2 cells that constitutively express a fusion protein of autophagosome marker protein Atg8 and green fluorescent protein (GFP), we observed the appearance of autophagosomes by fluorescence microscopy, which is a reliable morphological marker of autophagy, and the processing of the fusion protein to GFP, which is a biochemical marker of autophagy. Together, these results suggest the involvement of vacuole type H+-ATPase in the maturation step of autophagosomes to autolysosomes in the autophagic process of BY-2 cells. The accumulation of autophagic bodies in the central vacuole by concanamycin is a marker of the occurrence of autophagy; however, it does not necessarily mean that the central vacuole is the site of cytoplasm degradation. PMID:26368310

Dissection of autophagy in tobacco BY-2 cells under sucrose starvation conditions using the vacuolar H(+)-ATPase inhibitor concanamycin A and the autophagy-related protein Atg8.

PubMed

Yano, Kanako; Yanagisawa, Takahiro; Mukae, Kyosuke; Niwa, Yasuo; Inoue, Yuko; Moriyasu, Yuji

2015-01-01

Tobacco BY-2 cells undergo autophagy in sucrose-free culture medium, which is the process mostly responsible for intracellular protein degradation under these conditions. Autophagy was inhibited by the vacuolar H(+)-ATPase inhibitors concanamycin A and bafilomycin A1, which caused the accumulation of autophagic bodies in the central vacuoles. Such accumulation did not occur in the presence of the autophagy inhibitor 3-methyladenine, and concanamycin in turn inhibited the accumulation of autolysosomes in the presence of the cysteine protease inhibitor E-64c. Electron microscopy revealed not only that the autophagic bodies were accumulated in the central vacuole, but also that autophagosome-like structures were more frequently observed in the cytoplasm in treatments with concanamycin, suggesting that concanamycin affects the morphology of autophagosomes in addition to raising the pH of the central vacuole. Using BY-2 cells that constitutively express a fusion protein of autophagosome marker protein Atg8 and green fluorescent protein (GFP), we observed the appearance of autophagosomes by fluorescence microscopy, which is a reliable morphological marker of autophagy, and the processing of the fusion protein to GFP, which is a biochemical marker of autophagy. Together, these results suggest the involvement of vacuole type H(+)-ATPase in the maturation step of autophagosomes to autolysosomes in the autophagic process of BY-2 cells. The accumulation of autophagic bodies in the central vacuole by concanamycin is a marker of the occurrence of autophagy; however, it does not necessarily mean that the central vacuole is the site of cytoplasm degradation.

Hitchhiking vesicular transport routes to the vacuole: Amyloid recruitment to the Insoluble Protein Deposit (IPOD)

PubMed Central

Kumar, Rajesh; Neuser, Nicole; Tyedmers, Jens

2017-01-01

ABSTRACT Sequestration of aggregates into specialized deposition sites occurs in many species across all kingdoms of life ranging from bacteria to mammals and is commonly believed to have a cytoprotective function. Yeast cells possess at least 3 different spatially separated deposition sites, one of which is termed “Insoluble Protein Deposit (IPOD)” and harbors amyloid aggregates. We have recently discovered that recruitment of amyloid aggregates to the IPOD uses an actin cable based recruitment machinery that also involves vesicular transport.1 Here we discuss how different proteins known to be involved in vesicular transport processes to the vacuole might act to guide amyloid aggregates to the IPOD. These factors include the Myosin V motor protein Myo2 involved in transporting vacuolar vesicles along actin cables, the transmembrane protein Atg9 involved in the recruitment of large precursor hydrolase complexes to the vacuole, the phosphatidylinositol/ phosphatidylcholine (PI/PC) transfer protein Sec 14 and the SNARE chaperone Sec 18. Furthermore, we present new data suggesting that the yeast dynamin homolog Vps1 is also crucial for faithful delivery of the amyloid model protein PrD-GFP to the IPOD. This is in agreement with a previously identified role for Vps1 in recruitment of heat-denatured aggregates to a perivacuolar deposition site.2 PMID:28277942

The Role of the Progressive Ankylosis Protein (ANK) in Adipogenic/Osteogenic Fate Decision of Precursor Cells

PubMed Central

Minashima, Takeshi; Quirno, Martin; Lee, You Jin; Kirsch, Thorsten

2017-01-01

The progressive ankylosis protein (ANK) is a transmembrane protein that transports intracellular pyrophosphate (PPi) to the extracellular milieu. In this study we show increased fatty degeneration of the bone marrow of adult ank/ank mice, which lack a functional ANK protein. In addition, isolated bone marrow stromal cells (BMSCs) isolated from ank/ank mice showed a decreased proliferation rate and osteogenic differentiation potential, and an increased adipogenic differentiation potential compared to BMSCs isolated from wild type (WT) littermates. Wnt signaling pathway PCR array analysis revealed that Wnt ligands, Wnt receptors and Wnt signaling proteins that stimulate osteoblast differentiation were expressed at markedly lower levels in ank/ank BMSCs than in WT BMSCs. Lack of ANK function also resulted in impaired bone fracture healing, as indicated by a smaller callus formed and delayed bone formation in the callus site. Whereas 5 weeks after fracture, the fractured bone in WT mice was further remodeled and restored to original shape, the fractured bone in ank/ank mice was not fully restored and remodeled to original shape. In conclusion, our study provides evidence that ANK plays a critical role in the adipogenic/osteogenic fate decision of adult mesenchymal precursor cells. ANK functions in precursor cells are required for osteogenic differentiation of these cells during adult bone homeostasis and repair, whereas lack of ANK functions favors adipogenic differentiation. PMID:28286238

Split2 Protein-Ligation Generates Active IL-6-Type Hyper-Cytokines from Inactive Precursors.

PubMed

Moll, Jens M; Wehmöller, Melanie; Frank, Nils C; Homey, Lisa; Baran, Paul; Garbers, Christoph; Lamertz, Larissa; Axelrod, Jonathan H; Galun, Eithan; Mootz, Henning D; Scheller, Jürgen

2017-12-15

Trans-signaling of the major pro- and anti-inflammatory cytokines Interleukin (IL)-6 and IL-11 has the unique feature to virtually activate all cells of the body and is critically involved in chronic inflammation and regeneration. Hyper-IL-6 and Hyper-IL-11 are single chain designer trans-signaling cytokines, in which the cytokine and soluble receptor units are trapped in one complex via a flexible peptide linker. Albeit, Hyper-cytokines are essential tools to study trans-signaling in vitro and in vivo, the superior potency of these designer cytokines are accompanied by undesirable stress responses. To enable tailor-made generation of Hyper-cytokines, we developed inactive split-cytokine-precursors adapted for posttranslational reassembly by split-intein mediated protein trans-splicing (PTS). We identified cutting sites within IL-6 (E 134 /S 135 ) and IL-11 (G 116 /S 117 ) and obtained inactive split-Hyper-IL-6 and split-Hyper-IL-11 cytokine precursors. After fusion with split-inteins, PTS resulted in reconstitution of active Hyper-cytokines, which were efficiently secreted from transfected cells. Our strategy comprises the development of a background-free cytokine signaling system from reversibly inactivated precursor cytokines.

Nanoscale domain formation of phosphatidylinositol 4-phosphate in the plasma and vacuolar membranes of living yeast cells.

PubMed

Tomioku, Kan-Na; Shigekuni, Mikiko; Hayashi, Hiroki; Yoshida, Akane; Futagami, Taiki; Tamaki, Hisanori; Tanabe, Kenji; Fujita, Akikazu

2018-05-01

In budding yeast Saccharomyces cerevisiae, PtdIns(4)P serves as an essential signalling molecule in the Golgi complex, endosomal system, and plasma membrane, where it is involved in the control of multiple cellular functions via direct interactions with PtdIns(4)P-binding proteins. To analyse the distribution of PtdIns(4)P in yeast cells at a nanoscale level, we employed an electron microscopy technique that specifically labels PtdIns(4)P on the freeze-fracture replica of the yeast membrane. This method minimizes the possibility of artificial perturbation, because molecules in the membrane are physically immobilised in situ. We observed that PtdIns(4)P is localised on the cytoplasmic leaflet, but not the exoplasmic leaflet, of the plasma membrane, Golgi body, vacuole, and vesicular structure membranes. PtdIns(4)P labelling was not observed in the membrane of the endoplasmic reticulum, and in the outer and inner membranes of the nuclear envelope or mitochondria. PtdIns(4)P forms clusters of <100 nm in diameter in the plasma membrane and vacuolar membrane according to point pattern analysis of immunogold labelling. There are three kinds of compartments in the cytoplasmic leaflet of the plasma membrane. In the present study, we showed that PtdIns(4)P is specifically localised in the flat undifferentiated plasma membrane compartment. In the vacuolar membrane, PtdIns(4)P was concentrated in intramembrane particle (IMP)-deficient raft-like domains, which are tightly bound to lipid droplets, but not surrounding IMP-rich non-raft domains in geometrical IMP-distributed patterns in the stationary phase. This is the first report showing microdomain formations of PtdIns(4)P in the plasma membrane and vacuolar membrane of budding yeast cells at a nanoscale level, which will illuminate the functionality of PtdIns(4)P in each membrane. Copyright © 2018 Elsevier GmbH. All rights reserved.

Incorporation of DNA and Protein Precursors into Macromolecules by Bacteria at −15oC

PubMed Central

Christner, Brent C.

2002-01-01

DNA and protein precursors were incorporated into trichloroacetic acid-precipitated material by bacterial cell suspensions during incubation for 50 to 100 days at −15oC. Incorporation did not occur at −70oC and was inhibited by antibiotics. The results demonstrate that bacteria can perform macromolecular synthesis under conditions that mimic entrapment in glacial ice. PMID:12450874

Amyloid precursor protein modulates ERK-1 and -2 signaling.

PubMed

Venezia, Valentina; Nizzari, Mario; Repetto, Emanuela; Violani, Elisabetta; Corsaro, Alessandro; Thellung, Stefano; Villa, Valentina; Carlo, Pia; Schettini, Gennaro; Florio, Tullio; Russo, Claudio

2006-12-01

The amyloid precursor protein (APP) is a transmembrane protein with a short cytoplasmic tail whose physiological function is unclear, although it is well documented that the proteolytic processing of APP could influence the development of Alzheimer's disease (AD) through the formation of membrane-bound C-terminal fragments (CTFs) and of beta-amyloid peptides (Abeta). We have recently shown that tyrosine-phosphorylated APP and CTFs may interact with Grb2 and ShcA adaptor proteins and that this coupling occurs at a higher extent in AD subjects only. To study the interaction between APP or CTFs and ShcA/Grb2 and to investigate their molecular target we have used as experimental model two different cell lines: H4 human neuroglioma cells and APP/APLP null mouse embryonic fibroblast cells (MEFs). Here we show that in H4 cells APP interacts with Grb2; conversely in APP/APLP-null MEF cells this interaction is possible only after the reintroduction of human APP by transfection. We have also shown that in MEF cells the transfection of a plasmid encoding for human APP wild-type enhances the phosphorylation of ERK-1 and -2 as revealed by Western blotting and immunofluorescence experiments. Finally, also in H4 cells the overexpression of APP upregulates the levels of phospho-ERK-1 and -2. In summary our data suggest that APP may influence phospho-ERK-1 and -2 signaling through its binding with Grb2 and ShcA adaptors. The meaning of this event is not clear, but APP interaction with these adaptors could be relevant to regulate mitogenic pathway.

Modeling the vacuolar storage of malate shed lights on pre- and post-harvest fruit acidity.

PubMed

Etienne, Audrey; Génard, Michel; Lobit, Philippe; Bugaud, Christophe

2014-11-18

Malate is one of the most important organic acids in many fruits and its concentration plays a critical role in organoleptic properties. Several studies suggest that malate accumulation in fruit cells is controlled at the level of vacuolar storage. However, the regulation of vacuolar malate storage throughout fruit development, and the origins of the phenotypic variability of the malate concentration within fruit species remain to be clarified. In the present study, we adapted the mechanistic model of vacuolar storage proposed by Lobit et al. in order to study the accumulation of malate in pre and postharvest fruits. The main adaptation concerned the variation of the free energy of ATP hydrolysis during fruit development. Banana fruit was taken as a reference because it has the particularity of having separate growth and post-harvest ripening stages, during which malate concentration undergoes substantial changes. Moreover, the concentration of malate in banana pulp varies greatly among cultivars which make possible to use the model as a tool to analyze the genotypic variability. The model was calibrated and validated using data sets from three cultivars with contrasting malate accumulation, grown under different fruit loads and potassium supplies, and harvested at different stages. The model predicted the pre and post-harvest dynamics of malate concentration with fairly good accuracy for the three cultivars (mean RRMSE = 0.25-0.42). The sensitivity of the model to parameters and input variables was analyzed. According to the model, vacuolar composition, in particular potassium and organic acid concentrations, had an important effect on malate accumulation. The model suggested that rising temperatures depressed malate accumulation. The model also helped distinguish differences in malate concentration among the three cultivars and between the pre and post-harvest stages by highlighting the probable importance of proton pump activity and particularly of the free

Dietary (−)-epicatechin as a potent inhibitor of βγ-secretase amyloid precursor protein processing☆

PubMed Central

Cox, Carla J.; Choudhry, Fahd; Peacey, Eleanor; Perkinton, Michael S.; Richardson, Jill C.; Howlett, David R.; Lichtenthaler, Stefan F.; Francis, Paul T.; Williams, Robert J.

2015-01-01

Flavonoids, a group of dietary polyphenols have been shown to possess cognitive health benefits. Epidemiologic evidence suggests that they could play a role in risk reduction in dementia. Amyloid precursor protein processing and the subsequent generation of amyloid beta (Aβ) are central to the pathogenesis of Alzheimer's disease, as soluble, oligomeric Aβ is thought to be the toxic species driving disease progression. We undertook an in vitro screen to identify flavonoids with bioactivity at βγ-mediated amyloid precursor protein processing, which lead to identification of a number of flavonoids bioactive at 100 nM. Because of known bioavailability, we investigated the catechin family further and identified epigallocatechin and (−)-epicatechin as potent (nanomolar) inhibitors of amyloidogenic processing. Supporting this finding, we have shown reduced Aβ pathology and Aβ levels following short term, a 21-day oral delivery of (−)-epicatechin in 7-month-old TASTPM mice. Further, in vitro mechanistic studies suggest this is likely because of indirect BACE1 inhibition. Taken together, our results suggest that orally delivered (−)-epicatechin may be a potential prophylactic for Alzheimer's disease. PMID:25316600

Neurodegeneration in Alzheimer disease: role of amyloid precursor protein and presenilin 1 intracellular signaling.

PubMed

Nizzari, Mario; Thellung, Stefano; Corsaro, Alessandro; Villa, Valentina; Pagano, Aldo; Porcile, Carola; Russo, Claudio; Florio, Tullio

2012-01-01

Alzheimer disease (AD) is a heterogeneous neurodegenerative disorder characterized by (1) progressive loss of synapses and neurons, (2) intracellular neurofibrillary tangles, composed of hyperphosphorylated Tau protein, and (3) amyloid plaques. Genetically, AD is linked to mutations in few proteins amyloid precursor protein (APP) and presenilin 1 and 2 (PS1 and PS2). The molecular mechanisms underlying neurodegeneration in AD as well as the physiological function of APP are not yet known. A recent theory has proposed that APP and PS1 modulate intracellular signals to induce cell-cycle abnormalities responsible for neuronal death and possibly amyloid deposition. This hypothesis is supported by the presence of a complex network of proteins, clearly involved in the regulation of signal transduction mechanisms that interact with both APP and PS1. In this review we discuss the significance of novel finding related to cell-signaling events modulated by APP and PS1 in the development of neurodegeneration.

A multipurpose fusion tag derived from an unstructured and hyperacidic region of the amyloid precursor protein

PubMed Central

Sangawa, Takeshi; Tabata, Sanae; Suzuki, Kei; Saheki, Yasushi; Tanaka, Keiji; Takagi, Junichi

2013-01-01

Expression and purification of aggregation-prone and disulfide-containing proteins in Escherichia coli remains as a major hurdle for structural and functional analyses of high-value target proteins. Here, we present a novel gene-fusion strategy that greatly simplifies purification and refolding procedure at very low cost using a unique hyperacidic module derived from the human amyloid precursor protein. Fusion with this polypeptide (dubbed FATT for Flag-Acidic-Target Tag) results in near-complete soluble expression of variety of extracellular proteins, which can be directly refolded in the crude bacterial lysate and purified in one-step by anion exchange chromatography. Application of this system enabled preparation of functionally active extracellular enzymes and antibody fragments without the need for condition optimization. PMID:23526492

Wheat Vacuolar Iron Transporter TaVIT2 Transports Fe and Mn and Is Effective for Biofortification.

PubMed

Connorton, James M; Jones, Eleanor R; Rodríguez-Ramiro, Ildefonso; Fairweather-Tait, Susan; Uauy, Cristobal; Balk, Janneke

2017-08-01

Increasing the intrinsic nutritional quality of crops, known as biofortification, is viewed as a sustainable approach to alleviate micronutrient deficiencies. In particular, iron deficiency anemia is a major global health issue, but the iron content of staple crops such as wheat ( Triticum aestivum ) is difficult to change because of genetic complexity and homeostasis mechanisms. To identify target genes for the biofortification of wheat, we functionally characterized homologs of the VACUOLAR IRON TRANSPORTER ( VIT ). The wheat genome contains two VIT paralogs, TaVIT1 and TaVIT2 , which have different expression patterns but are both low in the endosperm. TaVIT2, but not TaVIT1, was able to rescue the growth of a yeast ( Saccharomyces cerevisiae ) mutant defective in vacuolar iron transport. TaVIT2 also complemented a manganese transporter mutant but not a vacuolar zinc transporter mutant. By overexpressing TaVIT2 under the control of an endosperm-specific promoter, we achieved a greater than 2-fold increase in iron in white flour fractions, exceeding minimum legal fortification levels in countries such as the United Kingdom. The antinutrient phytate was not increased and the iron in the white flour fraction was bioavailable in vitro, suggesting that food products made from the biofortified flour could contribute to improved iron nutrition. The single-gene approach impacted minimally on plant growth and also was effective in barley ( Hordeum vulgare ). Our results show that by enhancing vacuolar iron transport in the endosperm, this essential micronutrient accumulated in this tissue, bypassing existing homeostatic mechanisms. © 2017 American Society of Plant Biologists. All Rights Reserved.

The vacuolar channel VvALMT9 mediates malate and tartrate accumulation in berries of Vitis vinifera.

PubMed

De Angeli, Alexis; Baetz, Ulrike; Francisco, Rita; Zhang, Jingbo; Chaves, Maria Manuela; Regalado, Ana

2013-08-01

Vitis vinifera L. represents an economically important fruit species. Grape and wine flavour is made from a complex set of compounds. The acidity of berries is a major parameter in determining grape berry quality for wine making and fruit consumption. Despite the importance of malic and tartaric acid (TA) storage and transport for grape berry acidity, no vacuolar transporter for malate or tartrate has been identified so far. Some members of the aluminium-activated malate transporter (ALMT) anion channel family from Arabidopsis thaliana have been shown to be involved in mediating malate fluxes across the tonoplast. Therefore, we hypothesised that a homologue of these channels could have a similar role in V. vinifera grape berries. We identified homologues of the Arabidopsis vacuolar anion channel AtALMT9 through a TBLASTX search on the V. vinifera genome database. We cloned the closest homologue of AtALMT9 from grape berry cDNA and designated it VvALMT9. The expression profile revealed that VvALMT9 is constitutively expressed in berry mesocarp tissue and that its transcription level increases during fruit maturation. Moreover, we found that VvALMT9 is targeted to the vacuolar membrane. Using patch-clamp analysis, we could show that, besides malate, VvALMT9 mediates tartrate currents which are higher than in its Arabidopsis homologue. In summary, in the present study we provide evidence that VvALMT9 is a vacuolar malate channel expressed in grape berries. Interestingly, in V. vinifera, a tartrate-producing plant, the permeability of the channel is apparently adjusted to TA.

Creating Drought- and Salt-Tolerant Crops by Overexpressing a Vacuolar Pyrophosphatase Gene

USDA-ARS?s Scientific Manuscript database

Increased expression of an Arabidopsis vacuolar pyrophosphatase gene, AVP1, leads to increased drought and salt tolerance in transgenic plants, which has been demonstrated in laboratory and field conditions. The molecular mechanism of AVP1-mediated drought resistance is likely due to increased proto...

Transient anterior subcapsular vacuolar change of the crystalline lens in patients after posterior chamber phakic intraocular lens implantation.

PubMed

Chung, Jin Kwon; Shin, Jin Hee; Lee, Sung Jin

2013-10-25

We present two cases of transient vacuolar changes in the anterior subcapsular space of the crystalline lens in patients after posterior chamber phakic intraocular lens implantation. Implantable collamer lenses (ICL) were implanted in healthy myopic patients. Vacuolar changes developed just after the irrigating procedure through the narrow space between the ICL and the crystalline lens. Slit-lamp examinations and spectral domain optical coherence tomography showed bleb-like lesions in the anterior subcapsular space of one eye in each case, though the lesions gradually improved without visual deterioration. Consequently, the lesions turned into a few anterior subcapsular small faint opacities. Direct irrigation of the narrow space confined by the ICL and the crystalline lens is at risk for the development of vacuolar changes in the crystalline lens. The observed spontaneous reversal indicates that surgeons should not rush to surgical intervention but rather opt for close follow over several weeks.

Amyloid precursor protein and Presenilin1 interact with the adaptor GRB2 and modulate ERK 1,2 signaling.

PubMed

Nizzari, Mario; Venezia, Valentina; Repetto, Emanuela; Caorsi, Valentina; Magrassi, Raffaella; Gagliani, Maria Cristina; Carlo, Pia; Florio, Tullio; Schettini, Gennaro; Tacchetti, Carlo; Russo, Tommaso; Diaspro, Alberto; Russo, Claudio

2007-05-04

The amyloid precursor protein (APP) and the presenilins 1 and 2 are genetically linked to the development of familial Alzheimer disease. APP is a single-pass transmembrane protein and precursor of fibrillar and toxic amyloid-beta peptides, which are considered responsible for Alzheimer disease neurodegeneration. Presenilins are multipass membrane proteins, involved in the enzymatic cleavage of APP and other signaling receptors and transducers. The role of APP and presenilins in Alzheimer disease development seems to be related to the formation of amyloid-beta peptides; however, their physiological function, reciprocal interaction, and molecular mechanisms leading to neurodegeneration are unclear. APP and presenilins are also involved in multiple interactions with intracellular proteins, the significance of which is under investigation. Among the different APP-interacting proteins, we focused our interest on the GRB2 adaptor protein, which connects cell surface receptors to intracellular signaling pathways. In this study we provide evidence by co-immunoprecipitation experiments, confocal and electron microscopy, and by fluorescence resonance energy transfer experiments that both APP and presenilin1 interact with GRB2 in vesicular structures at the centrosome of the cell. The final target for these interactions is ERK1,2, which is activated in mitotic centrosomes in a PS1- and APP-dependent manner. These data suggest that both APP and presenilin1 can be part of a common signaling pathway that regulates ERK1,2 and the cell cycle.

ESCRT-III-Associated Protein ALIX Mediates High-Affinity Phosphate Transporter Trafficking to Maintain Phosphate Homeostasis in Arabidopsis

PubMed Central

Cardona-López, Ximena; Cuyas, Laura; Marín, Elena; Irigoyen, María Luisa; Gil, Erica; Puga, María Isabel; Bligny, Richard; Nussaume, Laurent; Geldner, Niko; Paz-Ares, Javier

2015-01-01

Prior to the release of their cargoes into the vacuolar lumen, sorting endosomes mature into multivesicular bodies (MVBs) through the action of ENDOSOMAL COMPLEX REQUIRED FOR TRANSPORT (ESCRT) protein complexes. MVB-mediated sorting of high-affinity phosphate transporters (PHT1) to the vacuole limits their plasma membrane levels under phosphate-sufficient conditions, a process that allows plants to maintain phosphate homeostasis. Here, we describe ALIX, a cytosolic protein that associates with MVB by interacting with ESCRT-III subunit SNF7 and mediates PHT1;1 trafficking to the vacuole in Arabidopsis thaliana. We show that the partial loss-of-function mutant alix-1 displays reduced vacuolar degradation of PHT1;1. ALIX derivatives containing the alix-1 mutation showed reduced interaction with SNF7, providing a simple molecular explanation for impaired cargo trafficking in alix-1 mutants. In fact, the alix-1 mutation also hampered vacuolar sorting of the brassinosteroid receptor BRI1. We also show that alix-1 displays altered vacuole morphogenesis, implying a new role for ALIX proteins in vacuolar biogenesis, likely acting as part of ESCRT-III complexes. In line with a presumed broad target spectrum, the alix-1 mutation is pleiotropic, leading to reduced plant growth and late flowering, with stronger alix mutations being lethal, indicating that ALIX participates in diverse processes in plants essential for their life. PMID:26342016

Inhibition of bone resorption in vitro by antisense RNA and DNA molecules targeted against carbonic anhydrase II or two subunits of vacuolar H(+)-ATPase.

PubMed Central

Laitala, T; Väänänen, H K

1994-01-01

The bone resorbing cells, osteoclasts, express high levels of carbonic anhydrase II (CA II) and vacuolar H(+)-ATPase (V-ATPase) during bone resorption. We have used antisense RNA and DNA molecules targeted against CA II, and against 16- and 60-kD subunits of vacuolar H(+)-ATPase (V-ATPase), to block the expression of these proteins in vitro. Osteoclastic bone resorption was studied in two in vitro culture systems: release of 45Calcium from prelabeled newborn mouse calvaria cultures, and resorption pit assays performed with rat osteoclasts cultured on bovine bone slices. Both antisense RNA and DNA against CA II and the V-ATPase were used to compare their specificities as regards inhibiting bone resorption in vitro. The antisense molecules inhibited the synthesis of these proteins by decreasing the amounts of mRNA in the cells in a highly specific manner. In osteoclast cultures treated with the 16-kD V-ATPase antisense RNA, acidification of an unknown population of intracellular vesicles was highly stimulated. The acidification of these vesicles was not sensitive to amiloride or bafilomycin A1. This suggests the existence of a back-up system for acidification of intracellular vesicles, when the expression of the V-ATPase is blocked. Our results further indicate that blocking the expression of CA II and V-ATPase with antisense RNA or DNA leads to decreased bone resorption. Images PMID:8200964

To Be Cytosolic or Vacuolar: The Double Life of Listeria monocytogenes.

PubMed

Bierne, Hélène; Milohanic, Eliane; Kortebi, Mounia

2018-01-01

Intracellular bacterial pathogens are generally classified into two types: those that exploit host membrane trafficking to construct specific niches in vacuoles (i.e., "vacuolar pathogens"), and those that escape from vacuoles into the cytosol, where they proliferate and often spread to neighboring cells (i.e., "cytosolic pathogens"). However, the boundary between these distinct intracellular phenotypes is tenuous and may depend on the timing of infection and on the host cell type. Here, we discuss recent progress highlighting this phenotypic duality in Listeria monocytogenes , which has long been a model for cytosolic pathogens, but now emerges as a bacterium also capable of residing in vacuoles, in a slow/non-growing state. The ability of L. monocytogenes to enter a persistence stage in vacuoles might play a role during the asymptomatic incubation period of listeriosis and/or the carriage of this pathogen in asymptomatic hosts. Moreover, persistent vacuolar Listeria could be less susceptible to antibiotics and more difficult to detect by routine techniques of clinical biology. These hypotheses deserve to be explored in order to better manage the risks related to this food-borne pathogen.

Vacuolar myelinopathy in waterfowl from a North Carolina impoundment

USGS Publications Warehouse

Augspurger, T.; Fischer, John R.; Thomas, Nancy; Sileo, L.; Brannian, Roger E.; Miller, Kimberli J.; Rocke, Tonie E.

2003-01-01

Vacuolar myelinopathy was confirmed by light and electron microscopic examination of mallards (Anas platyrhynchos), ring-necked ducks (Aythya collaris), and buffleheads (Bucephala albeola) collected during an epizootic at Lake Surf in central North Carolina (USA) between November 1998 and February 1999. Clinical signs of affected birds were consistent with central nervous system impairment of motor function (incoordination, abnormal movement and posture, weakness, paralysis). This is the first report of this disease in wild waterfowl (Anseriformes).Aug

Multi site polyadenylation and transcriptional response to stress of a vacuolar type H+-ATPase subunit A gene in Arabidopsis thaliana

PubMed Central

Magnotta, Scot M; Gogarten, Johann Peter

2002-01-01

Background Vacuolar type H+-ATPases play a critical role in the maintenance of vacuolar homeostasis in plant cells. V-ATPases are also involved in plants' defense against environmental stress. This research examined the expression and regulation of the catalytic subunit of the vacuolar type H+-ATPase in Arabidopsis thaliana and the effect of environmental stress on multiple transcripts generated by this gene. Results Evidence suggests that subunit A of the vacuolar type H+-ATPase is encoded by a single gene in Arabidopsis thaliana. Genome blot analysis showed no indication of a second subunit A gene being present. The single gene identified was shown by whole RNA blot analysis to be transcribed in all organs of the plant. Subunit A was shown by sequencing the 3' end of multiple cDNA clones to exhibit multi site polyadenylation. Four different poly (A) tail attachment sites were revealed. Experiments were performed to determine the response of transcript levels for subunit A to environmental stress. A PCR based strategy was devised to amplify the four different transcripts from the subunit A gene. Conclusions Amplification of cDNA generated from seedlings exposed to cold, salt stress, and etiolation showed that transcript levels for subunit A of the vacuolar type H+-ATPase in Arabidopsis were responsive to stress conditions. Cold and salt stress resulted in a 2–4 fold increase in all four subunit A transcripts evaluated. Etiolation resulted in a slight increase in transcript levels. All four transcripts appeared to behave identically with respect to stress conditions tested with no significant differential regulation. PMID:11985780

Arabidopsis Intracellular NHX-Type Sodium-Proton Antiporters are Required for Seed Storage Protein Processing.

PubMed

Ashnest, Joanne R; Huynh, Dung L; Dragwidge, Jonathan M; Ford, Brett A; Gendall, Anthony R

2015-11-01

The Arabidopsis intracellular sodium-proton exchanger (NHX) proteins AtNHX5 and AtNHX6 have a well-documented role in plant development, and have been used to improve salt tolerance in a variety of species. Despite evidence that intracellular NHX proteins are important in vacuolar trafficking, the mechanism of this role is poorly understood. Here we show that NHX5 and NHX6 are necessary for processing of the predominant seed storage proteins, and also influence the processing and activity of a vacuolar processing enzyme. Furthermore, we show by yeast two-hybrid and bimolecular fluorescence complementation (BiFC) technology that the C-terminal tail of NHX6 interacts with a component of Retromer, another component of the cell sorting machinery, and that this tail is critical for NHX6 activity. These findings demonstrate that NHX5 and NHX6 are important in processing and activity of vacuolar cargo, and suggest a mechanism by which NHX intracellular (IC)-II antiporters may be involved in subcellular trafficking. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Glycolytic control of vacuolar-type ATPase activity: A mechanism to regulate influenza viral infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kohio, Hinissan P.; Adamson, Amy L., E-mail: aladamso@uncg.edu

As new influenza virus strains emerge, finding new mechanisms to control infection is imperative. In this study, we found that we could control influenza infection of mammalian cells by altering the level of glucose given to cells. Higher glucose concentrations induced a dose-specific increase in influenza infection. Linking influenza virus infection with glycolysis, we found that viral replication was significantly reduced after cells were treated with glycolytic inhibitors. Addition of extracellular ATP after glycolytic inhibition restored influenza infection. We also determined that higher levels of glucose promoted the assembly of the vacuolar-type ATPase within cells, and increased vacuolar-type ATPase proton-transportmore » activity. The increase of viral infection via high glucose levels could be reversed by inhibition of the proton pump, linking glucose metabolism, vacuolar-type ATPase activity, and influenza viral infection. Taken together, we propose that altering glucose metabolism may be a potential new approach to inhibit influenza viral infection. - Highlights: • Increased glucose levels increase Influenza A viral infection of MDCK cells. • Inhibition of the glycolytic enzyme hexokinase inhibited Influenza A viral infection. • Inhibition of hexokinase induced disassembly the V-ATPase. • Disassembly of the V-ATPase and Influenza A infection was bypassed with ATP. • The state of V-ATPase assembly correlated with Influenza A infection of cells.« less

Trehalose Alters Subcellular Trafficking and the Metabolism of the Alzheimer-associated Amyloid Precursor Protein*

PubMed Central

Tien, Nguyen T.; Karaca, Ilker; Tamboli, Irfan Y.

2016-01-01

The disaccharide trehalose is commonly considered to stimulate autophagy. Cell treatment with trehalose could decrease cytosolic aggregates of potentially pathogenic proteins, including mutant huntingtin, α-synuclein, and phosphorylated tau that are associated with neurodegenerative diseases. Here, we demonstrate that trehalose also alters the metabolism of the Alzheimer disease-related amyloid precursor protein (APP). Cell treatment with trehalose decreased the degradation of full-length APP and its C-terminal fragments. Trehalose also reduced the secretion of the amyloid-β peptide. Biochemical and cell biological experiments revealed that trehalose alters the subcellular distribution and decreases the degradation of APP C-terminal fragments in endolysosomal compartments. Trehalose also led to strong accumulation of the autophagic marker proteins LC3-II and p62, and decreased the proteolytic activation of the lysosomal hydrolase cathepsin D. The combined data indicate that trehalose decreases the lysosomal metabolism of APP by altering its endocytic vesicular transport. PMID:26957541

A vacuolar carboxypeptidase mutant of Arabidopsis thaliana is degraded by the ERAD pathway independently of its N-glycan

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamamoto, Masaya; Kawanabe, Mitsuyoshi; Hayashi, Yoko

2010-03-12

Misfolded proteins produced in the endoplasmic reticulum (ER) are degraded by a mechanism, the ER-associated degradation (ERAD). Here we report establishment of the experimental system to analyze the ERAD in plant cells. Carboxypeptidase Y (CPY) is a vacuolar enzyme and its mutant CPY* is degraded by the ERAD in yeast. Since Arabidopsis thaliana has AtCPY, an ortholog of yeast CPY, we constructed and expressed fusion proteins consisting of AtCPY and GFP and of AtCPY*, which carries a mutation homologous to yeast CPY*, and GFP in A. thaliana cells. While AtCPY-GFP was efficiently transported to the vacuole, AtCPY*-GFP was retained inmore » the ER to be degraded in proteasome- and Cdc48-dependent manners. We also found that AtCPY*-GFP was degraded by the ERAD in yeast cells, but that its single N-glycan did not function as a degradation signal in yeast or plant cells. Therefore, AtCPY*-GFP can be used as a marker protein to analyze the ERAD pathway, likely for nonglycosylated substrates, in plant cells.« less

Post-translational regulation of acid invertase activity by vacuolar invertase inhibitor affects resistance to cold-induced sweetening of potato tubers.

PubMed

McKenzie, Marian J; Chen, Ronan K Y; Harris, John C; Ashworth, Matthew J; Brummell, David A

2013-01-01

Cold-induced sweetening (CIS) is a serious post-harvest problem for potato tubers, which need to be stored cold to prevent sprouting and pathogenesis in order to maintain supply throughout the year. During storage at cold temperatures (below 10 °C), many cultivars accumulate free reducing sugars derived from a breakdown of starch to sucrose that is ultimately cleaved by acid invertase to produce glucose and fructose. When affected tubers are processed by frying or roasting, these reducing sugars react with free asparagine by the Maillard reaction, resulting in unacceptably dark-coloured and bitter-tasting product and generating the probable carcinogen acrylamide as a by-product. We have previously identified a vacuolar invertase inhibitor (INH2) whose expression correlates both with low acid invertase activity and with resistance to CIS. Here we show that, during cold storage, overexpression of the INH2 vacuolar invertase inhibitor gene in CIS-susceptible potato tubers reduced acid invertase activity, the accumulation of reducing sugars and the generation of acrylamide in subsequent fry tests. Conversely, suppression of vacuolar invertase inhibitor expression in a CIS-resistant line increased susceptibility to CIS. The results show that post-translational regulation of acid invertase by the vacuolar invertase inhibitor is an important component of resistance to CIS. © 2012 Blackwell Publishing Ltd.

Quercetin prevents protein nitration and glycolytic block of proliferation in hydrogen peroxide insulted cultured neuronal precursor cells (NPCs): Implications on CNS regeneration.

PubMed

Sajad, Mir; Zargan, Jamil; Zargar, Mohammad Afzal; Sharma, Jyoti; Umar, Sadiq; Arora, Rajesh; Khan, Haider A

2013-05-01

Survival along with optimal proliferation of neuronal precursors determines the outcomes of the endogenous cellular repair in CNS. Cellular-oxidation based cell death has been described in several neurodegenerative disorders. Therefore, this study was aimed at the identification of the potent targets of oxidative damage to the neuronal precursors and its effective prevention by a natural flavonoid, Quercetin. Neuronal precursor cells (NPCs), Nestin+ and GFAP (Glial fibrillary acidic protein)+ were isolated and cultured from adult rat SVZ (subventricular zone). These cells were challenged with a single dose of H2O2 (50μM) and/or pre-treated with different concentrations of Quercetin. H2O2 severely limited the cellular viability and expansion of the neurospheres. Cellular-oxidation studies revealed reduction in glutathione dependent redox buffering along with depletion of enzymatic cellular antioxidants that might potentiate the nitrite (NO2(-)) and superoxide anion (O2(-)) mediated peroxynitrite (ONOO(-)) formation and irreversible protein nitration. We identified depleted PK-M2 (M2 isoform of pyruvate kinase) activity and apoptosis of NPCs revealed by the genomic DNA fragmentation and elevated PARP (poly ADP ribose polymerase) activity along with increased Caspase activity initiated by severely depolarised mitochondrial membranes. However, the pre-treatment of Quercetin in a dose-response manner prevented these changes and restored the expansion of neurospheres preferably by neutralizing the oxidative conditions and thereby reducing peroxynitrite formation, protein nitration and PK-M2 depletion. Our results unravel the potential interactions of oxidative environment and respiration in the survival and activation of precursors and offer a promise shown by a natural flavonoid in the protective strategy for neuronal precursors of adult brain. Copyright © 2013 Elsevier Inc. All rights reserved.

Expression, crystallization and phasing of vacuolar H(+)-ATPase subunit C (Vma5p) of Saccharomyces cerevisiae.

PubMed

Drory, Omri; Mor, Adi; Frolow, Felix; Nelson, Nathan

2004-10-01

The expression, crystallization and phasing of subunit C (Vma5p) of the yeast (Saccharomyces cerevisiae) vacuolar proton-translocating ATPase (V-ATPase) is described. The expressed protein consists of 412 residues: 392 from the reading frame of Vma5p and 20 N-terminal residues originating from the plasmid. Diffraction-quality crystals were obtained using the hanging-drop and sitting-drop vapour-diffusion methods assisted by streak-seeding, with PEG 3350 as precipitant. The crystals formed in hanging drops diffracted to 1.80 A and belong to space group P4(3)2(1)2(1), with unit-cell parameters a = b = 62.54, c = 327.37 A, alpha = beta = gamma = 90 degrees. The structure was solved using SIRAS with a Lu(O2C2H3)2 heavy-atom derivative.

Trehalose Alters Subcellular Trafficking and the Metabolism of the Alzheimer-associated Amyloid Precursor Protein.

PubMed

Tien, Nguyen T; Karaca, Ilker; Tamboli, Irfan Y; Walter, Jochen

2016-05-13

The disaccharide trehalose is commonly considered to stimulate autophagy. Cell treatment with trehalose could decrease cytosolic aggregates of potentially pathogenic proteins, including mutant huntingtin, α-synuclein, and phosphorylated tau that are associated with neurodegenerative diseases. Here, we demonstrate that trehalose also alters the metabolism of the Alzheimer disease-related amyloid precursor protein (APP). Cell treatment with trehalose decreased the degradation of full-length APP and its C-terminal fragments. Trehalose also reduced the secretion of the amyloid-β peptide. Biochemical and cell biological experiments revealed that trehalose alters the subcellular distribution and decreases the degradation of APP C-terminal fragments in endolysosomal compartments. Trehalose also led to strong accumulation of the autophagic marker proteins LC3-II and p62, and decreased the proteolytic activation of the lysosomal hydrolase cathepsin D. The combined data indicate that trehalose decreases the lysosomal metabolism of APP by altering its endocytic vesicular transport. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Tributyltin sensitivity of vacuolar-type Na(+)-transporting ATPase from Enterococcus hirae.

PubMed

Chardwiriyapreecha, Soracom; Inoue, Tomohiro; Sugimoto, Naoko; Sekito, Takayuki; Yamato, Ichiro; Murata, Takeshi; Homma, Michio; Kakinuma, Yoshimi

2009-10-01

Tributyltin chloride (TBT), an environmental pollutant, is toxic to a variety of eukaryotic and prokaryotic organisms. Some members of F-ATP synthase (F-ATPase)/vacuolar type ATPase (V-ATPase) superfamily have been identified as the molecular target of this compound. TBT inhibited the activities of H(+)-transporting or Na(+)-transporting F-ATPase as well as H(+)-transporting V-ATPase originated from various organisms. However, the sensitivity to TBT of Na(+)-transporting V-ATPase has not been investigated. We examined the effect of TBT on Na(+)-transporting V-ATPase from an eubacterium Enterococus hirae. The ATP hydrolytic activity of E. hirae V-ATPase in purified form as well as in membrane-bound form was little inhibited by less than 10 microM TBT; IC50 for TBT inhibition of purified enzyme was estimated to be about 35 microM. Active sodium transport by E. hirae cells, indicating the in vivo activity of this V-ATPase, was not inhibited by 20 microM TBT. By contrast, IC50 of H(+)-transporting V-ATPase of the vacuolar membrane vesicles from Saccharomyces cerevisiae was about 0.2 microM. E. hirae V-ATPase is thus extremely less sensitive to TBT.

The antiwrinkle effect of topical concentrated 2-dimethylaminoethanol involves a vacuolar cytopathology.

PubMed

Morissette, G; Germain, L; Marceau, F

2007-03-01

The 'cosmeceutical' agent 2-dimethylaminoethanol (DMAE) is a tertiary amine found in high concentration in numerous topical antiwrinkle preparations. We hypothesized that a 337 mmol L(-1) (3%) DMAE reservoir applied to the skin could reproduce the cytopathology induced by other amines by maintaining a millimolar drug concentration within a certain depth of the skin layers, and that vacuolar cell expansion could account for the very rapid effect on the apparent skin fullness. Morphological and functional assays were applied to cultured rabbit dermal fibroblasts treated with tertiary amines in vitro. A morphological verification of the vacuolization caused by topical DMAE was also attempted in vivo using the inner skin of the rabbit ear and in vitro using primary cultures of human cutaneous epithelial cells. Fibroblasts responded to DMAE (2.5-10 mmol L(-1)) by massive vacuolization (0.5-4 h; phase contrast observations). Triethanolamine, another chemical frequently used topically, was also active in this respect (10 mmol L(-1)). The vacuolar adenosine triphosphatase inhibitor bafilomycin A1 prevented DMAE- or triethanolamine-induced vacuolization; adding bafilomycin A1 or cell washout slowly reversed the established vacuolization induced by DMAE. Further effects of DMAE in cultured fibroblasts included a moderate cytotoxicity (10 mmol L(-1)) that was abated by bafilomycin A1 cotreatment, a concentration-dependent mitotic arrest (2.5 mmol L(-1)) and transient and mild effects on cell ploidy. The epidermis of the rabbit external ear was significantly thickened and exhibited clear perinuclear swelling indicative of vacuolization in response to 3% DMAE (1 h; paraffin tissue sections). Cultured human cutaneous epithelial cells responded to DMAE by vacuolization (inhibited by bafilomycin A1 cotreatment). The vacuolar cytopathology induced by concentrated organic amines may be the cellular basis of the antiwrinkle effect of DMAE.

Effects of acetylate hyperforin on the processing of amyloid precursor protein

PubMed Central

Chen, Xiang; Feng, Wenshang; Chen, Qing; Yang, Xiangling; Yang, Depo; Wang, Dongmei; Zhong, Ling

2009-01-01

Hyperforin (HF) is a phloroglucinol compound obtained from St. John's Wort (Hypericum perforatum). Recent studies have shown that Hyperforin can be used to improve psychopathologic symptoms of Alzheimer's disease but the mechanism is not clear. This may be partly due to the difficult in studying Hyperforin, since this chemical is unstable and is sensitive to light, oxygen, and heat. In this study, we explored the effects of acetylate hyperforin (ace-HF), a stable derivative of hyperforin, on the processing of amyloid precursor protein (APP). HEK293 cells transfected with pcDNA3.1APP695sw and SH-SY5Y cells were treated with ace-HF, followed by measuring the levels of APP and sAPPα. Twelve hours of treatment led to an increase in extracellular sAPPα, but APP mRNA and protein levels were unchanged. Further studies with α-secretase and a pan PKC inhibitor, Calphostin C, indicated that ace-HF's effect on extracellular sAPPα was closely related to PKC activities and α-secretase activities. Our findings suggest that ace-HF can modulate α-secretase-mediated APP processing via a PKC signaling pathway. PMID:21383880

Effects of acetylate hyperforin on the processing of amyloid precursor protein.

PubMed

Chen, Xiang; Feng, Wenshang; Chen, Qing; Yang, Xiangling; Yang, Depo; Wang, Dongmei; Zhong, Ling

2009-02-20

Hyperforin (HF) is a phloroglucinol compound obtained from St. John's Wort (Hypericum perforatum). Recent studies have shown that Hyperforin can be used to improve psychopathologic symptoms of Alzheimer's disease but the mechanism is not clear. This may be partly due to the difficult in studying Hyperforin, since this chemical is unstable and is sensitive to light, oxygen, and heat. In this study, we explored the effects of acetylate hyperforin (ace-HF), a stable derivative of hyperforin, on the processing of amyloid precursor protein (APP). HEK293 cells transfected with pcDNA3.1APP695sw and SH-SY5Y cells were treated with ace-HF, followed by measuring the levels of APP and sAPPα. Twelve hours of treatment led to an increase in extracellular sAPPα, but APP mRNA and protein levels were unchanged. Further studies with α-secretase and a pan PKC inhibitor, Calphostin C, indicated that ace-HF's effect on extracellular sAPPα was closely related to PKC activities and α-secretase activities. Our findings suggest that ace-HF can modulate α-secretase-mediated APP processing via a PKC signaling pathway.

Vacuolar invertase gene silencing in potato decreasing the frequency of sugar-end defects

USDA-ARS?s Scientific Manuscript database

Sugar-end defect is a tuber quality disorder and persistent problem for the French fry processing industry that causes unacceptable darkening of one end of French fries. This defect appears when environmental stress during tuber growth increases post-harvest vacuolar acid invertase activity at one e...

Distinct Requirements for Vacuolar Protein Sorting 34 Downstream Effector Phosphatidylinositol 3-Phosphate 5-Kinase in Podocytes Versus Proximal Tubular Cells

PubMed Central

Venkatareddy, Madhusudan; Verma, Rakesh; Kalinowski, Anne; Patel, Sanjeevkumar R.; Shisheva, Assia

2016-01-01

The mechanisms by which the glomerular filtration barrier prevents the loss of large macromolecules and simultaneously, maintains the filter remain poorly understood. Recent studies proposed that podocytes have an active role in both the endocytosis of filtered macromolecules and the maintenance of the filtration barrier. Deletion of a key endosomal trafficking regulator, the class 3 phosphatidylinositol (PtdIns) 3-kinase vacuolar protein sorting 34 (Vps34), in podocytes results in aberrant endosomal membrane morphology and podocyte dysfunction. We recently showed that the vacuolation phenotype in cultured Vps34–deficient podocytes is caused by the absence of a substrate for the Vps34 downstream effector PtdIns 3-phosphate 5-kinase (PIKfyve), which phosphorylates Vps34-generated PtdIns(3)P to produce PtdIns (3,5)P2. PIKfyve perturbation and PtdIns(3,5)P2 reduction result in massive membrane vacuolation along the endosomal system, but the cell-specific functions of PIKfyve in vivo remain unclear. We show here that the genetic deletion of PIKfyve in endocytically active proximal tubular cells resulted in the development of large cytoplasmic vacuoles caused by arrested endocytic traffic progression at a late-endosome stage. In contrast, deletion of PIKfyve in glomerular podocytes did not significantly alter the endosomal morphology, even in age 18-month-old mice. However, on culturing, the PIKfyve-deleted podocytes developed massive cytoplasmic vacuoles. In summary, these data suggest that glomerular podocytes and proximal tubules have different requirements for PIKfyve function, likely related to distinct in vivo needs for endocytic flux. PMID:26825532

A Common Genetic Variant in the 3′-UTR of Vacuolar H+-ATPase ATP6V0A1 Creates a Micro-RNA Motif to Alter Chromogranin A (CHGA) Processing and Hypertension Risk

PubMed Central

Wei, Zhiyun; Biswas, Nilima; Wang, Lei; Courel, Maite; Zhang, Kuixing; Soler-Jover, Alex; Taupenot, Laurent; O’Connor, Daniel T.

2012-01-01

Background The catecholamine release-inhibitor catestatin and its precursor chromogranin A (CHGA) may constitute “intermediate phenotypes” in analysis of genetic risk for cardiovascular disease such as hypertension. Previously, the vacuolar H+-ATPase subunit gene ATP6V0A1 was found within the confidence interval for linkage with catestatin secretion in a genome-wide study, and its 3′-UTR polymorphism T+3246C (rs938671) was associated with both catestatin processing from CHGA, as well as population blood pressure (BP). Here we explored the molecular mechanism of this effect by experiments with transfected chimeric photoproteins in chromaffin cells. Methods and Results Placing the ATP6V0A1 3′-UTR downstream of a luciferase reporter, we found that the C (variant) allele decreased overall gene expression. The 3′-UTR effect was verified by coupled in vitro transcription/translation of the entire/intact human ATP6V0A1 mRNA. Chromaffin granule pH, monitored by fluorescence a CHGA/EGFP chimera during vesicular H+-ATPase inhibition by bafilomycin A1, was more easily perturbed during co-expression of the ATP6V0A1 3′-UTR C-allele than the T-allele. After bafilomycin A1 treatment, the ratio of CHGA precursor to its catestatin fragments in PC12 cells was substantially diminished, though the qualitative composition of such fragments was not affected (on immunoblot or MALDI mass spectrometry). Bafilomycin A1 treatment also decreased exocytotic secretion from the regulated pathway, monitored by a CHGA chimera tagged with embryonic alkaline phosphatase (EAP). 3′-UTR T+3246C created a binding motif for micro-RNA hsa-miR-637; co-transfection of hsa-miR-637 precursor or antagomir/inhibitor oligonucleotides yielded the predicted changes in expression of luciferase reporter/ATP6V0A1-3′-UTR plasmids varying at T+3246C. Conclusions The results suggest a series of events whereby ATP6V0A1 3′-UTR variant T+3246C functioned: ATP6V0A1 expression was affected likely through

Wheat Vacuolar Iron Transporter TaVIT2 Transports Fe and Mn and Is Effective for Biofortification1[OPEN

PubMed Central

Jones, Eleanor R.; Rodríguez-Ramiro, Ildefonso

2017-01-01

Increasing the intrinsic nutritional quality of crops, known as biofortification, is viewed as a sustainable approach to alleviate micronutrient deficiencies. In particular, iron deficiency anemia is a major global health issue, but the iron content of staple crops such as wheat (Triticum aestivum) is difficult to change because of genetic complexity and homeostasis mechanisms. To identify target genes for the biofortification of wheat, we functionally characterized homologs of the VACUOLAR IRON TRANSPORTER (VIT). The wheat genome contains two VIT paralogs, TaVIT1 and TaVIT2, which have different expression patterns but are both low in the endosperm. TaVIT2, but not TaVIT1, was able to rescue the growth of a yeast (Saccharomyces cerevisiae) mutant defective in vacuolar iron transport. TaVIT2 also complemented a manganese transporter mutant but not a vacuolar zinc transporter mutant. By overexpressing TaVIT2 under the control of an endosperm-specific promoter, we achieved a greater than 2-fold increase in iron in white flour fractions, exceeding minimum legal fortification levels in countries such as the United Kingdom. The antinutrient phytate was not increased and the iron in the white flour fraction was bioavailable in vitro, suggesting that food products made from the biofortified flour could contribute to improved iron nutrition. The single-gene approach impacted minimally on plant growth and also was effective in barley (Hordeum vulgare). Our results show that by enhancing vacuolar iron transport in the endosperm, this essential micronutrient accumulated in this tissue, bypassing existing homeostatic mechanisms. PMID:28684433

Silencing of Amyloid Precursor Protein Expression Using a New Engineered Delta Ribozyme

PubMed Central

Ben Aissa, Manel; April, Marie-Claude; Bergeron, Lucien-Junior; Perreault, Jean-Pierre; Levesque, Georges

2012-01-01

Alzheimer's disease (AD) etiological studies suggest that an elevation in amyloid-β peptides (Aβ) level contributes to aggregations of the peptide and subsequent development of the disease. The major constituent of these amyloid peptides is the 1 to 40–42 residue peptide (Aβ 40−42) derived from amyloid protein precursor (APP). Most likely, reducing Aβ levels in the brain may block both its aggregation and neurotoxicity and would be beneficial for patients with AD. Among the several possible ways to lower Aβ accumulation in the cells, we have selectively chosen to target the primary step in the Aβ cascade, namely, to reduce APP gene expression. Toward this end, we engineered specific SOFA-HDV ribozymes, a new generation of catalytic RNA tools, to decrease APP mRNA levels. Additionally, we demonstrated that APP-ribozymes are effective at decreasing APP mRNA and protein levels as well as Aβ levels in neuronal cells. Our results could lay the groundwork for a new protective treatment for AD. PMID:22482079

The crystal structure of DR6 in complex with the amyloid precursor protein provides insight into death receptor activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Kai; Olsen, Olav; Tzvetkova-Robev, Dorothea

The amyloid precursor protein (APP) has garnered considerable attention due to its genetic links to Alzheimer's disease. Death receptor 6 (DR6) was recently shown to bind APP via the protein extracellular regions, stimulate axonal pruning, and inhibit synapse formation. Here, we report the crystal structure of the DR6 ectodomain in complex with the E2 domain of APP and show that it supports a model for APP-induced dimerization and activation of cell surface DR6.

The crystal structure of DR6 in complex with the amyloid precursor protein provides insight into death receptor activation

DOE PAGES

Xu, Kai; Olsen, Olav; Tzvetkova-Robev, Dorothea; ...

2015-04-02

The amyloid precursor protein (APP) has garnered considerable attention due to its genetic links to Alzheimer's disease. Death receptor 6 (DR6) was recently shown to bind APP via the protein extracellular regions, stimulate axonal pruning, and inhibit synapse formation. Here, we report the crystal structure of the DR6 ectodomain in complex with the E2 domain of APP and show that it supports a model for APP-induced dimerization and activation of cell surface DR6.

Cloning, localization and expression analysis of vacuolar sugar transporters in the CAM plant Ananas comosus (pineapple).

PubMed

Antony, Edna; Taybi, Tahar; Courbot, Mikaël; Mugford, Sam T; Smith, J Andrew C; Borland, Anne M

2008-01-01

In photosynthetic tissues of the CAM plant pineapple (Ananas comosus), storage of soluble sugars in the central vacuole during the daytime and their remobilization at night is required to provide carbon skeletons for nocturnal CO(2) fixation. However, soluble sugars produced photosynthetically must also be exported to support growth processes in heterotrophic tissues. To begin to address how vacuolar sugar storage and assimilate partitioning are regulated in A. comosus, degenerate PCR and cDNA library screening were used to clone three candidate sugar transporters from the leaves of this species. Subcellular localization of the three transporters was investigated via expression of YFP-fusion proteins in tobacco epidermal cells and their co-localization with subcellular markers by confocal microscopy. Using this strategy, a putative hexose transporter (AcMST1) and a putative inositol transporter (AcINT1) were identified that both localized to the tonoplast, whereas a putative sucrose transporter (AcSUT1) was found to localize to prevacuolar compartments. A cDNA (AcMST2) with high similarity to a recently characterized tonoplast hexose transporter in Arabidopsis was also identified from an A. comosus fruit EST database. Analyses of transcript abundance indicated that AcMST1 was more highly expressed in fruits compared to leaves of A. comosus, whilst transcripts of AcINT1, AcSUT1, and AcMST2 were more abundant in leaves. Transcript abundance of AcINT1, the putative inositol transporter, showed day-night changes comparable to those of other CAM-related transcripts described in Mesembryanthemum crystallinum. The results are discussed in terms of the role of vacuolar sugar transporters in regulating carbon flow during the diel cycle in CAM plants.

Immunoprecipitation and Characterization of Membrane Protein Complexes from Yeast

ERIC Educational Resources Information Center

Parra-Belky, Karlett; McCulloch, Kathryn; Wick, Nicole; Shircliff, Rebecca; Croft, Nicolas; Margalef, Katrina; Brown, Jamie; Crabill, Todd; Jankord, Ryan; Waldo, Eric

2005-01-01

In this undergraduate biochemistry laboratory experiment, the vacuolar ATPase protein complex is purified from yeast cell extracts by doing immunoprecipitations under nondenaturing conditions. Immunoprecipitations are performed using monoclonal antibodies to facilitate data interpretation, and subunits are separated on the basis of their molecular…

Vacuolar processing enzyme plays an essential role in the crystalline structure of glutelin in rice seed.

PubMed

Kumamaru, Toshihiro; Uemura, Yuji; Inoue, Yoshimi; Takemoto, Yoko; Siddiqui, Sadar Uddin; Ogawa, Masahiro; Hara-Nishimura, Ikuko; Satoh, Hikaru

2010-01-01

To identify the function of genes that regulate the processing of proglutelin, we performed an analysis of glup3 mutants, which accumulates excess amounts of proglutelin and lack the vacuolar processing enzyme (VPE). VPE activity in developing seeds from glup3 lines was reduced remarkably compared with the wild type. DNA sequencing of the VPE gene in glup3 mutants revealed either amino acid substitutions or the appearance of a stop codon within the coding region. Microscopic observations showed that alpha-globulin and proglutelin were distributed homogeneously within glup3 protein storage vacuoles (PSVs), and that glup3 PSVs lacked the crystalline lattice structure typical of wild-type PSVs. This suggests that the processing of proglutelin by VPE in rice is essential for proper PSV structure and compartmentalization of storage proteins. Growth retardation in glup3 seedlings was also observed, indicating that the processing of proglutelin influences early seedling development. These findings indicate that storage of glutelin in its mature form as a crystalline structure in PSVs is required for the rapid use of glutelin as a source of amino acids during early seedling development. In conclusion, VPE plays an important role in the formation of protein crystalline structures in PSVs.

Singlet Oxygen-Induced Membrane Disruption and Serpin-Protease Balance in Vacuolar-Driven Cell Death.

PubMed

Koh, Eugene; Carmieli, Raanan; Mor, Avishai; Fluhr, Robert

2016-07-01

Singlet oxygen plays a role in cellular stress either by providing direct toxicity or through signaling to initiate death programs. It was therefore of interest to examine cell death, as occurs in Arabidopsis, due to differentially localized singlet oxygen photosensitizers. The photosensitizers rose bengal (RB) and acridine orange (AO) were localized to the plasmalemma and vacuole, respectively. Their photoactivation led to cell death as measured by ion leakage. Cell death could be inhibited by the singlet oxygen scavenger histidine in treatments with AO but not with RB In the case of AO treatment, the vacuolar membrane was observed to disintegrate. Concomitantly, a complex was formed between a vacuolar cell-death protease, RESPONSIVE TO DESSICATION-21 and its cognate cytoplasmic protease inhibitor ATSERPIN1. In the case of RB treatment, the tonoplast remained intact and no complex was formed. Over-expression of AtSerpin1 repressed cell death, only under AO photodynamic treatment. Interestingly, acute water stress showed accumulation of singlet oxygen as determined by fluorescence of Singlet Oxygen Sensor Green, by electron paramagnetic resonance spectroscopy and the induction of singlet oxygen marker genes. Cell death by acute water stress was inhibited by the singlet oxygen scavenger histidine and was accompanied by vacuolar collapse and the appearance of serpin-protease complex. Over-expression of AtSerpin1 also attenuated cell death under this mode of cell stress. Thus, acute water stress damage shows parallels to vacuole-mediated cell death where the generation of singlet oxygen may play a role. © 2016 American Society of Plant Biologists. All Rights Reserved.

P2 receptor stimulation induces amyloid precursor protein production and secretion in rat cortical astrocytes.

PubMed

Tran, Minh D

2011-04-04

Amyloid precursor protein (APP) is ubiquitously expressed in a variety of tissues but is predominantly expressed in the brain. The expression of APP has been well studied in neurons but little is known about its presence in astrocytes. The study presented here shows that purinergic signaling is involved in the production and secretion of APP in primary cultures of rat cortical astrocytes. Extracellular ATP caused an increase in APP production and release in a time- and concentration-dependent manner and was inhibited by antagonists of P2 receptors. Further agonist and antagonist studies revealed involvement of P2Y2 and P2Y4 receptors in nucleotide-stimulated production and release of APP. In addition, signaling studies with various protein kinase inhibitors demonstrated that blockade of mitogen-activated protein kinases, but not Akt, inhibited nucleotide-stimulated APP expression and release. These results indicate that APP production and secretion can be regulated by activation of P2Y2/4 receptors coupled to protein kinase signaling pathways and suggest that astrocytes can be a potential source of APP. Published by Elsevier Ireland Ltd.

F-box only protein 2 (Fbxo2) regulates amyloid precursor protein levels and processing.

PubMed

Atkin, Graham; Hunt, Jack; Minakawa, Eiko; Sharkey, Lisa; Tipper, Nathan; Tennant, William; Paulson, Henry L

2014-03-07

The amyloid precursor protein (APP) is an integral membrane glycoprotein whose cleavage products, particularly amyloid-β, accumulate in Alzheimer disease (AD). APP is present at synapses and is thought to play a role in both the formation and plasticity of these critical neuronal structures. Despite the central role suggested for APP in AD pathogenesis, the mechanisms regulating APP in neurons and its processing into cleavage products remain incompletely understood. F-box only protein 2 (Fbxo2), a neuron-enriched ubiquitin ligase substrate adaptor that preferentially binds high-mannose glycans on glycoproteins, was previously implicated in APP processing by facilitating the degradation of the APP-cleaving β-secretase, β-site APP-cleaving enzyme. Here, we sought to determine whether Fbxo2 plays a similar role for other glycoproteins in the amyloid processing pathway. We present in vitro and in vivo evidence that APP is itself a substrate for Fbxo2. APP levels were decreased in the presence of Fbxo2 in non-neuronal cells, and increased in both cultured hippocampal neurons and brain tissue from Fbxo2 knock-out mice. The processing of APP into its cleavage products was also increased in hippocampi and cultured hippocampal neurons lacking Fbxo2. In hippocampal slices, this increase in cleavage products was accompanied by a significant reduction in APP at the cell surface. Taken together, these results suggest that Fbxo2 regulates APP levels and processing in the brain and may play a role in modulating AD pathogenesis.

Expression of an arabidopsis vacuolar H+-pyrophosphatase gene (AVP1) in cotton improves drought- and salt tolerance and increases fibre yield in the field conditions

USDA-ARS?s Scientific Manuscript database

The Arabidopsis gene AVP1 encodes a vacuolar pyrophosphatase that functions as a proton pump on the vacuolar membrane. Overexpression of AVP1 in Arabidopsis, tomato and rice enhances plant performance under salt and drought stress conditions, because up-regulation of the type I H+-PPase from Arabid...

Expression of an Arabidopsis Vacuolar H+-pyrophosphatase Gene (AVP1) in Cotton Improves Drought- and Salt Tolerance and Increases Fibre Yield in the Field Conditions.

USDA-ARS?s Scientific Manuscript database

The Arabidopsis gene AVP1 encodes a vacuolar pyrophosphatase that functions as a proton pump on the vacuolar membrane. Overexpression of AVP1 in Arabidopsis, tomato and rice enhances plant performance under salt and drought stress conditions, because up-regulation of the type I H+PPase from Arabido...

Amyloid precursor protein and endosomal-lysosomal dysfunction in Alzheimer's disease: inseparable partners in a multifactorial disease.

PubMed

Nixon, Ralph A

2017-07-01

Abnormalities of the endosomal-lysosomal network (ELN) are a signature feature of Alzheimer's disease (AD). These include the earliest known cytopathology that is specific to AD and that affects endosomes and induces the progressive failure of lysosomes, each of which are directly linked by distinct mechanisms to neurodegeneration. The origins of ELN dysfunction and β-amyloidogenesis closely overlap, which reflects their common genetic basis, the established early involvement of endosomes and lysosomes in amyloid precursor protein (APP) processing and clearance, and the pathologic effect of certain APP metabolites on ELN functions. Genes that promote β-amyloidogenesis in AD (APP, PSEN1/2, and APOE4) have primary effects on ELN function. The importance of primary ELN dysfunction to pathogenesis is underscored by the mutations in more than 35 ELN-related genes that, thus far, are known to cause familial neurodegenerative diseases even though different pathogenic proteins may be involved. In this article, I discuss growing evidence that implicates AD gene-driven ELN disruptions as not only the antecedent pathobiology that underlies β-amyloidogenesis but also as the essential partner with APP and its metabolites that drive the development of AD, including tauopathy, synaptic dysfunction, and neurodegeneration. The striking amelioration of diverse deficits in animal AD models by remediating ELN dysfunction further supports a need to integrate APP and ELN relationships, including the role of amyloid-β, into a broader conceptual framework of how AD arises, progresses, and may be effectively therapeutically targeted.-Nixon, R. A. Amyloid precursor protein and endosomal-lysosomal dysfunction in Alzheimer's disease: inseparable partners in a multifactorial disease. © FASEB.

[Function of transport H+-ATPases in plant cell plasma and vacuolar membranes of maize under salt stress conditions and effect of adaptogenic preparations].

PubMed

Rybchenko, Zh I; Palladina, T O

2011-01-01

Participations of electrogenic H+-pumps of plasma and vacuolar membranes represented by E1-E2 and V-type H+-ATPases in plant cell adaptation to salt stress conditions has been studied by determination of their transport activities. Experiments were carried out on corn seedlings exposed during 1 or 10 days at 0.1 M NaCl. Preparations Methyure and Ivine were used by seed soaking at 10(-7) M. Plasma and vacuolar membrane fractions were isolated from corn seedling roots. In variants without NaCl a hydrolytical activity of plasma membrane H+-ATPase was increased with seedling age and its transport one was changed insignificantly, wherease the response of the weaker vacuolar H+-ATPase was opposite. NaCl exposition decreased hydrolytical activities of both H+-ATPases and increased their transport ones. These results demonstrated amplification of H+-pumps function especially represented by vacuolar H+-ATPase. Both preparations, Methyure mainly, caused a further increase of transport activity which was more expressed in NaCl variants. Obtained results showed the important role of these H+-pumps in plant adaptation under salt stress conditions realized by energetical maintenance of the secondary active Na+/H+ -antiporters which remove Na+ from cytoplasm.

Amyloid Precursor-like Protein 2 Association with HLA Class I Molecules

PubMed Central

Tuli, Amit; Sharma, Mahak; Wang, Xiaojian; Simone, Laura C.; Capek, Haley L.; Cate, Steven; Hildebrand, William H.; Naslavsky, Naava; Caplan, Steve; Solheim, Joyce C.

2009-01-01

Amyloid precursor-like protein 2 (APLP2) is a ubiquitously expressed protein. The previously demonstrated functions for APLP2 include binding to the mouse major histocompatibility complex (MHC) class I molecule H-2Kd and down regulating its cell surface expression. In this study, we have investigated the interaction of APLP2 with the human leukocyte antigen (HLA) class I molecule in human tumor cell lines. APLP2 was readily detected in pancreatic, breast, and prostate tumor lines, although it was found only in very low amounts in lymphoma cell lines. In a pancreatic tumor cell line, HLA class I was extensively co-localized with APLP2 in vesicular compartments following endocytosis of HLA class I molecules. In pancreatic, breast, and prostate tumor lines, APLP2 was bound to the HLA class I molecule. APLP2 was found to bind to HLA-A24, and more strongly to HLA-A2. Increased expression of APLP2 resulted in reduced surface expression of HLA-A2 and HLA-A24. Overall, these studies demonstrate that APLP2 binds to the HLA class I molecule, co-localizes with it in intracellular vesicles, and reduces the level of HLA class I molecule cell surface expression. PMID:19184004

The Arabidopsis Vacuolar Sorting Receptor1 Is Required for Osmotic Stress-Induced Abscisic Acid Biosynthesis1[OPEN

PubMed Central

Wang, Zhen-Yu; Gehring, Chris; Zhu, Jianhua; Li, Feng-Min; Zhu, Jian-Kang; Xiong, Liming

2015-01-01

Osmotic stress activates the biosynthesis of the phytohormone abscisic acid (ABA) through a pathway that is rate limited by the carotenoid cleavage enzyme 9-cis-epoxycarotenoid dioxygenase (NCED). To understand the signal transduction mechanism underlying the activation of ABA biosynthesis, we performed a forward genetic screen to isolate mutants defective in osmotic stress regulation of the NCED3 gene. Here, we identified the Arabidopsis (Arabidopsis thaliana) Vacuolar Sorting Receptor1 (VSR1) as a unique regulator of ABA biosynthesis. The vsr1 mutant not only shows increased sensitivity to osmotic stress, but also is defective in the feedback regulation of ABA biosynthesis by ABA. Further analysis revealed that vacuolar trafficking mediated by VSR1 is required for osmotic stress-responsive ABA biosynthesis and osmotic stress tolerance. Moreover, under osmotic stress conditions, the membrane potential, calcium flux, and vacuolar pH changes in the vsr1 mutant differ from those in the wild type. Given that manipulation of the intracellular pH is sufficient to modulate the expression of ABA biosynthesis genes, including NCED3, and ABA accumulation, we propose that intracellular pH changes caused by osmotic stress may play a signaling role in regulating ABA biosynthesis and that this regulation is dependent on functional VSR1. PMID:25416474

The formation of Anthocyanic Vacuolar Inclusions in Arabidopsis thaliana and implications for the sequestration of anthocyanin pigments.

PubMed

Pourcel, Lucille; Irani, Niloufer G; Lu, Yuhua; Riedl, Ken; Schwartz, Steve; Grotewold, Erich

2010-01-01

Anthocyanins are flavonoid pigments that accumulate in the large central vacuole of most plants. Inside the vacuole, anthocyanins can be found uniformly distributed or as part of sub-vacuolar pigment bodies, the Anthocyanic Vacuolar Inclusions (AVIs). Using Arabidopsis seedlings grown under anthocyanin-inductive conditions as a model to understand how AVIs are formed, we show here that the accumulation of AVIs strongly correlates with the formation of cyanidin 3-glucoside (C3G) and derivatives. Arabidopsis mutants that fail to glycosylate anthocyanidins at the 5-O position (5gt mutant) accumulate AVIs in almost every epidermal cell of the cotyledons, as compared to wild-type seedlings, where only a small fraction of the cells show AVIs. A similar phenomenon is observed when seedlings are treated with vanadate. Highlighting a role for autophagy in the formation of the AVIs, we show that various mutants that interfere with the autophagic process (atg mutants) display lower numbers of AVIs, in addition to a reduced accumulation of anthocyanins. Interestingly, vanadate increases the numbers of AVIs in the atg mutants, suggesting that several pathways might participate in AVI formation. Taken together, our results suggest novel mechanisms for the formation of sub-vacuolar compartments capable of accumulating anthocyanin pigments.

Glucose Starvation Inhibits Autophagy via Vacuolar Hydrolysis and Induces Plasma Membrane Internalization by Down-regulating Recycling*

PubMed Central

Lang, Michael J.; Martinez-Marquez, Jorge Y.; Prosser, Derek C.; Ganser, Laura R.; Buelto, Destiney; Wendland, Beverly; Duncan, Mara C.

2014-01-01

Cellular energy influences all aspects of cellular function. Although cells can adapt to a gradual reduction in energy, acute energy depletion poses a unique challenge. Because acute depletion hampers the transport of new energy sources into the cell, the cell must use endogenous substrates to replenish energy after acute depletion. In the yeast Saccharomyces cerevisiae, glucose starvation causes an acute depletion of intracellular energy that recovers during continued glucose starvation. However, how the cell replenishes energy during the early phase of glucose starvation is unknown. In this study, we investigated the role of pathways that deliver proteins and lipids to the vacuole during glucose starvation. We report that in response to glucose starvation, plasma membrane proteins are directed to the vacuole through reduced recycling at the endosomes. Furthermore, we found that vacuolar hydrolysis inhibits macroautophagy in a target of rapamycin complex 1-dependent manner. Accordingly, we found that endocytosis and hydrolysis are required for survival in glucose starvation, whereas macroautophagy is dispensable. Together, these results suggest that hydrolysis of components delivered to the vacuole independent of autophagy is the cell survival mechanism used by S. cerevisiae in response to glucose starvation. PMID:24753258

Internalization and vacuolar targeting of the brassinosteroid hormone receptor BRI1 are regulated by ubiquitination.

PubMed

Martins, Sara; Dohmann, Esther M N; Cayrel, Anne; Johnson, Alexander; Fischer, Wolfgang; Pojer, Florence; Satiat-Jeunemaître, Béatrice; Jaillais, Yvon; Chory, Joanne; Geldner, Niko; Vert, Grégory

2015-01-21

Brassinosteroids are plant steroid hormones that control many aspects of plant growth and development, and are perceived at the cell surface by the plasma membrane-localized receptor kinase BRI1. Here we show that BRI1 is post-translationally modified by K63 polyubiquitin chains in vivo. Using both artificial ubiquitination of BRI1 and generation of an ubiquitination-defective BRI1 mutant form, we demonstrate that ubiquitination promotes BRI1 internalization from the cell surface and is essential for its recognition at the trans-Golgi network/early endosomes (TGN/EE) for vacuolar targeting. Finally, we demonstrate that the control of BRI1 protein dynamics by ubiquitination is an important control mechanism for brassinosteroid responses in plants. Altogether, our results identify ubiquitination and K63-linked polyubiquitin chain formation as a dual targeting signal for BRI1 internalization and sorting along the endocytic pathway, and highlight its role in hormonally controlled plant development.

Bidirectional Regulation of Amyloid Precursor Protein-Induced Memory Defects by Nebula/DSCR1: A Protein Upregulated in Alzheimer's Disease and Down Syndrome

PubMed Central

Shaw, Jillian L.; Zhang, Shixing

2015-01-01

Aging individuals with Down syndrome (DS) have an increased risk of developing Alzheimer's disease (AD), a neurodegenerative disorder characterized by impaired memory. Memory problems in both DS and AD individuals usually develop slowly and progressively get worse with age, but the cause of this age-dependent memory impairment is not well understood. This study examines the functional interactions between Down syndrome critical region 1 (DSCR1) and amyloid-precursor protein (APP), proteins upregulated in both DS and AD, in regulating memory. Using Drosophila as a model, we find that overexpression of nebula (fly homolog of DSCR1) initially protects against APP-induced memory defects by correcting calcineurin and cAMP signaling pathways but accelerates the rate of memory loss and exacerbates mitochondrial dysfunction in older animals. We report that transient upregulation of Nebula/DSCR1 or acute pharmacological inhibition of calcineurin in aged flies protected against APP-induced memory loss. Our data suggest that calcineurin dyshomeostasis underlies age-dependent memory impairments and further imply that chronic Nebula/DSCR1 upregulation may contribute to age-dependent memory impairments in AD in DS. SIGNIFICANCE STATEMENT Most Down syndrome (DS) individuals eventually develop Alzheimer's disease (AD)-like dementia, but mechanisms underlying this age-dependent memory impairment remain poorly understood. This study examines Nebula/Down syndrome critical region 1 (DSCR1) and amyloid-precursor protein (APP), proteins upregulated in both DS and AD, in regulating memory. We uncover a previously unidentified role for Nebula/DSCR1 in modulating APP-induced memory defects during aging. We show that upregulation of Nebula/DSCR1, an inhibitor of calcineurin, rescues APP-induced memory defects in young flies but enhances memory loss of older flies. Excitingly, transient Nebula/DSCR1 overexpression or calcineurin inhibition in aged flies ameliorates APP-mediated memory

Bidirectional Regulation of Amyloid Precursor Protein-Induced Memory Defects by Nebula/DSCR1: A Protein Upregulated in Alzheimer's Disease and Down Syndrome.

PubMed

Shaw, Jillian L; Zhang, Shixing; Chang, Karen T

2015-08-12

Aging individuals with Down syndrome (DS) have an increased risk of developing Alzheimer's disease (AD), a neurodegenerative disorder characterized by impaired memory. Memory problems in both DS and AD individuals usually develop slowly and progressively get worse with age, but the cause of this age-dependent memory impairment is not well understood. This study examines the functional interactions between Down syndrome critical region 1 (DSCR1) and amyloid-precursor protein (APP), proteins upregulated in both DS and AD, in regulating memory. Using Drosophila as a model, we find that overexpression of nebula (fly homolog of DSCR1) initially protects against APP-induced memory defects by correcting calcineurin and cAMP signaling pathways but accelerates the rate of memory loss and exacerbates mitochondrial dysfunction in older animals. We report that transient upregulation of Nebula/DSCR1 or acute pharmacological inhibition of calcineurin in aged flies protected against APP-induced memory loss. Our data suggest that calcineurin dyshomeostasis underlies age-dependent memory impairments and further imply that chronic Nebula/DSCR1 upregulation may contribute to age-dependent memory impairments in AD in DS. Most Down syndrome (DS) individuals eventually develop Alzheimer's disease (AD)-like dementia, but mechanisms underlying this age-dependent memory impairment remain poorly understood. This study examines Nebula/Down syndrome critical region 1 (DSCR1) and amyloid-precursor protein (APP), proteins upregulated in both DS and AD, in regulating memory. We uncover a previously unidentified role for Nebula/DSCR1 in modulating APP-induced memory defects during aging. We show that upregulation of Nebula/DSCR1, an inhibitor of calcineurin, rescues APP-induced memory defects in young flies but enhances memory loss of older flies. Excitingly, transient Nebula/DSCR1 overexpression or calcineurin inhibition in aged flies ameliorates APP-mediated memory problems. These results

The Cellular Chaperone Heat Shock Protein 90 Is Required for Foot-and-Mouth Disease Virus Capsid Precursor Processing and Assembly of Capsid Pentamers.

PubMed

Newman, Joseph; Asfor, Amin S; Berryman, Stephen; Jackson, Terry; Curry, Stephen; Tuthill, Tobias J

2018-03-01

Productive picornavirus infection requires the hijacking of host cell pathways to aid with the different stages of virus entry, synthesis of the viral polyprotein, and viral genome replication. Many picornaviruses, including foot-and-mouth disease virus (FMDV), assemble capsids via the multimerization of several copies of a single capsid precursor protein into a pentameric subunit which further encapsidates the RNA. Pentamer formation is preceded by co- and posttranslational modification of the capsid precursor (P1-2A) by viral and cellular enzymes and the subsequent rearrangement of P1-2A into a structure amenable to pentamer formation. We have developed a cell-free system to study FMDV pentamer assembly using recombinantly expressed FMDV capsid precursor and 3C protease. Using this assay, we have shown that two structurally different inhibitors of the cellular chaperone heat shock protein 90 (hsp90) impeded FMDV capsid precursor processing and subsequent pentamer formation. Treatment of FMDV permissive cells with the hsp90 inhibitor prior to infection reduced the endpoint titer by more than 10-fold while not affecting the activity of a subgenomic replicon, indicating that translation and replication of viral RNA were unaffected by the drug. IMPORTANCE FMDV of the Picornaviridae family is a pathogen of huge economic importance to the livestock industry due to its effect on the restriction of livestock movement and necessary control measures required following an outbreak. The study of FMDV capsid assembly, and picornavirus capsid assembly more generally, has tended to be focused upon the formation of capsids from pentameric intermediates or the immediate cotranslational modification of the capsid precursor protein. Here, we describe a system to analyze the early stages of FMDV pentameric capsid intermediate assembly and demonstrate a novel requirement for the cellular chaperone hsp90 in the formation of these pentameric intermediates. We show the added complexity

Hygromycin B hypersensitive (hhy) mutants implicate an intact trans-Golgi and late endosome interface in efficient Tor1 vacuolar localization and TORC1 function.

PubMed

Ejzykowicz, Daniele E; Locken, Kristopher M; Ruiz, Fiona J; Manandhar, Surya P; Olson, Daniel K; Gharakhanian, Editte

2017-06-01

Saccharomyces cerevisiae vacuoles are functionally analogous to mammalian lysosomes. Both also serve as physical platforms for Tor Complex 1 (TORC1) signal transduction, the master regulator of cellular growth and proliferation. Hygromycin B is a eukaryotic translation inhibitor. We recently reported on hygromycin B hypersensitive (hhy) mutants that fail to grow at subtranslation inhibitory concentrations of the drug and exhibit vacuolar defects (Banuelos et al. in Curr Genet 56:121-137, 2010). Here, we show that hhy phenotype is not due to increased sensitivity to translation inhibition and establish a super HHY (s-HHY) subgroup of genes comprised of ARF1, CHC1, DRS2, SAC1, VPS1, VPS34, VPS45, VPS52, and VPS54 that function exclusively or inclusively at trans-Golgi and late endosome interface. Live cell imaging of s-hhy mutants revealed that hygromycin B treatment disrupts vacuolar morphology and the localization of late endosome marker Pep12, but not that of late endosome-independent vacuolar SNARE Vam3. This, along with normal post-late endosome trafficking of the vital dye FM4-64, establishes that severe hypersensitivity to hygromycin B correlates specifically with compromised trans-Golgi and late endosome interface. We also show that Tor1p vacuolar localization and TORC1 anabolic functions, including growth promotion and phosphorylation of its direct substrate Sch9, are compromised in s-hhy mutants. Thus, an intact trans-Golgi and late endosome interface is a requisite for efficient Tor1 vacuolar localization and TORC1 function.

Different haplotypes encode the same protein for independent sources of zucchini yellow mosaic virus resistance in cucumber

USDA-ARS?s Scientific Manuscript database

Cucumber (Cucumis sativus) production is negatively affected by zucchini yellow mosaic virus (ZYMV). Three sources of ZYMV resistance have been commercially deployed and all three resistances are conditioned by a single recessive gene. A vacuolar protein sorting-associated protein 4-like (VPS4-like)...

Structures of Trypanosome Vacuolar Soluble Pyrophosphatases: Anti-Parasitic Drug Targets

PubMed Central

Yang, Yunyun; Ko, Tzu-Ping; Chen, Chun-Chi; Huang, Guozhong; Zheng, Yingying; Liu, Weidong; Wang, Iren; Ho, Meng-Ru; Danny Hsu, Shang-Te; O’Dowd, Bing; Huff, Hannah C.; Huang, Chun-Hsiang; Docampo, Roberto; Oldfield, Eric; Guo, Rey-Ting

2016-01-01

Trypanosomatid parasites are the causative agents of many neglected tropical diseases including the leishmaniases, Chagas disease, and human African trypanosomiasis. They exploit unusual vacuolar soluble pyrophosphatases (VSPs), absent in humans, for cell growth and virulence and as such, are drug targets. Here, we report the crystal structures of VSP1s from Trypanosoma cruzi and T. brucei, together with that of the T. cruzi protein bound to a bisphosphonate inhibitor. Both VSP1s form a hybrid structure containing an (N-terminal) EF-hand domain fused to a (C-terminal) pyrophosphatase domain. The two domains are connected via an extended loop of about 17 residues. Crystallographic analysis and size exclusion chromatography indicate that the VSP1s form tetramers containing head-to-tail dimers. Phosphate and diphosphate ligands bind in the PPase substrate-binding pocket and interact with several conserved residues, and a bisphosphonate inhibitor (BPH-1260) binds to the same site. Based on Cytoscape and other bioinformatics analyses it is apparent that similar folds will be found in most if not all trypanosomatid VSP1s, including those found in insects (Angomonas deanei, Strigomonas culicis), plant pathogens (Phytomonas spp.) and Leishmania spp. Overall, the results are of general interest since they open the way to structure-based drug design for many of the neglected tropical diseases. PMID:26907161

Allelic differences in a vacuolar invertase affect Arabidopsis growth at early plant development.

PubMed

Leskow, Carla Coluccio; Kamenetzky, Laura; Dominguez, Pia Guadalupe; Díaz Zirpolo, José Antonio; Obata, Toshihiro; Costa, Hernán; Martí, Marcelo; Taboga, Oscar; Keurentjes, Joost; Sulpice, Ronan; Ishihara, Hirofumi; Stitt, Mark; Fernie, Alisdair Robert; Carrari, Fernando

2016-07-01

Improving carbon fixation in order to enhance crop yield is a major goal in plant sciences. By quantitative trait locus (QTL) mapping, it has been demonstrated that a vacuolar invertase (vac-Inv) plays a key role in determining the radical length in Arabidopsis. In this model, variation in vac-Inv activity was detected in a near isogenic line (NIL) population derived from a cross between two divergent accessions: Landsberg erecta (Ler) and Cape Verde Island (CVI), with the CVI allele conferring both higher Inv activity and longer radicles. The aim of the current work is to understand the mechanism(s) underlying this QTL by analyzing structural and functional differences of vac-Inv from both accessions. Relative transcript abundance analyzed by quantitative real-time PCR (qRT-PCR) showed similar expression patterns in both accessions; however, DNA sequence analyses revealed several polymorphisms that lead to changes in the corresponding protein sequence. Moreover, activity assays revealed higher vac-Inv activity in genotypes carrying the CVI allele than in those carrying the Ler allele. Analyses of purified recombinant proteins showed a similar K m for both alleles and a slightly higher V max for that of Ler. Treatment of plant extracts with foaming to release possible interacting Inv inhibitory protein(s) led to a large increase in activity for the Ler allele, but no changes for genotypes carrying the CVI allele. qRT-PCR analyses of two vac-Inv inhibitors in seedlings from parental and NIL genotypes revealed different expression patterns. Taken together, these results demonstrate that the vac-Inv QTL affects root biomass accumulation and also carbon partitioning through a differential regulation of vac-Inv inhibitors at the mRNA level. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Membrane microdomain switching: a regulatory mechanism of amyloid precursor protein processing.

PubMed

Sakurai, Takashi; Kaneko, Kumi; Okuno, Misako; Wada, Koji; Kashiyama, Taku; Shimizu, Hideaki; Akagi, Takumi; Hashikawa, Tsutomu; Nukina, Nobuyuki

2008-10-20

Neuronal activity has an impact on beta cleavage of amyloid precursor protein (APP) by BACE1 to generate amyloid-beta peptide (Abeta). However, the molecular mechanisms underlying this effect remain to be elucidated. Cholesterol dependency of beta cleavage prompted us to analyze immunoisolated APP-containing detergent-resistant membranes from rodent brains. We found syntaxin 1 as a key molecule for activity-dependent regulation of APP processing in cholesterol-dependent microdomains. In living cells, APP associates with syntaxin 1-containing microdomains through X11-Munc18, which inhibits the APP-BACE1 interaction and beta cleavage via microdomain segregation. Phosphorylation of Munc18 by cdk5 causes a shift of APP to BACE1-containing microdomains. Neuronal hyperactivity, implicated in Abeta overproduction, promotes the switching of APP microdomain association as well as beta cleavage in a partially cdk5-dependent manner. We propose that microdomain switching is a mechanism of cholesterol- and activity-dependent regulation of APP processing in neurons.

Membrane microdomain switching: a regulatory mechanism of amyloid precursor protein processing

PubMed Central

Sakurai, Takashi; Kaneko, Kumi; Okuno, Misako; Wada, Koji; Kashiyama, Taku; Shimizu, Hideaki; Akagi, Takumi; Hashikawa, Tsutomu; Nukina, Nobuyuki

2008-01-01

Neuronal activity has an impact on β cleavage of amyloid precursor protein (APP) by BACE1 to generate amyloid-β peptide (Aβ). However, the molecular mechanisms underlying this effect remain to be elucidated. Cholesterol dependency of β cleavage prompted us to analyze immunoisolated APP-containing detergent-resistant membranes from rodent brains. We found syntaxin 1 as a key molecule for activity-dependent regulation of APP processing in cholesterol-dependent microdomains. In living cells, APP associates with syntaxin 1–containing microdomains through X11–Munc18, which inhibits the APP–BACE1 interaction and β cleavage via microdomain segregation. Phosphorylation of Munc18 by cdk5 causes a shift of APP to BACE1-containing microdomains. Neuronal hyperactivity, implicated in Aβ overproduction, promotes the switching of APP microdomain association as well as β cleavage in a partially cdk5-dependent manner. We propose that microdomain switching is a mechanism of cholesterol- and activity-dependent regulation of APP processing in neurons. PMID:18936252

Tyrosine Binding Protein Sites Regulate the Intracellular Trafficking and Processing of Amyloid Precursor Protein through a Novel Lysosome-Directed Pathway.

PubMed

Tam, Joshua H K; Cobb, M Rebecca; Seah, Claudia; Pasternak, Stephen H

2016-01-01

The amyloid hypothesis posits that the production of β-amyloid (Aβ) aggregates leads to neurodegeneration and cognitive decline associated with AD. Aβ is produced by sequential cleavage of the amyloid precursor protein (APP) by β- and γ-secretase. While nascent APP is well known to transit to the endosomal/ lysosomal system via the cell surface, we have recently shown that APP can also traffic to lysosomes intracellularly via its interaction with AP-3. Because AP-3 interacts with cargo protein via interaction with tyrosine motifs, we mutated the three tyrosines motif in the cytoplasmic tail of APP. Here, we show that the YTSI motif interacts with AP-3, and phosphorylation of the serine in this motif disrupts the interaction and decreases APP trafficking to lysosomes. Furthermore, we show that phosphorylation at this motif can decrease the production of neurotoxic Aβ 42. This demonstrates that reducing APP trafficking to lysosomes may be a strategy to reduce Aβ 42 in Alzheimer's disease.

Tyrosine Binding Protein Sites Regulate the Intracellular Trafficking and Processing of Amyloid Precursor Protein through a Novel Lysosome-Directed Pathway

PubMed Central

Tam, Joshua H. K.; Cobb, M. Rebecca; Seah, Claudia; Pasternak, Stephen H.

2016-01-01

The amyloid hypothesis posits that the production of β-amyloid (Aβ) aggregates leads to neurodegeneration and cognitive decline associated with AD. Aβ is produced by sequential cleavage of the amyloid precursor protein (APP) by β- and γ-secretase. While nascent APP is well known to transit to the endosomal/ lysosomal system via the cell surface, we have recently shown that APP can also traffic to lysosomes intracellularly via its interaction with AP-3. Because AP-3 interacts with cargo protein via interaction with tyrosine motifs, we mutated the three tyrosines motif in the cytoplasmic tail of APP. Here, we show that the YTSI motif interacts with AP-3, and phosphorylation of the serine in this motif disrupts the interaction and decreases APP trafficking to lysosomes. Furthermore, we show that phosphorylation at this motif can decrease the production of neurotoxic Aβ 42. This demonstrates that reducing APP trafficking to lysosomes may be a strategy to reduce Aβ 42 in Alzheimer’s disease. PMID:27776132

Two forms of Vibrio cholerae O1 El Tor hemolysin derived from identical precursor protein.

PubMed

Ikigai, H; Ono, T; Nakae, T; Otsuru, H; Shimamura, T

1999-01-08

Vibrio cholerae O1 grown in heart infusion broth produces two forms of El Tor hemolysin (ETH) monomers of 65 and 50 kDa. These monomers form several different sizes of mixed oligomers ranging from 180 to 280 kDa in the liposomal membranes. We found that the N-terminal amino acid sequences, NH2-Trp-Pro-Ala-Pro-Ala-Asn-Ser-Glu, of both the 65- and 50-kDa toxins were identical. We assumed, therefore, that the 65- and 50-kDa toxins were derivatives of the identical precursor protein and the 50-kDa protein was a truncated derivative of 65-kDa ETH. To substantiate this assumption, we treated the 260-kDa oligomer with trypsin and obtained a 190-kDa oligomer. This 190-kDa oligomer consisted of only the 50-kDa subunits. Both 260- and 190-kDa oligomers formed ion channels indistinguishable from each other in planar lipid bilayers. These results suggest that the essential part of the ETH in forming the membrane-damaging aggregate is a 50-kDa protein.

Characterization of the beta amyloid precursor protein-like gene in the central nervous system of the crab Chasmagnathus. Expression during memory consolidation.

PubMed

Fustiñana, Maria Sol; Ariel, Pablo; Federman, Noel; Freudenthal, Ramiro; Romano, Arturo

2010-09-01

Human β-amyloid, the main component in the neuritic plaques found in patients with Alzheimer's disease, is generated by cleavage of the β-amyloid precursor protein. Beyond the role in pathology, members of this protein family are synaptic proteins and have been associated with synaptogenesis, neuronal plasticity and memory, both in vertebrates and in invertebrates. Consolidation is necessary to convert a short-term labile memory to a long-term and stable form. During consolidation, gene expression and de novo protein synthesis are regulated in order to produce key proteins for the maintenance of plastic changes produced during the acquisition of new information. Here we partially cloned and sequenced the beta-amyloid precursor protein like gene homologue in the crab Chasmagnathus (cappl), showing a 37% of identity with the fruit fly Drosophila melanogaster homologue and 23% with Homo sapiens but with much higher degree of sequence similarity in certain regions. We observed a wide distribution of cappl mRNA in the nervous system as well as in muscle and gills. The protein localized in all tissues analyzed with the exception of muscle. Immunofluorescence revealed localization of cAPPL in associative and sensory brain areas. We studied gene and protein expression during long-term memory consolidation using a well characterized memory model: the context-signal associative memory in this crab species. mRNA levels varied at different time points during long-term memory consolidation and correlated with cAPPL protein levels cAPPL mRNA and protein is widely distributed in the central nervous system of the crab and the time course of expression suggests a role of cAPPL during long-term memory formation.

Amyloid beta precursor protein regulates male sexual behavior.

PubMed

Park, Jin Ho; Bonthius, Paul J; Tsai, Houng-Wei; Bekiranov, Stefan; Rissman, Emilie F

2010-07-28

Sexual behavior is variable between individuals, ranging from celibacy to sexual addictions. Within normal populations of individual men, ranging from young to middle aged, testosterone levels do not correlate with libido. To study the genetic mechanisms that contribute to individual differences in male sexual behavior, we used hybrid B6D2F1 male mice, which are a cross between two common inbred strains (C57BL/6J and DBA/2J). Unlike most laboratory rodent species in which male sexual behavior is highly dependent upon gonadal steroids, sexual behavior in a large proportion of these hybrid male mice after castration is independent of gonadal steroid hormones and their receptors; thus, we have the ability to discover novel genes involved in this behavior. Gene expression arrays, validation of gene candidates, and transgenic mice that overexpress one of the genes of interest were used to reveal genes involved in maintenance of male sexual behavior. Several genes related to neuroprotection and neurodegeneration were differentially expressed in the hypothalamus of males that continued to mate after castration. Male mice overexpressing the human form of one of these candidate genes, amyloid beta precursor protein (APP), displayed enhanced sexual behavior before castration and maintained sexual activity for a longer duration after castration compared with controls. Our results reveal a novel and unexpected relationship between APP and male sexual behavior. We speculate that declining APP during normal aging in males may contribute to the loss of sexual function.

COPS5 (Jab1) protein increases β site processing of amyloid precursor protein and amyloid β peptide generation by stabilizing RanBP9 protein levels.

PubMed

Wang, Hongjie; Dey, Debleena; Carrera, Ivan; Minond, Dmitriy; Bianchi, Elisabetta; Xu, Shaohua; Lakshmana, Madepalli K

2013-09-13

Increased processing of amyloid precursor protein (APP) and accumulation of neurotoxic amyloid β peptide (Aβ) in the brain is central to the pathogenesis of Alzheimer's disease (AD). Therefore, the identification of molecules that regulate Aβ generation is crucial for future therapeutic approaches for AD. We demonstrated previously that RanBP9 regulates Aβ generation in a number of cell lines and primary neuronal cultures by forming tripartite protein complexes with APP, low-density lipoprotein-related protein, and BACE1, consequently leading to increased amyloid plaque burden in the brain. RanBP9 is a scaffold protein that exists and functions in multiprotein complexes. To identify other proteins that may bind RanBP9 and regulate Aβ levels, we used a two-hybrid analysis against a human brain cDNA library and identified COPS5 as a novel RanBP9-interacting protein. This interaction was confirmed by coimmunoprecipitation experiments in both neuronal and non-neuronal cells and mouse brain. Colocalization of COPS5 and RanBP9 in the same subcellular compartments further supported the interaction of both proteins. Furthermore, like RanBP9, COPS5 robustly increased Aβ generation, followed by increased soluble APP-β (sAPP-β) and decreased soluble-APP-α (sAPP-α) levels. Most importantly, down-regulation of COPS5 by siRNAs reduced Aβ generation, implying that endogenous COPS5 regulates Aβ generation. Finally, COPS5 levels were increased significantly in AD brains and APΔE9 transgenic mice, and overexpression of COPS5 strongly increased RanBP9 protein levels by increasing its half-life. Taken together, these results suggest that COPS5 increases Aβ generation by increasing RanBP9 levels. Thus, COPS5 is a novel RanBP9-binding protein that increases APP processing and Aβ generation by stabilizing RanBP9 protein levels.

Identification of a 170-kDa protein associated with the vacuolar Na+/H+ antiport of Beta vulgaris.

PubMed Central

Barkla, B J; Blumwald, E

1991-01-01

The effect of the addition of amiloride to the growth medium was tested on the Na+/H+ antiport activity of tonoplast vesicles isolated from sugar beet (beta vulgaris L.) cell suspensions. Cells grown in the presence of NaCl and amiloride displayed an increased antiport activity. Analysis of the kinetic data showed that while the affinity of the antiport for Na+ ions did not change, the maximal velocity of the Na+/H+ exchange increased markedly. These results suggest the addition of more antiport molecules to the tonoplast and/or an increase in the turnover rate of the Na+/H+ exchange. The increase in activity of the antiport by the presence of amiloride was correlated with the enhanced synthesis of a tonoplast 170-kDa polypeptide. The increased synthesis of this polypeptide was detected not only upon exposure of the cells to amiloride but also when the cells were exposed to high NaCl concentrations. Polyclonal antibodies against the 170-kDa polypeptide almost completely inhibited the antiport activity. These results suggest the association of the 170-kDa polypeptide with the vacuolar Na+/H+ antiport. Images PMID:1662387

Identification of a 170-kDa protein associated with the vacuolar Na+/H+ antiport of Beta vulgaris.

PubMed

Barkla, B J; Blumwald, E

1991-12-15

The effect of the addition of amiloride to the growth medium was tested on the Na+/H+ antiport activity of tonoplast vesicles isolated from sugar beet (beta vulgaris L.) cell suspensions. Cells grown in the presence of NaCl and amiloride displayed an increased antiport activity. Analysis of the kinetic data showed that while the affinity of the antiport for Na+ ions did not change, the maximal velocity of the Na+/H+ exchange increased markedly. These results suggest the addition of more antiport molecules to the tonoplast and/or an increase in the turnover rate of the Na+/H+ exchange. The increase in activity of the antiport by the presence of amiloride was correlated with the enhanced synthesis of a tonoplast 170-kDa polypeptide. The increased synthesis of this polypeptide was detected not only upon exposure of the cells to amiloride but also when the cells were exposed to high NaCl concentrations. Polyclonal antibodies against the 170-kDa polypeptide almost completely inhibited the antiport activity. These results suggest the association of the 170-kDa polypeptide with the vacuolar Na+/H+ antiport.

beta. -Amyloid precursor protein of Alzheimer disease occurs as 110- to 135-kilodalton membrane-associated proteins in neural and nonneural tissues

DOE Office of Scientific and Technical Information (OSTI.GOV)

Selkoe, D.J.; Podlisny, M.B.; Joachim, C.L.

1988-10-01

Progressive cerebral deposition of extracellular filaments composed of the {beta}-amyloid protein ({beta}AP) is a constant feature of Alzheimer disease (AD). Since the gene on chromosome 21 encoding the {beta}AP precursor ({beta}APP) is not known to be altered in AD, transcriptional or posttranslational changes may underlie accelerated {beta}AP deposition. Using two antibodies to the predicted carboxyl terminus of {beta}APP, the authors have identified the native {beta}APP in brain and nonneural human tissues as a 110- to 135-kDa protein complex that is insoluble in buffer and found in various membrane-rich subcellular fractions. These proteins are relatively uniformly distributed in adult brain, abundantmore » in fetal brain, and detected in nonneural tissues that contain {beta}APP mRNA. Similarly sized proteins occur in rat, cow, and monkey brain and in cultured human HL-60 and HeLa cells; the precise patterns in the 110- to 135-kDa range are heterogeneous among various tissues and cell lines. They conclude that the highly conserved {beta}APP molecule occurs in mammalian tissues as a heterogeneous group of membrane-associated proteins of {approx} 120 kDa. Detection of the nonamyloidogenic carboxyl terminus within plaques suggests that proteolytic processing of the {beta}APP into insoluble filaments occurs locally in cortical regions that develop {beta}-amyloid deposits with age.« less

Amyloid-β protein precursor regulates phosphorylation and cellular compartmentalization of microtubule associated protein tau.

PubMed

Nizzari, Mario; Barbieri, Federica; Gentile, Maria Teresa; Passarella, Daniela; Caorsi, Calentina; Diaspro, Alberto; Taglialatela, Maurizio; Pagano, Aldo; Colucci-D'Amato, Luca; Florio, Tullio; Russo, Claudio

2012-01-01

Tau is a multifunctional protein detected in different cellular compartments in neuronal and non-neuronal cells. When hyperphosphorylated and aggregated in atrophic neurons, tau is considered the culprit for neuronal death in familial and sporadic tauopathies. With regards to Alzheimer's disease (AD) pathogenesis, it is not yet established whether entangled tau represents a cause or a consequence of neurodegeneration. In fact, it is unquestionably accepted that amyloid-β protein precursor (AβPP) plays a pivotal role in the genesis of the disease, and it is postulated that the formation of toxic amyloid-β peptides from AβPP is the primary event that subsequently induces abnormal tau phosphorylation. In this work, we show that in the brain of AD patients there is an imbalance between the nuclear and the cytoskeletal pools of phospho-tau. We observed that in non-AD subjects, there is a stable pool of phospho-tau which remains strictly confined to neuronal nuclei, while nuclear localization of phospho-tau is significantly underrepresented in neurons of AD patients bearing neurofibrillary tangles. A specific phosphorylation of tau is required during mitosis in vitro and in vivo, likely via a Grb2-ERK1/2 signaling cascade. In differentiated neuronal A1 cells, the overexpression of AβPP modulates tau phosphorylation, altering the ratio between cytoskeletal and nuclear pools, and correlates with cell death. Altogether our data provide evidence that AβPP, in addition to amyloid formation, modulates the phosphorylation of tau and its subcellular compartmentalization, an event that may lead to the formation of neurofibrillary tangles and to neurodegeneration when occurring in postmitotic neurons.

A canine model of Alzheimer's disease generated by overexpressing a mutated human amyloid precursor protein.

PubMed

Lee, Geun-Shik; Jeong, Yeon Woo; Kim, Joung Joo; Park, Sun Woo; Ko, Kyeong Hee; Kang, Mina; Kim, Yu Kyung; Jung, Eui-Man; Moon, Changjong; Hyun, Sang Hwan; Hwang, Kyu-Chan; Kim, Nam-Hyung; Shin, Taeyoung; Jeung, Eui-Bae; Hwang, Woo Suk

2014-04-01

Canines are considered the most authentic model for studying multifactorial human diseases, as these animals typically share a common environment with man. Somatic cell nuclear transfer (SCNT) technology along with genetic engineering of nuclear donor cells provides a unique opportunity for examining human diseases using transgenic canines. In the present study, we generated transgenic canines that overexpressed the human amyloid precursor protein (APP) gene containing well-characterized familial Alzheimer's disease (AD) mutations. We successfully obtained five out of six live puppies by SCNT. This was confirmed by observing the expression of green fluorescence protein in the body as a visual transgenic marker and the overexpression of the mutated APP gene in the brain. The transgenic canines developed AD-like symptoms, such as enlarged ventricles, an atrophied hippocampus, and β-amyloid plaques in the brain. Thus, the transgenic canines we created can serve as a novel animal model for studying human AD.

Regulatory assembly of the vacuolar proton pump VoV1-ATPase in yeast cells by FLIM-FRET

NASA Astrophysics Data System (ADS)

Ernst, Stefan; Batisse, Claire; Zarrabi, Nawid; Böttcher, Bettina; Börsch, Michael

2010-02-01

We investigate the reversible disassembly of VOV1-ATPase in life yeast cells by time resolved confocal FRET imaging. VOV1-ATPase in the vacuolar membrane pumps protons from the cytosol into the vacuole. VOV1-ATPase is a rotary biological nanomotor driven by ATP hydrolysis. The emerging proton gradient is used for secondary transport processes as well as for pH and Ca2+ homoeostasis in the cell. The activity of the VOV1-ATPase is regulated through assembly / disassembly processes. During starvation the two parts of VOV1-ATPase start to disassemble. This process is reversed after addition of glucose. The exact mechanisms are unknown. To follow the disassembly / reassembly in vivo we tagged two subunits C and E with different fluorescent proteins. Cellular distributions of C and E were monitored using a duty cycle-optimized alternating laser excitation scheme (DCO-ALEX) for time resolved confocal FRET-FLIM measurements.

Caspase-6 Activation in Familial Alzheimer Disease Brains Carrying Amyloid Precursor Protein, Presenilin I or Presenilin II Mutations

PubMed Central

Albrecht, Steffen; Bogdanovic, Nenad; Ghetti, Bernardino; Winblad, Bengt; LeBlanc, Andréa C.

2010-01-01

We previously demonstrated the activation of Caspase-6 in the hippocampus and cortex in cases of mild, moderate, severe and very severe Alzheimer disease (AD). To determine whether Caspase-6 is also activated in familial AD, we performed an immunohistochemical analysis of active Caspase-6 and Tau cleaved by Caspase-6 in temporal cortex and hippocampal tissue sections from cases of familial AD. The cases included 5 carrying the amyloid precursor protein K670N, M671L Swedish mutation, 1 carrying the amyloid precursor protein E693G Arctic mutation, 2 each carrying the Presenilin I M146V, F105L, A431E, V261F, Y115C mutations, and 1 with the Presenilin II N141I mutation. Active Caspase-6 immunoreactivity was found in all cases. Caspase-6 immunoreactivity was observed in neuritic plaques or cotton wool plaques in some cases, neuropil threads and neurofibrillary tangles. These results indicate that Caspase-6 is activated in familial forms of AD, as previously observed in sporadic forms. Since sporadic and familial AD cases have similar pathological features, these results support a fundamental role of Caspase-6 in the pathophysiology of both familial and sporadic AD. PMID:19915487

Immunological demonstration of intestinal absorption and digestion of protein macromolecules in the trout (Salmo gairdneri).

PubMed

Georgopoulou, U; Sire, M F; Vernier, J M

1986-01-01

An immunofluorescence technique using antibodies against the Fc and Fab fragments of human IgG (IgGH) was used to study the absorption of proteins by the intestinal epithelial cells of rainbow trout after oral or anal administration. Cellular absorption of a high molecular weight protein, hepatitis-B surface antigen (HBsAg), was also studied by using two monoclonal antibodies, one specific for the confirmation of the antigen (implying disulfide bridges), and the other that reacts with the constituent polypeptides. Both absorbed IgGH and HBsAg were seen to be segregated in the apical vacuolar system, a characteristic feature of intestinal epithelial cells. The same antibodies were used with an everted sac technique in conjunction with immunofluorescence, to show the intravacuolar degradation of IgGH and HBsAg following absorption. By using an antibody against cathepsin D, it was possible to demonstrate, by immunofluorescence, the localization of this enzyme in the same vacuolar system. After coupling the antibody to peroxidase or to the protein A/colloidalgold complex, the ultrastructural antigenic sites of cathepsin D could be seen to be localized in the interior of the vacuoles. The vacuolar localization of a cathepsin B activity was determined by incubating sections of intestinal mucosa, or isolated epithelial cells, with a specific synthetic substrate (Z-Ala-Arg-Arg-methoxynaphthylamide). The supranuclear hyaloplasmic vacuoles of intestinal epithelial cells may be considered to be phagolysosomes that assure the degradation of absorbed proteins. This function may be of fundamental importance in the in the nutritional processes of this species.

Vacuolar H+-ATPase Protects Saccharomyces cerevisiae Cells against Ethanol-Induced Oxidative and Cell Wall Stresses

PubMed Central

Charoenbhakdi, Sirikarn; Dokpikul, Thanittra; Burphan, Thanawat; Techo, Todsapol

2016-01-01

ABSTRACT During fermentation, increased ethanol concentration is a major stress for yeast cells. Vacuolar H+-ATPase (V-ATPase), which plays an important role in the maintenance of intracellular pH homeostasis through vacuolar acidification, has been shown to be required for tolerance to straight-chain alcohols, including ethanol. Since ethanol is known to increase membrane permeability to protons, which then promotes intracellular acidification, it is possible that the V-ATPase is required for recovery from alcohol-induced intracellular acidification. In this study, we show that the effects of straight-chain alcohols on membrane permeabilization and acidification of the cytosol and vacuole are strongly dependent on their lipophilicity. These findings suggest that the membrane-permeabilizing effect of straight-chain alcohols induces cytosolic and vacuolar acidification in a lipophilicity-dependent manner. Surprisingly, after ethanol challenge, the cytosolic pH in Δvma2 and Δvma3 mutants lacking V-ATPase activity was similar to that of the wild-type strain. It is therefore unlikely that the ethanol-sensitive phenotype of vma mutants resulted from severe cytosolic acidification. Interestingly, the vma mutants exposed to ethanol exhibited a delay in cell wall remodeling and a significant increase in intracellular reactive oxygen species (ROS). These findings suggest a role for V-ATPase in the regulation of the cell wall stress response and the prevention of endogenous oxidative stress in response to ethanol. IMPORTANCE The yeast Saccharomyces cerevisiae has been widely used in the alcoholic fermentation industry. Among the environmental stresses that yeast cells encounter during the process of alcoholic fermentation, ethanol is a major stress factor that inhibits yeast growth and viability, eventually leading to fermentation arrest. This study provides evidence for the molecular mechanisms of ethanol tolerance, which is a desirable characteristic for yeast strains

Vacuolar H+-ATPase Protects Saccharomyces cerevisiae Cells against Ethanol-Induced Oxidative and Cell Wall Stresses.

PubMed

Charoenbhakdi, Sirikarn; Dokpikul, Thanittra; Burphan, Thanawat; Techo, Todsapol; Auesukaree, Choowong

2016-05-15

During fermentation, increased ethanol concentration is a major stress for yeast cells. Vacuolar H(+)-ATPase (V-ATPase), which plays an important role in the maintenance of intracellular pH homeostasis through vacuolar acidification, has been shown to be required for tolerance to straight-chain alcohols, including ethanol. Since ethanol is known to increase membrane permeability to protons, which then promotes intracellular acidification, it is possible that the V-ATPase is required for recovery from alcohol-induced intracellular acidification. In this study, we show that the effects of straight-chain alcohols on membrane permeabilization and acidification of the cytosol and vacuole are strongly dependent on their lipophilicity. These findings suggest that the membrane-permeabilizing effect of straight-chain alcohols induces cytosolic and vacuolar acidification in a lipophilicity-dependent manner. Surprisingly, after ethanol challenge, the cytosolic pH in Δvma2 and Δvma3 mutants lacking V-ATPase activity was similar to that of the wild-type strain. It is therefore unlikely that the ethanol-sensitive phenotype of vma mutants resulted from severe cytosolic acidification. Interestingly, the vma mutants exposed to ethanol exhibited a delay in cell wall remodeling and a significant increase in intracellular reactive oxygen species (ROS). These findings suggest a role for V-ATPase in the regulation of the cell wall stress response and the prevention of endogenous oxidative stress in response to ethanol. The yeast Saccharomyces cerevisiae has been widely used in the alcoholic fermentation industry. Among the environmental stresses that yeast cells encounter during the process of alcoholic fermentation, ethanol is a major stress factor that inhibits yeast growth and viability, eventually leading to fermentation arrest. This study provides evidence for the molecular mechanisms of ethanol tolerance, which is a desirable characteristic for yeast strains used in alcoholic

Singlet Oxygen-Induced Membrane Disruption and Serpin-Protease Balance in Vacuolar-Driven Cell Death1[OPEN

PubMed Central

Carmieli, Raanan; Mor, Avishai; Fluhr, Robert

2016-01-01

Singlet oxygen plays a role in cellular stress either by providing direct toxicity or through signaling to initiate death programs. It was therefore of interest to examine cell death, as occurs in Arabidopsis, due to differentially localized singlet oxygen photosensitizers. The photosensitizers rose bengal (RB) and acridine orange (AO) were localized to the plasmalemma and vacuole, respectively. Their photoactivation led to cell death as measured by ion leakage. Cell death could be inhibited by the singlet oxygen scavenger histidine in treatments with AO but not with RB. In the case of AO treatment, the vacuolar membrane was observed to disintegrate. Concomitantly, a complex was formed between a vacuolar cell-death protease, RESPONSIVE TO DESSICATION-21 and its cognate cytoplasmic protease inhibitor ATSERPIN1. In the case of RB treatment, the tonoplast remained intact and no complex was formed. Over-expression of AtSerpin1 repressed cell death, only under AO photodynamic treatment. Interestingly, acute water stress showed accumulation of singlet oxygen as determined by fluorescence of Singlet Oxygen Sensor Green, by electron paramagnetic resonance spectroscopy and the induction of singlet oxygen marker genes. Cell death by acute water stress was inhibited by the singlet oxygen scavenger histidine and was accompanied by vacuolar collapse and the appearance of serpin-protease complex. Over-expression of AtSerpin1 also attenuated cell death under this mode of cell stress. Thus, acute water stress damage shows parallels to vacuole-mediated cell death where the generation of singlet oxygen may play a role. PMID:26884487

Amyloid precursor protein controls cholesterol turnover needed for neuronal activity

PubMed Central

Pierrot, Nathalie; Tyteca, Donatienne; D'auria, Ludovic; Dewachter, Ilse; Gailly, Philippe; Hendrickx, Aurélie; Tasiaux, Bernadette; Haylani, Laetitia El; Muls, Nathalie; N'Kuli, Francisca; Laquerrière, Annie; Demoulin, Jean-Baptiste; Campion, Dominique; Brion, Jean-Pierre; Courtoy, Pierre J; Kienlen-Campard, Pascal; Octave, Jean-Noël

2013-01-01

Perturbation of lipid metabolism favours progression of Alzheimer disease, in which processing of Amyloid Precursor Protein (APP) has important implications. APP cleavage is tightly regulated by cholesterol and APP fragments regulate lipid homeostasis. Here, we investigated whether up or down regulation of full-length APP expression affected neuronal lipid metabolism. Expression of APP decreased HMG-CoA reductase (HMGCR)-mediated cholesterol biosynthesis and SREBP mRNA levels, while its down regulation had opposite effects. APP and SREBP1 co-immunoprecipitated and co-localized in the Golgi. This interaction prevented Site-2 protease-mediated processing of SREBP1, leading to inhibition of transcription of its target genes. A GXXXG motif in APP sequence was critical for regulation of HMGCR expression. In astrocytes, APP and SREBP1 did not interact nor did APP affect cholesterol biosynthesis. Neuronal expression of APP decreased both HMGCR and cholesterol 24-hydroxylase mRNA levels and consequently cholesterol turnover, leading to inhibition of neuronal activity, which was rescued by geranylgeraniol, generated in the mevalonate pathway, in both APP expressing and mevastatin treated neurons. We conclude that APP controls cholesterol turnover needed for neuronal activity. PMID:23554170

Amyloid precursor protein at node of Ranvier modulates nodal formation

PubMed Central

Xu, De-En; Zhang, Wen-Min; Yang, Zara Zhuyun; Zhu, Hong-Mei; Yan, Ke; Li, Shao; Bagnard, Dominique; Dawe, Gavin S; Ma, Quan-Hong; Xiao, Zhi-Cheng

2014-01-01

Amyloid precursor protein (APP), commonly associated with Alzheimer disease, is upregulated and distributes evenly along the injured axons, and therefore, also known as a marker of demyelinating axonal injury and axonal degeneration. However, the physiological distribution and function of APP along myelinated axons was unknown. We report that APP aggregates at nodes of Ranvier (NOR) in the myelinated central nervous system (CNS) axons but not in the peripheral nervous system (PNS). At CNS NORs, APP expression co-localizes with tenascin-R and is flanked by juxtaparanodal potassium channel expression demonstrating that APP localized to NOR. In APP-knockout (KO) mice, nodal length is significantly increased, while sodium channels are still clustered at NORs. Moreover, APP KO and APP-overexpressing transgenic (APP TG) mice exhibited a decreased and an increased thickness of myelin in spinal cords, respectively, although the changes are limited in comparison to their littermate WT mice. The thickness of myelin in APP KO sciatic nerve also increased in comparison to that in WT mice. Our observations indicate that APP acts as a novel component at CNS NORs, modulating nodal formation and has minor effects in promoting myelination. PMID:25482638

Incorporation of Deoxyribonucleic Acid Precursors by T4 Deoxyribonucleic Acid-Protein Complexes Retained on Glass Fiber Filters

PubMed Central

Miller, Robert C.; Kozinski, Andrzej W.

1970-01-01

Bacteriophage T4 deoxyribonucleic acid (DNA)-protein complexes were retained preferentially on glass fiber filters. DNA polymerase activity in the complex was detected through the incorporation of 3H-labeled DNA precursors. The primer-product DNA hybridized with both phage and Escherichia coli DNA. Density labeling experiments showed that about 30% of incorporated 3H-deoxyadenosine triphosphate was found in DNA which hybridized with phage DNA; this DNA was found to be covalently attached to the primer DNA. PMID:5497903

Vacuolar processing enzyme: an executor of plant cell death.

PubMed

Hara-Nishimura, Ikuko; Hatsugai, Noriyuki; Nakaune, Satoru; Kuroyanagi, Miwa; Nishimura, Mikio

2005-08-01

Apoptotic cell death in animals is regulated by cysteine proteinases called caspases. Recently, vacuolar processing enzyme (VPE) was identified as a plant caspase. VPE deficiency prevents cell death during hypersensitive response and cell death of limited cell layers at the early stage of embryogenesis. Because plants do not have macrophages, dying cells must degrade their materials by themselves. VPE plays an essential role in the regulation of the lytic system of plants during the processes of defense and development. VPE is localized in the vacuoles, unlike animal caspases, which are localized in the cytosol. Thus, plants might have evolved a regulated cellular suicide strategy that, unlike animal apoptosis, is mediated by VPE and the vacuoles.

The early mature part of bacterial twin-arginine translocation (Tat) precursor proteins contributes to TatBC receptor binding.

PubMed

Ulfig, Agnes; Freudl, Roland

2018-05-11

The twin-arginine translocation (Tat) pathway transports folded proteins across bacterial membranes. Tat precursor proteins possess a conserved twin-arginine (RR) motif in their signal peptides that is involved in the binding of the proteins to the membrane-associated TatBC receptor complex. In addition, the hydrophobic region in the Tat signal peptides also contributes to TatBC binding, but whether regions beyond the signal-peptide cleavage site are involved in this process is unknown. Here, we analyzed the contribution of the early mature protein part of the Escherichia coli trimethylamine N -oxide reductase (TorA) to productive TatBC receptor binding. We identified substitutions in the 30 amino acids immediately following the TorA signal peptide (30aa-region) that restored export of a transport-defective TorA[KQ]-30aa-MalE precursor, in which the RR residues had been replaced by a lysine-glutamine pair. Some of these substitutions increased the hydrophobicity of the N-terminal part of the 30aa-region and thereby likely enhanced hydrophobic substrate-receptor interactions within the hydrophobic TatBC substrate-binding cavity. Another class of substitutions increased the positive net charge of the region's C-terminal part, presumably leading to strengthened electrostatic interactions between the mature substrate part and the cytoplasmic TatBC regions. Furthermore, we identified substitutions in the C-terminal domains of TatB following the transmembrane segment that restored transport of various transport-defective TorA-MalE derivatives. Some of these substitutions most likely affected the orientation or conformation of the flexible, carboxy-proximal helices of TatB. Therefore, we propose that a tight accommodation of the folded mature region by TatB contributes to productive binding of Tat substrates to TatBC. © 2018 Ulfig and Freudl.

Vacuolar status and water relations in embryonic axes of recalcitrant Aesculus hippocastanum seeds during stratification and early germination.

PubMed

Obroucheva, Natalie V; Lityagina, Snezhana V; Novikova, Galina V; Sin'kevich, Irina A

2012-01-01

In tropical recalcitrant seeds, their rapid transition from shedding to germination at high hydration level is of physiological interest but difficult to study because of the time constraint. In recalcitrant horse chestnut seeds produced in central Russia, this transition is much longer and extends through dormancy and dormancy release. This extended time period permits studies of the water relations in embryonic axes during the long recalcitrant period in terms of vacuolar status and water transport. Horse chestnut (Aesculus hippocastanum) seeds sampled in Moscow were stratified in cold wet sand for 4 months. Vacuole presence and development in embryonic axes were examined by vital staining, light and electron microscopy. Aquaporins and vacuolar H(+)-ATPase were identified immunochemically. Water channel operation was tested by water inflow rate. Vacuolar acid invertase was estimated in terms of activity and electrophoretic properties. Throughout the long recalcitrant period after seed shedding, cells of embryonic axes maintained active vacuoles and a high water content. Preservation of enzyme machinery in vacuoles was evident from retention of invertase activity, substrate specificity, molecular mass and subunit composition. Plasmalemma and tonoplast aquaporins and the E subunit of vacuolar H(+)-ATPase were also present. In non-dormant seeds prior to growth initiation, vacuoles enlarged at first in hypocotyls, and then in radicles, with their biogenesis being similar. Vacuolation was accompanied by increasing invertase activity, leading to sugar accumulation and active osmotic functioning. After growth initiation, vacuole enlargement was favoured by enhanced water inflow through water channels formed by aquaporins. Maintenance of high water content and desiccation sensitivity, as well as preservation of active vacuoles in embryonic axes after shedding, can be considered a specific feature of recalcitrant seeds, overlooked when studying tropical recalcitrants due

Vacuolar status and water relations in embryonic axes of recalcitrant Aesculus hippocastanum seeds during stratification and early germination

PubMed Central

Obroucheva, Natalie V.; Lityagina, Snezhana V.; Novikova, Galina V.; Sin'kevich, Irina A.

2012-01-01

Backgrounds and aims In tropical recalcitrant seeds, their rapid transition from shedding to germination at high hydration level is of physiological interest but difficult to study because of the time constraint. In recalcitrant horse chestnut seeds produced in central Russia, this transition is much longer and extends through dormancy and dormancy release. This extended time period permits studies of the water relations in embryonic axes during the long recalcitrant period in terms of vacuolar status and water transport. Methodology Horse chestnut (Aesculus hippocastanum) seeds sampled in Moscow were stratified in cold wet sand for 4 months. Vacuole presence and development in embryonic axes were examined by vital staining, light and electron microscopy. Aquaporins and vacuolar H+-ATPase were identified immunochemically. Water channel operation was tested by water inflow rate. Vacuolar acid invertase was estimated in terms of activity and electrophoretic properties. Principal results Throughout the long recalcitrant period after seed shedding, cells of embryonic axes maintained active vacuoles and a high water content. Preservation of enzyme machinery in vacuoles was evident from retention of invertase activity, substrate specificity, molecular mass and subunit composition. Plasmalemma and tonoplast aquaporins and the E subunit of vacuolar H+-ATPase were also present. In non-dormant seeds prior to growth initiation, vacuoles enlarged at first in hypocotyls, and then in radicles, with their biogenesis being similar. Vacuolation was accompanied by increasing invertase activity, leading to sugar accumulation and active osmotic functioning. After growth initiation, vacuole enlargement was favoured by enhanced water inflow through water channels formed by aquaporins. Conclusions Maintenance of high water content and desiccation sensitivity, as well as preservation of active vacuoles in embryonic axes after shedding, can be considered a specific feature of recalcitrant

Possible roles of vacuolar H+-ATPase and mitochondrial function in tolerance to air-drying stress revealed by genome-wide screening of Saccharomyces cerevisiae deletion strains.

PubMed

Shima, Jun; Ando, Akira; Takagi, Hiroshi

2008-03-01

Yeasts used in bread making are exposed to air-drying stress during dried yeast production processes. To clarify the genes required for air-drying tolerance, we performed genome-wide screening using the complete deletion strain collection of diploid Saccharomyces cerevisiae. The screening identified 278 gene deletions responsible for air-drying sensitivity. These genes were classified based on their cellular function and on the localization of their gene products. The results showed that the genes required for air-drying tolerance were frequently involved in mitochondrial functions and in connection with vacuolar H(+)-ATPase, which plays a role in vacuolar acidification. To determine the role of vacuolar acidification in air-drying stress tolerance, we monitored intracellular pH. The results showed that intracellular acidification was induced during air-drying and that this acidification was amplified in a deletion mutant of the VMA2 gene encoding a component of vacuolar H(+)-ATPase, suggesting that vacuolar H(+)-ATPase helps maintain intracellular pH homeostasis, which is affected by air-drying stress. To determine the effects of air-drying stress on mitochondria, we analysed the mitochondrial membrane potential under air-drying stress conditions using MitoTracker. The results showed that mitochondria were extremely sensitive to air-drying stress, suggesting that a mitochondrial function is required for tolerance to air-drying stress. We also analysed the correlation between oxidative-stress sensitivity and air-drying-stress sensitivity. The results suggested that oxidative stress is a critical determinant of sensitivity to air-drying stress, although ROS-scavenging systems are not necessary for air-drying stress tolerance. (c) 2008 John Wiley & Sons, Ltd.

Spider silk MASP1 and MASP2 proteins as carbon fiber precursors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lewis, Randolph V

The objective of this project is to develop an unconventional non-petroleum based carbon fiber precursor which has the potential to be produced in high yield and quantities. Methods will be developed to produce pilot-scale quantities of fibers from spider silk proteins with mechanical properties at least 75% that of the natural dragline silk fibers in tensile strength and elongations of less than 5%. The precursor fibers will be converted to carbon fibers, with a goal of >250Ksi strength and 1-2% elongation. Cost analysis will be performed and the process optimized. Task 1: Subtask 1. Protein production: We exceeded the go/more » no go milestone of 1.0g/L of one of the spider silk protein (MSp2) purified last FY and have now increased from 5L to 500L fermentations. We have made a series of changes to the purification protocol from the initial report last FY. These led to a reduction in the time needed for the purification and reduced the purification costs by nearly 90%. Subtask 2. Fiber spinning: The major focus has been to produce more material to send 24 fiber thread to ONRL. We are still developing the methodology to successfully spin 24 fiber yarns. This involves both the spinning dope solutions as well as the methods to keep the fibers from fusing during the post spin stretch. The second area of focus has been to standardize the spin dopes for making the fibers. We now know that the conductivity (indicative of salt remaining with the protein after purification) is an important factor in successful spinning as is the pH. We now know that we need to be below 600 uS conductivity and that the most effective pH is protein dependent. Subtask 3. Silkworm silk: We have found the transgenic silkworms made using gene replacement at the fibroin light chain instead of heavy chain as we did previously have a higher tensile strength. See figures below showing the curve for the top end of the cocoon fibers. This tensile strength is the same as the average for spider

Environmental Factors Influencing Blooms of a Neurotoxic Stigonematalan Cyanobacterium Responsible for Avian Vacuolar Myelinopathy

DTIC Science & Technology

2013-01-01

aquatic plants and subsequent ecological consequences. The authors of this technical note have linked avian vacuolar myelinopathy (AVM), a disease...additional cyanobacteria sequences to determine designations for probe development, to advance understanding of the species’ phylogeny , and to lay...groundwork for its formal description. Phylogeny data confirm that the species is in section V, order Stigonematales. Phylogeny also infers that the

Curcumin decreases amyloid-beta peptide levels by attenuating the maturation of amyloid-beta precursor protein.

PubMed

Zhang, Can; Browne, Andrew; Child, Daniel; Tanzi, Rudolph E

2010-09-10

Alzheimer disease (AD) is a devastating neurodegenerative disease with no cure. The pathogenesis of AD is believed to be driven primarily by amyloid-beta (Abeta), the principal component of senile plaques. Abeta is an approximately 4-kDa peptide generated via cleavage of the amyloid-beta precursor protein (APP). Curcumin is a compound in the widely used culinary spice, turmeric, which possesses potent and broad biological activities, including anti-inflammatory and antioxidant activities, chemopreventative effects, and effects on protein trafficking. Recent in vivo studies indicate that curcumin is able to reduce Abeta-related pathology in transgenic AD mouse models via unknown molecular mechanisms. Here, we investigated the effects of curcumin on Abeta levels and APP processing in various cell lines and mouse primary cortical neurons. We show for the first time that curcumin potently lowers Abeta levels by attenuating the maturation of APP in the secretory pathway. These data provide a mechanism of action for the ability of curcumin to attenuate amyloid-beta pathology.

Curcumin Decreases Amyloid-β Peptide Levels by Attenuating the Maturation of Amyloid-β Precursor Protein*

PubMed Central

Zhang, Can; Browne, Andrew; Child, Daniel; Tanzi, Rudolph E.

2010-01-01

Alzheimer disease (AD) is a devastating neurodegenerative disease with no cure. The pathogenesis of AD is believed to be driven primarily by amyloid-β (Aβ), the principal component of senile plaques. Aβ is an ∼4-kDa peptide generated via cleavage of the amyloid-β precursor protein (APP). Curcumin is a compound in the widely used culinary spice, turmeric, which possesses potent and broad biological activities, including anti-inflammatory and antioxidant activities, chemopreventative effects, and effects on protein trafficking. Recent in vivo studies indicate that curcumin is able to reduce Aβ-related pathology in transgenic AD mouse models via unknown molecular mechanisms. Here, we investigated the effects of curcumin on Aβ levels and APP processing in various cell lines and mouse primary cortical neurons. We show for the first time that curcumin potently lowers Aβ levels by attenuating the maturation of APP in the secretory pathway. These data provide a mechanism of action for the ability of curcumin to attenuate amyloid-β pathology. PMID:20622013

[Mutations of amyloid precursor protein in early-onset familial Alzheimer's disease].

PubMed

Naruse, S; Tsuji, S; Miyatake, T

1992-09-01

Genetic linkage studies of familial Alzheimer's disease (FAD) have suggested that some form of early-onset FAD is linked to proximal long arm of chromosome 21. It has been also suggested that some form of late-onset FAD is linked to long arm of chromosome 19. Goate et al have identified a mis-sense mutation (Val to Ile) in exon 17 of the amyloid precursor protein (APP) gene in 2 of 16 early-onset FAD families, and have shown that the FAD locus in an FAD family is tightly linked to the mis-sense mutation. To determine if the mis-sense mutation is observed in different ethnic origine, we have studied some early-onset FAD families. Two early-onset FAD families showed the existence of the mutation. As the mutation has been identified in different ethnic origine and the mutation has not been observed in normal individuals, it strengthen hypothesis that the mutation is pathogenic. Recently, Val to Phe and Val to Gly mutations have been also identified at the same codon (Codon 717) of the APP gene.

The amyloid precursor protein locus and very-late-onset Alzheimer disease.

PubMed

Olson, J M; Goddard, K A; Dudek, D M

2001-10-01

Although mutations in the amyloid-beta precursor protein (APP) gene are known to confer high risk of Alzheimer disease (AD) to a small percentage of families in which it has early onset, convincing evidence of a major role for the APP locus in late-onset AD has not been forthcoming. In this report, we have used a covariate-based affected-sib-pair linkage method to analyze the chromosome 21 clinical and genetic data obtained on affected sibships by the National Institute of Mental Health Alzheimer Disease Genetics Initiative. The baseline model (without covariates) gave a LOD score of 0.02, which increases to 1.43 when covariates representing the additive effects of E2 and E4 are added. Larger increases in LOD scores were found when age at last examination/death (LOD score 5.54; P=.000002) or age at onset plus disease duration (LOD score 5.63; P=.000006) were included in the linkage model. We conclude that the APP locus may predispose to AD in the very elderly.

The proteolytic processing site of the precursor of lysyl oxidase.

PubMed Central

Cronshaw, A D; Fothergill-Gilmore, L A; Hulmes, D J

1995-01-01

The precise cleavage site of the N-terminal propeptide region of the precursor of lysyl oxidase has not yet been established, due to N-terminal blocking of the mature protein. Using a combination of peptide fragmentation, amino acid sequencing, time-of-flight m.s. and partial chemical unblocking procedures, it is shown that the mature form of lysyl oxidase begins at residue Asp-169 of the precursor protein (numbered according to the human sequence). The cleavage site is 28 residues to the C-terminal side of the site previously suggested on the basis of apparant molecular mass by SDS/PAGE, with the consequence that the two putative, N-linked glycosylation sites and the position of the Arg/Gln sequence polymorphism are now all in the precursor region. PMID:7864821

Human NKCC2 cation–Cl– co-transporter complements lack of Vhc1 transporter in yeast vacuolar membranes.

PubMed

Petrezselyova, Silvia; Dominguez, Angel; Herynkova, Pavla; Macias, Juan F; Sychrova, Hana

2013-10-01

Cation–chloride co-transporters serve to transport Cl– and alkali metal cations. Whereas a large family of these exists in higher eukaryotes, yeasts only possess one cation–chloride co-transporter, Vhc1, localized to the vacuolar membrane. In this study, the human cation–chloride co-transporter NKCC2 complemented the phenotype of VHC1 deletion in Saccharomyces cerevisiae and its activity controlled the growth of salt-sensitive yeast cells in the presence of high KCl, NaCl and LiCl. A S. cerevisiae mutant lacking plasma-membrane alkali–metal cation exporters Nha1 and Ena1-5 and the vacuolar cation–chloride co-transporter Vhc1 is highly sensitive to increased concentrations of alkali–metal cations, and it proved to be a suitable model for characterizing the substrate specificity and transport activity of human wild-type and mutated cation–chloride co-transporters. Copyright © 2013 John Wiley & Sons, Ltd.

Model for Stress-induced Protein Degradation in Lemna minor1

PubMed Central

Cooke, Robert J.; Roberts, Keith; Davies, David D.

1980-01-01

Transfer of Lemna minor fronds to adverse or stress conditions produces a large increase in the rate of protein degradation. Cycloheximide partially inhibits stress-induced protein degradation and also partially inhibits the protein degradation which occurs in the absence of stress. The increased protein degradation does not appear to be due to an increase in activity of soluble proteolytic enzymes. Biochemical evidence indicates that stress, perhaps acting via hormones, affects the permeability of certain membranes, particularly the tonoplast. A general model for stress-induced protein degradation is presented in which changes in membrane properties allow vacuolar proteolytic enzymes increased access to cytoplasmic proteins. PMID:16661588

Root bacterial endophytes confer drought resistance and enhance expression and activity of a vacuolar H+ -pumping pyrophosphatase in pepper plants.

PubMed

Vigani, Gianpiero; Rolli, Eleonora; Marasco, Ramona; Dell'Orto, Marta; Michoud, Grégoire; Soussi, Asma; Raddadi, Noura; Borin, Sara; Sorlini, Claudia; Zocchi, Graziano; Daffonchio, Daniele

2018-05-22

It has been previously shown that the transgenic overexpression of the plant root vacuolar proton pumps H + -ATPase (V-ATPase) and H + -PPase (V-PPase) confer tolerance to drought. Since plant-root endophytic bacteria can also promote drought tolerance, we hypothesize that such promotion can be associated to the enhancement of the host vacuolar proton pumps expression and activity. To test this hypothesis, we selected two endophytic bacteria endowed with an array of in vitro plant growth promoting traits. Their genome sequences confirmed the presence of traits previously shown to confer drought resistance to plants, such as the synthesis of nitric oxide and of organic volatile organic compounds. We used the two strains on pepper (Capsicuum annuum L.) because of its high sensitivity to drought. Under drought conditions, both strains stimulated a larger root system and enhanced the leaves' photosynthetic activity. By testing the expression and activity of the vacuolar proton pumps, H + -ATPase (V-ATPase) and H + -PPase (V-PPase), we found that bacterial colonization enhanced V-PPase only. We conclude that the enhanced expression and activity of V-PPase can be favoured by the colonization of drought-tolerance-inducing bacterial endophytes. This article is protected by copyright. All rights reserved. © 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

Amyloid precursor protein mRNA levels in Alzheimer's disease brain.

PubMed

Preece, Paul; Virley, David J; Costandi, Moheb; Coombes, Robert; Moss, Stephen J; Mudge, Anne W; Jazin, Elena; Cairns, Nigel J

2004-03-17

Insoluble beta-amyloid deposits in Alzheimer's disease (AD) brain are proteolytically derived from the membrane bound amyloid precursor protein (APP). The APP gene is differentially spliced to produce isoforms that can be classified into those containing a Kunitz-type serine protease inhibitor domain (K(+), APP(751), APP(770), APRP(365) and APRP(563)), and those without (K(-), APP(695) and APP(714)). Given the hypothesis that Abeta is a result of aberrant catabolism of APP, differential expression of mRNA isoforms containing protease inhibitors might play an active role in the pathology of AD. We took 513 cerebral cortex samples from 90 AD and 81 control brains and quantified the mRNA isoforms of APP with TaqMan real-time RT-PCR. After adjustment for age at death, brain pH and gender we found a change in the ratio of KPI(+) to KPI(-) mRNA isoforms of APP. Three separate probes, designed to recognise only KPI(+) mRNA species, gave increases of between 28% and 50% in AD brains relative to controls (p=0.002). There was no change in the mRNA levels of KPI-(APP 695) (p=0.898). Therefore, whilst KPI-mRNA levels remained stable the KPI(+) species increased specifically in the AD brains.

Precursor-product discrimination by La protein during tRNA metabolism

PubMed Central

Bayfield, Mark A.; Maraia, Richard J.

2009-01-01

SUMMARY La proteins bind pre-tRNAs at their UUU-3'OH ends, facilitating their maturation. While the mechanism by which La binds pre-tRNA 3' trailers is known, the function of the RNA-binding β-sheet surface of RRM1 is unknown. How La dissociates from UUU-3'OH-containing trailers after 3' processing is also unknown. La preferentially binds pre-tRNAs over processed tRNAs or 3' trailer products through coupled use of two sites: one on the La motif and another on the RRM1 β surface that binds elsewhere on tRNA. Two sites provide stable pre-tRNA binding while processed tRNA and 3' trailer are released from their single sites relatively fast. RRM1 loop-3 mutations decrease affinity for pre-tRNA and tRNA but not UUU-3'OH trailer, and impair tRNA maturation in vivo. We propose that RRM1 functions in activities that are more complex than UUU-3'OH binding. Accordingly, the RRM1 mutations also impair a RNA chaperone activity of La. The results suggest how La distinguishes precursor from product RNAs, allowing it to recycle onto a new pre-tRNA. PMID:19287396

Precursor-product discrimination by La protein during tRNA metabolism.

PubMed

Bayfield, Mark A; Maraia, Richard J

2009-04-01

La proteins bind pre-tRNAs at their UUU-3'OH ends, facilitating their maturation. Although the mechanism by which La binds pre-tRNA 3' trailers is known, the function of the RNA binding beta-sheet surface of the RNA-recognition motif (RRM1) is unknown. How La dissociates from UUU-3'OH-containing trailers after 3' processing is also unknown. Here we show that La preferentially binds pre-tRNAs over processed tRNAs or 3' trailer products through coupled use of two sites: one on the La motif and another on the RRM1 beta-surface that binds elsewhere on tRNA. Two sites provide stable pre-tRNA binding, whereas the processed tRNA and 3' trailer are released from their single sites relatively fast. RRM1 loop-3 mutations decrease affinity for pre-tRNA and tRNA, but not for the UUU-3'OH trailer, and impair tRNA maturation in vivo. We propose that RRM1 functions in activities that are more complex than UUU-3'OH binding. Accordingly, the RRM1 mutations also impair an RNA chaperone activity of La. The results suggest how La distinguishes precursor from product RNAs, allowing it to recycle onto a new pre-tRNA.

Arabidopsis adaptor protein 1G is critical for pollen development.

PubMed

Feng, Chong; Wang, Jia-Gang; Liu, Hai-Hong; Li, Sha; Zhang, Yan

2017-09-01

Pollen development is a pre-requisite for sexual reproduction of angiosperms, during which various cellular activities are involved. Pollen development accompanies dynamic remodeling of vacuoles through fission and fusion, disruption of which often compromises pollen viability. We previously reported that the Y subunit of adaptor protein 1 (AP1G) mediates synergid degeneration during pollen tube reception. Here, we demonstrate that AP1G is essential for pollen development. AP1G loss-of-function resulted in male gametophytic lethality due to defective pollen development. By ultrastructural analysis and fluorescence labeling, we demonstrate that AP1G loss-of-function compromised dynamic vacuolar remodeling during pollen development and impaired vacuolar acidification of pollen. Results presented here support a key role of vacuoles in gametophytic pollen development. © 2017 Institute of Botany, Chinese Academy of Sciences.

Glycosylphosphatidylinositol membrane anchors in Saccharomyces cerevisiae: absence of ceramides from complete precursor glycolipids.

PubMed Central

Sipos, G; Puoti, A; Conzelmann, A

1994-01-01

Glycosylphosphatidylinositol (GPI) anchoring of membrane proteins occurs through two distinct steps, namely the assembly of a precursor glycolipid and its subsequent transfer onto newly synthesized proteins. To analyze the structure of the yeast precursor glycolipid we made use of the pmi40 mutant that incorporates very high amounts of [3H]mannose. Two very polar [3H]mannose-labeled glycolipids named CP1 and CP2 qualified as GPI precursor lipids since their carbohydrate head group, Man alpha 1,2(X-->PO4-->6)Man alpha 1,2Man alpha 1,6Man alpha-GlcN-inositol (with X most likely being ethanolamine) comprises the core structure which is common to all GPI anchors described so far. CP1 predominates in cells grown at 24 degrees C whereas CP2 is induced by stress conditions. The apparent structural identity of the head groups suggests that CP1 and CP2 contain different lipid moieties. The lipid moieties of both CP1 and CP2 can be removed by mild alkaline hydrolysis although the protein-bound GPI anchors made by the pmi40 cells under identical labeling conditions contain mild base resistant ceramides. These findings imply that the ceramide moiety found on the majority of yeast GPI anchored proteins is added through a lipid remodeling step that occurs after the addition of the GPI precursor glycolipids to proteins. Images PMID:8026463

GRA12, a Toxoplasma dense granule protein associated with the intravacuolar membranous nanotubular network.

PubMed

Michelin, Adeline; Bittame, Amina; Bordat, Yann; Travier, Laetitia; Mercier, Corinne; Dubremetz, Jean-François; Lebrun, Maryse

2009-02-01

The intracellular protozoan parasite Toxoplasma gondii develops within the parasitophorous vacuole (PV), an intracellular niche in which it secretes proteins from secretory organelles named dense granules and rhoptries. Here, we describe a new dense granule protein that should now be referred to as GRA12, and that displays no homology with other proteins. Immunofluorescence and immuno-electron microscopy showed that GRA12 behaves similarly to both GRA2 and GRA6. It is secreted into the PV from the anterior pole of the parasite soon after the beginning of invasion, transits to the posterior invaginated pocket of the parasite where a membranous tubulovesicular network is first assembled, and finally resides throughout the vacuolar space, associated with the mature membranous nanotubular network. GRA12 fails to localise at the parasite posterior end in the absence of GRA2. Within the vacuolar space, like the other GRA proteins, GRA12 exists in both a soluble and a membrane-associated form. Using affinity chromatography experiments, we showed that in both the parasite and the PV soluble fractions, GRA12 is purified with the complex of GRA proteins associated with a tagged version of GRA2 and that this association is lost in the PV membranous fraction.

One precursor, three apolipoproteins: the relationship between two crustacean lipoproteins, the large discoidal lipoprotein and the high density lipoprotein/β-glucan binding protein.

PubMed

Stieb, Stefanie; Roth, Ziv; Dal Magro, Christina; Fischer, Sabine; Butz, Eric; Sagi, Amir; Khalaila, Isam; Lieb, Bernhard; Schenk, Sven; Hoeger, Ulrich

2014-12-01

The novel discoidal lipoprotein (dLp) recently detected in the crayfish, differs from other crustacean lipoproteins in its large size, apoprotein composition and high lipid binding capacity, We identified the dLp sequence by transcriptome analyses of the hepatopancreas and mass spectrometry. Further de novo assembly of the NGS data followed by BLAST searches using the sequence of the high density lipoprotein/1-glucan binding protein (HDL-BGBP) of Astacus leptodactylus as query revealed a putative precursor molecule with an open reading frame of 14.7 kb and a deduced primary structure of 4889 amino acids. The presence of an N-terminal lipid bind- ing domain and a DUF 1943 domain suggests the relationship with the large lipid transfer proteins. Two-putative dibasic furin cleavage sites were identified bordering the sequence of the HDL-BGBP. When subjected to mass spectroscopic analyses, tryptic peptides of the large apoprotein of dLp matched the N-terminal part of the precursor, while the peptides obtained for its small apoprotein matched the C-terminal part. Repeating the analysis in the prawn Macrobrachium rosenbergii revealed a similar protein with identical domain architecture suggesting that our findings do not represent an isolated instance. Our results indicate that the above three apolipoproteins (i.e HDL-BGBP and both the large and the small subunit of dLp) are translated as a large precursor. Cleavage at the furin type sites releases two subunits forming a heterodimeric dLP particle, while the remaining part forms an HDL-BGBP whose relationship with other lipoproteins as well as specific functions are yet to be elucidated.

The Prenylated Rab GTPase Receptor PRA1.F4 Contributes to Protein Exit from the Golgi Apparatus.

PubMed

Lee, Myoung Hui; Yoo, Yun-Joo; Kim, Dae Heon; Hanh, Nguyen Hong; Kwon, Yun; Hwang, Inhwan

2017-07-01

Prenylated Rab acceptor1 (PRA1) functions in the recruitment of prenylated Rab proteins to their cognate organelles. Arabidopsis ( Arabidopsis thaliana ) contains a large number of proteins belonging to the AtPRA1 family. However, their physiological roles remain largely unknown. Here, we investigated the physiological role of AtPRA1.F4, a member of the AtPRA1 family. A T-DNA insertion knockdown mutant of AtPRA1.F4 , atpra1.f4 , was smaller in stature than parent plants and possessed shorter roots, whereas transgenic plants overexpressing HA:AtPRA1.F4 showed enhanced development of secondary roots and root hairs. However, both overexpression and knockdown plants exhibited increased sensitivity to high-salt stress, lower vacuolar Na + /K + -ATPase and plasma membrane ATPase activities, lower and higher pH in the vacuole and apoplast, respectively, and highly vesiculated Golgi apparatus. HA:AtPRA1.F4 localized to the Golgi apparatus and assembled into high-molecular-weight complexes. atpra1.f4 plants displayed a defect in vacuolar trafficking, which was complemented by low but not high levels of HA : AtPRA1.F4 Overexpression of HA:AtPRA1.F4 also inhibited protein trafficking at the Golgi apparatus, albeit differentially depending on the final destination or type of protein: trafficking of vacuolar proteins, plasma membrane proteins, and trans-Golgi network (TGN)-localized SYP61 was strongly inhibited; trafficking of TGN-localized SYP51 was slightly inhibited; and trafficking of secretory proteins and TGN-localized SYP41 was negligibly or not significantly inhibited. Based on these results, we propose that Golgi-localized AtPRA1.F4 is involved in the exit of many but not all types of post-Golgi proteins from the Golgi apparatus. Additionally, an appropriate level of AtPRA1.F4 is crucial for its function at the Golgi apparatus. © 2017 American Society of Plant Biologists. All Rights Reserved.

Interaction of the amyloid precursor protein-like protein 1 (APLP1) E2 domain with heparan sulfate involves two distinct binding modes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dahms, Sven O., E-mail: sdahms@fli-leibniz.de; Mayer, Magnus C.; Miltenyi Biotec GmbH, Robert-Koch-Strasse 1, 17166 Teterow

2015-03-01

Two X-ray structures of APLP1 E2 with and without a heparin dodecasaccharide are presented, revealing two distinct binding modes of the protein to heparan sulfate. The data provide a mechanistic explanation of how APP-like proteins bind to heparan sulfates and how they specifically recognize nonreducing structures of heparan sulfates. Beyond the pathology of Alzheimer’s disease, the members of the amyloid precursor protein (APP) family are essential for neuronal development and cell homeostasis in mammals. APP and its paralogues APP-like protein 1 (APLP1) and APP-like protein 2 (APLP2) contain the highly conserved heparan sulfate (HS) binding domain E2, which effects variousmore » (patho)physiological functions. Here, two crystal structures of the E2 domain of APLP1 are presented in the apo form and in complex with a heparin dodecasaccharide at 2.5 Å resolution. The apo structure of APLP1 E2 revealed an unfolded and hence flexible N-terminal helix αA. The (APLP1 E2){sub 2}–(heparin){sub 2} complex structure revealed two distinct binding modes, with APLP1 E2 explicitly recognizing the heparin terminus but also interacting with a continuous heparin chain. The latter only requires a certain register of the sugar moieties that fits to a positively charged surface patch and contributes to the general heparin-binding capability of APP-family proteins. Terminal binding of APLP1 E2 to heparin specifically involves a structure of the nonreducing end that is very similar to heparanase-processed HS chains. These data reveal a conserved mechanism for the binding of APP-family proteins to HS and imply a specific regulatory role of HS modifications in the biology of APP and APP-like proteins.« less

Cleavage of amyloid precursor protein by an archaeal presenilin homologue PSH

PubMed Central

Dang, Shangyu; Wu, Shenjie; Wang, Jiawei; Li, Hongbo; Huang, Min; He, Wei; Li, Yue-Ming; Wong, Catherine C. L.; Shi, Yigong

2015-01-01

Aberrant cleavage of amyloid precursor protein (APP) by γ-secretase contributes to the development of Alzheimer’s disease. More than 200 disease-derived mutations have been identified in presenilin (the catalytic subunit of γ-secretase), making modulation of γ-secretase activity a potentially attractive therapeutic opportunity. Unfortunately, the technical challenges in dealing with intact γ-secretase have hindered discovery of modulators and demand a convenient substitute approach. Here we report that, similar to γ-secretase, the archaeal presenilin homolog PSH faithfully processes the substrate APP C99 into Aβ42, Aβ40, and Aβ38. The molar ratio of the cleavage products Aβ42 over Aβ40 by PSH is nearly identical to that by γ-secretase. The proteolytic activity of PSH is specifically suppressed by presenilin-specific inhibitors. Known modulators of γ-secretase also modulate PSH similarly in terms of the Aβ42/Aβ40 ratio. Structural analysis reveals association of a known γ-secretase inhibitor with PSH between its two catalytic aspartate residues. These findings identify PSH as a surrogate protease for the screening of agents that may regulate the protease activity and the cleavage preference of γ-secretase. PMID:25733893

Epigenetic Induction of EGR-1 Expression by the Amyloid Precursor Protein during Exposure to Novelty

PubMed Central

Hendrickx, Aurélie; Pierrot, Nathalie; Tasiaux, Bernadette; Schakman, Olivier; Brion, Jean-Pierre; Kienlen-Campard, Pascal; De Smet, Charles; Octave, Jean-Noël

2013-01-01

Following transcriptome comparison of primary cultures isolated from brain of mice expressing or not the amyloid precursor protein APP, we found transcription of the EGR-1 gene to be regulated by APP. In primary cultures of cortical neurons, APP significantly down regulated EGR-1 expression at both mRNA and protein levels in a γ-secretase independent manner. The intracellular domain of APP did not interact with EGR-1 gene promoter, but enrichment of acetylated histone H4 at the EGR-1 promoter region was measured in APP-/- neurons, as well as in brain of APP-/- mice, in which increase in EGR-1 expression was also measured. These results argue for an important function of APP in the epigenetic regulation of EGR-1 gene transcription both in vitro and in vivo. In APP-/- mice, constitutive overexpression of EGR-1 in brain impaired epigenetic induction of this early transcriptional regulator during exposure to novelty. Altogether, these results indicate an important function of APP in the epigenetic regulation of the transcription of EGR-1, known to be important for memory formation. PMID:24066134

A fluorescent reporter protein containing AtRMR1 domains is targeted to the storage and central vacuoles in Arabidopsis thaliana and tobacco leaf cells.

PubMed

Scabone, Camila María; Frigerio, Lorenzo; Petruccelli, Silvana

2011-10-01

To develop a new strategy to target recombinant proteins to the vacuolar storage system in transgenic plants, the ability of the transmembrane and cytosolic domains of Arabidopsis receptor homology-transmembrane-RING H2-1 (AtRMR1) was evaluated. A secreted version of RFP (secRFP) and a fusion of it to the transmembrane and cytosolic domains of AtRMR1 (RFP-TMCT) were produced and studied both in transient and stable expression assays. Transient expression in leaves of Nicotiana tabacum showed that secRFP is secreted to the apoplast while its fusion to TMCT of AtRMR1 is sufficient to prevent secretion of the reporter. In tobacco leaves, RFP-TMCT reporter showed an endoplasmic reticulum pattern in early expression stages while in late expression stages, it was found in the vacuolar lumen. For the first time, the role of TM and CT domains of AtRMR1 in stable expression in Arabidopsis thaliana is presented; the fusion of TMCT to secRFP is sufficient to sort RFP to the lumen of the central vacuoles in leaves and roots and to the lumen of PSV in cotyledons of mature embryos. In addition, biochemical studies performed in extract from transgenic plants showed that RFP-TMCT is an integral membrane protein. Full-length RFP-TMCT was also found in the vacuolar lumen, suggesting internalization into destination vacuole. Not colocalization of RFP-TMCT with tonoplast and plasma membrane markers were observed. This membrane vacuolar determinant sorting signal could be used for future application in molecular pharming as an alternative means to sort proteins of interest to vacuoles.

A Dual Microscopy-Based Assay To Assess Listeria monocytogenes Cellular Entry and Vacuolar Escape.

PubMed

Quereda, Juan J; Pizarro-Cerdá, Javier; Balestrino, Damien; Bobard, Alexandre; Danckaert, Anne; Aulner, Nathalie; Shorte, Spencer; Enninga, Jost; Cossart, Pascale

2016-01-01

Listeria monocytogenes is a Gram-positive bacterium and a facultative intracellular pathogen that invades mammalian cells, disrupts its internalization vacuole, and proliferates in the host cell cytoplasm. Here, we describe a novel image-based microscopy assay that allows discrimination between cellular entry and vacuolar escape, enabling high-content screening to identify factors specifically involved in these two steps. We first generated L. monocytogenes and Listeria innocua strains expressing a β-lactamase covalently attached to the bacterial cell wall. These strains were then incubated with HeLa cells containing the Förster resonance energy transfer (FRET) probe CCF4 in their cytoplasm. The CCF4 probe was cleaved by the bacterial surface β-lactamase only in cells inoculated with L. monocytogenes but not those inoculated with L. innocua, thereby demonstrating bacterial access to the host cytoplasm. Subsequently, we performed differential immunofluorescence staining to distinguish extracellular versus total bacterial populations in samples that were also analyzed by the FRET-based assay. With this two-step analysis, bacterial entry can be distinguished from vacuolar rupture in a single experiment. Our novel approach represents a powerful tool for identifying factors that determine the intracellular niche of L. monocytogenes. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Neurotoxicity of a Fragment of the Amyloid Precursor Associated with Alzheimer's Disease

NASA Astrophysics Data System (ADS)

Yankner, Bruce A.; Dawes, Linda R.; Fisher, Shannon; Villa-Komaroff, Lydia; Oster-Granite, Mary Lou; Neve, Rachael L.

1989-07-01

Amyloid deposition in senile plaques and the cerebral vasculature is a marker of Alzheimer's disease. Whether amyloid itself contributes to the neurodegenerative process or is simply a by-product of that process is unknown. Pheochromocytoma (PC12) and fibroblast (NIH 3T3) cell lines were transfected with portions of the gene for the human amyloid precursor protein. Stable PC12 cell transfectants expressing a specific amyloid-containing fragment of the precursor protein gradually degenerated when induced to differentiate into neuronal cells with nerve growth factor. Conditioned medium from these cells was toxic to neurons in primary hippocampal cultures, and the toxic agent could be removed by immunoabsorption with an antibody directed against the amyloid polypeptide. Thus, a peptide derived from the amyloid precursor may be neurotoxic.

Metabolic Characterization of Intact Cells Reveals Intracellular Amyloid Beta but Not Its Precursor Protein to Reduce Mitochondrial Respiration

PubMed Central

Schaefer, Patrick M.; von Einem, Bjoern; Walther, Paul; Calzia, Enrico; von Arnim, Christine A. F.

2016-01-01

One hallmark of Alzheimer´s disease are senile plaques consisting of amyloid beta (Aβ), which derives from the processing of the amyloid precursor protein (APP). Mitochondrial dysfunction has been linked to the pathogenesis of Alzheimer´s disease and both Aβ and APP have been reported to affect mitochondrial function in isolated systems. However, in intact cells, considering a physiological localization of APP and Aβ, it is pending what triggers the mitochondrial defect. Thus, the aim of this study was to dissect the impact of APP versus Aβ in inducing mitochondrial alterations with respect to their subcellular localization. We performed an overexpression of APP or beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), increasing APP and Aβ levels or Aβ alone, respectively. Conducting a comprehensive metabolic characterization we demonstrate that only APP overexpression reduced mitochondrial respiration, despite lower extracellular Aβ levels compared to BACE overexpression. Surprisingly, this could be rescued by a gamma secretase inhibitor, oppositionally indicating an Aβ-mediated mitochondrial toxicity. Analyzing Aβ localization revealed that intracellular levels of Aβ and an increased spatial association of APP/Aβ with mitochondria are associated with reduced mitochondrial respiration. Thus, our data provide marked evidence for a prominent role of intracellular Aβ accumulation in Alzheimer´s disease associated mitochondrial dysfunction. Thereby it highlights the importance of the localization of APP processing and intracellular transport as a decisive factor for mitochondrial function, linking two prominent hallmarks of neurodegenerative diseases. PMID:28005987

Differential regulation of amyloid precursor protein sorting with pathological mutations results in a distinct effect on amyloid-β production.

PubMed

Lin, Yen-Chen; Wang, Jia-Yi; Wang, Kai-Chen; Liao, Jhih-Ying; Cheng, Irene H

2014-11-01

The deposition of amyloid-β (Aβ) peptide, which is generated from amyloid precursor protein (APP), is the pathological hallmark of Alzheimer's disease (AD). Three APP familial AD mutations (D678H, D678N, and H677R) located at the sixth and seventh amino acid of Aβ have distinct effect on Aβ aggregation, but their influence on the physiological and pathological roles of APP remain unclear. We found that the D678H mutation strongly enhances amyloidogenic cleavage of APP, thus increasing the production of Aβ. This enhancement of amyloidogenic cleavage is likely because of the acceleration of APPD678H sorting into the endosomal-lysosomal pathway. In contrast, the APPD678N and APPH677R mutants do not cause the same effects. Therefore, this study indicates a regulatory role of D678H in APP sorting and processing, and provides genetic evidence for the importance of APP sorting in AD pathogenesis. The internalization of amyloid precursor protein (APP) increases its opportunity to be processed by β-secretase and to produce Amyloid-β (Aβ) that causes Alzheimer's disease (AD). We report a pathogenic APPD678H mutant that enhances APP internalization into the endosomal-lysosomal pathway and thus promotes the β-secretase cleavage and Aβ production. This study provides genetic evidence for the importance of APP sorting in AD pathogenesis. © 2014 International Society for Neurochemistry.

Supplementing glycosylation: A review of applying nucleotide-sugar precursors to growth medium to affect therapeutic recombinant protein glycoform distributions.

PubMed

Blondeel, Eric J M; Aucoin, Marc G

2018-06-15

Glycosylation is a critical quality attribute (CQA) of many therapeutic proteins, particularly monoclonal antibodies (mAbs), and is a major consideration in the approval of biosimilar biologics due to its effects to therapeutic efficacy. Glycosylation generates a distribution of glycoforms, resulting in glycoproteins with inherent molecule-to-molecule heterogeneity, capable of activating (or failing to activate) different effector functions of the immune system. Glycoforms can be affected by the supplementation of nucleotide-sugar precursors, and related components, to culture growth medium, affecting the metabolism of glycosylation. These supplementations has been demonstrated to increase nucleotide-sugar intracellular pools, and impact glycoform distributions, but with varied results. These variations can be attributed to five key factors: Differences between cell platforms (enzyme/transporter expression levels); differences between recombinant proteins produced (glycan-site accessibility); the fermentation and sampling timeline (glucose availability and exoglycosidase accumulation); glutamine levels (affecting ammonia levels, which impact Golgi pH, as well as UDP-GlcNAc pools); and finally, a lack of standardized metrics for observing shifts in glycoform distributions (glycosylation indices) across different experiments. The purpose of this review is to provide detail and clarity on the state of the art of supplementation strategies for nucleotide-sugar precursors for affecting glycosylation in cell culture processes, and to apply glycosylation indices for standardized comparisons across the field. Copyright © 2018. Published by Elsevier Inc.

Overexpression of amyloid precursor protein increases copper content in HEK293 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Suazo, Miriam; Hodar, Christian; Morgan, Carlos

2009-05-15

Amyloid precursor protein (APP) is a transmembrane glycoprotein widely expressed in mammalian tissues and plays a central role in Alzheimer's disease. However, its physiological function remains elusive. Cu{sup 2+} binding and reduction activities have been described in the extracellular APP135-156 region, which might be relevant for cellular copper uptake and homeostasis. Here, we assessed Cu{sup 2+} reduction and {sup 64}Cu uptake in two human HEK293 cell lines overexpressing APP. Our results indicate that Cu{sup 2+} reduction increased and cells accumulated larger levels of copper, maintaining cell viability at supra-physiological levels of Cu{sup 2+} ions. Moreover, wild-type cells exposed to bothmore » Cu{sup 2+} ions and APP135-155 synthetic peptides increased copper reduction and uptake. Complementation of function studies in human APP751 transformed Fre1 defective Saccharomyces cerevisiae cells rescued low Cu{sup 2+} reductase activity and increased {sup 64}Cu uptake. We conclude that Cu{sup 2+} reduction activity of APP facilitates copper uptake and may represent an early step in cellular copper homeostasis.« less

In vivo neuronal synthesis and axonal transport of Kunitz protease inhibitor (KPI)-containing forms of the amyloid precursor protein.

PubMed

Moya, K L; Confaloni, A M; Allinquant, B

1994-11-01

We have shown previously that the amyloid precursor protein (APP) is synthesized in retinal ganglion cells and is rapidly transported down the axons, and that different molecular weight forms of the precursor have different developmental time courses. Some APP isoforms contain a Kunitz protease inhibitor (KPI) domain, and APP that lacks the KPI domain is considered the predominant isoform in neurons. We now show that, among the various rapidly transported APPs, a 140-kDa isoform contains the KPI domain. This APP isoform is highly expressed in rapidly growing retinal axons, and it is also prominent in adult axon endings. This 140-kDa KPI-containing APP is highly sulfated compared with other axonally transported isoforms. These results show that APP with the KPI domain is a prominent isoform synthesized in neurons in vivo, and they suggest that the regulation of protease activity may be an important factor during the establishment of neuronal connections.

Inactivation of Protein Tyrosine Phosphatase Receptor Type Z by Pleiotrophin Promotes Remyelination through Activation of Differentiation of Oligodendrocyte Precursor Cells.

PubMed

Kuboyama, Kazuya; Fujikawa, Akihiro; Suzuki, Ryoko; Noda, Masaharu

2015-09-02

Multiple sclerosis (MS) is a progressive neurological disorder associated with myelin destruction and neurodegeneration. Oligodendrocyte precursor cells (OPCs) present in demyelinated lesions gradually fail to differentiate properly, so remyelination becomes incomplete. Protein tyrosine phosphatase receptor type Z (PTPRZ), one of the most abundant protein tyrosine phosphatases expressed in OPCs, is known to suppress oligodendrocyte differentiation and maintain their precursor cell stage. In the present study, we examined the in vivo mechanisms for remyelination using a cuprizone-induced demyelination model. Ptprz-deficient and wild-type mice both exhibited severe demyelination and axonal damage in the corpus callosum after cuprizone feeding. The similar accumulation of OPCs was observed in the lesioned area in both mice; however, remyelination was significantly accelerated in Ptprz-deficient mice after the removal of cuprizone. After demyelination, the expression of pleiotrophin (PTN), an inhibitory ligand for PTPRZ, was transiently increased in mouse brains, particularly in the neurons involved, suggesting its role in promoting remyelination by inactivating PTPRZ activity. In support of this view, oligodendrocyte differentiation was augmented in a primary culture of oligodendrocyte-lineage cells from wild-type mice in response to PTN. In contrast, these cells from Ptprz-deficient mice showed higher oligodendrocyte differentiation without PTN and differentiation was not enhanced by its addition. We further demonstrated that PTN treatment increased the tyrosine phosphorylation of p190 RhoGAP, a PTPRZ substrate, using an established line of OPCs. Therefore, PTPRZ inactivation in OPCs by PTN, which is secreted from demyelinated axons, may be the mechanism responsible for oligodendrocyte differentiation during reparative remyelination in the CNS. Multiple sclerosis (MS) is an inflammatory disease of the CNS that destroys myelin, the insulation that surrounds axons

Activation of PPARδ prevents endothelial dysfunction induced by overexpression of amyloid-β precursor protein.

PubMed

d'Uscio, Livius V; Das, Pritam; Santhanam, Anantha V R; He, Tongrong; Younkin, Steven G; Katusic, Zvonimir S

2012-12-01

Existing evidence suggests that amyloid-β precursor protein (APP) causes endothelial dysfunction and contributes to pathogenesis of atherosclerosis. In the present study, experiments were designed to: (1) determine the mechanisms underlying endothelial dysfunction and (2) define the effects of peroxisome proliferator-activated receptor delta (PPARδ) ligand on endothelial function in transgenic Tg2576 mice overexpressing mutated human APP. Confocal microscopy and western blot analyses of wild-type mice aortas provided evidence that APP protein is mainly present in endothelial cells. Overexpression of APP significantly impaired endothelium-dependent relaxations to acetylcholine and phosphorylation of endothelial nitric oxide synthase at Ser(1177) in aortas. HPLC analysis revealed that tetrahydrobiopterin (BH(4)) levels were reduced in Tg2576 mice aortas. This was caused by increased oxidation of BH(4) and reduced expression and activity of GTP-cyclohydrolase I. Furthermore, gp91phox protein expression and superoxide anion production were increased in aortas of Tg2576 mice. This augmented superoxide formation was completely prevented by the NADPH oxidase inhibitor VAS2870. Expression of copper-/zinc-superoxide dismutase (Cu/ZnSOD) and extracellular SOD was downregulated. Treatment with PPARδ ligand GW501516 (2 mg/kg/day) for 14 days significantly increased BH(4) bioavailability and improved endothelium-dependent relaxations in Tg2576 mice aortas. GW501516 also normalized protein expression of gp91(phox) and SODs, thereby reducing production of superoxide anion in the aortas. Our results suggest that in APP transgenic mice loss of nitric oxide and increased oxidative stress are the major causes of endothelial dysfunction. The vascular protective effects of GW501516 in Tg2576 mice appear to be critically dependent on prevention of superoxide anion production.

LegC3, an Effector Protein from Legionella pneumophila, Inhibits Homotypic Yeast Vacuole Fusion In Vivo and In Vitro

PubMed Central

Bennett, Terry L.; Kraft, Shannon M.; Reaves, Barbara J.; Mima, Joji; O’Brien, Kevin M.; Starai, Vincent J.

2013-01-01

During infection, the intracellular pathogenic bacterium Legionella pneumophila causes an extensive remodeling of host membrane trafficking pathways, both in the construction of a replication-competent vacuole comprised of ER-derived vesicles and plasma membrane components, and in the inhibition of normal phagosome:endosome/lysosome fusion pathways. Here, we identify the LegC3 secreted effector protein from L. pneumophila as able to inhibit a SNARE- and Rab GTPase-dependent membrane fusion pathway in vitro, the homotypic fusion of yeast vacuoles (lysosomes). This vacuole fusion inhibition appeared to be specific, as similar secreted coiled-coiled domain containing proteins from L. pneumophila, LegC7/YlfA and LegC2/YlfB, did not inhibit vacuole fusion. The LegC3-mediated fusion inhibition was reversible by a yeast cytosolic extract, as well as by a purified soluble SNARE, Vam7p. LegC3 blocked the formation of trans-SNARE complexes during vacuole fusion, although we did not detect a direct interaction of LegC3 with the vacuolar SNARE protein complexes required for fusion. Additionally, LegC3 was incapable of inhibiting a defined synthetic model of vacuolar SNARE-driven membrane fusion, further suggesting that LegC3 does not directly inhibit the activity of vacuolar SNAREs, HOPS complex, or Sec17p/18p during membrane fusion. LegC3 is likely utilized by Legionella to modulate eukaryotic membrane fusion events during pathogenesis. PMID:23437241

Relationship of the Membrane ATPase from Halobacterium saccharovorum to Vacuolar ATPases

NASA Technical Reports Server (NTRS)

Stan-Lotter, Helga; Bowman, Emma J.; Hochstein, Lawrence I.

1991-01-01

Polyclonal antiserum against subunit A (67 kDa) of the vacuolar ATPase from Neurospora crassa reacted with subunit I (87 kDa) from a membrane ATPase of the extremely halophilic archaebacterium Halobacterium saccharovorum. The halobacterial ATPase was inhibited by nitrate and N-ethylmaleimide; the extent of the latter inhibition was diminished in the presence of adenosine di- or triphosphates. 4-Chloro-7-nitrobenzofurazan in- hibited the hatobacterial ATPase also in a nucleotide- protectable manner; the bulk of inhibitor was associated with subunit II (60 kDa). The data suggested that this halobacterial ATPase may have conserved structural features from both the vacuotar and the F-type ATPases.

Trans fatty acids enhance amyloidogenic processing of the Alzheimer amyloid precursor protein (APP).

PubMed

Grimm, Marcus O W; Rothhaar, Tatjana L; Grösgen, Sven; Burg, Verena K; Hundsdörfer, Benjamin; Haupenthal, Viola J; Friess, Petra; Kins, Stefan; Grimm, Heike S; Hartmann, Tobias

2012-10-01

Hydrogenation of oils and diary products of ruminant animals leads to an increasing amount of trans fatty acids in the human diet. Trans fatty acids are incorporated in several lipids and accumulate in the membrane of cells. Here we systematically investigate whether the regulated intramembrane proteolysis of the amyloid precursor protein (APP) is affected by trans fatty acids compared to the cis conformation. Our experiments clearly show that trans fatty acids compared to cis fatty acids increase amyloidogenic and decrease nonamyloidogenic processing of APP, resulting in an increased production of amyloid beta (Aβ) peptides, main components of senile plaques, which are a characteristic neuropathological hallmark for Alzheimer's disease (AD). Moreover, our results show that oligomerization and aggregation of Aβ are increased by trans fatty acids. The mechanisms identified by this in vitro study suggest that the intake of trans fatty acids potentially increases the AD risk or causes an earlier onset of the disease. Copyright © 2012 Elsevier Inc. All rights reserved.

Intermolecular cleavage of hepatitis A virus (HAV) precursor protein P1-P2 by recombinant HAV proteinase 3C.

PubMed Central

Kusov, Y Y; Sommergruber, W; Schreiber, M; Gauss-Müller, V

1992-01-01

Active proteinase 3C of hepatitis A virus (HAV) was expressed in bacteria either as a mature enzyme or as a protein fused to the entire polymerase 3D or to a part of it, and their identities were shown by immunoblot analysis. Intermolecular cleavage activity was demonstrated by incubating in vitro-translated and radiolabeled HAV precursor protein P1-P2 with extracts of bacteria transformed with plasmids containing recombinant HAV 3C. Identification of cleavage products P1, VP1, and VPO-VP3 by immunoprecipitation clearly demonstrates that HAV 3C can cleave between P1 and P2 as well as within P1 and thus shows an activity profile similar to that of cardiovirus 3C. Images PMID:1328690

A complex between contactin-1 and the protein tyrosine phosphatase PTPRZ controls the development of oligodendrocyte precursor cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lamprianou, Smaragda; Chatzopoulou, Elli; Thomas, Jean-Léon

The six members of the contactin (CNTN) family of neural cell adhesion molecules are involved in the formation and maintenance of the central nervous system (CNS) and have been linked to mental retardation and neuropsychiatric disorders such as autism. Five of the six CNTNs bind to the homologous receptor protein tyrosine phosphatases gamma (PTPRG) and zeta (PTPRZ), but the biological roles of these interactions remain unclear. We report here the cocrystal structure of the carbonic anhydrase-like domain of PTPRZ bound to tandem Ig repeats of CNTN1 and combine these structural data with binding assays to show that PTPRZ binds specificallymore » to CNTN1 expressed at the surface of oligodendrocyte precursor cells. Furthermore, analyses of glial cell populations in wild-type and PTPRZ-deficient mice show that the binding of PTPRZ to CNTN1 expressed at the surface of oligodendrocyte precursor cells inhibits their proliferation and promotes their development into mature oligodendrocytes. Overall, these results implicate the PTPRZ/CNTN1 complex as a previously unknown modulator of oligodendrogenesis.« less

The Amyloid Precursor Protein (APP) Triplicated Gene Impairs Neuronal Precursor Differentiation and Neurite Development through Two Different Domains in the Ts65Dn Mouse Model for Down Syndrome*

PubMed Central

Trazzi, Stefania; Fuchs, Claudia; Valli, Emanuele; Perini, Giovanni; Bartesaghi, Renata; Ciani, Elisabetta

2013-01-01

Intellectual disability in Down syndrome (DS) appears to be related to severe proliferation impairment during brain development. Recent evidence shows that it is not only cellular proliferation that is heavily compromised in DS, but also cell fate specification and dendritic maturation. The amyloid precursor protein (APP), a gene that is triplicated in DS, plays a key role in normal brain development by influencing neural precursor cell proliferation, cell fate specification, and neuronal maturation. APP influences these processes via two separate domains, the APP intracellular domain (AICD) and the soluble secreted APP. We recently found that the proliferation impairment of neuronal precursors (NPCs) from the Ts65Dn mouse model for DS was caused by derangement of the Shh pathway due to overexpression of patched1(Ptch1), its inhibitory regulator. Ptch1 overexpression was related to increased levels within the APP/AICD system. The overall goal of this study was to determine whether APP contributes to neurogenesis impairment in DS by influencing in addition to proliferation, cell fate specification, and neurite development. We found that normalization of APP expression restored the reduced neuronogenesis, the increased astrogliogenesis, and the reduced neurite length of trisomic NPCs, indicating that APP overexpression underpins all aspects of neurogenesis impairment. Moreover, we found that two different domains of APP impair neuronal differentiation and maturation in trisomic NPCs. The APP/AICD system regulates neuronogenesis and neurite length through the Shh pathway, whereas the APP/secreted AP system promotes astrogliogenesis through an IL-6-associated signaling cascade. These results provide novel insight into the mechanisms underlying brain development alterations in DS. PMID:23740250

The Bimodal Lifestyle of Intracellular Salmonella in Epithelial Cells: Replication in the Cytosol Obscures Defects in Vacuolar Replication

PubMed Central

Steele-Mortimer, Olivia

2012-01-01

Salmonella enterica serovar Typhimurium invades and proliferates within epithelial cells. Intracellular bacteria replicate within a membrane bound vacuole known as the Salmonella containing vacuole. However, this bacterium can also replicate efficiently in the cytosol of epithelial cells and net intracellular growth is a product of both vacuolar and cytosolic replication. Here we have used semi-quantitative single-cell analyses to investigate the contribution of each of these replicative niches to intracellular proliferation in cultured epithelial cells. We show that cytosolic replication can account for the majority of net replication even though it occurs in less than 20% of infected cells. Consequently, assays for net growth in a population of infected cells, for example by recovery of colony forming units, are not good indicators of vacuolar proliferation. We also show that the Salmonella Type III Secretion System 2, which is required for SCV biogenesis, is not required for cytosolic replication. Altogether this study illustrates the value of single cell analyses when studying intracellular pathogens. PMID:22719929

Activities of Vacuolar Cysteine Proteases in Plant Senescence.

PubMed

Martínez, Dana E; Costa, Lorenza; Guiamét, Juan José

2018-01-01

Plant senescence is accompanied by a marked increase in proteolytic activities, and cysteine proteases (Cys-protease) represent the prevailing class among the responsible proteases. Cys-proteases predominantly locate to lytic compartments, i.e., to the central vacuole (CV) and to senescence-associated vacuoles (SAVs), the latter being specific to the photosynthetic cells of senescing leaves. Cellular fractionation of vacuolar compartments may facilitate Cys-proteases purification and their concentration for further analysis. Active Cys-proteases may be analyzed by different, albeit complementary approaches: (1) in vivo examination of proteolytic activity by fluorescence microscopy using specific substrates which become fluorescent upon cleavage by Cys-proteases, (2) protease labeling with specific probes that react irreversibly with the active enzymes, and (3) zymography, whereby protease activities are detected in polyacrylamide gels copolymerized with a substrate for proteases. Here we describe the three methods mentioned above for detection of active Cys-proteases and a cellular fractionation technique to isolate SAVs.

Hereditary vacuolar internal anal sphincter myopathy causing proctalgia fugax and constipation: a new case contribution.

PubMed

de la Portilla, Fernando; Borrero, Juan José; Rafel, Enrique

2005-03-01

Hereditary anal sphincter myopathy is rare. We present a family with one affected member with proctalgia fugax, constipation and internal anal sphincter hypertrophy. Ultrastructural findings show vacuolization of smooth muscle cells without the characteristic polyglucosan inclusion. Further relief of symptoms was obtained using an oral calcium antagonist. Based on clinical presentation, endosonography and morphological findings, we consider our case is a histological variant of the vacuolar myopathy originally described.

Processing of the precursor of protamine P2 in mouse. Peptide mapping and N-terminal sequence analysis of intermediates.

PubMed Central

Carré-Eusèbe, D; Lederer, F; Lê, K H; Elsevier, S M

1991-01-01

Protamine P2, the major basic chromosomal protein of mouse spermatozoa, is synthesized as a precursor almost twice as long as the mature protein, its extra length arising from an N-terminal extension of 44 amino acid residues. This precursor is integrated into chromatin of spermatids, and the extension is processed during chromatin condensation in the haploid cells. We have studied processing in the mouse and have identified two intermediates generated by proteolytic cleavage of the precursor. H.p.l.c. separated protamine P2 from four other spermatid proteins, including the precursor and three proteins known to possess physiological characteristics expected of processing intermediates. Peptide mapping indicated that all of these proteins were structurally similar. Two major proteins were further purified by PAGE, transferred to poly(vinylidene difluoride) membranes and submitted to automated N-terminal sequence analysis. Both sequences were found within the deduced sequence of the precursor extension. The N-terminus of the larger intermediate, PP2C, was Gly-12, whereas the N-terminus of the smaller, PP2D, was His-21. Both processing sites involved a peptide bond in which the carbonyl function was contributed by an acidic amino acid. Images Fig. 1. Fig. 3. Fig. 4. PMID:1854346

Amyloid precursor protein-induced axonopathies are independent of amyloid-beta peptides.

PubMed

Stokin, Gorazd B; Almenar-Queralt, Angels; Gunawardena, Shermali; Rodrigues, Elizabeth M; Falzone, Tomás; Kim, Jungsu; Lillo, Concepción; Mount, Stephanie L; Roberts, Elizabeth A; McGowan, Eileen; Williams, David S; Goldstein, Lawrence S B

2008-11-15

Overexpression of amyloid precursor protein (APP), as well as mutations in the APP and presenilin genes, causes rare forms of Alzheimer's disease (AD). These genetic changes have been proposed to cause AD by elevating levels of amyloid-beta peptides (Abeta), which are thought to be neurotoxic. Since overexpression of APP also causes defects in axonal transport, we tested whether defects in axonal transport were the result of Abeta poisoning of the axonal transport machinery. Because directly varying APP levels also alters APP domains in addition to Abeta, we perturbed Abeta generation selectively by combining APP transgenes in Drosophila and mice with presenilin-1 (PS1) transgenes harboring mutations that cause familial AD (FAD). We found that combining FAD mutant PS1 with FAD mutant APP increased Abeta42/Abeta40 ratios and enhanced amyloid deposition as previously reported. Surprisingly, however, this combination suppressed rather than increased APP-induced axonal transport defects in both Drosophila and mice. In addition, neuronal apoptosis induced by expression of FAD mutant human APP in Drosophila was suppressed by co-expressing FAD mutant PS1. We also observed that directly elevating Abeta with fusions to the Familial British and Danish Dementia-related BRI protein did not enhance axonal transport phenotypes in APP transgenic mice. Finally, we observed that perturbing Abeta ratios in the mouse by combining FAD mutant PS1 with FAD mutant APP did not enhance APP-induced behavioral defects. A potential mechanism to explain these findings was suggested by direct analysis of axonal transport in the mouse, which revealed that axonal transport or entry of APP into axons is reduced by FAD mutant PS1. Thus, we suggest that APP-induced axonal defects are not caused by Abeta.

Regulation of Vacuolar H+-ATPase (V-ATPase) Reassembly by Glycolysis Flow in 6-Phosphofructo-1-kinase (PFK-1)-deficient Yeast Cells*

PubMed Central

Chan, Chun-Yuan; Dominguez, Dennis; Parra, Karlett J.

2016-01-01

Yeast 6-phosphofructo-1-kinase (PFK-1) has two subunits, Pfk1p and Pfk2p. Deletion of Pfk2p alters glucose-dependent V-ATPase reassembly and vacuolar acidification (Chan, C. Y., and Parra, K. J. (2014) Yeast phosphofructokinase-1 subunit Pfk2p is necessary for pH homeostasis and glucose-dependent vacuolar ATPase reassembly. J. Biol. Chem. 289, 19448–19457). This study capitalized on the mechanisms suppressing vacuolar H+-ATPase (V-ATPase) in pfk2Δ to gain new knowledge of the mechanisms underlying glucose-dependent V-ATPase regulation. Because V-ATPase is fully assembled in pfk2Δ, and glycolysis partially suppressed at steady state, we manipulated glycolysis and assessed its direct involvement on V-ATPase function. At steady state, the ratio of proton transport to ATP hydrolysis increased 24% after increasing the glucose concentration from 2% to 4% to enhance the glycolysis flow in pfk2Δ. Tighter coupling restored vacuolar pH when glucose was abundant and glycolysis operated below capacity. After readdition of glucose to glucose-deprived cells, glucose-dependent V1Vo reassembly was proportional to the glycolysis flow. Readdition of 2% glucose to pfk2Δ cells, which restored 62% of ethanol concentration, led to equivalent 60% V1Vo reassembly levels. Steady-state level of assembly (100% reassembly) was reached at 4% glucose when glycolysis reached a threshold in pfk2Δ (≥40% the wild-type flow). At 4% glucose, the level of Pfk1p co-immunoprecipitated with V-ATPase decreased 58% in pfk2Δ, suggesting that Pfk1p binding to V-ATPase may be inhibitory in the mutant. We concluded that V-ATPase activity at steady state and V-ATPase reassembly after readdition of glucose to glucose-deprived cells are controlled by the glycolysis flow. We propose a new mechanism by which glucose regulates V-ATPase catalytic activity that occurs at steady state without changing V1Vo assembly. PMID:27226568

A functional aquaporin co-localizes with the vacuolar proton pyrophosphatase to acidocalcisomes and the contractile vacuole complex of Trypanosoma cruzi.

PubMed

Montalvetti, Andrea; Rohloff, Peter; Docampo, Roberto

2004-09-10

We cloned an aquaporin gene from Trypanosoma cruzi (TcAQP) that encodes a protein of 231 amino acids, which is highly hydrophobic. The protein has six putative transmembrane domains and the two signature motifs asparagine-proline-alanine (NPA) which have been shown, in other aquaporins, to be involved in the formation of an aqueous channel spanning the bilayer. TcAQP was sensitive to endo H treatment, suggesting that the protein is N-glycosylated. Oocytes of Xenopus laevis expressing TcAQP swelled under hyposmotic conditions indicating water permeability, which was abolished after preincubating oocytes with very low concentrations of the AQP inhibitors HgCl(2) and AgNO(3). glycerol transport was detected. No Immunofluorescence microscopy of T. cruzi expressing GFP-TcAQP showed co-localization of TcAQP with the vacuolar proton pyrophosphatase (V-H(+)-PPase), a marker of acidocalcisomes. This localization was confirmed by Western blotting and immunofluorescence staining using polyclonal antibodies against a C-terminal peptide of TcAQP. In addition, there was a strong anterior labeling in a vacuole, close to the flagellar pocket, that was distinct from the acidocalcisomes and that was identified by immunogold electron microscopy as the contractile vacuole complex. Taking together, the presence of an aquaporin in acidocalcisomes and the contractile vacuole complex of T. cruzi, provides support for the role of these organelles in osmotic adaptations of these parasites.

Abscisic acid induction of vacuolar H+-ATPase activity in mesembryanthemum crystallinum is developmentally regulated

PubMed

Barkla; Vera-Estrella; Maldonado-Gama; Pantoja

1999-07-01

Abscisic acid (ABA) has been implicated as a key component in water-deficit-induced responses, including those triggered by drought, NaCl, and low- temperature stress. In this study a role for ABA in mediating the NaCl-stress-induced increases in tonoplast H+-translocating ATPase (V-ATPase) and Na+/H+ antiport activity in Mesembryanthemum crystallinum, leading to vacuolar Na+ sequestration, were investigated. NaCl or ABA treatment of adult M. crystallinum plants induced V-ATPase H+ transport activity, and when applied in combination, an additive effect on V-ATPase stimulation was observed. In contrast, treatment of juvenile plants with ABA did not induce V-ATPase activity, whereas NaCl treatment resulted in a similar response to that observed in adult plants. Na+/H+ antiport activity was induced in both juvenile and adult plants by NaCl, but ABA had no effect at either developmental stage. Results indicate that ABA-induced changes in V-ATPase activity are dependent on the plant reaching its adult phase, whereas NaCl-induced increases in V-ATPase and Na+/H+ antiport activity are independent of plant age. This suggests that ABA-induced V-ATPase activity may be linked to the stress-induced, developmentally programmed switch from C3 metabolism to Crassulacean acid metabolism in adult plants, whereas, vacuolar Na+ sequestration, mediated by the V-ATPase and Na+/H+ antiport, is regulated through ABA-independent pathways.

Modulation of beta-amyloid precursor protein trafficking and processing by the low density lipoprotein receptor family.

PubMed

Cam, Judy A; Bu, Guojun

2006-08-18

Amyloid-beta peptide (Abeta) accumulation in the brain is an early, toxic event in the pathogenesis of Alzheimer's disease (AD). Abeta is produced by proteolytic processing of a transmembrane protein, beta-amyloid precursor protein (APP), by beta- and gamma-secretases. Mounting evidence has demonstrated that alterations in APP cellular trafficking and localization directly impact its processing to Abeta. Recent studies have shown that members of the low-density lipoprotein receptor family, including LRP, LRP1B, SorLA/LR11, and apolipoprotein E (apoE) receptor 2, interact with APP and regulate its endocytic trafficking. Another common feature of these receptors is their ability to bind apoE, which exists in three isoforms in humans and the presence of the epsilon4 allele represents a genetic risk factor for AD. In this review, we summarize the current understanding of the function of these apoE receptors with a focus on their role in APP trafficking and processing. Knowledge of the interactions between these distinct low-density lipoprotein receptor family members and APP may ultimately influence future therapies for AD.

Activation of PPARδ prevents endothelial dysfunction induced by overexpression of amyloid-β precursor protein

PubMed Central

d'Uscio, Livius V.; Das, Pritam; Santhanam, Anantha V.R.; He, Tongrong; Younkin, Steven G.; Katusic, Zvonimir S.

2012-01-01

Aims Existing evidence suggests that amyloid-β precursor protein (APP) causes endothelial dysfunction and contributes to pathogenesis of atherosclerosis. In the present study, experiments were designed to: (1) determine the mechanisms underlying endothelial dysfunction and (2) define the effects of peroxisome proliferator-activated receptor delta (PPARδ) ligand on endothelial function in transgenic Tg2576 mice overexpressing mutated human APP. Methods and results Confocal microscopy and western blot analyses of wild-type mice aortas provided evidence that APP protein is mainly present in endothelial cells. Overexpression of APP significantly impaired endothelium-dependent relaxations to acetylcholine and phosphorylation of endothelial nitric oxide synthase at Ser1177 in aortas. HPLC analysis revealed that tetrahydrobiopterin (BH4) levels were reduced in Tg2576 mice aortas. This was caused by increased oxidation of BH4 and reduced expression and activity of GTP-cyclohydrolase I. Furthermore, gp91phox protein expression and superoxide anion production were increased in aortas of Tg2576 mice. This augmented superoxide formation was completely prevented by the NADPH oxidase inhibitor VAS2870. Expression of copper-/zinc-superoxide dismutase (Cu/ZnSOD) and extracellular SOD was downregulated. Treatment with PPARδ ligand GW501516 (2 mg/kg/day) for 14 days significantly increased BH4 bioavailability and improved endothelium-dependent relaxations in Tg2576 mice aortas. GW501516 also normalized protein expression of gp91phox and SODs, thereby reducing production of superoxide anion in the aortas. Conclusion Our results suggest that in APP transgenic mice loss of nitric oxide and increased oxidative stress are the major causes of endothelial dysfunction. The vascular protective effects of GW501516 in Tg2576 mice appear to be critically dependent on prevention of superoxide anion production. PMID:22886847

A Toxoplasma gondii protein with homology to intracellular type Na{sup +}/H{sup +} exchangers is important for osmoregulation and invasion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Francia, Maria E.; Wicher, Sarah; Pace, Douglas A.

2011-06-10

The obligate intracellular parasite Toxoplasma gondii is exposed to a variety of physiological conditions while propagating in an infected organism. The mechanisms by which Toxoplasma overcomes these dramatic changes in its environment are not known. In yeast and plants, ion detoxification and osmotic regulation are controlled by vacuolar compartments. A novel compartment named the plant-like vacuole or vacuolar compartment (PLV/VAC) has recently been described in T.gondii, which could potentially protect extracellular tachyzoites against salt and other ionic stresses. Here, we report the molecular characterization of the vacuolar type Na{sup +}/H{sup +} exchanger in T. gondii, TgNHE3, and its co-localization withmore » the PLV/VAC proton-pyrophosphatase (TgVP1). We have created a TgNHE3 knockout strain, which is more sensitive to hyperosmotic shock and toxic levels of sodium, possesses a higher intracellular Ca{sup 2+} concentration [Ca{sup 2+}]{sub i}, and exhibits a reduced host invasion efficiency. The defect in invasion correlates with a measurable reduction in the secretion of the adhesin TgMIC2. Overall, our results suggest that the PLV/VAC has functions analogous to those of the vacuolar compartments of plants and yeasts, providing the parasite with a mechanism to resist ionic fluctuations and, potentially, regulate protein trafficking.« less

Experiences and Perspectives of Polycystic Kidney Disease Patients following a Diet of Reduced Osmoles, Protein, and Acid Precursors Supplemented with Water: A Qualitative Study.

PubMed

Taylor, Jacob M; Ptomey, Lauren; Hamilton-Reeves, Jill M; Sullivan, Debra K; Creed, Catherine; Carlson, Susan E; Wesson, Donald E; Grantham, Jared J; Gibson, Cheryl A

2016-01-01

Salt, protein, acid precursors, and fluid intake have been identified as factors that influence cyst growth in ADPKD. Unfortunately, the feasibility of following these dietary restrictions/enhancements from a patient's point-of-view has yet to be studied. The purpose of this study is to understand better the experiences of patients following a relatively complex dietary prescription targeting these factors. Twelve adults with ADPKD and kidney function >30ml/min/1.73m2 were recruited from the University of Kansas Medical Center Polycystic Kidney Disease clinic. In a qualitative design, semi-structured interviews of participants were conducted following a four week dietary intervention (experimental diet lower in sodium, protein, and acid precursors, and supplemented with water) either face-to-face or by telephone. All interviews were recorded, transcribed verbatim, and checked for accuracy. Transcripts were analyzed thematically for emerging themes. Participants reported that eating less meat and more fruits and vegetables were the easiest components of the diet, whereas reaching the daily goal amount of fruits and vegetables and tracking the diet constantly were the most difficult components. Participants had little difficulty with fluid intake and reported the prescribed fluid goal as achievable. The tracking system for fruits and vegetables and protein was reported to be both helpful and intuitive, but tracking their intake on paper was tedious. Eating out was the most significant barrier to following the diet with some individuals avoiding restaurants in order to comply with the dietary prescription. Participants on the experimental diet heightened their awareness of the consumption of dietary salt, protein, acid precursors, and fluid intake. Additionally, most participants believed adherence to the prescribed diet was feasible. However, participants wanted less cumbersome ways to track and monitor the diet, especially given that the prescribed diet is designed

The Na+(K+)/H+ exchanger Nhx1 controls multivesicular body-vacuolar lysosome fusion.

PubMed

Karim, Mahmoud Abdul; Brett, Christopher Leonard

2018-02-01

Loss-of-function mutations in human endosomal Na + (K + )/H + exchangers (NHEs) NHE6 and NHE9 are implicated in neurological disorders including Christianson syndrome, autism, and attention deficit and hyperactivity disorder. These mutations disrupt retention of surface receptors within neurons and glial cells by affecting their delivery to lysosomes for degradation. However, the molecular basis of how these endosomal NHEs control endocytic trafficking is unclear. Using Saccharomyces cerevisiae as a model, we conducted cell-free organelle fusion assays to show that transport activity of the orthologous endosomal NHE Nhx1 is important for multivesicular body (MVB)-vacuolar lysosome fusion, the last step of endocytosis required for surface protein degradation. We find that deleting Nhx1 disrupts the fusogenicity of the MVB, not the vacuole, by targeting pH-sensitive machinery downstream of the Rab-GTPase Ypt7 needed for SNARE-mediated lipid bilayer merger. All contributing mechanisms are evolutionarily conserved offering new insight into the etiology of human disorders linked to loss of endosomal NHE function. © 2018 Karim and Brett. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Novel Tonoplast Transporters Identified Using a Proteomic Approach with Vacuoles Isolated from Cauliflower Buds1[W][OA

PubMed Central

Schmidt, Ulrike G.; Endler, Anne; Schelbert, Silvia; Brunner, Arco; Schnell, Magali; Neuhaus, H. Ekkehard; Marty-Mazars, Daniéle; Marty, Francis; Baginsky, Sacha; Martinoia, Enrico

2007-01-01

Young meristematic plant cells contain a large number of small vacuoles, while the largest part of the vacuome in mature cells is composed by a large central vacuole, occupying 80% to 90% of the cell volume. Thus far, only a limited number of vacuolar membrane proteins have been identified and characterized. The proteomic approach is a powerful tool to identify new vacuolar membrane proteins. To analyze vacuoles from growing tissues we isolated vacuoles from cauliflower (Brassica oleracea) buds, which are constituted by a large amount of small cells but also contain cells in expansion as well as fully expanded cells. Here we show that using purified cauliflower vacuoles and different extraction procedures such as saline, NaOH, acetone, and chloroform/methanol and analyzing the data against the Arabidopsis (Arabidopsis thaliana) database 102 cauliflower integral proteins and 214 peripheral proteins could be identified. The vacuolar pyrophosphatase was the most prominent protein. From the 102 identified proteins 45 proteins were already described. Nine of these, corresponding to 46% of peptides detected, are known vacuolar proteins. We identified 57 proteins (55.9%) containing at least one membrane spanning domain with unknown subcellular localization. A comparison of the newly identified proteins with expression profiles from in silico data revealed that most of them are highly expressed in young, developing tissues. To verify whether the newly identified proteins were indeed localized in the vacuole we constructed and expressed green fluorescence protein fusion proteins for five putative vacuolar membrane proteins exhibiting three to 11 transmembrane domains. Four of them, a putative organic cation transporter, a nodulin N21 family protein, a membrane protein of unknown function, and a senescence related membrane protein were localized in the vacuolar membrane, while a white-brown ATP-binding cassette transporter homolog was shown to reside in the plasma membrane

Amyloid precursor protein and Presenilin 1 interaction studied by FRET in human H4 cells.

PubMed

Nizzari, Mario; Venezia, Valentina; Bianchini, Paolo; Caorsi, Valentina; Diaspro, Alberto; Repetto, Emanuela; Thellung, Stefano; Corsaro, Alessandro; Carlo, Pia; Schettini, Gennaro; Florio, Tullio; Russo, Claudio

2007-01-01

The mayor pathologic hallmarks of Alzheimer's disease (AD) are senile plaque and neurofibrillary tangles. Senile plaque are primarily made up of deposits of amyloid-beta protein, a proteolytic product derived from the amyloid precursor protein (APP). APP is a transmembrane protein detected into the endoplasmic reticulum, in the Golgi apparatus, at the cell surface, recycled by endocytosis to endosomes, whose physiological function is unclear. Presenilins (PS), are a component of gamma-secretase complex that cleave alpha-CTFs (carboxy-terminal fragment), or beta-CTFs, leaving 40 or 42 amino acids amyloid-beta peptides and 58 or 56 amino acids intracellular domains (AICD). Where the amyloid-beta peptides is generated is not clear. The study of APP-PS interaction in specific cell compartments provides a good opportunity to light upon the molecular mechanisms regulating the activity of the "gamma-secretase complex," and where beta-amyloid is generated. In our study we used a biophysical assay of protein proximity: fluorescence resonance energy transfer (FRET), that can provide information about molecular interactions when two proteins are in the close proximity (<10 nm), to examine the subcellular localization and interaction between APP and PS1 in human neuroglioma cells (H4). Confocal microscopic analysis reveals extensive colocalization in different cells' compartment, and centrosomal or microtubule organizing center (MTOC) localization of APP and PS1, but not necessarily a close molecular interaction. We used FRET to determine if APP and PS1 interact at the cell centrosome. FRET data suggest a close interaction between APP and PS1 in subcellular compartments and at the centrosome of H4 cells. Using this approach we show that APP and PS1 are closely associated in the centrosomes of the H4 cell.

Intracellular trafficking of the β-secretase and processing of amyloid precursor protein.

PubMed

Zhi, Pei; Chia, Pei Zhi Cheryl; Chia, Cheryl; Gleeson, Paul A

2011-09-01

The main component of the amyloid plaques found in the brains of those with Alzheimer's disease (AD) is a polymerized form of the β-amyloid peptide (Aβ) and is considered to play a central role in the pathogenesis of this neurodegenerative disorder. Aβ is derived from the proteolytic processing of the amyloid precursor protein (APP). Beta site APP-cleaving enzyme, BACE1 (also known as β-secretase) is a membrane-bound aspartyl protease responsible for the initial step in the generation of Aβ peptide and is thus a prime target for therapeutic intervention. Substantive evidence now indicates that the processing of APP by BACE1 is regulated by the intracellular sorting of the enzyme and, moreover, perturbations in these intracellular trafficking pathways have been linked to late-onset AD. In this review, we highlight the recent advances in the understanding of the regulation of the intracellular sorting of BACE1 and APP and illustrate why the trafficking of these cargos represent a key issue for understanding the membrane-mediated events associated with the generation of the neurotoxic Aβ products in AD. Copyright © 2011 International Union of Biochemistry and Molecular Biology, Inc.

Vacuolar CAX1 and CAX3 influence auxin transport in guard cells via regulation of apoplastic pH

USDA-ARS?s Scientific Manuscript database

Cation exchangers CAX1 and CAX3 are vacuolar ion transporters involved in ion homeostasis in plants. Widely expressed in the plant, they mediate calcium transport from the cytosol to the vacuole lumen using the proton gradient across the tonoplast. Here, we report an unexpected role of CAX1 and CAX3...

Role of Tim50 in the Transfer of Precursor Proteins from the Outer to the Inner Membrane of Mitochondria

PubMed Central

Sichting, Martin; Popov-Čeleketić, Dušan; Mapa, Koyeli; Gevorkyan-Airapetov, Lada; Zohary, Keren; Hell, Kai; Azem, Abdussalam

2009-01-01

Transport of essentially all matrix and a number of inner membrane proteins is governed, entirely or in part, by N-terminal presequences and requires a coordinated action of the translocases of outer and inner mitochondrial membranes (TOM and TIM23 complexes). Here, we have analyzed Tim50, a subunit of the TIM23 complex that is implicated in transfer of precursors from TOM to TIM23. Tim50 is recruited to the TIM23 complex via Tim23 in an interaction that is essentially independent of the rest of the translocase. We find Tim50 in close proximity to the intermembrane space side of the TOM complex where it recognizes both types of TIM23 substrates, those that are to be transported into the matrix and those destined to the inner membrane, suggesting that Tim50 recognizes presequences. This function of Tim50 depends on its association with TIM23. We conclude that the efficient transfer of precursors between TOM and TIM23 complexes requires the concerted action of Tim50 with Tim23. PMID:19144822

Role of Tim50 in the transfer of precursor proteins from the outer to the inner membrane of mitochondria.

PubMed

Mokranjac, Dejana; Sichting, Martin; Popov-Celeketić, Dusan; Mapa, Koyeli; Gevorkyan-Airapetov, Lada; Zohary, Keren; Hell, Kai; Azem, Abdussalam; Neupert, Walter

2009-03-01

Transport of essentially all matrix and a number of inner membrane proteins is governed, entirely or in part, by N-terminal presequences and requires a coordinated action of the translocases of outer and inner mitochondrial membranes (TOM and TIM23 complexes). Here, we have analyzed Tim50, a subunit of the TIM23 complex that is implicated in transfer of precursors from TOM to TIM23. Tim50 is recruited to the TIM23 complex via Tim23 in an interaction that is essentially independent of the rest of the translocase. We find Tim50 in close proximity to the intermembrane space side of the TOM complex where it recognizes both types of TIM23 substrates, those that are to be transported into the matrix and those destined to the inner membrane, suggesting that Tim50 recognizes presequences. This function of Tim50 depends on its association with TIM23. We conclude that the efficient transfer of precursors between TOM and TIM23 complexes requires the concerted action of Tim50 with Tim23.

Spatial and Temporal Resolution of Global Protein Synthesis during HSV Infection Using Bioorthogonal Precursors and Click Chemistry

PubMed Central

Serwa, Remigiusz A.; O’Hare, Peter

2016-01-01

We used pulse-labeling with the methionine analogue homopropargylglycine (HPG) to investigate spatiotemporal aspects of protein synthesis during herpes simplex virus (HSV) infection. In vivo incorporation of HPG enables subsequent selective coupling of fluorochrome-capture reagents to newly synthesised proteins. We demonstrate that HPG labeling had no effect on cell viability, on accumulation of test early or late viral proteins, or on overall virus yields. HPG pulse-labeling followed by SDS-PAGE analysis confirmed incorporation into newly synthesised proteins, while parallel processing by in situ cycloaddition revealed new insight into spatiotemporal aspects of protein localisation during infection. A striking feature was the rapid accumulation of newly synthesised proteins not only in a general nuclear pattern but additionally in newly forming sub-compartments represented by small discrete foci. These newly synthesised protein domains (NPDs) were similar in size and morphology to PML domains but were more numerous, and whereas PML domains were progressively disrupted, NPDs were progressively induced and persisted. Immediate-early proteins ICP4 and ICP0 were excluded from NPDs, but using an ICP0 mutant defective in PML disruption, we show a clear spatial relationship between NPDs and PML domains with NPDs frequently forming immediately adjacent and co-joining persisting PML domains. Further analysis of location of the chaperone Hsc70 demonstrated that while NPDs formed early in infection without overt Hsc70 recruitment, later in infection Hsc70 showed pronounced recruitment frequently in a coat-like fashion around NPDs. Moreover, while ICP4 and ICP0 were excluded from NPDs, ICP22 showed selective recruitment. Our data indicate that NPDs represent early recruitment of host and viral de novo translated protein to distinct structural entities which are precursors to the previously described VICE domains involved in protein quality control in the nucleus, and reveal

CLC-Nt1, a putative chloride channel protein of tobacco, co-localizes with mitochondrial membrane markers.

PubMed Central

Lurin, C; Güclü, J; Cheniclet, C; Carde, J P; Barbier-Brygoo, H; Maurel, C

2000-01-01

The voltage-dependent chloride channel (CLC) family of membrane proteins has cognates in animals, yeast, bacteria and plants, and chloride-channel activity has been assigned to most of the animal homologues. Lack of evidence of CLC functions in plants prompted us to characterize the cellular localization of the tobacco CLC-Nt1 protein. Specific polyclonal antibodies were raised against an N-terminal polypeptide of CLC-Nt1. These antibodies were used to probe membrane proteins prepared by various cell-fractionation methods. These included aqueous two-phase partitioning (for plasma membranes), free-flow electrophoresis (for vacuolar and plasma membranes), intact vacuole isolation, Percoll-gradient centrifugation (for plastids and mitochondria) and stepped, linear, sucrose-density-gradient centrifugation (for mitochondria). Each purified membrane fraction was characterized with specific marker enzyme activities or antibodies. Our studies ruled out the possibility that the major cell localization of CLC-Nt1 was the vacuolar or plasma membranes, the endoplasmic reticulum, the Golgi apparatus or the plastids. In contrast, we showed that the tobacco CLC-Nt1 specifically co-localized with the markers of the mitochondrial inner membrane, cytochrome c oxidase and NAD9 protein. CLC-Nt1 may correspond to the inner membrane anion channel ('IMAC') described previously in animal and plant mitochondria. PMID:10816421

CLC-Nt1, a putative chloride channel protein of tobacco, co-localizes with mitochondrial membrane markers.

PubMed

Lurin, C; Güclü, J; Cheniclet, C; Carde, J P; Barbier-Brygoo, H; Maurel, C

2000-06-01

The voltage-dependent chloride channel (CLC) family of membrane proteins has cognates in animals, yeast, bacteria and plants, and chloride-channel activity has been assigned to most of the animal homologues. Lack of evidence of CLC functions in plants prompted us to characterize the cellular localization of the tobacco CLC-Nt1 protein. Specific polyclonal antibodies were raised against an N-terminal polypeptide of CLC-Nt1. These antibodies were used to probe membrane proteins prepared by various cell-fractionation methods. These included aqueous two-phase partitioning (for plasma membranes), free-flow electrophoresis (for vacuolar and plasma membranes), intact vacuole isolation, Percoll-gradient centrifugation (for plastids and mitochondria) and stepped, linear, sucrose-density-gradient centrifugation (for mitochondria). Each purified membrane fraction was characterized with specific marker enzyme activities or antibodies. Our studies ruled out the possibility that the major cell localization of CLC-Nt1 was the vacuolar or plasma membranes, the endoplasmic reticulum, the Golgi apparatus or the plastids. In contrast, we showed that the tobacco CLC-Nt1 specifically co-localized with the markers of the mitochondrial inner membrane, cytochrome c oxidase and NAD9 protein. CLC-Nt1 may correspond to the inner membrane anion channel ('IMAC') described previously in animal and plant mitochondria.

Consequences of inhibiting amyloid precursor protein processing enzymes on synaptic function and plasticity.

PubMed

Wang, Hui; Megill, Andrea; He, Kaiwen; Kirkwood, Alfredo; Lee, Hey-Kyoung

2012-01-01

Alzheimer's disease (AD) is a neurodegenerative disease, one of whose major pathological hallmarks is the accumulation of amyloid plaques comprised of aggregated β-amyloid (Aβ) peptides. It is now recognized that soluble Aβ oligomers may lead to synaptic dysfunctions early in AD pathology preceding plaque deposition. Aβ is produced by a sequential cleavage of amyloid precursor protein (APP) by the activity of β- and γ-secretases, which have been identified as major candidate therapeutic targets of AD. This paper focuses on how Aβ alters synaptic function and the functional consequences of inhibiting the activity of the two secretases responsible for Aβ generation. Abnormalities in synaptic function resulting from the absence or inhibition of the Aβ-producing enzymes suggest that Aβ itself may have normal physiological functions which are disrupted by abnormal accumulation of Aβ during AD pathology. This interpretation suggests that AD therapeutics targeting the β- and γ-secretases should be developed to restore normal levels of Aβ or combined with measures to circumvent the associated synaptic dysfunction(s) in order to have minimal impact on normal synaptic function.

Amyloid precursor protein expression and processing are differentially regulated during cortical neuron differentiation.

PubMed

Bergström, Petra; Agholme, Lotta; Nazir, Faisal Hayat; Satir, Tugce Munise; Toombs, Jamie; Wellington, Henrietta; Strandberg, Joakim; Bontell, Thomas Olsson; Kvartsberg, Hlin; Holmström, Maria; Boreström, Cecilia; Simonsson, Stina; Kunath, Tilo; Lindahl, Anders; Blennow, Kaj; Hanse, Eric; Portelius, Erik; Wray, Selina; Zetterberg, Henrik

2016-07-07

Amyloid precursor protein (APP) and its cleavage product amyloid β (Aβ) have been thoroughly studied in Alzheimer's disease. However, APP also appears to be important for neuronal development. Differentiation of induced pluripotent stem cells (iPSCs) towards cortical neurons enables in vitro mechanistic studies on human neuronal development. Here, we investigated expression and proteolytic processing of APP during differentiation of human iPSCs towards cortical neurons over a 100-day period. APP expression remained stable during neuronal differentiation, whereas APP processing changed. α-Cleaved soluble APP (sAPPα) was secreted early during differentiation, from neuronal progenitors, while β-cleaved soluble APP (sAPPβ) was first secreted after deep-layer neurons had formed. Short Aβ peptides, including Aβ1-15/16, peaked during the progenitor stage, while processing shifted towards longer peptides, such as Aβ1-40/42, when post-mitotic neurons appeared. This indicates that APP processing is regulated throughout differentiation of cortical neurons and that amyloidogenic APP processing, as reflected by Aβ1-40/42, is associated with mature neuronal phenotypes.

The intact Kunitz domain protects the amyloid precursor protein from being processed by matriptase-2.

PubMed

Beckmann, Anna-Madeleine; Glebov, Konstantin; Walter, Jochen; Merkel, Olaf; Mangold, Martin; Schmidt, Frederike; Becker-Pauly, Christoph; Gütschow, Michael; Stirnberg, Marit

2016-08-01

Proteolytic processing of the amyloid precursor protein (APP) leads to amyloid-β (Aβ) peptides. So far, the mechanism of APP processing is insufficiently characterized at the molecular level. Whereas the knowledge of Aβ generation by several proteases has been expanded, the contribution of the Kunitz-type protease inhibitor domain (KPI) present in two major APP isoforms to the complex proteolytic processing of APP is poorly understood. In this study, we have identified KPI-containing APP as a very potent, slow-binding inhibitor for the membrane-bound proteolytic regulator of iron homeostasis matriptase-2 by forming stable complexes with its target protease in HEK cells. Inhibition and complex formation depend on the intact KPI domain. By inhibiting matriptase-2, KPI-containing APP is protected from matriptase-2-mediated proteolysis within the Aβ region, thus preventing the generation of N-terminally truncated Aβ.

CSF soluble amyloid precursor proteins in the diagnosis of incipient Alzheimer disease.

PubMed

Perneczky, R; Tsolakidou, A; Arnold, A; Diehl-Schmid, J; Grimmer, T; Förstl, H; Kurz, A; Alexopoulos, P

2011-07-05

To explore if soluble amyloid precursor proteins (sAPP) in CSF improve the identification of patients with incipient Alzheimer disease (AD) in a group of patients with mild cognitive impairment (MCI). A cohort study with follow-up assessments of 58 patients with MCI with baseline CSF sampling was conducted: 21 patients had progressed to probable AD (MCI-AD), 27 still had MCI, 8 had reverted to normal (MCI-NAD), and 2 patients with frontotemporal dementia (FTD) were excluded. Sixteen additional patients with FTD were included to explore the specificity of the CSF markers. CSF concentrations of sAPPα, sAPPβ, tau, and Aβ(1-42) were measured with sensitive and specific ELISAs. Associations between diagnostic status, CSF protein concentrations, and other patient characteristics were explored using multiple logistic regression analyses with stepwise variable selection. The optimal sensitivity and specificity of the best models were derived from receiver operating characteristic curves. The MCI-AD group had significantly higher sAPPβ concentrations than the MCI-NAD and the FTD groups. A combination of sAPPβ, tau, and age differentiated the MCI-AD and the MCI-NAD groups with a sensitivity of 80.00% and a specificity of 81.00%. The best model for the differentiation of the MCI-AD and the FTD groups included sAPPβ and tau, and showed a sensitivity of 95.20% and a specificity of 81.20%. Aβ(1-42) and sAPPα did not significantly contribute to the models. These findings suggest that sAPPβ may be clinically useful, and superior to Aβ(1-42), in the early and differential diagnosis of incipient AD.

Membrane proteins involved in transport, vesicle traffic and Ca(2+) signaling increase in beetroots grown in saline soils.

PubMed

Lino, Bárbara; Chagolla, Alicia; E González de la Vara, Luis

2016-07-01

By separating plasma membrane proteins according to their hydropathy from beetroots grown in saline soils, several proteins probably involved in salt tolerance were identified by mass spectrometry. Beetroots, as a salt-tolerant crop, have developed mechanisms to cope with stresses associated with saline soils. To observe which plasma membrane (PM) proteins were more abundant in beet roots grown in saline soils, beet root plants were irrigated with water or 0.2 M NaCl. PM-enriched membrane preparations were obtained from these plants, and their proteins were separated according to their hydropathy by serial phase partitioning with Triton X-114. Some proteins whose abundance increased visibly in membranes from salt-grown beetroots were identified by mass spectrometry. Among them, there was a V-type H(+)-ATPase (probably from contaminating vacuolar membranes), which increased with salt at all stages of beetroots' development. Proteins involved in solute transport (an H(+)-transporting PPase and annexins), vesicle traffic (clathrin and synaptotagmins), signal perception and transduction (protein kinases and phospholipases, mostly involved in calcium signaling) and metabolism, appeared to increase in salt-grown beetroot PM-enriched membranes. These results suggest that PM and vacuolar proteins involved in transport, metabolism and signal transduction increase in beet roots adapted to saline soils. In addition, these results show that serial phase partitioning with Triton X-114 is a useful method to separate membrane proteins for their identification by mass spectrometry.

A recombinant pseudorabies virus co-expressing capsid proteins precursor P1-2A of FMDV and VP2 protein of porcine parvovirus: a trivalent vaccine candidate.

PubMed

Hong, Qi; Qian, Ping; Li, Xiang-Min; Yu, Xiao-Lan; Chen, Huan-Chun

2007-11-01

Pseudorabies (PR), foot-and-mouth disease (FMD), and porcine parvovirus disease are three important infectious diseases in swine worldwide. The gene-deleted pseudorabies virus (PRV) has been used as a live-viral vector to develop multivalent genetic engineering vaccine. In this study, a recombinant PRV, which could co-express protein precursor P1-2A of FMDV and VP2 protein of PPV, was constructed using PRV TK(-)/gE(-)/LacZ(+) mutant as the vector. After homologous recombination and plaque purification, recombinant virus PRV TK(-)/gE(-)/P1-2A-VP2 was acquired and identified. Immunogenicity, safety of the recombinant PRV and its protection against PRV were confirmed in a mouse model by indirect ELISA and serum neutralization test. The results show that the recombinant PRV is a candidate vaccine strain to develop a novel trivalent vaccine against PRV, FMDV and PPV in swine.

Reduced vacuolar β-1,3-glucan synthesis affects carbohydrate metabolism as well as plastid homeostasis and structure in Phaeodactylum tricornutum.

PubMed

Huang, Weichao; Haferkamp, Ilka; Lepetit, Bernard; Molchanova, Mariia; Hou, Shengwei; Jeblick, Wolfgang; Río Bártulos, Carolina; Kroth, Peter G

2018-05-01

The β-1,3-glucan chrysolaminarin is the main storage polysaccharide of diatoms. In contrast to plants and green algae, diatoms and most other algal groups do not accumulate storage polysaccharides in their plastids. The diatom Phaeodactylum tricornutum possesses only a single gene encoding a putative β-1,3-glucan synthase ( Pt BGS). Here, we characterize this enzyme by expressing GFP fusion proteins in P. tricornutum and by creating and investigating corresponding gene silencing mutants. We demonstrate that Pt BGS is a vacuolar protein located in the tonoplast. Metabolite analyses of two mutant strains with reduced amounts of Pt BGS reveal a reduction in their chrysolaminarin content and an increase of soluble sugars and lipids. This indicates that carbohydrates are shunted into alternative pathways when chrysolaminarin production is impaired. The mutant strains show reduced growth and lower photosynthetic capacities, while possessing higher photoprotective abilities than WT cells. Interestingly, a strong reduction in Pt BGS expression also results in aberrations of the usually very regular thylakoid membrane patterns, including increased thylakoid thickness, reduced numbers of thylakoids per plastid, and increased numbers of lamellae per thylakoid stack. Our data demonstrate the complex intertwinement of carbohydrate storage in the vacuoles with carbohydrate metabolism, photosynthetic homeostasis, and plastid morphology.

Identification of a novel amyloid precursor protein processing pathway that generates secreted N-terminal fragments.

PubMed

Vella, Laura J; Cappai, Roberto

2012-07-01

Alzheimer's disease (AD) is a neurodegenerative disorder of the central nervous system. The proteolytic processing of the amyloid precursor protein (APP) into the β-amyloid (Aβ) peptide is a central event in AD. While the pathway that generates Aβ is well described, many questions remain concerning general APP metabolism and its metabolites. It is becoming clear that the amino-terminal region of APP can be processed to release small N-terminal fragments (NTFs). The purpose of this study was to investigate the occurrence and generation of APP NTFs in vivo and in cell culture (SH-SY5Y) in order to delineate the cellular pathways implicated in their generation. We were able to detect 17- to 28-kDa APP NTFs in human and mouse brain tissue that are distinct from N-APP fragments previously reported. We show that the 17- to 28-kDa APP NTFs were highly expressed in mice from the age of 2 wk to adulthood. SH-SY5Y studies indicate the generation of APP NTFs involves a novel APP processing pathway, regulated by protein kinase C, but independent of α-secretase or β-secretase 1 (BACE) activity. These results identify a novel, developmentally regulated APP processing pathway that may play an important role in the physiological function of APP.

The Marine Natural Product Manzamine A Targets Vacuolar ATPases and Inhibits Autophagy in Pancreatic Cancer Cells

PubMed Central

Kallifatidis, Georgios; Hoepfner, Dominic; Jaeg, Tiphaine; Guzmán, Esther A.; Wright, Amy E.

2013-01-01

Manzamine A, a member of the manzamine alkaloids, was originally isolated from marine sponges of the genus Haliclona. It was recently shown to have activity against pancreatic cancer cells, but the precise mechanism of action remained unclear. To further our understanding of the mechanism of action of manzamine A, chemogenomic profiling in the yeast S. cerevisiae was performed, suggesting that manzamine A is an uncoupler of vacuolar ATPases. Fluorescence microscopy confirmed this effect on yeast vacuoles, where manzamine A produced a phenotype very similar to that of the established v-ATPase inhibitor bafilomycin A1. In pancreatic cancer cells, 10 µM manzamine A affected vacuolar ATPase activity and significantly increased the level of autophagosome marker LC3-II and p62/SQSTM1 as observed by western blot analysis. Treatment with manzamine A in combination with bafilomycin A1 (inhibitor of autophagosome-lysosome fusion) did not change the levels of LC3-II when compared to cells treated with bafilomycin A1 alone, suggesting that manzamine A is a potential inhibitor of autophagy by preventing autophagosome turnover. As autophagy is essential for pancreatic tumor growth, blocking this pathway with manzamine A suggests a promising strategy for the treatment of pancreatic cancer. PMID:24048269

Amyloplasts and Vacuolar Membrane Dynamics in the Living Graviperceptive Cell of the Arabidopsis Inflorescence StemW⃞

PubMed Central

Saito, Chieko; Morita, Miyo T.; Kato, Takehide; Tasaka, Masao

2005-01-01

We developed an adequate method for the in vivo analysis of organelle dynamics in the gravity-perceptive cell (endodermis) of the Arabidopsis thaliana inflorescence stem, revealing behavior of amyloplasts and vacuolar membranes in those cells. Amyloplasts in the endodermis showed saltatory movements even before gravistimulation by reorientation, and these movements were confirmed as microfilament dependent. From our quantitative analysis in the wild type, the gravity-oriented movement of amyloplasts mainly occurred during 0 to 3 min after gravistimulation by reorientation, supporting findings from our previous physiological study. Even after microfilament disruption, the gravity-oriented movement of amyloplasts remained. By contrast, in zig/sgr4 mutants, where a SNARE molecule functioning in vacuole biogenesis has been disrupted, the movement of amyloplasts in the endodermis is severely restricted both before and after gravistimulation by reorientation. Here, we describe vacuolar membrane behavior in these cells in the wild-type, actin filament–disrupted, and zig/sgr4 mutants and discuss its putatively important features for the perception of gravity. We also discuss the data on the two kinds of movements of amyloplasts that may play an important role in gravitropism: (1) the leading edge amyloplasts and (2) the en mass movement of amyloplasts. PMID:15689424

Long-term live-cell imaging reveals new roles for Salmonella effector proteins SseG and SteA.

PubMed

McQuate, Sarah E; Young, Alexandra M; Silva-Herzog, Eugenia; Bunker, Eric; Hernandez, Mateo; de Chaumont, Fabrice; Liu, Xuedong; Detweiler, Corrella S; Palmer, Amy E

2017-01-01

Salmonella Typhimurium is an intracellular bacterial pathogen that infects both epithelial cells and macrophages. Salmonella effector proteins, which are translocated into the host cell and manipulate host cell components, control the ability to replicate and/or survive in host cells. Due to the complexity and heterogeneity of Salmonella infections, there is growing recognition of the need for single-cell and live-cell imaging approaches to identify and characterize the diversity of cellular phenotypes and how they evolve over time. Here, we establish a pipeline for long-term (17 h) live-cell imaging of infected cells and subsequent image analysis methods. We apply this pipeline to track bacterial replication within the Salmonella-containing vacuole in epithelial cells, quantify vacuolar replication versus survival in macrophages and investigate the role of individual effector proteins in mediating these parameters. This approach revealed that dispersed bacteria can coalesce at later stages of infection, that the effector protein SseG influences the propensity for cytosolic hyper-replication in epithelial cells, and that while SteA only has a subtle effect on vacuolar replication in epithelial cells, it has a profound impact on infection parameters in immunocompetent macrophages, suggesting differential roles for effector proteins in different infection models. © 2016 John Wiley & Sons Ltd.

Long-Term Live Cell Imaging Reveals New Roles For Salmonella Effector Proteins SseG and SteA

PubMed Central

McQuate, Sarah E.; Young, Alexandra M.; Silva-Herzog, Eugenia; Bunker, Eric; Hernandez, Mateo; de Chaumont, Fabrice; Liu, Xuedong; Detweiler, Corrella S.; Palmer, Amy E.

2016-01-01

Summary Salmonella Typhimurium is an intracellular bacterial pathogen that infects both epithelial cells and macrophages. Salmonella effector proteins, which are translocated into the host cell and manipulate host cell components, control the ability to replicate and/or survive in host cells. Due to the complexity and heterogeneity of Salmonella infections, there is growing recognition of the need for single cell and live-cell imaging approaches to identify and characterize the diversity of cellular phenotypes and how they evolve over time. Here we establish a pipeline for long-term (16 hours) live-cell imaging of infected cells and subsequent image analysis methods. We apply this pipeline to track bacterial replication within the Salmonella-containing vacuole in epithelial cells, quantify vacuolar replication versus survival in macrophages, and investigate the role of individual effector proteins in mediating these parameters. This approach revealed that dispersed bacteria can coalesce at later stages of infection, that the effector protein SseG influences the propensity for cytosolic hyperreplication in epithelial cells, and that while SteA only has a subtle effect on vacuolar replication in epithelial cells, it has a profound impact on infection parameters in immunocompetent macrophages, suggesting differential roles for effector proteins in different infection models. PMID:27376507

Silencing of vacuolar invertase and asparagine synthetase genes and its impact on acrylamide formation of fried potato products.

PubMed

Zhu, Xiaobiao; Gong, Huiling; He, Qunyan; Zeng, Zixian; Busse, James S; Jin, Weiwei; Bethke, Paul C; Jiang, Jiming

2016-02-01

Acrylamide is produced in a wide variety of carbohydrate-rich foods during high-temperature cooking. Dietary acrylamide is a suspected human carcinogen, and health concerns related to dietary acrylamide have been raised worldwide. French fries and potato chips contribute a significant proportion to the average daily intake of acrylamide, especially in developed countries. One way to mitigate health concerns related to acrylamide is to develop potato cultivars that have reduced contents of the acrylamide precursors asparagine, glucose and fructose in tubers. We generated a large number of silencing lines of potato cultivar Russet Burbank by targeting the vacuolar invertase gene VInv and the asparagine synthetase genes StAS1 and StAS2 with a single RNA interference construct. The transcription levels of these three genes were correlated with reducing sugar (glucose and fructose) and asparagine content in tubers. Fried potato products from the best VInv/StAS1/StAS2-triple silencing lines contained only one-fifteenth of the acrylamide content of the controls. Interestingly, the extent of acrylamide reduction of the best triple silencing lines was similar to that of the best VInv-single silencing lines developed previously from the same potato cultivar Russet Burbank. These results show that an acrylamide mitigation strategy focused on developing potato cultivars with low reducing sugars is likely to be an effective and sufficient approach for minimizing the acrylamide-forming potential of French fry processing potatoes. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Vacuolar invertase gene silencing in potato (Solanum tuberosum L.) improves processing quality by decreasing the frequency of sugar-end defects

USDA-ARS?s Scientific Manuscript database

Sugar-end defect is a tuber quality disorder that causes unacceptable darkening of one end of French fries. This defect appears when environmental stress during tuber growth increases post-harvest vacuolar acid invertase activity at one end of the tuber. Reducing sugars produced by invertase form da...

Co-overexpressing a plasma membrane and a vacuolar membrane sodium/proton antiporter significantly improves salt tolerance in transgenic Arabidopsis plants.

USDA-ARS?s Scientific Manuscript database

The Arabidopsis gene AtNHX1 encodes a vacuolar membrane bound sodium/proton (Sodium/Hydrogen) antiporter that transports sodium into the vacuole and exports hydrogen into the cytoplasm. The Arabidopsis gene SOS1 encodes a plasma membrane bound sodium/hydrogen antiporter that exports sodium to the ex...

The h-region of twin-arginine signal peptides supports productive binding of bacterial Tat precursor proteins to the TatBC receptor complex.

PubMed

Ulfig, Agnes; Fröbel, Julia; Lausberg, Frank; Blümmel, Anne-Sophie; Heide, Anna Katharina; Müller, Matthias; Freudl, Roland

2017-06-30

The twin-arginine translocation (Tat) pathway transports folded proteins across bacterial membranes. Tat precursor proteins possess a conserved twin-arginine (RR) motif in their signal peptides that is involved in their binding to the Tat translocase, but some facets of this interaction remain unclear. Here, we investigated the role of the hydrophobic (h-) region of the Escherichia coli trimethylamine N -oxide reductase (TorA) signal peptide in TatBC receptor binding in vivo and in vitro We show that besides the RR motif, a minimal, functional h-region in the signal peptide is required for Tat-dependent export in Escherichia coli Furthermore, we identified mutations in the h-region that synergistically suppressed the export defect of a TorA[KQ]-30aa-MalE Tat reporter protein in which the RR motif was replaced with a lysine-glutamine pair. Strikingly, all suppressor mutations increased the hydrophobicity of the h-region. By systematically replacing a neutral residue in the h-region with various amino acids, we detected a positive correlation between the hydrophobicity of the h-region and the translocation efficiency of the resulting reporter variants. In vitro cross-linking of residues located in the periplasmically-oriented part of the TatBC receptor to TorA[KQ]-30aa-MalE reporter variants harboring a more hydrophobic h-region in their signal peptides confirmed that unlike in TorA[KQ]-30aa-MalE with an unaltered h-region, the mutated reporters moved deep into the TatBC-binding cavity. Our results clearly indicate that, besides the Tat motif, the h-region of the Tat signal peptides is another important binding determinant that significantly contributes to the productive interaction of Tat precursor proteins with the TatBC receptor complex. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Disruption of the vacuolar calcium-ATPases in Arabidopsis results in the activation of a salicylic acid-dependent programmed cell death pathway.

PubMed

Boursiac, Yann; Lee, Sang Min; Romanowsky, Shawn; Blank, Robert; Sladek, Chris; Chung, Woo Sik; Harper, Jeffrey F

2010-11-01

Calcium (Ca(2+)) signals regulate many aspects of plant development, including a programmed cell death pathway that protects plants from pathogens (hypersensitive response). Cytosolic Ca(2+) signals result from a combined action of Ca(2+) influx through channels and Ca(2+) efflux through pumps and cotransporters. Plants utilize calmodulin-activated Ca(2+) pumps (autoinhibited Ca(2+)-ATPase [ACA]) at the plasma membrane, endoplasmic reticulum, and vacuole. Here, we show that a double knockout mutation of the vacuolar Ca(2+) pumps ACA4 and ACA11 in Arabidopsis (Arabidopsis thaliana) results in a high frequency of hypersensitive response-like lesions. The appearance of macrolesions could be suppressed by growing plants with increased levels (greater than 15 mm) of various anions, providing a method for conditional suppression. By removing plants from a conditional suppression, lesion initials were found to originate primarily in leaf mesophyll cells, as detected by aniline blue staining. Initiation and spread of lesions could also be suppressed by disrupting the production or accumulation of salicylic acid (SA), as shown by combining aca4/11 mutations with a sid 2 (for salicylic acid induction-deficient2) mutation or expression of the SA degradation enzyme NahG. This indicates that the loss of the vacuolar Ca(2+) pumps by itself does not cause a catastrophic defect in ion homeostasis but rather potentiates the activation of a SA-dependent programmed cell death pathway. Together, these results provide evidence linking the activity of the vacuolar Ca(2+) pumps to the control of a SA-dependent programmed cell death pathway in plants.

Vacuolar H(+)-Pyrophosphatase AVP1 is Involved in Amine Fungicide Tolerance in Arabidopsis thaliana and Provides Tridemorph Resistance in Yeast.

PubMed

Hernández, Agustín; Herrera-Palau, Rosana; Madroñal, Juan M; Albi, Tomás; López-Lluch, Guillermo; Perez-Castiñeira, José R; Navas, Plácido; Valverde, Federico; Serrano, Aurelio

2016-01-01

Amine fungicides are widely used as crop protectants. Their success is believed to be related to their ability to inhibit postlanosterol sterol biosynthesis in fungi, in particular sterol-Δ(8),Δ(7)-isomerases and sterol-Δ(14)-reductases, with a concomitant accumulation of toxic abnormal sterols. However, their actual cellular effects and mechanisms of death induction are still poorly understood. Paradoxically, plants exhibit a natural resistance to amine fungicides although they have similar enzymes in postcicloartenol sterol biosynthesis that are also susceptible to fungicide inhibition. A major difference in vacuolar ion homeostasis between plants and fungi is the presence of a dual set of primary proton pumps in the former (V-ATPase and H(+)-pyrophosphatase), but only the V-ATPase in the latter. Abnormal sterols affect the proton-pumping capacity of V-ATPases in fungi and this has been proposed as a major determinant in fungicide action. Using Saccharomyces cerevisiae as a model fungus, we provide evidence that amine fungicide treatment induced cell death by apoptosis. Cell death was concomitant with impaired H(+)-pumping capacity in vacuole vesicles and dependent on vacuolar proteases. Also, the heterologous expression of the Arabidopsis thaliana main H(+)-pyrophosphatase (AVP1) at the fungal vacuolar membrane reduced apoptosis levels in yeast and increased resistance to amine fungicides. Consistently, A. thaliana avp1 mutant seedlings showed increased susceptibility to this amine fungicide, particularly at the level of root development. This is in agreement with AVP1 being nearly the sole H(+)-pyrophosphatase gene expressed at the root elongation zones. All in all, the present data suggest that H(+)-pyrophosphatases are major determinants of plant tolerance to amine fungicides.

Specific Inhibition of β-Secretase Processing of the Alzheimer Disease Amyloid Precursor Protein.

PubMed

Ben Halima, Saoussen; Mishra, Sabyashachi; Raja, K Muruga Poopathi; Willem, Michael; Baici, Antonio; Simons, Kai; Brüstle, Oliver; Koch, Philipp; Haass, Christian; Caflisch, Amedeo; Rajendran, Lawrence

2016-03-08

Development of disease-modifying therapeutics is urgently needed for treating Alzheimer disease (AD). AD is characterized by toxic β-amyloid (Aβ) peptides produced by β- and γ-secretase-mediated cleavage of the amyloid precursor protein (APP). β-secretase inhibitors reduce Aβ levels, but mechanism-based side effects arise because they also inhibit β-cleavage of non-amyloid substrates like Neuregulin. We report that β-secretase has a higher affinity for Neuregulin than it does for APP. Kinetic studies demonstrate that the affinities and catalytic efficiencies of β-secretase are higher toward non-amyloid substrates than toward APP. We show that non-amyloid substrates are processed by β-secretase in an endocytosis-independent manner. Exploiting this compartmentalization of substrates, we specifically target the endosomal β-secretase by an endosomally targeted β-secretase inhibitor, which blocked cleavage of APP but not non-amyloid substrates in many cell systems, including induced pluripotent stem cell (iPSC)-derived neurons. β-secretase inhibitors can be designed to specifically inhibit the Alzheimer process, enhancing their potential as AD therapeutics without undesired side effects. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Comparative Protein Profiling of Intraphagosomal Expressed Proteins of Mycobacterium bovis BCG.

PubMed

Singhal, Neelja; Kumar, Manish; Sharma, Divakar; Bisht, Deepa

2016-01-01

BCG, the only available vaccine against tuberculosis affords a variable protection which wanes with time. In this study we have analyzed and compared the proteins which are expressed differentially during broth-culture and intraphagosomal growth of M.bovis BCG. Eight proteins which showed increased expression during the intraphagosomal growth were identified by MALDI-TOF/MS. These were - a precursor of alanine and proline-rich secreted protein apa, isoforms of malate dehydrogenase, large subunit alpha (Alpha-ETF) of electron transfer flavoprotein, immunogenic protein MPB64 precursor, UPF0036 protein, and two proteins with unknown function. Based on these findings we speculate that higher expression of these proteins has a probable role in intracellular survival, adaptation and/or immunoprotective effect of BCG. Further, these proteins might also be used as gene expression markers for endosome trafficking events of BCG.

Vacuolar sequestration capacity and long-distance metal transport in plants

PubMed Central

Peng, Jia-Shi; Gong, Ji-Ming

2014-01-01

The vacuole is a pivotal organelle functioning in storage of metabolites, mineral nutrients, and toxicants in higher plants. Accumulating evidence indicates that in addition to its storage role, the vacuole contributes essentially to long-distance transport of metals, through the modulation of Vacuolar sequestration capacity (VSC) which is shown to be primarily controlled by cytosolic metal chelators and tonoplast-localized transporters, or the interaction between them. Plants adapt to their environments by dynamic regulation of VSC for specific metals and hence targeting metals to specific tissues. Study of VSC provides not only a new angle to understand the long-distance root-to-shoot transport of minerals in plants, but also an efficient way to biofortify essential mineral nutrients or to phytoremediate non-essential metal pollution. The current review will focus on the most recent proceedings on the interaction mechanisms between VSC regulation and long-distance metal transport. PMID:24550927

Vacuolar sequestration capacity and long-distance metal transport in plants.

PubMed

Peng, Jia-Shi; Gong, Ji-Ming

2014-01-01

The vacuole is a pivotal organelle functioning in storage of metabolites, mineral nutrients, and toxicants in higher plants. Accumulating evidence indicates that in addition to its storage role, the vacuole contributes essentially to long-distance transport of metals, through the modulation of Vacuolar sequestration capacity (VSC) which is shown to be primarily controlled by cytosolic metal chelators and tonoplast-localized transporters, or the interaction between them. Plants adapt to their environments by dynamic regulation of VSC for specific metals and hence targeting metals to specific tissues. Study of VSC provides not only a new angle to understand the long-distance root-to-shoot transport of minerals in plants, but also an efficient way to biofortify essential mineral nutrients or to phytoremediate non-essential metal pollution. The current review will focus on the most recent proceedings on the interaction mechanisms between VSC regulation and long-distance metal transport.

Inhibition of Wnt signaling induces amyloidogenic processing of amyloid precursor protein and the production and aggregation of Amyloid-β (Aβ)42 peptides.

PubMed

Tapia-Rojas, Cheril; Burgos, Patricia V; Inestrosa, Nibaldo C

2016-12-01

Alzheimer's disease (AD) is the most common neurodegenerative disorder and the most frequent cause of dementia in the aged population. According to the amyloid hypothesis, the amyloid-β (Aβ) peptide plays a key role in the pathogenesis of AD. Aβ is generated from the amyloidogenic processing of amyloid precursor protein and can aggregate to form oligomers, which have been described as a major synaptotoxic agent in neurons. Dysfunction of Wnt signaling has been linked to increased Aβ formation; however, several other studies have argued against this possibility. Herein, we use multiple experimental approaches to confirm that the inhibition of Wnt signaling promoted the amyloidogenic proteolytic processing of amyloid precursor protein. We also demonstrate that inhibiting Wnt signaling increases the production of the Aβ 42 peptide, the Aβ 42 /Aβ 40 ratio, and the levels of Aβ oligomers such as trimers and tetramers. Moreover, we show that activating Wnt signaling reduces the levels of Aβ 42 and its aggregates, increases Aβ 40 levels, and reduces the Aβ 42 /Aβ 40 ratio. Finally, we show that the protective effects observed in response to activation of the Wnt pathway rely on β-catenin-dependent transcription, which is demonstrated experimentally via the expression of various 'mutant forms of β-catenin'. Together, our findings indicate that loss of the Wnt signaling pathway may contribute to the pathogenesis of AD. © 2016 International Society for Neurochemistry.

Differentiating the Influences of Aging and Adiposity on Brain Weights, Levels of Serum and Brain Cytokines, Gastrointestinal Hormones, and Amyloid Precursor Protein.

PubMed

Banks, William A; Abrass, Christine K; Hansen, Kim M

2016-01-01

Aging and obesity exert important effects on disease. Differentiating these effects is difficult, however, because weight gain often accompanies aging. Here, we used a nested design of aged, calorically restricted, and refed rats to measure changes in brain and blood levels of cytokines and gastrointestinal hormones, brain amyloid precursor protein levels, and brain and body weights. By comparing groups and using path analysis, we found divergent influences of chronological aging versus body weight, our main findings being (i) changes in whole brain weight and serum macrophage colony-stimulating factor levels correlated better with body weight than with chronological aging, (ii) a decrease in brain cytokines and brain plasminogen activator inhibitor levels correlated better with chronological aging than with body weight, (iii) serum erythropoietin levels were influenced by both body weight and aging, (iv) serum plasminogen activator inhibitor, serum cytokines, and brain tumor necrosis factor were not influenced by aging or body weight, and (v) brain amyloid precursor protein more closely related to body weight and serum levels of gastrointestinal hormones than to brain weight, chronological aging, or cytokines. These findings show that although aging and body weight interact, their influences are distinct not only among various cytokines and hormones but also between the central nervous system and the peripheral tissue compartments. Published by Oxford University Press on behalf of the Gerontological Society of America 2014.

Regulation of transport processes across the tonoplast

PubMed Central

Neuhaus, H. Ekkehard; Trentmann, Oliver

2014-01-01

In plants, the vacuole builds up the cellular turgor and represents an important component in cellular responses to diverse stress stimuli. Rapid volume changes of cells, particularly of motor cells, like guard cells, are caused by variation of osmolytes and consequently of the water contents in the vacuole. Moreover, directed solute uptake into or release out of the large central vacuole allows adaptation of cytosolic metabolite levels according to the current physiological requirements and specific cellular demands. Therefore, solute passage across the vacuolar membrane, the tonoplast, has to be tightly regulated. Important principles in vacuolar transport regulation are changes of tonoplast transport protein abundances by differential expression of genes or changes of their activities, e.g., due to post-translational modification or by interacting proteins. Because vacuolar transport is in most cases driven by an electro-chemical gradient altered activities of tonoplast proton pumps significantly influence vacuolar transport capacities. Intense studies on individual tonoplast proteins but also unbiased system biological approaches have provided important insights into the regulation of vacuolar transport. This short review refers to selected examples of tonoplast proteins and their regulation, with special focus on protein phosphorylation. PMID:25309559

Amyloid Precursor-like Protein 2 Increases the Endocytosis, Instability, and Turnover of the H2-Kd MHC Class I Molecule1

PubMed Central

Tuli, Amit; Sharma, Mahak; McIlhaney, Mary M.; Talmadge, James E.; Naslavsky, Naava; Caplan, Steve; Solheim, Joyce C.

2008-01-01

The defense against the invasion of viruses and tumors relies on the presentation of viral and tumor-derived peptides to cytotoxic T lymphocytes by cell surface major histocompatibility complex (MHC) class I molecules. Previously, we showed that the ubiquitously expressed protein amyloid precursor-like protein 2 (APLP2) associates with the folded form of the MHC class I molecule Kd. In the current study, APLP2 was found to associate with folded Kd molecules following their endocytosis and to increase the amount of endocytosed Kd. In addition, increased expression of APLP2 was shown to decrease Kd surface expression and thermostability. Correspondingly, Kd thermostability and surface expression were increased by down-regulation of APLP2 expression. Overall, these data suggest that APLP2 modulates the stability and endocytosis of Kd molecules. PMID:18641335

Protein import into isolated pea root leucoplasts.

PubMed

Chu, Chiung-Chih; Li, Hsou-Min

2015-01-01

Leucoplasts are important organelles for the synthesis and storage of starch, lipids and proteins. However, molecular mechanism of protein import into leucoplasts and how it differs from that of import into chloroplasts remain unknown. We used pea seedlings for both chloroplast and leucoplast isolations to compare within the same species. We further optimized the isolation and import conditions to improve import efficiency and to permit a quantitative comparison between the two plastid types. The authenticity of the import was verified using a mitochondrial precursor protein. Our results show that, when normalized to Toc75, most translocon proteins are less abundant in leucoplasts than in chloroplasts. A precursor shown to prefer the receptor Toc132 indeed had relatively more similar import efficiencies between chloroplasts and leucoplasts compared to precursors that prefer Toc159. Furthermore we found two precursors that exhibited very high import efficiency into leucoplasts. Their transit peptides may be candidates for delivering transgenic proteins into leucoplasts and for analyzing motifs important for leucoplast import.

Abscisic Acid Induction of Vacuolar H+-ATPase Activity in Mesembryanthemum crystallinum Is Developmentally Regulated1

PubMed Central

Barkla, Bronwyn J.; Vera-Estrella, Rosario; Maldonado-Gama, Minerva; Pantoja, Omar

1999-01-01

Abscisic acid (ABA) has been implicated as a key component in water-deficit-induced responses, including those triggered by drought, NaCl, and low- temperature stress. In this study a role for ABA in mediating the NaCl-stress-induced increases in tonoplast H+-translocating ATPase (V-ATPase) and Na+/H+ antiport activity in Mesembryanthemum crystallinum, leading to vacuolar Na+ sequestration, were investigated. NaCl or ABA treatment of adult M. crystallinum plants induced V-ATPase H+ transport activity, and when applied in combination, an additive effect on V-ATPase stimulation was observed. In contrast, treatment of juvenile plants with ABA did not induce V-ATPase activity, whereas NaCl treatment resulted in a similar response to that observed in adult plants. Na+/H+ antiport activity was induced in both juvenile and adult plants by NaCl, but ABA had no effect at either developmental stage. Results indicate that ABA-induced changes in V-ATPase activity are dependent on the plant reaching its adult phase, whereas NaCl-induced increases in V-ATPase and Na+/H+ antiport activity are independent of plant age. This suggests that ABA-induced V-ATPase activity may be linked to the stress-induced, developmentally programmed switch from C3 metabolism to Crassulacean acid metabolism in adult plants, whereas, vacuolar Na+ sequestration, mediated by the V-ATPase and Na+/H+ antiport, is regulated through ABA-independent pathways. PMID:10398716

Full-length amyloid precursor protein regulates lipoprotein metabolism and amyloid-β clearance in human astrocytes.

PubMed

Fong, Lauren K; Yang, Max M; Dos Santos Chaves, Rodrigo; Reyna, Sol M; Langness, Vanessa F; Woodruff, Grace; Roberts, Elizabeth A; Young, Jessica E; Goldstein, Lawrence S B

2018-06-01

Mounting evidence suggests that alterations in cholesterol homeostasis are involved in Alzheimer's disease (AD) pathogenesis. Amyloid precursor protein (APP) or multiple fragments generated by proteolytic processing of APP have previously been implicated in the regulation of cholesterol metabolism. However, the physiological function of APP in regulating lipoprotein homeostasis in astrocytes, which are responsible for de novo cholesterol biosynthesis and regulation in the brain, remains unclear. To address this, here we used CRISPR/Cas9 genome editing to generate isogenic APP-knockout (KO) human induced pluripotent stem cells (hiPSCs) and differentiated them into human astrocytes. We found that APP-KO astrocytes have reduced cholesterol and elevated levels of sterol regulatory element-binding protein (SREBP) target gene transcripts and proteins, which were both downstream consequences of reduced lipoprotein endocytosis. To elucidate which APP fragments regulate cholesterol homeostasis and examine whether familial AD mutations in APP affect lipoprotein metabolism, we analyzed an isogenic allelic series harboring the APP Swedish and APP V717F variants. Only astrocytes homozygous for the APP Swedish (APP Swe/Swe ) mutation, which had reduced full-length APP (FL APP) due to increased β-secretase cleavage, recapitulated the APP-KO phenotypes. Astrocytic internalization of amyloid-β (Aβ), another ligand for low-density lipoprotein (LDL) receptors, was also impaired in APP-KO and APP Swe/Swe astrocytes. Finally, impairing cleavage of FL APP through β-secretase inhibition in APP Swe/Swe astrocytes reversed the LDL and Aβ endocytosis defects. In conclusion, FL APP is involved in the endocytosis of LDL receptor ligands and required for proper cholesterol homeostasis and Aβ clearance in human astrocytes. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Vacuolar amino acid transporter Avt5p is responsible for lithium uptake in Schizosaccharomyces pombe.

PubMed

Iwaki, Tomoko; Sekito, Takayuki; Kakinuma, Yoshimi

2010-01-01

The fission yeast Schizosaccharomyces pombe was sensitive to salinity; cell growth was stopped by 0.5 M NaCl and by 10 mM LiCl. The avt5+ gene encodes a vacuolar transporter with a broad specificity for amino acids. We found that the avt5Delta mutant became highly tolerant of Li+ and Na+ in growth. Concanamycin A-sensitive Li+ uptake as well as cellular Li+ content was lower in the avt5 mutant, suggesting a role of Avt5p in cellular uptake of toxic Li+.

Processing of alkaline phosphatase precursor to the mature enzyme by an Escherichia coli inner membrane preparation.

PubMed Central

Chang, C N; Inouye, H; Model, P; Beckwith, J

1980-01-01

An inner membrane preparation co-translationally cleaved both the alkaline phosphatase and bacteriophage f1 coat protein precursors to the mature proteins. Post-translational outer membrane proteolysis of pre-alkaline phosphatase generated a protein smaller than the authentic monomer. Images PMID:6991486

Vacuolar H+-ATPase Is Expressed in Response to Gibberellin during Tomato Seed Germination1

PubMed Central

Cooley, Michael B.; Yang, Hong; Dahal, Peetambar; Mella, R. Alejandra; Downie, A. Bruce; Haigh, Anthony M.; Bradford, Kent J.

1999-01-01

Completion of germination (radicle emergence) by gibberellin (GA)-deficient (gib-1) mutant tomato (Lycopersicon esculentum Mill.) seeds is dependent upon exogenous GA, because weakening of the endosperm tissue enclosing the radicle tip requires GA. To investigate genes that may be involved in endosperm weakening or embryo growth, differential cDNA display was used to identify mRNAs differentially expressed in gib-1 seeds imbibed in the presence or absence of GA4+7. Among these was a GA-responsive mRNA encoding the 16-kD hydrophobic subunit c of the V0 membrane sector of vacuolar H+-translocating ATPases (V-ATPase), which we termed LVA-P1. LVA-P1 mRNA expression in gib-1 seeds was dependent on GA and was particularly abundant in the micropylar region prior to radicle emergence. Both GA dependence and tissue localization of LVA-P1 mRNA expression were confirmed directly in individual gib-1 seeds using tissue printing. LVA-P1 mRNA was also expressed in wild-type seeds during development and germination, independent of exogenous GA. Specific antisera detected protein subunits A and B of the cytoplasmic V1 sector of the V-ATPase holoenzyme complex in gib-1 seeds only in the presence of GA, and expression was localized to the micropylar region. The results suggest that V-ATPase plays a role in GA-regulated germination of tomato seeds. PMID:10594121

The emerging structure of vacuolar ATPases.

PubMed

Drory, Omri; Nelson, Nathan

2006-10-01

Bioenergetics and physiology of primary pumps have been revitalized by new insights into the mechanism of energizing biomembranes. Structural information is becoming available, and the three-dimensional structure of F-ATPase is being resolved. The growing understanding of the fundamental mechanism of energy coupling may revolutionize our view of biological processes. The F- and V-ATPases (vacuolar-type ATPase) exhibit a common mechanical design in which nucleotide-binding on the catalytic sector, through a cycle of conformation changes, drives the transmembrane passage of protons by turning a membrane-embedded rotor. This motor can run in forward or reverse directions, hydrolyzing ATP as it pumps protons uphill or creating ATP as protons flow downhill. In contrast to F-ATPases, whose primary function in eukaryotic cells is to form ATP at the expense of the proton-motive force (pmf), V-ATPases function exclusively as an ATP-dependent proton pump. The pmf generated by V-ATPases in organelles and membranes of eukaryotic cells is utilized as a driving force for numerous secondary transport processes. V- and F-ATPases have similar structure and mechanism of action, and several of their subunits evolved from common ancestors. Electron microscopy studies of V-ATPase revealed its general structure at low resolution. Recently, several structures of V-ATPase subunits, solved by X-ray crystallography with atomic resolution, were published. This, together with electron microscopy low-resolution maps of the whole complex, and biochemistry cross-linking experiments, allows construction of a structural model for a part of the complex that may be used as a working hypothesis for future research.

NGFFFamide and echinotocin: structurally unrelated myoactive neuropeptides derived from neurophysin-containing precursors in sea urchins.

PubMed

Elphick, Maurice R; Rowe, Matthew L

2009-04-01

The myoactive neuropeptide NGIWYamide was originally isolated from the holothurian (sea cucumber) Apostichopus japonicus but there is evidence that NGIWYamide-like peptides also occur in other echinoderms. Here we report the discovery of a gene in the sea urchin Strongylocentrotus purpuratus that encodes two copies of an NGIWYamide-like peptide: Asn-Gly-Phe-Phe-Phe-(NH(2)) or NGFFFamide. Interestingly, the C-terminal region of the NGFFFamide precursor shares sequence similarity with neurophysins, carrier proteins hitherto uniquely associated with precursors of vasopressin/oxytocin-like neuropeptides. Thus, the NGFFFamide precursor is the first neurophysin-containing neuropeptide precursor to be discovered that does not contain a vasopressin/oxytocin-like peptide. However, it remains to be determined whether neurophysin acts as a carrier protein for NGFFFamide. The S. purpuratus genome also contains a gene encoding a precursor comprising a neurophysin polypeptide and 'echinotocin' (CFISNCPKGamide) - the first vasopressin/oxytocin-like peptide to be identified in an echinoderm. Therefore, in S. purpuratus there are two genes encoding precursors that have a neurophysin domain but which encode neuropeptides that are structurally unrelated. Furthermore, both NGFFFamide and echinotocin cause contraction of tube foot and oesophagus preparations from the sea urchin Echinus esculentus, consistent with the myoactivity of NGIWYamide in sea cucumbers and the myoactivity of vasopressin/oxytocin-like peptides in other animal phyla. Presumably the NGFFFamide precursor acquired its neurophysin domain following partial or complete duplication of a gene encoding a vasopressin/oxytocin-like peptide, but it remains to be determined when in evolutionary history this occurred.

RNAi Mediated curcin precursor gene silencing in Jatropha (Jatropha curcas L.).

PubMed

Patade, Vikas Yadav; Khatri, Deepti; Kumar, Kamal; Grover, Atul; Kumari, Maya; Gupta, Sanjay Mohan; Kumar, Devender; Nasim, Mohammed

2014-07-01

Curcin, a type I ribosomal inhibiting protein-RIP, encoded by curcin precursor gene, is a phytotoxin present in Jatropha (Jatropha curcas L.). Here, we report designing of RNAi construct for the curcin precursor gene and further its genetic transformation of Jatropha to reduce its transcript expression. Curcin precursor gene was first cloned from Jatropha strain DARL-2 and part of the gene sequence was cloned in sense and antisense orientation separated by an intron sequence in plant expression binary vector pRI101 AN. The construction of the RNAi vector was confirmed by double digestion and nucleotide sequencing. The vector was then mobilized into Agrobacterium tumefaciens strain GV 3101 and used for tissue culture independent in planta transformation protocol optimized for Jatropha. Germinating seeds were injured with a needle before infection with Agrobacterium and then transferred to sterilized sand medium. The seedlings were grown for 90 days and genomic DNA was isolated from leaves for transgenic confirmation based on real time PCR with NPT II specific dual labeled probe. Result of the transgenic confirmation analysis revealed presence of the gene silencing construct in ten out of 30 tested seedlings. Further, quantitative transcript expression analysis of the curcin precursor gene revealed reduction in the transcript abundance by more than 98% to undetectable level. The transgenic plants are being grown in containment for further studies on reduction in curcin protein content in Jatropha seeds.

Distinct amyloid precursor protein processing machineries of the olfactory system.

PubMed

Kim, Jae Yeon; Rasheed, Ameer; Yoo, Seung-Jun; Kim, So Yeun; Cho, Bongki; Son, Gowoon; Yu, Seong-Woon; Chang, Keun-A; Suh, Yoo-Hun; Moon, Cheil

2018-01-01

Processing of amyloid precursor protein (APP) occurs through sequential cleavages first by β-secretase and then by the γ-secretase complex. However, abnormal processing of APP leads to excessive production of β-amyloid (Aβ) in the central nervous system (CNS), an event which is regarded as a primary cause of Alzheimer's disease (AD). In particular, gene mutations of the γ-secretase complex-which contains presenilin 1 or 2 as the catalytic core-could trigger marked Aβ accumulation. Olfactory dysfunction usually occurs before the onset of typical AD-related symptoms (eg, memory loss or muscle retardation), suggesting that the olfactory system may be one of the most vulnerable regions to AD. To date however, little is known about why the olfactory system is affected so early by AD prior to other regions. Thus, we examined the distribution of secretases and levels of APP processing in the olfactory system under either healthy or pathological conditions. Here, we show that the olfactory system has distinct APP processing machineries. In particular, we identified higher expressions levels and activity of γ-secretase in the olfactory epithelium (OE) than other regions of the brain. Moreover, APP c-terminal fragments (CTF) are markedly detected. During AD progression, we note increased expression of presenilin2 of γ-secretases in the OE, not in the OB, and show that neurotoxic Aβ*56 accumulates more quickly in the OE. Taken together, these results suggest that the olfactory system has distinct APP processing machineries under healthy and pathological conditions. This finding may provide a crucial understanding of the unique APP-processing mechanisms in the olfactory system, and further highlights the correlation between olfactory deficits and AD symptoms. Copyright © 2017 Elsevier Inc. All rights reserved.

HMC-1 human mast cells synthesize neurotensin (NT) precursor, secrete bioactive NT-like peptide(s) and express NT receptor NTS1.

PubMed

Cochrane, David E; Carraway, Robert E; Harrington, Kimberly; Laudano, Melissa; Rawlings, Stephen; Feldberg, Ross S

2011-12-01

To determine if mast cells synthesize the inflammatory peptide, neurotensin (NT), secrete immunoreactive and bioactive NT, and express the NT receptor NTS1. HMC-1 cells, pleural mast cells from Sprague-Dawley rats, LAD2 mast cells, and human cord blood mast cells were used. HMC-1 cells were stimulated with NT, C48/80, mastoparan, or PGE(2). For changes in cutaneous vascular permeability, anesthetized rats were injected intravenously with Evans Blue dye and intradermally with saline, NT, histamine, diphenhydramine, and C48/80. RT-PCR was used to identify RNA transcripts. Histamine was measured by fluorometric assay. In vivo cutaneous vascular permeability assays, radio-immunoassays for NT, Western blotting for the NT precursor protein and NTS1 protein from HMC-1 cells and tissues from rats were used. Immunohistochemistry was used to identify NT precursor-like proteins in HMC-1 mast cells. HMC-1 cells express mRNAs for NT precursor, PC5A processing enzyme and NTS1 receptor. Human cord blood mast cells and LAD2 mast cells express mRNA transcripts for NT precursor and NTS1. Western blotting showed NT precursor and NTS1 receptor in HMC1. Rat tissues with high numbers of mast cells contained NT precursor proteins. NT-like peptides from HMC-1 displayed NT-like bioactivity. HMC-1 mast cells synthesize and secrete immunoreactive and bioactive NT-like peptide(s) and express the NT receptor, suggesting that NT from mast cells might serve autocrine and paracrine roles.

Fibrinogen gamma-A chain precursor in CSF: a candidate biomarker for Alzheimer's disease

PubMed Central

Lee, Joung Wook; Namkoong, Hong; Kim, Hyun Kee; Kim, Sanghee; Hwang, Dong Whi; Na, Hae Ri; Ha, Seon-Ah; Kim, Jae-Ryong; Kim, Jin Woo

2007-01-01

Background Cerebrospinal fluid (CSF) may be valuable for exploring protein markers for the diagnosis of Alzheimer's disease (AD). The prospect of early detection and treatment, to slow progression, holds hope for aging populations with increased average lifespan. The aim of the present study was to investigate candidate CSF biological markers in patients with mild cognitive impairment (MCI) and AD and compare them with age-matched normal control subjects. Methods We applied proteomics approaches to analyze CSF samples derived from 27 patients with AD, 3 subjects with MCI and 30 controls. The AD group was subdivided into three groups by clinical severity according to clinical dementia rating (CDR), a well known clinical scale for dementia. Results We demonstrated an elevated level of fibrinogen gamma-A chain precursor protein in CSF from patients with mild cognitive impairment and AD compared to the age-matched normal subjects. Moreover, its expression was more prominent in the AD group than in the MCI and correlated with disease severity and progression. In contrast, fibrinogen gamma-A chain precursor protein was detected very low in the age-matched normal group. Conclusion These findings suggest that the CSF level of fibrinogen gamma-A chain precursor may be a candidate biomarker for AD. PMID:17565664

Acute γ-secretase Inhibition of Nonhuman Primate CNS Shifts Amyloid Precursor Protein (APP) Metabolism from Amyloid-β Production to Alternative APP Fragments without Amyloid-β Rebound

PubMed Central

Cook, Jacquelynn J.; Wildsmith, Kristin R.; Gilberto, David B.; Holahan, Marie A.; Kinney, Gene G.; Mathers, Parker D.; Michener, Maria S.; Price, Eric A.; Shearman, Mark S.; Simon, Adam J.; Wang, Jennifer X.; Wu, Guoxin; Yarasheski, Kevin E.; Bateman, Randall J.

2010-01-01

The accumulation of amyloid beta (Aβ) in Alzheimer’s disease is caused by an imbalance of production and clearance, which leads to increased soluble Aβ species and extracellular plaque formation in the brain. Multiple Aβ-lowering therapies are currently in development: an important goal is to characterize the molecular mechanisms of action and effects on physiological processing of Aβ, as well as other amyloid precursor protein (APP) metabolites, in models which approximate human Aβ physiology. To this end, we report the translation of the human in vivo stable-isotope-labeling kinetics (SILK) method to a rhesus monkey cisterna magna ported (CMP) nonhuman primate model, and use the model to test the mechanisms of action of a γ-secretase inhibitor (GSI). A major concern of inhibiting the enzymes which produce Aβ (β- and γ-secretase) is that precursors of Aβ may accumulate and cause a rapid increase in Aβ production when enzyme inhibition discontinues. In this study, the GSI MK-0752 was administered to conscious CMP rhesus monkeys in conjunction with in vivo stable isotope labeling, and dose-dependently reduced newly generated CNS Aβ. In contrast to systemic Aβ metabolism, CNS Aβ production was not increased after the GSI was cleared. These results indicate that most of the CNS APP was metabolized to products other than Aβ, including C-terminal truncated forms of Aβ: 1–14, 1–15 and 1–16; this demonstrates an alternative degradation pathway for CNS amyloid precursor protein during γ-secretase inhibition. PMID:20463236

Investigation of copper(II) binding to the protein precursor of Non-Amyloid-Beta Component of Alzheimer Disease Amyloid Plaque

NASA Astrophysics Data System (ADS)

Rose, Francis; Hodak, Miroslav; Bernholc, Jerry

2007-03-01

The Non-Amyloid-Beta Component Precursor (NACP) is a natively unfolded synaptic protein that is implicated in Alzheimers and Parkinsons diseases. Its aggregation into fibrillar structures is accelerated by the binding of copper(II). Experimental studies suggest that the dominant copper binding site is located at the histidine residue in NACP. Based on this evidence we assembled a model fragment of the binding site and used DFT to analyze the conformational details of the most probable binding motifs. We investigated the overall conformational effects with classical MD by constraining the copper binding site to the most energetically favorable geometry obtained from the DFT calculations. These results are compared and contrasted with those of the unbound NACP.

Contribution of Kunitz protease inhibitor and transmembrane domains to amyloid precursor protein homodimerization.

PubMed

Ben Khalifa, N; Tyteca, D; Courtoy, P J; Renauld, J C; Constantinescu, S N; Octave, J N; Kienlen-Campard, P

2012-01-01

The two major isoforms of the human amyloid precursor protein (APP) are APP695 and APP751. They differ by the insertion of a Kunitz-type protease inhibitor (KPI) sequence in the extracellular domain of APP751. APP-KPI isoforms are increased in Alzheimer's disease brains, and they could be associated with disease progression. Recent studies have shown that APP processing to Aβ is regulated by homodimerization, which involves both extracellular and juxtamembrane/transmembrane (JM/TM) regions. Our aim is to understand the mechanisms controlling APP dimerization and the contribution of the ectodomain and JM/TM regions to this process. We used bimolecular fluorescence complementation approaches coupled to fluorescence-activated cell sorting analysis to measure the dimerization level of different APP isoforms and APP C-terminal fragments (C99) mutated in their JM/TM region. APP751 was found to form significantly more homodimers than APP695. Mutation of dimerization motifs in the TM domain of APP or C99 did not significantly affect fluorescence complementation. These findings indicate that the KPI domain plays a major role in APP dimerization. They set the basis for further investigation of the relation between dimerization, metabolism and function of APP. Copyright © 2012 S. Karger AG, Basel.

Conformational Stability of the NH2-Terminal Propeptide of the Precursor of Pulmonary Surfactant Protein SP-B

PubMed Central

Bañares-Hidalgo, Ángeles; Estrada, Pilar

2016-01-01

Assembly of pulmonary surfactant lipid-protein complexes depends on conformational changes coupled with proteolytic maturation of proSP-B, the precursor of pulmonary surfactant protein B (SP-B), along the surfactant biogenesis pathway in pneumocytes. Conformational destabilization of the N-terminal propeptide of proSP-B (SP-BN) triggers exposure of the mature SP-B domain for insertion into surfactant lipids. We have studied the conformational stability during GdmCl- or urea-promoted unfolding of SP-BN with trp fluorescence and circular dichroism spectroscopies. Binding of the intermediate states to bis-ANS suggests their molten globule-like character. ΔG0H2O was ~ 12.7 kJ·mol-1 either with urea or GdmCl. None of the thermal transitions of SP-BN detected by CD correspond to protein unfolding. Differential scanning calorimetry of SP-BN evidenced two endothermic peaks involved in oligomer dissociation as confirmed with 2 M urea. Ionic strength was relevant since at 150 mM NaCl, the process originating the endotherm at the highest temperature was irreversible (Tm2 = 108.5°C) with an activation energy of 703.8 kJ·mol-1. At 500 mM NaCl the process became reversible (Tm2 = 114.4°C) and data were fitted to the Non-two States model with two subpeaks. No free thiols in the propeptide could be titrated by DTNB with or without 5.7 M GdmCl, indicating disulfide bonds establishment. PMID:27380171

Amyloid Precursor Protein Translation Is Regulated by a 3’UTR Guanine Quadruplex

PubMed Central

Sharoni, Michal; Olson, Kalee; Sebastian, Neeraj P.; Ansaloni, Sara; Schweitzer-Stenner, Reinhard; Akins, Michael R.; Bevilacqua, Philip C.; Saunders, Aleister J.

2015-01-01

A central event in Alzheimer’s disease is the accumulation of amyloid β (Aβ) peptides generated by the proteolytic cleavage of the amyloid precursor protein (APP). APP overexpression leads to increased Aβ generation and Alzheimer’s disease in humans and altered neuronal migration and increased long term depression in mice. Conversely, reduction of APP expression results in decreased Aβ levels in mice as well as impaired learning and memory and decreased numbers of dendritic spines. Together these findings indicate that therapeutic interventions that aim to restore APP and Aβ levels must do so within an ideal range. To better understand the effects of modulating APP levels, we explored the mechanisms regulating APP expression focusing on post-transcriptional regulation. Such regulation can be mediated by RNA regulatory elements such as guanine quadruplexes (G-quadruplexes), non-canonical structured RNA motifs that affect RNA stability and translation. Via a bioinformatics approach, we identified a candidate G-quadruplex within the APP mRNA in its 3’UTR (untranslated region) at residues 3008–3027 (NM_201414.2). This sequence exhibited characteristics of a parallel G-quadruplex structure as revealed by circular dichroism spectrophotometry. Further, as with other G-quadruplexes, the formation of this structure was dependent on the presence of potassium ions. This G-quadruplex has no apparent role in regulating transcription or mRNA stability as wild type and mutant constructs exhibited equivalent mRNA levels as determined by real time PCR. Instead, we demonstrate that this G-quadruplex negatively regulates APP protein expression using dual luciferase reporter and Western blot analysis. Taken together, our studies reveal post-transcriptional regulation by a 3’UTR G-quadruplex as a novel mechanism regulating APP expression. PMID:26618502

Amyloid precursor protein regulates migration and metalloproteinase gene expression in prostate cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miyazaki, Toshiaki; Ikeda, Kazuhiro; Horie-Inoue, Kuniko

Highlights: • APP knockdown reduced proliferation and migration of prostate cancer cells. • APP knockdown reduced expression of metalloproteinase and EMT-related genes. • APP overexpression promoted LNCaP cell migration. • APP overexpression increased expression of metalloproteinase and EMT-related genes. - Abstract: Amyloid precursor protein (APP) is a type I transmembrane protein, and one of its processed forms, β-amyloid, is considered to play a central role in the development of Alzheimer’s disease. We previously showed that APP is a primary androgen-responsive gene in prostate cancer and that its increased expression is correlated with poor prognosis for patients with prostate cancer. APPmore » has also been implicated in several human malignancies. Nevertheless, the mechanism underlying the pro-proliferative effects of APP on cancers is still not well-understood. In the present study, we explored a pathophysiological role for APP in prostate cancer cells using siRNA targeting APP (siAPP). The proliferation and migration of LNCaP and DU145 prostate cancer cells were significantly suppressed by siAPP. Differentially expressed genes in siAPP-treated cells compared to control siRNA-treated cells were identified by microarray analysis. Notably, several metalloproteinase genes, such as ADAM10 and ADAM17, and epithelial–mesenchymal transition (EMT)-related genes, such as VIM, and SNAI2, were downregulated in siAPP-treated cells as compared to control cells. The expression of these genes was upregulated in LNCaP cells stably expressing APP when compared with control cells. APP-overexpressing LNCaP cells exhibited enhanced migration in comparison to control cells. These results suggest that APP may contribute to the proliferation and migration of prostate cancer cells by modulating the expression of metalloproteinase and EMT-related genes.« less

Nicotinamide Riboside Is a Major NAD+ Precursor Vitamin in Cow Milk.

PubMed

Trammell, Samuel Aj; Yu, Liping; Redpath, Philip; Migaud, Marie E; Brenner, Charles

2016-05-01

Nicotinamide riboside (NR) is a recently discovered NAD(+) precursor vitamin with a unique biosynthetic pathway. Although the presence of NR in cow milk has been known for more than a decade, the concentration of NR with respect to the other NAD(+) precursors was unknown. We aimed to determine NAD(+) precursor vitamin concentration in raw samples of milk from individual cows and from commercially available cow milk. LC tandem mass spectrometry and isotope dilution technologies were used to quantify NAD(+) precursor vitamin concentration and to measure NR stability in raw and commercial milk. Nuclear magnetic resonance (NMR) spectroscopy was used to test for NR binding to substances in milk. Cow milk typically contained ∼12 μmol NAD(+) precursor vitamins/L, of which 60% was present as nicotinamide and 40% was present as NR. Nicotinic acid and other NAD(+) metabolites were below the limits of detection. Milk from samples testing positive for Staphylococcus aureus contained lower concentrations of NR (Spearman ρ = -0.58, P = 0.014), and NR was degraded by S. aureus Conventional milk contained more NR than milk sold as organic. Nonetheless, NR was stable in organic milk and exhibited an NMR spectrum consistent with association with a protein fraction in skim milk. NR is a major NAD(+) precursor vitamin in cow milk. Control of S. aureus may be important to preserve the NAD(+) precursor vitamin concentration of milk. © 2016 American Society for Nutrition.

Amyloid beta precursor protein and ubiquitin epitopes in human and experimental dystrophic axons. Ultrastructural localization.

PubMed Central

Bacci, B.; Cochran, E.; Nunzi, M. G.; Izeki, E.; Mizutani, T.; Patton, A.; Hite, S.; Sayre, L. M.; Autilio-Gambetti, L.; Gambetti, P.

1994-01-01

Dystrophic axons (DA) represent a major pathological feature of several neurodegenerative disorders, including infantile neuroaxonal dystrophy (INAD) and Alzheimer disease. We have previously presented evidence that amyloid beta precursor protein (BPP) and ubiquitin (Ub) are present in DA of different origin. We have now characterized the immunoreactivity of DA experimentally induced in rat by the administration of parabromophenylacetylurea (BPAU) and examined the subcellular localization of Ub and BPP in BPAU-induced DA and in DA present in subjects affected by INAD. BPAU-induced DA strongly immunoreacted with antisera to Ub and to COOH- and NH2-terminal regions of BPP. Immunoblots of DA-enriched brain regions were consistent with an increase in the amount of Ub and BPP in DA. Moreover, BPAU-induced DA immunoreacted with antibodies to PGP 9.5, a neuronal-specific Ub COOH-terminal hydrolase, and to the inducible heat shock protein 70. Antigenic characterization also indicated that the tubulovesicular membranes within DA derived largely from the smooth endoplasmic reticulum rather than from the Golgi system or the synaptic vesicles. Subcellular immunolocalization of Ub and BPP in both INAD- and BPAU-induced DA revealed that Ub and BPP colocalize in granulovesicular material in both conditions. In INAD DA intense Ub immunoreactivity was also detected in nonmembranous electron dense structures that were present only in these DA, probably because of the chronic course of INAD. Although BPP immunostaining may be related to accumulation of BPP-containing membranes in DA, Ub immunostaining is likely to result from activation of the Ub system by the neuron in the attempt to remove excessive and possibly abnormal proteins. A similar pathogenesis can be postulated for DA of Alzheimer disease. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:7512790

The m6A methyltransferase Ime4 epitranscriptionally regulates triacylglycerol metabolism and vacuolar morphology in haploid yeast cells.

PubMed

Yadav, Pradeep Kumar; Rajasekharan, Ram

2017-08-18

N 6 -Methyladenosine (m 6 A) is among the most common modifications in eukaryotic mRNA. The role of yeast m 6 A methyltransferase, Ime4, in meiosis and sporulation in diploid strains is very well studied, but its role in haploid strains has remained unknown. Here, with the help of an immunoblotting strategy and Ime4-GFP protein localization studies, we establish the physiological role of Ime4 in haploid cells. Our data showed that Ime4 epitranscriptionally regulates triacylglycerol metabolism and vacuolar morphology through the long-chain fatty acyl-CoA synthetase Faa1, independently of the RNA methylation complex (MIS complex). The MIS complex consists of the Ime4, Mum2, and Slz1 proteins. Our affinity enrichment strategy (methylated RNA immunoprecipitation assays) using m 6 A polyclonal antibodies coupled with mRNA isolation, quantitative real-time PCR, and standard PCR analyses confirmed the presence of m 6 A-modified FAA1 transcripts in haploid yeast cells. The term "epitranscriptional regulation" encompasses the RNA modification-mediated regulation of genes. Moreover, we demonstrate that the Aft2 transcription factor up-regulates FAA1 expression. Because the m 6 A methylation machinery is fundamentally conserved throughout eukaryotes, our findings will help advance the rapidly emerging field of RNA epitranscriptomics. The metabolic link identified here between m 6 A methylation and triacylglycerol metabolism via the Ime4 protein provides new insights into lipid metabolism and the pathophysiology of lipid-related metabolic disorders, such as obesity. Because the yeast vacuole is an analogue of the mammalian lysosome, our findings pave the way to better understand the role of m 6 A methylation in lysosome-related functions and diseases. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The biosynthetic gene cluster for the cyanogenic glucoside dhurrin in Sorghum bicolor contains its co-expressed vacuolar MATE transporter

PubMed Central

Darbani, Behrooz; Motawia, Mohammed Saddik; Olsen, Carl Erik; Nour-Eldin, Hussam H.; Møller, Birger Lindberg; Rook, Fred

2016-01-01

Genomic gene clusters for the biosynthesis of chemical defence compounds are increasingly identified in plant genomes. We previously reported the independent evolution of biosynthetic gene clusters for cyanogenic glucoside biosynthesis in three plant lineages. Here we report that the gene cluster for the cyanogenic glucoside dhurrin in Sorghum bicolor additionally contains a gene, SbMATE2, encoding a transporter of the multidrug and toxic compound extrusion (MATE) family, which is co-expressed with the biosynthetic genes. The predicted localisation of SbMATE2 to the vacuolar membrane was demonstrated experimentally by transient expression of a SbMATE2-YFP fusion protein and confocal microscopy. Transport studies in Xenopus laevis oocytes demonstrate that SbMATE2 is able to transport dhurrin. In addition, SbMATE2 was able to transport non-endogenous cyanogenic glucosides, but not the anthocyanin cyanidin 3-O-glucoside or the glucosinolate indol-3-yl-methyl glucosinolate. The genomic co-localisation of a transporter gene with the biosynthetic genes producing the transported compound is discussed in relation to the role self-toxicity of chemical defence compounds may play in the formation of gene clusters. PMID:27841372

Epizootiologic studies of avian vacuolar myelinopathy in waterbirds

USGS Publications Warehouse

Rocke, Tonie E.; Thomas, N.J.; Augspurger, T.; Miller, Kimberli J.

2002-01-01

Epizootic avian vacuolar myelinopathy (AVM) was first recognized as a neurologic disease in bald eagles (Haliaeetus leucocephalus) and American coots (Fulica americana) in Arkansas, USA in 1994 and 1996, respectively, but attempts to identify the etiology of the disease have been unsuccessful to date. Between 1998 and 2001, wing clipped sentinel birds (wild American coots and game farm mallards [Anas platyrhynchos]) were released at Lake Surf, North Carolina, a lake with recurrent outbreaks of AVM, in order to gain a better understanding of the epizootiology of the disease. As early as 5-7 days post-release, sentinel coots and mallards showed neurologic signs of disease and were confirmed with AVM upon histologic examination of their brains. Serial releases of sentinel mallards during the summer, fall, and winter of 2000-01 demonstrated that exposure to the causative agent at a threshold sufficient to manifest disease was seasonal and occurred over about a 2 mo period, during November and December. Our findings that disease onset can be very rapid (5-7 days) and that exposure to the causative agent of AVM is site-specific, seasonal (late fall to early winter), and occurs over a relatively short duration (several months) supports the hypothesis that the disease is caused by a chemical substance, most likely of natural origin.

Planar Cell Polarity Breaks the Symmetry of PAR Protein Distribution prior to Mitosis in Drosophila Sensory Organ Precursor Cells.

PubMed

Besson, Charlotte; Bernard, Fred; Corson, Francis; Rouault, Hervé; Reynaud, Elodie; Keder, Alyona; Mazouni, Khalil; Schweisguth, François

2015-04-20

During development, cell-fate diversity can result from the unequal segregation of fate determinants at mitosis. Polarization of the mother cell is essential for asymmetric cell division (ACD). It often involves the formation of a cortical domain containing the PAR complex proteins Par3, Par6, and atypical protein kinase C (aPKC). In the fly notum, sensory organ precursor cells (SOPs) divide asymmetrically within the plane of the epithelium and along the body axis to generate two distinct cells. Fate asymmetry depends on the asymmetric localization of the PAR complex. In the absence of planar cell polarity (PCP), SOPs divide with a random planar orientation but still asymmetrically, showing that PCP is dispensable for PAR asymmetry at mitosis. To study when and how the PAR complex localizes asymmetrically, we have used a quantitative imaging approach to measure the planar polarization of the proteins Bazooka (Baz, fly Par3), Par6, and aPKC in living pupae. By using imaging of functional GFP-tagged proteins with image processing and computational modeling, we find that Baz, Par6, and aPKC become planar polarized prior to mitosis in a manner independent of the AuroraA kinase and that PCP is required for the planar polarization of Baz, Par6, and aPKC during interphase. This indicates that a "mitosis rescue" mechanism establishes asymmetry at mitosis in PCP mutants. This study therefore identifies PCP as the initial symmetry-breaking signal for the planar polarization of PAR proteins in asymmetrically dividing SOPs. Copyright © 2015 Elsevier Ltd. All rights reserved.

Vacuolar SPX-MFS transporters are essential for phosphate adaptation in plants.

PubMed

Liu, Jinlong; Fu, Shaomin; Yang, Lei; Luan, Mingda; Zhao, Fugeng; Luan, Sheng; Lan, Wenzhi

2016-08-02

To survive in most soils in which inorganic phosphate (Pi) levels are limited and constantly changing, plants universally use the vacuoles as cellular Pi "sink" and "source" to maintain Pi homeostasis. However, the transporters that mediate Pi sequestration into the vacuoles remain unknown. Recently, we and other 2 groups independently identified the members of SPS-MSF family as the candidates for tonoplast Pi transporters in Arabidopsis thaliana and Oryza sativa. We and Liu et al. demonstrated that one of SPS-MSF member, VPT1 (Vacuolar Phosphate Transporter 1), also named as PHT5;1 (Phosphate Transporter 5;1), plays a predominant role in Pi sequestration of vacuoles in Arabidopsis. Here we show that vpt1 mutants and VPT1-GFP overexpressing lines displayed sensitive to Pi stress under the hydroponic system containing the medium with low iron, supporting that VPT1 is essential for Arabidopsis to adapt phosphate stress.

An Intracellular Threonine of Amyloid-β Precursor Protein Mediates Synaptic Plasticity Deficits and Memory Loss

PubMed Central

Lombino, Franco; Biundo, Fabrizio; Tamayev, Robert; Arancio, Ottavio; D’Adamio, Luciano

2013-01-01

Mutations in Amyloid-ß Precursor Protein (APP) and BRI2/ITM2b genes cause Familial Alzheimer and Danish Dementias (FAD/FDD), respectively. APP processing by BACE1, which is inhibited by BRI2, yields sAPPß and ß-CTF. ß-CTF is cleaved by gamma-secretase to produce Aß. A knock-in mouse model of FDD, called FDDKI, shows deficits in memory and synaptic plasticity, which can be attributed to sAPPß/ß-CTF but not Aß. We have investigated further the pathogenic function of ß-CTF focusing on Thr668 of ß-CTF because phosphorylation of Thr668 is increased in AD cases. We created a knock-in mouse bearing a Thr668Ala mutation (APPTA mice) that prevents phosphorylation at this site. This mutation prevents the development of memory and synaptic plasticity deficits in FDDKI mice. These data are consistent with a role for the carboxyl-terminal APP domain in the pathogenesis of dementia and suggest that averting the noxious role of Thr668 is a viable therapeutic strategy for human dementias. PMID:23451158

Alterations in the Balance of Amyloid-β Protein Precursor Species in the Cerebrospinal Fluid of Alzheimer's Disease Patients.

PubMed

Lopez-Font, Inmaculada; Boix, Claudia P; Zetterberg, Henrik; Blennow, Kaj; Sáez-Valero, Javier

2017-01-01

We recently demonstrated that soluble forms of the amyloid-β protein precursor (sAβPP) assemble into multimeric complexes in cerebrospinal fluid (CSF), which contributes to the underestimation of specific sAβPP species when assessed by ELISA. To circumvent this issue, we analyzed by SDS-PAGE large fragments of sAβPP and their variants in the CSF from Alzheimer's disease (AD; n = 20) and control (n = 20) subjects, probing with specific antibodies against particular domains. Similar levels of sAβPPα and sAβPPβ protein were found in CSF samples from AD and controls, yet there appeared to be a shift in the balance of the soluble full-length AβPP (sAβPPf) species in AD samples, with a decrease in the proportion of the lower (∼100 kDa) band relative to the upper (∼120 kDa) band. Similar differences were observed in the contribution of the major KPI-immunoreactive AβPP species. CSF samples also displayed differences in the correlations of AβPP species with classical AD biomarkers, particularly with respect to the Aβ42 peptide. The differences reveal alterations that probably reflect pathophysiological changes in the brain.

Novel Roles of Amyloid-Beta Protein Precursor Metabolites in Fragile X Syndrome and Autism

PubMed Central

Westmark, Cara J.; Sokol, Deborah K.; Maloney, Bryan; Lahiri, Debomoy K.

2017-01-01

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and is associated with up to 5% of autism cases. Several promising drugs are in preclinical testing for FXS; however, bench-to-bedside plans for the clinic are severely limited due to lack of validated biomarkers and outcome measures. Published work from our laboratories has demonstrated altered levels of amyloid-beta (Aβ) protein precursor (APP) and its metabolites in FXS and idiopathic autism. Westmark and colleagues have focused on β-secretase (amyloidogenic) processing and the accumulation of Aβ peptides in adult FXS models while Lahiri and Sokol have studied α-secretase (nonamyloidogenic or anabolic) processing and altered levels of sAPPα and Aβ in pediatric autism and FXS. Thus, our groups have hypothesized a pivotal role for these Alzheimer’s disease (AD)-related proteins in the neurodevelopmental disorders of FXS and autism. In this review, we discuss the contribution of APP metabolites to FXS and autism pathogenesis as well as the potential use of these metabolites as blood-based biomarkers and therapeutic targets. Our future focus is to identify key underlying mechanisms through which APP metabolites contribute to FXS and autism condition-to-disease pathology. Positive outcomes will support utilizing APP metabolites as blood-based biomarkers in clinical trials as well as testing drugs that modulate APP processing as potential disease therapeutics. Our studies to understand the role of APP metabolites in developmental conditions such as FXS and autism are a quantum leap for the neuroscience field, which has traditionally restricted any role of APP to AD and aging. PMID:27573877

Crystallization and preliminary X-ray diffraction study of the protealysin precursor belonging to the peptidase family M4

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gromova, T. Yu., E-mail: duk@img.ras.ru; Demidyuk, I. V.; Kostrov, S. V.

2008-09-15

A protealysin precursor (the enzyme of the peptidase family M4) was crystallized for the first time. The crystal-growth conditions were found, and single crystals of the protein with dimensions of 0.3-0.5 mm were grown. The preliminary X-ray diffraction study of the enzyme was performed. The protealysin precursor was shown to crystallize in two crystal modifications suitable for the X-ray diffraction study of the three-dimensional structure of the protein molecule at atomic resolution.

Failure to transmit avian vacuolar myelinopathy to mallard ducks

USGS Publications Warehouse

Larsen, R.S.; Nutter, F.B.; Augspurger, T.; Rocke, T.E.; Thomas, N.J.; Stoskopf, M.K.

2003-01-01

Avian vacuolar myelinopathy (AVM) is a neurologic disease that has been diagnosed in free-ranging birds in the southeastern United States. Bald eagles (Haliaeetus leuocephalus), American coots (Fulica americana), and mallards (Anas platyrhynchos) have been affected. Previous investigations have not determined the etiology of this disease. In November and December 2002, we attempted to induce AVM in game-farmed mallards through four, 7-day exposure trials. Mallards were housed in six groups of eight, with two of these groups serving as controls. One group was housed with AVM-affected coots; one group was tube fed daily with water from the lake where affected coots were captured; one group was tube fed daily with aquatic vegetation (Hydrilla verticillata) from the same lake; and another group was tube fed daily with sediment from the lake. No ducks exhibited clinical neurologic abnormalities consistent with AVM and no evidence of AVM was present at histopathologic examination of brain tissue. Although limitations in sample size, quantity of individual doses, frequency of dose administration, duration of exposure, and timing of these trials restrict the interpretation of the findings, AVM was not readily transmitted by direct contact, water, hydrilla, or sediment in this investigation.

Protein secretion and surface display in Gram-positive bacteria

PubMed Central

Schneewind, Olaf; Missiakas, Dominique M.

2012-01-01

The cell wall peptidoglycan of Gram-positive bacteria functions as a surface organelle for the transport and assembly of proteins that interact with the environment, in particular, the tissues of an infected host. Signal peptide-bearing precursor proteins are secreted across the plasma membrane of Gram-positive bacteria. Some precursors carry C-terminal sorting signals with unique sequence motifs that are cleaved by sortase enzymes and linked to the cell wall peptidoglycan of vegetative forms or spores. The sorting signals of pilin precursors are cleaved by pilus-specific sortases, which generate covalent bonds between proteins leading to the assembly of fimbrial structures. Other precursors harbour surface (S)-layer homology domains (SLH), which fold into a three-pronged spindle structure and bind secondary cell wall polysaccharides, thereby associating with the surface of specific Gram-positive microbes. Type VII secretion is a non-canonical secretion pathway for WXG100 family proteins in mycobacteria. Gram-positive bacteria also secrete WXG100 proteins and carry unique genes that either contribute to discrete steps in secretion or represent distinctive substrates for protein transport reactions. PMID:22411983

Molecular and functional characterization of vacuolar-ATPase from the American dog tick Dermacentor variabilis.

PubMed

Petchampai, N; Sunyakumthorn, P; Guillotte, M L; Thepparit, C; Kearney, M T; Mulenga, A; Azad, A F; Macaluso, K R

2014-02-01

Vacuolar (V)-ATPase is a proton-translocating enzyme that acidifies cellular compartments for various functions such as receptor-mediated endocytosis, intracellular trafficking and protein degradation. Previous studies in Dermacentor variabilis chronically infected with Rickettsia montanensis have identified V-ATPase as one of the tick-derived molecules transcribed in response to rickettsial infection. To examine the role of the tick V-ATPase in tick-Rickettsia interactions, a full-length 2887-bp cDNA (2532-bp open reading frame) clone corresponding to the transcript of the V0 domain subunit a of D. variabilis V-ATPase (DvVATPaseV0a) gene encoding an 843 amino acid protein with an estimated molecular weight of ~96 kDa was isolated from D. variabilis. Amino acid sequence analysis of DvVATPaseV0a showed the highest similarity to VATPaseV0a from Ixodes scapularis. A potential N-glycosylation site and eight putative transmembrane segments were identified in the sequence. Western blot analysis of tick tissues probed with polyclonal antibody raised against recombinant DvVATPaseV0a revealed the expression of V-ATPase in the tick ovary. Transcriptional profiles of DvVATPaseV0a demonstrated a greater mRNA expression in the tick ovary, compared with the midgut and salivary glands; however, the mRNA level in each of these tick tissues remained unchanged after infection with R. montanensis for 1 h. V-ATPase inhibition bioassays resulted in a significant decrease in the ability of R. montanensis to invade tick cells in vitro, suggesting a role of V-ATPase in rickettsial infection of tick cells. Characterization of tick-derived molecules involved in rickettsial infection is essential for a thorough understanding of rickettsial transmission within tick populations and the ecology of tick-borne rickettsial diseases. © 2013 The Authors. Insect Molecular Biology published by John Wiley & Sons Ltd on behalf of The Royal Entomological Society.

Rapid endocytosis of the low density lipoprotein receptor-related protein modulates cell surface distribution and processing of the beta-amyloid precursor protein.

PubMed

Cam, Judy A; Zerbinatti, Celina V; Li, Yonghe; Bu, Guojun

2005-04-15

The low density lipoprotein receptor-related protein (LRP) is a approximately 600-kDa multifunctional endocytic receptor that is highly expressed in the brain. LRP and its ligands apolipoprotein E, alpha2-macroglobulin, and beta-amyloid precursor protein (APP), are genetically linked to Alzheimer disease and are found in characteristic plaque deposits in brains of patients with Alzheimer disease. To identify which extracellular domains of LRP interact with APP, we used minireceptors of each of the individual LRP ligand binding domains and assessed their ability to bind and degrade a soluble APP fragment. LRP minireceptors containing ligand binding domains II and IV, but not I or III, interacted with APP. To test whether APP trafficking is directly related to the rapid endocytosis of LRP, we generated stable Chinese hamster ovary cell lines expressing either a wild-type LRP minireceptor or its endocytosis mutants. Chinese hamster ovary cells stably expressing wild-type LRP minireceptor had less cell surface APP than pcDNA3 vector-transfected cells, whereas those stably expressing endocytosis-defective LRP minireceptors accumulated APP at the cell surface. We also found that the steady-state levels of the amyloid beta-peptides (Abeta) is dictated by the relative expression levels of APP and LRP, probably reflecting the dual roles of LRP in both Abeta production and clearance. Together, these data establish a relationship between LRP rapid endocytosis and APP trafficking and proteolytic processing to generate Abeta.

Genome-Wide Analysis Reveals the Vacuolar pH-Stat of Saccharomyces cerevisiae

PubMed Central

Brett, Christopher L.; Kallay, Laura; Hua, Zhaolin; Green, Richard; Chyou, Anthony; Zhang, Yongqiang; Graham, Todd R.; Donowitz, Mark; Rao, Rajini

2011-01-01

Protons, the smallest and most ubiquitous of ions, are central to physiological processes. Transmembrane proton gradients drive ATP synthesis, metabolite transport, receptor recycling and vesicle trafficking, while compartmental pH controls enzyme function. Despite this fundamental importance, the mechanisms underlying pH homeostasis are not entirely accounted for in any organelle or organism. We undertook a genome-wide survey of vacuole pH (pHv) in 4,606 single-gene deletion mutants of Saccharomyces cerevisiae under control, acid and alkali stress conditions to reveal the vacuolar pH-stat. Median pHv (5.27±0.13) was resistant to acid stress (5.28±0.14) but shifted significantly in response to alkali stress (5.83±0.13). Of 107 mutants that displayed aberrant pHv under more than one external pH condition, functional categories of transporters, membrane biogenesis and trafficking machinery were significantly enriched. Phospholipid flippases, encoded by the family of P4-type ATPases, emerged as pH regulators, as did the yeast ortholog of Niemann Pick Type C protein, implicated in sterol trafficking. An independent genetic screen revealed that correction of pHv dysregulation in a neo1ts mutant restored viability whereas cholesterol accumulation in human NPC1−/− fibroblasts diminished upon treatment with a proton ionophore. Furthermore, while it is established that lumenal pH affects trafficking, this study revealed a reciprocal link with many mutants defective in anterograde pathways being hyperacidic and retrograde pathway mutants with alkaline vacuoles. In these and other examples, pH perturbations emerge as a hitherto unrecognized phenotype that may contribute to the cellular basis of disease and offer potential therapeutic intervention through pH modulation. PMID:21423800

Substance P activates ADAM9 mRNA expression and induces α-secretase-mediated amyloid precursor protein cleavage.

PubMed

Marolda, R; Ciotti, M T; Matrone, C; Possenti, R; Calissano, P; Cavallaro, S; Severini, C

2012-04-01

Altered levels of Substance P (SP), a neuropeptide endowed with neuroprotective and anti-apoptotic properties, were found in brain areas and spinal fluid of Alzheimer's disease (AD) patients. One of the hallmarks of AD is the abnormal extracellular deposition of neurotoxic beta amyloid (Aβ) peptides, derived from the proteolytic processing of amyloid precursor protein (APP). In the present study, we confirmed, the neurotrophic action of SP in cultured rat cerebellar granule cells (CGCs) and investigated its effects on APP metabolism. Incubation with low (5 mM) potassium induced apoptotic cell death of CGCs and amyloidogenic processing of APP, whereas treatment with SP (200 nM) reverted these effects via NK1 receptors. The non-amyloidogenic effect of SP consisted of reduction of Aβ(1-42), increase of sAPPα and enhanced α-secretase activity, without a significant change in steady-state levels of cellular APP. The intracellular mechanisms whereby SP alters APP metabolism were further investigated by measuring mRNA and/or steady-state protein levels of key enzymes involved with α-, β- and γ-secretase activity. Among them, Adam9, both at the mRNA and protein level, was the only enzyme to be significantly down-regulated following the induction of apoptosis (K5) and up-regulated after SP treatment. In addition to its neuroprotective properties, this study shows that SP is able to stimulate non-amyloidogenic APP processing, thereby reducing the possibility of generation of toxic Aβ peptides in brain. Copyright © 2011 Elsevier Ltd. All rights reserved.

Involvement of Vacuolar Sequestration and Active Transport in Tolerance of Saccharomyces cerevisiae to Hop Iso-α-Acids▿ † ¶

PubMed Central

Hazelwood, Lucie A.; Walsh, Michael C.; Pronk, Jack T.; Daran, Jean-Marc

2010-01-01

The hop plant, Humulus lupulus L., has an exceptionally high content of secondary metabolites, the hop α-acids, which possess a range of beneficial properties, including antiseptic action. Studies performed on the mode of action of hop iso-α-acids have hitherto been restricted to lactic acid bacteria. The present study investigated molecular mechanisms of hop iso-α-acid resistance in the model eukaryote Saccharomyces cerevisiae. Growth inhibition occurred at concentrations of hop iso-α-acids that were an order of magnitude higher than those found with hop-tolerant prokaryotes. Chemostat-based transcriptome analysis and phenotype screening of the S. cerevisiae haploid gene deletion collection were used as complementary methods to screen for genes involved in hop iso-α-acid detoxification and tolerance. This screening and further analysis of deletion mutants confirmed that yeast tolerance to hop iso-α-acids involves three major processes, active proton pumping into the vacuole by the vacuolar-type ATPase to enable vacuolar sequestration of iso-α-acids and alteration of cell wall structure and, to a lesser extent, active export of iso-α-acids across the plasma membrane. Furthermore, iso-α-acids were shown to affect cellular metal homeostasis by acting as strong zinc and iron chelators. PMID:19915041

Multisite tyrosine phosphorylation of the N-terminus of Mint1/X11α by Src kinase regulates the trafficking of amyloid precursor protein.

PubMed

Dunning, Christopher J R; Black, Hannah L; Andrews, Katie L; Davenport, Elizabeth C; Conboy, Michael; Chawla, Sangeeta; Dowle, Adam A; Ashford, David; Thomas, Jerry R; Evans, Gareth J O

2016-05-01

Mint/X11 is one of the four neuronal trafficking adaptors that interact with amyloid precursor protein (APP) and are linked with its cleavage to generate β-amyloid peptide, a key player in the pathology of Alzheimer's disease. How APP switches between adaptors at different stages of the secretory pathway is poorly understood. Here, we show that tyrosine phosphorylation of Mint1 regulates the destination of APP. A canonical SH2-binding motif ((202) YEEI) was identified in the N-terminus of Mint1 that is phosphorylated on tyrosine by C-Src and recruits the active kinase for sequential phosphorylation of further tyrosines (Y191 and Y187). A single Y202F mutation in the Mint1 N-terminus inhibits C-Src binding and tyrosine phosphorylation. Previous studies observed that co-expression of wild-type Mint1 and APP causes accumulation of APP in the trans-Golgi. Unphosphorylatable Mint1 (Y202F) or pharmacological inhibition of Src reduced the accumulation of APP in the trans-Golgi of heterologous cells. A similar result was observed in cultured rat hippocampal neurons where Mint1(Y202F) permitted the trafficking of APP to more distal neurites than the wild-type protein. These data underline the importance of the tyrosine phosphorylation of Mint1 as a critical switch for determining the destination of APP. The regulation of amyloid precursor protein (APP) trafficking is poorly understood. We have discovered that the APP adapter, Mint1, is phosphorylated by C-Src kinase. Mint1 causes APP accumulation in the trans-Golgi network, whereas inhibition of Src or mutation of Mint1-Y202 permits APP recycling. The phosphorylation status of Mint1 could impact on the pathological trafficking of APP in Alzheimer's disease. © 2016 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.

Non-selective cation channels in plasma and vacuolar membranes and their contribution to K+ transport.

PubMed

Pottosin, Igor; Dobrovinskaya, Oxana

2014-05-15

Both in vacuolar and plasma membranes, in addition to truly K(+)-selective channels there is a variety of non-selective channels, which conduct K(+) and other ions with little preference. Many non-selective channels in the plasma membrane are active at depolarized potentials, thus, contributing to K(+) efflux rather than to K(+) uptake. They may play important roles in xylem loading or contribute to a K(+) leak, induced by salt or oxidative stress. Here, three currents, expressed in root cells, are considered: voltage-insensitive cation current, non-selective outwardly rectifying current, and low-selective conductance, activated by reactive oxygen species. The latter two do not only poorly discriminate between different cations (like K(+)vs Na(+)), but also conduct anions. Such solute channels may mediate massive electroneutral transport of salts and might be involved in osmotic adjustment or volume decrease, associated with cell death. In the tonoplast two major currents are mediated by SV (slow) and FV (fast) vacuolar channels, respectively, which are virtually impermeable for anions. SV channels conduct mono- and divalent cations indiscriminately and are activated by high cytosolic Ca(2+) and depolarized voltages. FV channels are inhibited by micromolar cytosolic Ca(2+), Mg(2+), and polyamines, and conduct a variety of monovalent cations, including K(+). Strikingly, both SV and FV channels sense the K(+) content of vacuoles, which modulates their voltage dependence, and in case of SV, also alleviates channel's inhibition by luminal Ca(2+). Therefore, SV and FV channels may operate as K(+)-sensing valves, controlling K(+) distribution between the vacuole and the cytosol. Copyright © 2014 Elsevier GmbH. All rights reserved.

SOS2 Promotes Salt Tolerance in Part by Interacting with the Vacuolar H+-ATPase and Upregulating Its Transport Activity▿

PubMed Central

Batelli, Giorgia; Verslues, Paul E.; Agius, Fernanda; Qiu, Quansheng; Fujii, Hiroaki; Pan, Songqin; Schumaker, Karen S.; Grillo, Stefania; Zhu, Jian-Kang

2007-01-01

The salt overly sensitive (SOS) pathway is critical for plant salt stress tolerance and has a key role in regulating ion transport under salt stress. To further investigate salt tolerance factors regulated by the SOS pathway, we expressed an N-terminal fusion of the improved tandem affinity purification tag to SOS2 (NTAP-SOS2) in sos2-2 mutant plants. Expression of NTAP-SOS2 rescued the salt tolerance defect of sos2-2 plants, indicating that the fusion protein was functional in vivo. Tandem affinity purification of NTAP-SOS2-containing protein complexes and subsequent liquid chromatography-tandem mass spectrometry analysis indicated that subunits A, B, C, E, and G of the peripheral cytoplasmic domain of the vacuolar H+-ATPase (V-ATPase) were present in a SOS2-containing protein complex. Parallel purification of samples from control and salt-stressed NTAP-SOS2/sos2-2 plants demonstrated that each of these V-ATPase subunits was more abundant in NTAP-SOS2 complexes isolated from salt-stressed plants, suggesting that the interaction may be enhanced by salt stress. Yeast two-hybrid analysis showed that SOS2 interacted directly with V-ATPase regulatory subunits B1 and B2. The importance of the SOS2 interaction with the V-ATPase was shown at the cellular level by reduced H+ transport activity of tonoplast vesicles isolated from sos2-2 cells relative to vesicles from wild-type cells. In addition, seedlings of the det3 mutant, which has reduced V-ATPase activity, were found to be severely salt sensitive. Our results suggest that regulation of V-ATPase activity is an additional key function of SOS2 in coordinating changes in ion transport during salt stress and in promoting salt tolerance. PMID:17875927

Clathrin coat controls synaptic vesicle acidification by blocking vacuolar ATPase activity

PubMed Central

Farsi, Zohreh; Rammner, Burkhard; Woehler, Andrew; Lafer, Eileen M; Mim, Carsten; Jahn, Reinhard

2018-01-01

Newly-formed synaptic vesicles (SVs) are rapidly acidified by vacuolar adenosine triphosphatases (vATPases), generating a proton electrochemical gradient that drives neurotransmitter loading. Clathrin-mediated endocytosis is needed for the formation of new SVs, yet it is unclear when endocytosed vesicles acidify and refill at the synapse. Here, we isolated clathrin-coated vesicles (CCVs) from mouse brain to measure their acidification directly at the single vesicle level. We observed that the ATP-induced acidification of CCVs was strikingly reduced in comparison to SVs. Remarkably, when the coat was removed from CCVs, uncoated vesicles regained ATP-dependent acidification, demonstrating that CCVs contain the functional vATPase, yet its function is inhibited by the clathrin coat. Considering the known structures of the vATPase and clathrin coat, we propose a model in which the formation of the coat surrounds the vATPase and blocks its activity. Such inhibition is likely fundamental for the proper timing of SV refilling. PMID:29652249

The yeast vps class E mutants: the beginning of the molecular genetic analysis of multivesicular body biogenesis.

PubMed

Coonrod, Emily M; Stevens, Tom H

2010-12-01

In 1992, Raymond et al. published a compilation of the 41 yeast vacuolar protein sorting (vps) mutant groups and described a large class of mutants (class E vps mutants) that accumulated an exaggerated prevacuolar endosome-like compartment. Further analysis revealed that this "class E compartment" contained soluble vacuolar hydrolases, vacuolar membrane proteins, and Golgi membrane proteins unable to recycle back to the Golgi complex, yet these class E vps mutants had what seemed to be normal vacuoles. The 13 class E VPS genes were later shown to encode the proteins that make up the complexes required for formation of intralumenal vesicles in late endosomal compartments called multivesicular bodies, and for the sorting of ubiquitinated cargo proteins into these internal vesicles for eventual delivery to the vacuole or lysosome.

Impact of membrane lipid composition on the structure and stability of the transmembrane domain of amyloid precursor protein

PubMed Central

Dominguez, Laura; Foster, Leigh; Straub, John E.; Thirumalai, D.

2016-01-01

Cleavage of the amyloid precursor protein (APP) by γ-secretase is a crucial first step in the evolution of Alzheimer’s disease. To discover the cleavage mechanism, it is urgent to predict the structures of APP monomers and dimers in varying membrane environments. We determined the structures of the C9923−55 monomer and homodimer as a function of membrane lipid composition using a multiscale simulation approach that blends atomistic and coarse-grained models. We demonstrate that the C9923−55 homodimer structures form a heterogeneous ensemble with multiple conformational states, each stabilized by characteristic interpeptide interactions. The relative probabilities of each conformational state are sensitive to the membrane environment, leading to substantial variation in homodimer peptide structure as a function of membrane lipid composition or the presence of an anionic lipid environment. In contrast, the helicity of the transmembrane domain of monomeric C991−55 is relatively insensitive to the membrane lipid composition, in agreement with experimental observations. The dimer structures of human EphA2 receptor depend on the lipid environment, which we show is linked to the location of the structural motifs in the dimer interface, thereby establishing that both sequence and membrane composition modulate the complete energy landscape of membrane-bound proteins. As a by-product of our work, we explain the discrepancy in structures predicted for C99 congener homodimers in membrane and micelle environments. Our study provides insight into the observed dependence of C99 protein cleavage by γ-secretase, critical to the formation of amyloid-β protein, on membrane thickness and lipid composition. PMID:27559086

Autophagic flux is required for the synthesis of triacylglycerols and ribosomal protein turnover in Chlamydomonas.

PubMed

Couso, Inmaculada; Pérez-Pérez, María Esther; Martínez-Force, Enrique; Kim, Hee-Sik; He, Yonghua; Umen, James G; Crespo, José L

2018-03-14

Autophagy is an intracellular catabolic process that allows cells to recycle unneeded or damaged material to maintain cellular homeostasis. This highly dynamic process is characterized by the formation of double-membrane vesicles called autophagosomes, which engulf and deliver the cargo to the vacuole. Flow of material through the autophagy pathway and its degradation in the vacuole is known as autophagic flux, and reflects the autophagic degradation activity. A number of assays have been developed to determine autophagic flux in yeasts, mammals, and plants, but it has not been examined yet in algae. Here we analyzed autophagic flux in the model green alga Chlamydomonas reinhardtii. By monitoring specific autophagy markers such as ATG8 lipidation and using immunofluorescence and electron microscopy techniques, we show that concanamycin A, a vacuolar ATPase inhibitor, blocks autophagic flux in Chlamydomonas. Our results revealed that vacuolar lytic function is needed for the synthesis of triacylglycerols and the formation of lipid bodies in nitrogen- or phosphate-starved cells. Moreover, we found that concanamycin A treatment prevented the degradation of ribosomal proteins RPS6 and RPL37 under nitrogen or phosphate deprivation. These results indicate that autophagy might play an important role in the regulation of lipid metabolism and the recycling of ribosomal proteins under nutrient limitation in Chlamydomonas.

Multifaceted plant responses to circumvent Phe hyperaccumulation by downregulation of flux through the shikimate pathway and by vacuolar Phe sequestration.

PubMed

Lynch, Joseph H; Orlova, Irina; Zhao, Chengsong; Guo, Longyun; Jaini, Rohit; Maeda, Hiroshi; Akhtar, Tariq; Cruz-Lebron, Junellie; Rhodes, David; Morgan, John; Pilot, Guillaume; Pichersky, Eran; Dudareva, Natalia

2017-12-01

Detrimental effects of hyperaccumulation of the aromatic amino acid phenylalanine (Phe) in animals, known as phenylketonuria, are mitigated by excretion of Phe derivatives; however, how plants endure Phe accumulating conditions in the absence of an excretion system is currently unknown. To achieve Phe hyperaccumulation in a plant system, we simultaneously decreased in petunia flowers expression of all three Phe ammonia lyase (PAL) isoforms that catalyze the non-oxidative deamination of Phe to trans-cinnamic acid, the committed step for the major pathway of Phe metabolism. A total decrease in PAL activity by 81-94% led to an 18-fold expansion of the internal Phe pool. Phe accumulation had multifaceted intercompartmental effects on aromatic amino acid metabolism. It resulted in a decrease in the overall flux through the shikimate pathway, and a redirection of carbon flux toward the shikimate-derived aromatic amino acids tyrosine and tryptophan. Accumulation of Phe did not lead to an increase in flux toward phenylacetaldehyde, for which Phe is a direct precursor. Metabolic flux analysis revealed this to be due to the presence of a distinct metabolically inactive pool of Phe, likely localized in the vacuole. We have identified a vacuolar cationic amino acid transporter (PhCAT2) that contributes to sequestering excess of Phe in the vacuole. In vitro assays confirmed PhCAT2 can transport Phe, and decreased PhCAT2 expression in PAL-RNAi transgenic plants resulted in 1.6-fold increase in phenylacetaldehyde emission. These results demonstrate mechanisms by which plants maintain intercompartmental aromatic amino acid homeostasis, and provide critical insight for future phenylpropanoid metabolic engineering strategies. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Relative increase in Alzheimer's disease of soluble forms of cerebral Abeta amyloid protein precursor containing the Kunitz protease inhibitory domain.

PubMed

Moir, R D; Lynch, T; Bush, A I; Whyte, S; Henry, A; Portbury, S; Multhaup, G; Small, D H; Tanzi, R E; Beyreuther, K; Masters, C L

1998-02-27

Although a number of studies have examined amyloid precursor protein (APP) mRNA levels in Alzheimer's disease (AD), no clear consensus has emerged as to whether the levels of transcripts for isoforms containing a Kunitz protease inhibitory (KPI)-encoded region are increased or decreased in AD. Here we compare AD and control brain for the relative amounts of APP protein containing KPI to APP protein lacking this domain. APP protein was purified from the soluble subcellular fraction and Triton X-100 membrane pellet extract of one hemisphere of AD (n = 10), normal (n = 7), and neurological control (n = 5) brains. The amount of KPI-containing APP in the purified protein samples was determined using two independent assay methods. The first assay exploited the inhibitory action of KPI-containing APP on trypsin. The second assay employed reflectance analysis of Western blots. The proportion of KPI-containing forms of APP in the soluble subcellular fraction of AD brains is significantly elevated (p < 0.01) compared with controls. Species containing a KPI domain comprise 32-41 and 76-77% of purified soluble APP from control and AD brains, respectively. For purified membrane-associated APP, 72-77 and 65-82% of control and AD samples, respectively, contain a KPI domain. Since KPI-containing species of APP may be more amyloidogenic (Ho, L., Fukuchi, K., and Yonkin, S. G. (1996) J. Biol. Chem. 271, 30929-30934), our findings support an imbalance of isoforms as one possible mechanism for amyloid deposition in sporadic AD.

HAL-2 Promotes Homologous Pairing during Caenorhabditis elegans Meiosis by Antagonizing Inhibitory Effects of Synaptonemal Complex Precursors

PubMed Central

Zhang, Weibin; Miley, Natasha; Zastrow, Michael S.; MacQueen, Amy J.; Sato, Aya; Nabeshima, Kentaro; Martinez-Perez, Enrique; Mlynarczyk-Evans, Susanna; Carlton, Peter M.; Villeneuve, Anne M.

2012-01-01

During meiosis, chromosomes align with their homologous pairing partners and stabilize this alignment through assembly of the synaptonemal complex (SC). Since the SC assembles cooperatively yet is indifferent to homology, pairing and SC assembly must be tightly coordinated. We identify HAL-2 as a key mediator in this coordination, showing that HAL-2 promotes pairing largely by preventing detrimental effects of SC precursors (SYP proteins). hal-2 mutants fail to establish pairing and lack multiple markers of chromosome movement mediated by pairing centers (PCs), chromosome sites that link chromosomes to cytoplasmic microtubules through nuclear envelope-spanning complexes. Moreover, SYP proteins load inappropriately along individual unpaired chromosomes in hal-2 mutants, and markers of PC-dependent movement and function are restored in hal-2; syp double mutants. These and other data indicate that SYP proteins can impede pairing and that HAL-2 promotes pairing predominantly but not exclusively by counteracting this inhibition, thereby enabling activation and regulation of PC function. HAL-2 concentrates in the germ cell nucleoplasm and colocalizes with SYP proteins in nuclear aggregates when SC assembly is prevented. We propose that HAL-2 functions to shepherd SYP proteins prior to licensing of SC assembly, preventing untimely interactions between SC precursors and chromosomes and allowing sufficient accumulation of precursors for rapid cooperative assembly upon homology verification. PMID:22912597

HAL-2 promotes homologous pairing during Caenorhabditis elegans meiosis by antagonizing inhibitory effects of synaptonemal complex precursors.

PubMed

Zhang, Weibin; Miley, Natasha; Zastrow, Michael S; MacQueen, Amy J; Sato, Aya; Nabeshima, Kentaro; Martinez-Perez, Enrique; Mlynarczyk-Evans, Susanna; Carlton, Peter M; Villeneuve, Anne M

2012-01-01

During meiosis, chromosomes align with their homologous pairing partners and stabilize this alignment through assembly of the synaptonemal complex (SC). Since the SC assembles cooperatively yet is indifferent to homology, pairing and SC assembly must be tightly coordinated. We identify HAL-2 as a key mediator in this coordination, showing that HAL-2 promotes pairing largely by preventing detrimental effects of SC precursors (SYP proteins). hal-2 mutants fail to establish pairing and lack multiple markers of chromosome movement mediated by pairing centers (PCs), chromosome sites that link chromosomes to cytoplasmic microtubules through nuclear envelope-spanning complexes. Moreover, SYP proteins load inappropriately along individual unpaired chromosomes in hal-2 mutants, and markers of PC-dependent movement and function are restored in hal-2; syp double mutants. These and other data indicate that SYP proteins can impede pairing and that HAL-2 promotes pairing predominantly but not exclusively by counteracting this inhibition, thereby enabling activation and regulation of PC function. HAL-2 concentrates in the germ cell nucleoplasm and colocalizes with SYP proteins in nuclear aggregates when SC assembly is prevented. We propose that HAL-2 functions to shepherd SYP proteins prior to licensing of SC assembly, preventing untimely interactions between SC precursors and chromosomes and allowing sufficient accumulation of precursors for rapid cooperative assembly upon homology verification.

Transcriptomic identification of starfish neuropeptide precursors yields new insights into neuropeptide evolution

PubMed Central

Semmens, Dean C.; Mirabeau, Olivier; Moghul, Ismail; Pancholi, Mahesh R.; Wurm, Yannick; Elphick, Maurice R.

2016-01-01

Neuropeptides are evolutionarily ancient mediators of neuronal signalling in nervous systems. With recent advances in genomics/transcriptomics, an increasingly wide range of species has become accessible for molecular analysis. The deuterostomian invertebrates are of particular interest in this regard because they occupy an ‘intermediate' position in animal phylogeny, bridging the gap between the well-studied model protostomian invertebrates (e.g. Drosophila melanogaster, Caenorhabditis elegans) and the vertebrates. Here we have identified 40 neuropeptide precursors in the starfish Asterias rubens, a deuterostomian invertebrate from the phylum Echinodermata. Importantly, these include kisspeptin-type and melanin-concentrating hormone-type precursors, which are the first to be discovered in a non-chordate species. Starfish tachykinin-type, somatostatin-type, pigment-dispersing factor-type and corticotropin-releasing hormone-type precursors are the first to be discovered in the echinoderm/ambulacrarian clade of the animal kingdom. Other precursors identified include vasopressin/oxytocin-type, gonadotropin-releasing hormone-type, thyrotropin-releasing hormone-type, calcitonin-type, cholecystokinin/gastrin-type, orexin-type, luqin-type, pedal peptide/orcokinin-type, glycoprotein hormone-type, bursicon-type, relaxin-type and insulin-like growth factor-type precursors. This is the most comprehensive identification of neuropeptide precursor proteins in an echinoderm to date, yielding new insights into the evolution of neuropeptide signalling systems. Furthermore, these data provide a basis for experimental analysis of neuropeptide function in the unique context of the decentralized, pentaradial echinoderm bauplan. PMID:26865025

Protein accumulation in aleurone cells, sub-aleurone cells and the center starch endosperm of cereals.

PubMed

Zheng, Yankun; Wang, Zhong

2014-10-01

There are mainly three endosperm storage tissues in the cereal endosperm: aleurone cells, sub-aleurone cells and the center starch endosperm. The protein accumulation is very different in the three endosperm storage tissues. The aleurone cells accumulate protein in aleurone granules. The sub-aleurone cells and the center starch endosperm accumulate protein in endoplasmic reticulum-derived protein bodies and vacuolar protein bodies. Proteins are deposited in different patterns within different endosperm storage tissues probably because of the special storage properties of these tissues. There are several special genes and other molecular factors to mediate the protein accumulation in these tissues. Different proteins have distinct functions in the protein body formation and the protein interactions determine protein body assembly. There are both cooperation and competition relationships between protein, starch and lipid in the cereal endosperm. This paper reviews the latest investigations on protein accumulation in aleurone cells, sub-aleurone cells and the center starch endosperm. Useful information will be supplied for future investigations on the cereal endosperm development.

Plasma protein profiling of patients with intraductal papillary mucinous neoplasm of the pancreas as potential precursor lesions of pancreatic cancer.

PubMed

Ilies, Maria; Sappa, Praveen Kumar; Iuga, Cristina Adela; Loghin, Felicia; Gesell Salazar, Manuela; Weiss, Frank Ulrich; Beyer, Georg; Lerch, Markus M; Völker, Uwe; Mayerle, Julia; Hammer, Elke

2018-02-01

Efforts for the early diagnosis of the pancreatic ductal adenocarcinoma (PDAC) have recently been driven to one of the precursor lesions, namely intraductal papillary mucinous neoplasm of the pancreas (IPMN). Only a few studies have focused on IPMN molecular biology and its overall progression to cancer. Therefore, IPMN lacks comprehensive characterization which makes its clinical management controversial. In this study, we characterized plasma proteins in the presence of IPMNs in comparison to healthy controls, chronic pancreatitis, and PDAC by a proteomics approach using data-independent acquisition based mass spectrometry. We describe several protein sets that could aid IPMN diagnosis, but also differentiation of IPMN from healthy controls, as well as from benign and malignant diseases. Among all, high levels of carbonic anhydrases and hemoglobins were characteristic for the IPMN group. By employing ELISA based quantification we validated our results for human tissue inhibitor of metalloproteinase inhibitor 1 (TIMP-1). We consider IPMN management directed towards an early potential cancer development a crucial opportunity before PDAC initiation and thus its early detection and cure. Copyright © 2017 Elsevier B.V. All rights reserved.

Host and Bacterial Proteins That Repress Recruitment of LC3 to Shigella Early during Infection

PubMed Central

Baxt, Leigh A.; Goldberg, Marcia B.

2014-01-01

Shigella spp. are intracytosolic gram-negative pathogens that cause disease by invasion and spread through the colonic mucosa, utilizing host cytoskeletal components to form propulsive actin tails. We have previously identified the host factor Toca-1 as being recruited to intracellular S. flexneri and being required for efficient bacterial actin tail formation. We show that at early times during infection (40 min.), the type three-secreted effector protein IcsB recruits Toca-1 to intracellular bacteria and that recruitment of Toca-1 is associated with repression of recruitment of LC3, as well as with repression of recruitment of the autophagy marker NDP52, around these intracellular bacteria. LC3 is best characterized as a marker of autophagosomes, but also marks phagosomal membranes in the process LC3-associated phagocytosis. IcsB has previously been demonstrated to be required for S. flexneri evasion of autophagy at late times during infection (4–6 hr) by inhibiting binding of the autophagy protein Atg5 to the Shigella surface protein IcsA (VirG). Our results suggest that IcsB and Toca-1 modulation of LC3 recruitment restricts LC3-associated phagocytosis and/or LC3 recruitment to vacuolar membrane remnants. Together with published results, our findings suggest that IcsB inhibits innate immune responses in two distinct ways, first, by inhibiting LC3-associated phagocytosis and/or LC3 recruitment to vacuolar membrane remnants early during infection, and second, by inhibiting autophagy late during infection. PMID:24722587

The C-terminal propeptide of a plant defensin confers cytoprotective and subcellular targeting functions

PubMed Central

2014-01-01

Background Plant defensins are small (45–54 amino acids), basic, cysteine-rich proteins that have a major role in innate immunity in plants. Many defensins are potent antifungal molecules and are being evaluated for their potential to create crop plants with sustainable disease resistance. Defensins are produced as precursor molecules which are directed into the secretory pathway and are divided into two classes based on the absence (class I) or presence (class II) of an acidic C-terminal propeptide (CTPP) of about 33 amino acids. The function of this CTPP had not been defined. Results By transgenically expressing the class II plant defensin NaD1 with and without its cognate CTPP we have demonstrated that NaD1 is phytotoxic to cotton plants when expressed without its CTPP. Transgenic cotton plants expressing constructs encoding the NaD1 precursor with the CTPP had the same morphology as non-transgenic plants but expression of NaD1 without the CTPP led to plants that were stunted, had crinkled leaves and were less viable. Immunofluorescence microscopy and transient expression of a green fluorescent protein (GFP)-CTPP chimera were used to confirm that the CTPP is sufficient for vacuolar targeting. Finally circular dichroism and NMR spectroscopy were used to show that the CTPP adopts a helical confirmation. Conclusions In this report we have described the role of the CTPP on NaD1, a class II defensin from Nicotiana alata flowers. The CTPP of NaD1 is sufficient for vacuolar targeting and plays an important role in detoxification of the defensin as it moves through the plant secretory pathway. This work may have important implications for the use of defensins for disease protection in transgenic crops. PMID:24495600

The endosomal protein CHARGED MULTIVESICULAR BODY PROTEIN1 regulates the autophagic turnover of plastids in Arabidopsis.

PubMed

Spitzer, Christoph; Li, Faqiang; Buono, Rafael; Roschzttardtz, Hannetz; Chung, Taijoon; Zhang, Min; Osteryoung, Katherine W; Vierstra, Richard D; Otegui, Marisa S

2015-02-01

Endosomal Sorting Complex Required for Transport (ESCRT)-III proteins mediate membrane remodeling and the release of endosomal intraluminal vesicles into multivesicular bodies. Here, we show that the ESCRT-III subunit paralogs CHARGED MULTIVESICULAR BODY PROTEIN1 (CHMP1A) and CHMP1B are required for autophagic degradation of plastid proteins in Arabidopsis thaliana. Similar to autophagy mutants, chmp1a chmp1b (chmp1) plants hyperaccumulated plastid components, including proteins involved in plastid division. The autophagy machinery directed the release of bodies containing plastid material into the cytoplasm, whereas CHMP1A and B were required for delivery of these bodies to the vacuole. Autophagy was upregulated in chmp1 as indicated by an increase in vacuolar green fluorescent protein (GFP) cleavage from the autophagic reporter GFP-ATG8. However, autophagic degradation of the stromal cargo RECA-GFP was drastically reduced in the chmp1 plants upon starvation, suggesting that CHMP1 mediates the efficient delivery of autophagic plastid cargo to the vacuole. Consistent with the compromised degradation of plastid proteins, chmp1 plastids show severe morphological defects and aberrant division. We propose that CHMP1 plays a direct role in the autophagic turnover of plastid constituents. © 2015 American Society of Plant Biologists. All rights reserved.

The HOPS/Class C Vps Complex Tethers High-Curvature Membranes via a Direct Protein-Membrane Interaction.

PubMed

Ho, Ruoya; Stroupe, Christopher

2016-10-01

Membrane tethering is a physical association of two membranes before their fusion. Many membrane tethering factors have been identified, but the interactions that mediate inter-membrane associations remain largely a matter of conjecture. Previously, we reported that the homotypic fusion and protein sorting/Class C vacuolar protein sorting (HOPS/Class C Vps) complex, which has two binding sites for the yeast vacuolar Rab GTPase Ypt7p, can tether two low-curvature liposomes when both membranes bear Ypt7p. Here, we show that HOPS tethers highly curved liposomes to Ypt7p-bearing low-curvature liposomes even when the high-curvature liposomes are protein-free. Phosphorylation of the curvature-sensing amphipathic lipid-packing sensor (ALPS) motif from the Vps41p HOPS subunit abrogates tethering of high-curvature liposomes. A HOPS complex without its Vps39p subunit, which contains one of the Ypt7p binding sites in HOPS, lacks tethering activity, though it binds high-curvature liposomes and Ypt7p-bearing low-curvature liposomes. Thus, HOPS tethers highly curved membranes via a direct protein-membrane interaction. Such high-curvature membranes are found at the sites of vacuole tethering and fusion. There, vacuole membranes bend sharply, generating large areas of vacuole-vacuole contact. We propose that HOPS localizes via the Vps41p ALPS motif to these high-curvature regions. There, HOPS binds via Vps39p to Ypt7p in an apposed vacuole membrane. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

An Alzheimer Disease-linked Rare Mutation Potentiates Netrin Receptor Uncoordinated-5C-induced Signaling That Merges with Amyloid β Precursor Protein Signaling.

PubMed

Hashimoto, Yuichi; Toyama, Yuka; Kusakari, Shinya; Nawa, Mikiro; Matsuoka, Masaaki

2016-06-03

A missense mutation (T835M) in the uncoordinated-5C (UNC5C) netrin receptor gene increases the risk of late-onset Alzheimer disease (AD) and also the vulnerability of neurons harboring the mutation to various insults. The molecular mechanisms underlying T835M-UNC5C-induced death remain to be elucidated. In this study, we show that overexpression of wild-type UNC5C causes low-grade death, which is intensified by an AD-linked mutation T835M. An AD-linked survival factor, calmodulin-like skin protein (CLSP), and a natural ligand of UNC5C, netrin1, inhibit this death. T835M-UNC5C-induced neuronal cell death is mediated by an intracellular death-signaling cascade, consisting of death-associated protein kinase 1/protein kinase D/apoptosis signal-regulating kinase 1 (ASK1)/JNK/NADPH oxidase/caspases, which merges at ASK1 with a death-signaling cascade, mediated by amyloid β precursor protein (APP). Notably, netrin1 also binds to APP and partially inhibits the death-signaling cascade, induced by APP. These results may provide new insight into the amyloid β-independent pathomechanism of AD. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Molecular characterization of an ependymin precursor from goldfish brain.

PubMed

Königstorfer, A; Sterrer, S; Eckerskorn, C; Lottspeich, F; Schmidt, R; Hoffmann, W

1989-01-01

Ependymins are thought to be implicated in fundamental processes involved in plasticity of the goldfish CNS. Gas-phase sequencing of purified ependymins beta and gamma revealed that they share the same N-terminal sequence. Each sequence displays microheterogeneities at several positions. Based on the protein sequences obtained, we constructed synthetic oligonucleotides and used them as hybridization probes for screening cDNA libraries of goldfish brain. In this article we describe the full-length sequence of a mRNA encoding a precursor of ependymins. A cleavable signal sequence characteristic of secretory proteins is located at the N-terminal end, followed directly by the ependymin sequence. Also, two potential N-glycosylation sites were detected. A computer search revealed that ependymins form a novel family of unique proteins.

Structural modification of serum vitamin D3-binding protein and immunosuppression in AIDS patients.

PubMed

Yamamoto, N; Naraparaju, V R; Srinivasula, S M

1995-11-01

A serum glycoprotein, vitamin D3-binding protein (Gc protein), can be converted by beta-galactosidase of stimulated B lymphocytes and sialidase of T lymphocytes to a potent macrophage-activating factor (MAF), a protein with N-acetylgalactosamine as the remaining sugar moiety. Thus, Gc protein is a precursor for MAF. Treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generates an extremely high-titered MAF (GcMAF). When peripheral blood monocytes/macrophages of 46 HIV-infected patients were treated with GcMAF (100 pg/ml), the monocytes/macrophages of all patients were efficiently activated. However, the MAF precursor activity of plasma Gc protein was low in 16 (35%) of of these patients. Loss of the MAF precursor activity appeared to be due to deglycosylation of plasma Gc protein by alpha-N-acetylgalactosaminidase found in the patient blood stream. Levels of plasma alpha-N-acetylgalactosaminidase activity in individual patients had an inverse correlation with the MAF precursor activity of their plasma Gc protein. Thus, precursor activity of Gc protein and alpha-N-acetylgalactosaminidase activity in patient blood can serve as diagnostic and prognostic indices.

Proteolytic processing of the vitellogenin precursor in the boll weevil, Anthonomus grandis.

PubMed

Heilmann, L J; Trewitt, P M; Kumaran, A K

1993-01-01

The soluble proteins of the eggs of the coleopteran insect Anthonomus grandis Boheman, the cotton boll weevil, consist almost entirely of two vitellin types with M(r)s of 160,000 and 47,000. We sequenced their N-terminal ends and one internal cyanogen bromide fragment of the large vitellin and compared these sequences with the deduced amino acid sequence from the vitellogenin gene. The results suggest that both the boll weevil vitellin proteins are products of the proteolytic cleavage of a single precursor protein. The smaller 47,000 M(r) vitellin protein is derived from the N-terminal portion of the precursor adjacent to an 18 amino acid signal peptide. The cleavage site between the large and small vitellins at amino acid 362 is adjacent to a pentapeptide sequence containing two pairs of arginine residues. Comparison of the boll weevil sequences with limited known sequences from the single 180,000 M(r) honey bee protein show that the honey bee vitellin N-terminal exhibits sequence homology to the N-terminal of the 47,000 M(r) boll weevil vitellin. Treatment of the vitellins with an N-glycosidase results in a decrease in molecular weight of both proteins, from 47,000 to 39,000 and from 160,000 to 145,000, indicating that about 10-15% of the molecular weight of each vitellin consists of N-linked carbohydrate. The molecular weight of the deglycosylated large vitellin is smaller than that predicted from the gene sequence, indicating possible further proteolytic processing at the C-terminal of that protein.

The low density lipoprotein receptor-related protein 1B retains beta-amyloid precursor protein at the cell surface and reduces amyloid-beta peptide production.

PubMed

Cam, Judy A; Zerbinatti, Celina V; Knisely, Jane M; Hecimovic, Silva; Li, Yonghe; Bu, Guojun

2004-07-09

The low density lipoprotein (LDL) receptor-related protein 1B (LRP1B) is a newly identified member of the LDL receptor family that shares high homology with the LDL receptor-related protein (LRP). LRP1B was originally described as a putative tumor suppressor in lung cancer cells; however, its expression profile in several regions of adult human brain suggests it may have additional functions in the central nervous system. Since LRP1B has overlapping ligand binding properties with LRP, we investigated whether LRP1B, like LRP, could interact with the beta-amyloid precursor protein (APP) and modulate its processing to amyloid-beta peptides (Abetas). Using an LRP1B minireceptor (mLRP1B4) generated to study the trafficking of LRP1B, we found that mLRP1B4 and APP form an immunoprecipitable complex. Furthermore mLRP1B4 bound and facilitated the degradation of a soluble isoform of APP containing a Kunitz proteinase inhibitor domain but not soluble APP lacking a Kunitz proteinase inhibitor domain. A functional consequence of mLRP1B4 expression was a significant accumulation of APP at the cell surface, which is likely related to the slow endocytosis rate of LRP1B. More importantly, mLRP1B4-expressing cells that accumulated cell surface APP produced less Abeta and secreted more soluble APP. These findings reveal that LRP1B is a novel binding partner of APP that functions to decrease APP processing to Abeta. Consequently LRP1B expression could function to protect against the pathogenesis of Alzheimer's disease.

Mitosis-specific phosphorylation of amyloid precursor protein at Threonine 668 leads to its altered processing and association with centrosomes

PubMed Central

2011-01-01

Background Atypical expression of cell cycle regulatory proteins has been implicated in Alzheimer's disease (AD), but the molecular mechanisms by which they induce neurodegeneration are not well understood. We examined transgenic mice expressing human amyloid precursor protein (APP) and presenilin 1 (PS1) for changes in cell cycle regulatory proteins to determine whether there is a correlation between cell cycle activation and pathology development in AD. Results Our studies in the AD transgenic mice show significantly higher levels of cyclin E, cyclin D1, E2F1, and P-cdc2 in the cells in the vicinity of the plaques where maximum levels of Threonine 668 (Thr668)-phosphorylated APP accumulation was observed. This suggests that the cell cycle regulatory proteins might be influencing plaque pathology by affecting APP phosphorylation. Using neuroglioma cells overexpressing APP we demonstrate that phosphorylation of APP at Thr668 is mitosis-specific. Cells undergoing mitosis show altered cellular distribution and localization of P-APP at the centrosomes. Also, Thr668 phosphorylation in mitosis correlates with increased processing of APP to generate Aβ and the C-terminal fragment of APP, which is prevented by pharmacological inhibitors of the G1/S transition. Conclusions The data presented here suggests that cell cycle-dependent phosphorylation of APP may affect its normal cellular function. For example, association of P-APP with the centrosome may affect spindle assembly and cell cycle progression, further contributing to the development of pathology in AD. The experiments with G1/S inhibitors suggest that cell cycle inhibition may impede the development of Alzheimer's pathology by suppressing modification of βAPP, and thus may represent a novel approach to AD treatment. Finally, the cell cycle regulated phosphorylation and processing of APP into Aβ and the C-terminal fragment suggest that these proteins may have a normal function during mitosis. PMID:22112898

Trafficking of cell surface beta-amyloid precursor protein: retrograde and transcytotic transport in cultured neurons

PubMed Central

1995-01-01

Amyloid beta-protein (A beta), the principal constituent of senile plaques seen in Alzheimer's disease (AD), is derived by proteolysis from the beta-amyloid precursor protein (beta PP). The mechanism of A beta production in neurons, which are hypothesized to be a rich source of A beta in brain, remains to be defined. In this study, we describe a detailed localization of cell surface beta PP and its subsequent trafficking in primary cultured neurons. Full-length cell surface beta PP was present primarily on perikarya and axons, the latter with a characteristic discontinuous pattern. At growth cones, cell surface beta PP was inconsistently detected. By visualizing the distribution of beta PP monoclonal antibodies added to intact cultures, beta PP was shown to be internalized from distal axons or terminals and retrogradely transported back to perikarya in organelles which colocalized with fluid-phase endocytic markers. Retrograde transport of beta PP was shown in both hippocampal and peripheral sympathetic neurons, the latter using a compartment culture system that isolated cell bodies from distal axons and terminals. In addition, we demonstrated that beta PP from distal axons was transcytotically transported to the surface of perikarya from distal axons in sympathetic neurons. Indirect evidence of this transcytotic pathway was obtained in hippocampal neurons using antisense oligonucleotide to the kinesin heavy chain to inhibit anterograde beta PP transport. Taken together, these results demonstrate novel aspects of beta PP trafficking in neurons, including retrograde axonal transport and transcytosis. Moreover, the axonal predominance of cell surface beta PP is unexpected in view of the recent report of polarized sorting of beta PP to the basolateral domain of MDCK cells. PMID:7721945

Cerebrospinal fluid levels of amyloid precursor protein are associated with ventricular size in post-hemorrhagic hydrocephalus of prematurity.

PubMed

Morales, Diego M; Holubkov, Richard; Inder, Terri E; Ahn, Haejun C; Mercer, Deanna; Rao, Rakesh; McAllister, James P; Holtzman, David M; Limbrick, David D

2015-01-01

Neurological outcomes of preterm infants with post-hemorrhagic hydrocephalus (PHH) remain among the worst in infancy, yet there remain few instruments to inform the treatment of PHH. We previously observed PHH-associated elevations in cerebrospinal fluid (CSF) amyloid precursor protein (APP), neural cell adhesion molecule-L1 (L1CAM), neural cell adhesion molecule-1 (NCAM-1), and other protein mediators of neurodevelopment. The objective of this study was to examine the association of CSF APP, L1CAM, and NCAM-1 with ventricular size as an early step toward developing CSF markers of PHH. CSF levels of APP, L1CAM, NCAM-1, and total protein (TP) were measured in 12 preterm infants undergoing PHH treatment. Ventricular size was determined using cranial ultrasounds. The relationships between CSF APP, L1CAM, and NCAM-1, occipitofrontal circumference (OFC), volume of CSF removed, and ventricular size were examined using correlation and regression analyses. CSF levels of APP, L1CAM, and NCAM-1 but not TP paralleled treatment-related changes in ventricular size. CSF APP demonstrated the strongest association with ventricular size, estimated by frontal-occipital horn ratio (FOR) (Pearson R = 0.76, p = 0.004), followed by NCAM-1 (R = 0.66, p = 0.02) and L1CAM (R = 0.57,p = 0.055). TP was not correlated with FOR (R = 0.02, p = 0.95). Herein, we report the novel observation that CSF APP shows a robust association with ventricular size in preterm infants treated for PHH. The results from this study suggest that CSF APP and related proteins at once hold promise as biomarkers of PHH and provide insight into the neurological consequences of PHH in the preterm infant.

Deglycosylation of serum vitamin D3-binding protein leads to immunosuppression in cancer patients.

PubMed

Yamamoto, N; Naraparaju, V R; Asbell, S O

1996-06-15

Serum vitamin D3-binding protein (Gc protein) can be converted by beta-galactosidase of B cells and sialidase of T cells to a potent macrophage activating factor, a protein with N-acetylgalactosamine as the remaining sugar moiety. Thus, Gc protein is the precursor of the macrophage activating factor (MAF). Treatment of Gc protein with immobilized beta-galactosidase and sialidase generates an extremely high titered MAF, Gc-MAF. When peripheral blood monocytes/macrophages of 52 patients bearing various types of cancer were incubated with 100 pg/ml of GcMAF, the monocytes/macrophages of all patients were efficiently activated. However, the MAF precursor activity of patient plasma Gc protein was found to be severely reduced in about 25% of this patient population. About 45% of the patients had moderately reduced MAF precursor activities. Loss of the precursor activity was found to be due to deglycosylation of plasma Gc protein by alpha-N-acetylgalactosaminidase detected in the patient's bloodstream. The source of the enzyme appeared to be cancerous cells. Radiation therapy decreased plasma alpha-N-acetylgalactosaminidase activity with concomitant increase of precursor activity. This implies that radiation therapy decreases the number of cancerous cells capable of secreting alpha-N-acetylgalactosaminidase. Both alpha-N-acetylgalactosaminidase activity and MAF precursor activity of Gc protein in patient bloodstream can serve as diagnostic and prognostic indices.

Platelet amyloid precursor protein isoform expression in Alzheimer's disease: evidence for peripheral marker.

PubMed

Vignini, A; Sartini, D; Morganti, S; Nanetti, L; Luzzi, S; Provinciali, L; Mazzanti, L; Emanuelli, M

2011-01-01

Alzheimer's disease (AD) is a chronic neurodegenerative disorder characterized by a progressive cognitive and memory decline. Among peripheral markers of AD, great interest has been focused on the amyloid precursor protein (APP). In this regard, platelets represent an important peripheral source of APP since it has been demonstrated that the three major isoforms, that are constituted of 770, 751 and 695 aa residues, are inserted in the membrane of resting platelets. APP 751 and APP 770 contain a Kunitz-type serine protease inhibitor domain (APP KPI) and APP 695 lacks this domain. To address this issue, we first examined the platelet APP isoform mRNAs prospectively as biomarker for the diagnosis of AD by means of real-time quantitative PCR, and then evaluated the correlation between APP mRNA expression levels and cognitive impairment of enrolled subjects. Differential gene expression measurements in the AD patient group (n=18) revealed a significant up-regulation of APP TOT (1.52-fold), APP KPI (1.32-fold), APP 770 (1.33-fold) and APP 751 (1.26-fold) compared to controls (n=22). Moreover, a statistically significant positive correlation was found between APP mRNA levels (TOT, KPI, 770 and 751) and cognitive impairment. Since AD definitive diagnosis still relies on pathological evaluation at autopsy, the present results are consistent with the hypothesis that platelet APP could be considered a potential reliable peripheral marker for studying AD and could contribute to define a signature for the presence of AD pathology.

Sel1 repeat protein LpnE is a Legionella pneumophila virulence determinant that influences vacuolar trafficking.

PubMed

Newton, Hayley J; Sansom, Fiona M; Dao, Jenny; McAlister, Adrian D; Sloan, Joan; Cianciotto, Nicholas P; Hartland, Elizabeth L

2007-12-01

The environmental pathogen Legionella pneumophila possesses five proteins with Sel1 repeats (SLRs) from the tetratricopeptide repeat protein family. Three of these proteins, LpnE, EnhC, and LidL, have been implicated in the ability of L. pneumophila to efficiently establish infection and/or manipulate host cell trafficking events. Previously, we showed that LpnE is important for L. pneumophila entry into macrophages and epithelial cells. In further virulence studies here, we show that LpnE is also required for efficient infection of Acanthamoeba castellanii by L. pneumophila and for replication of L. pneumophila in the lungs of A/J mice. In addition, we found that the role of LpnE in host cell invasion is dependent on the eight SLR regions of the protein. A truncated form of LpnE lacking the two C-terminal SLR domains was unable to complement the invasion defect of an lpnE mutant of L. pneumophila 130b in both the A549 and THP-1 cell lines. The lpnE mutant displayed impaired avoidance of LAMP-1 association, suggesting that LpnE influenced trafficking of the L. pneumophila vacuole, similar to the case for EnhC and LidL. We also found that LpnE was present in L. pneumophila culture supernatants and that its export was independent of both the Lsp type II secretion system and the Dot/Icm type IV secretion system. The fact that LpnE was exported suggested that the protein may interact with a eukaryotic protein. Using LpnE as bait, we screened a HeLa cell cDNA library for interacting partners, using the yeast two-hybrid system. Examination of the protein-protein interaction between LpnE and a eukaryotic protein, obscurin-like protein 1, suggested that LpnE can interact with eukaryotic proteins containing immunoglobulin-like folds via the SLR regions. This investigation has further characterized the contribution of LpnE to L. pneumophila virulence and, more specifically, the importance of the SLR regions to LpnE function.

Inorganic polyphosphate in the yeast Saccharomyces cerevisiae with a mutation disturbing the function of vacuolar ATPase.

PubMed

Tomaschevsky, A A; Ryasanova, L P; Kulakovskaya, T V; Kulaev, I S

2010-08-01

A mutation in the vma2 gene disturbing V-ATPase function in the yeast Saccharomyces cerevisiae results in a five- and threefold decrease in inorganic polyphosphate content in the stationary and active phases of growth on glucose, respectively. The average polyphosphate chain length in the mutant cells is decreased. The mutation does not prevent polyphosphate utilization during cultivation in a phosphate-deficient medium and recovery of its level on reinoculation in complete medium after phosphate deficiency. The content of short chain acid-soluble polyphosphates is recovered first. It is supposed that these polyphosphates are less dependent on the electrochemical gradient on the vacuolar membrane.

Biomineralization of a Self-assembled, Soft-Matrix Precursor: Enamel

NASA Astrophysics Data System (ADS)

Snead, Malcolm L.

2015-04-01

Enamel is the bioceramic covering of teeth, a composite tissue composed of hierarchical organized hydroxyapatite crystallites fabricated by cells under physiologic pH and temperature. Enamel material properties resist wear and fracture to serve a lifetime of chewing. Understanding the cellular and molecular mechanisms for enamel formation may allow a biology-inspired approach to material fabrication based on self-assembling proteins that control form and function. A genetic understanding of human diseases exposes insight from nature's errors by exposing critical fabrication events that can be validated experimentally and duplicated in mice using genetic engineering to phenocopy the human disease so that it can be explored in detail. This approach led to an assessment of amelogenin protein self-assembly that, when altered, disrupts fabrication of the soft enamel protein matrix. A misassembled protein matrix precursor results in loss of cell-to-matrix contacts essential to fabrication and mineralization.

Rescue of Hypovitaminosis A Induces Non-Amyloidogenic Amyloid Precursor Protein (APP) Processing.

PubMed

Reinhardt, Sven; Grimm, Marcus O W; Stahlmann, Christoph; Hartmann, Tobias; Shudo, Koichi; Tomita, Taisuke; Endres, Kristina

2016-01-01

Retinoic acid, the bioactive metabolite of beta-carotene or vitamin A, plays a pleiotropic, multifunctional role in vertebrate development. Studies in rodents revealed that a diet deficient in vitamin A results in a complex neonatal syndrome (the VAD syndrome), manifested in many organs. In humans, the function of retinoic acid (RA) extends into adulthood, where it has important roles in fertility, vision, and suppression of neoplastic growth. In recent years, it has also been suggested that retinoic acid might potentially act as a therapeutically relevant drug in attenuating or even preventing neurodegenerative diseases such as Alzheimer's disease (AD). Here, we report that VAD leads to an increase in A-beta peptide levels while only minor effects were observed on expression levels of the amyloid precursor protein (APP) processing proteinases in wild type mice. In line with these findings, rescue of hypovitaminosis reduced A-beta amount to baseline and induced sApp-alpha secretion in combination with an increase of alpha-secretase Adam10. By comparing retinoic acid treatment starting from a full nutrition status and a "VAD" situation in human neuroblastoma cells, we show that while intensities of differential gene expression were higher in replenished cells, a large overlap in AD-related, regulated genes was observed. Our data suggest that hypovitaminosis A can contribute to onset or progression of AD by increasing synthesis of A-beta peptides and that several AD-related genes such as ADAM10 or BDNF are regulated by retinoic acid. We suggest that dietary supplementation with retinoic acid derivatives is likely to have a beneficial effect on AD-pathology in individuals with hypovitaminosis and patients with normal vitamin A status.

Design and application of a data-independent precursor and product ion repository.

PubMed

Thalassinos, Konstantinos; Vissers, Johannes P C; Tenzer, Stefan; Levin, Yishai; Thompson, J Will; Daniel, David; Mann, Darrin; DeLong, Mark R; Moseley, M Arthur; America, Antoine H; Ottens, Andrew K; Cavey, Greg S; Efstathiou, Georgios; Scrivens, James H; Langridge, James I; Geromanos, Scott J

2012-10-01

The functional design and application of a data-independent LC-MS precursor and product ion repository for protein identification, quantification, and validation is conceptually described. The ion repository was constructed from the sequence search results of a broad range of discovery experiments investigating various tissue types of two closely related mammalian species. The relative high degree of similarity in protein complement, ion detection, and peptide and protein identification allows for the analysis of normalized precursor and product ion intensity values, as well as standardized retention times, creating a multidimensional/orthogonal queryable, qualitative, and quantitative space. Peptide ion map selection for identification and quantification is primarily based on replication and limited variation. The information is stored in a relational database and is used to create peptide- and protein-specific fragment ion maps that can be queried in a targeted fashion against the raw or time aligned ion detections. These queries can be conducted either individually or as groups, where the latter affords pathway and molecular machinery analysis of the protein complement. The presented results also suggest that peptide ionization and fragmentation efficiencies are highly conserved between experiments and practically independent of the analyzed biological sample when using similar instrumentation. Moreover, the data illustrate only minor variation in ionization efficiency with amino acid sequence substitutions occurring between species. Finally, the data and the presented results illustrate how LC-MS performance metrics can be extracted and utilized to ensure optimal performance of the employed analytical workflows.

Vitamin D receptor is present on the neuronal plasma membrane and is co-localized with amyloid precursor protein, ADAM10 or Nicastrin.

PubMed

Dursun, Erdinç; Gezen-Ak, Duygu

2017-01-01

Our recent study indicated that vitamin D and its receptors are important parts of the amyloid processing pathway in neurons. Yet the role of vitamin D receptor (VDR) in amyloid pathogenesis is complex and all regulations over the production of amyloid beta cannot be explained solely with the transcriptional regulatory properties of VDR. Given that we hypothesized that VDR might exist on the neuronal plasma membrane in close proximity with amyloid precursor protein (APP) and secretase complexes. The present study primarily focused on the localization of VDR in neurons and its interaction with amyloid pathology-related proteins. The localization of VDR on neuronal membranes and its co-localization with target proteins were investigated with cell surface staining followed by immunofluorescence labelling. The FpClass was used for protein-protein interaction prediction. Our results demonstrated the localization of VDR on the neuronal plasma membrane and the co-localization of VDR and APP or ADAM10 or Nicastrin and limited co-localization of VDR and PS1. E-cadherin interaction with APP or the γ-secretase complex may involve NOTCH1, NUMB, or FHL2, according to FpClass. This suggested complex might also include VDR, which greatly contributes to Ca+2 hemostasis with its ligand vitamin D. Consequently, we suggested that VDR might be a member of this complex also with its own non-genomic action and that it can regulate the APP processing pathway in this way in neurons.

Structure of the vacuolar H+-ATPase rotary motor reveals new mechanistic insights.

PubMed

Rawson, Shaun; Phillips, Clair; Huss, Markus; Tiburcy, Felix; Wieczorek, Helmut; Trinick, John; Harrison, Michael A; Muench, Stephen P

2015-03-03

Vacuolar H(+)-ATPases are multisubunit complexes that operate with rotary mechanics and are essential for membrane proton transport throughout eukaryotes. Here we report a ∼ 1 nm resolution reconstruction of a V-ATPase in a different conformational state from that previously reported for a lower-resolution yeast model. The stator network of the V-ATPase (and by implication that of other rotary ATPases) does not change conformation in different catalytic states, and hence must be relatively rigid. We also demonstrate that a conserved bearing in the catalytic domain is electrostatic, contributing to the extraordinarily high efficiency of rotary ATPases. Analysis of the rotor axle/membrane pump interface suggests how rotary ATPases accommodate different c ring stoichiometries while maintaining high efficiency. The model provides evidence for a half channel in the proton pump, supporting theoretical models of ion translocation. Our refined model therefore provides new insights into the structure and mechanics of the V-ATPases. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Enrichment of skin-derived neural precursor cells from dermal cell populations by altering culture conditions.

PubMed

Bayati, Vahid; Gazor, Rohoullah; Nejatbakhsh, Reza; Negad Dehbashi, Fereshteh

2016-01-01

As stem cells play a critical role in tissue repair, their manipulation for being applied in regenerative medicine is of great importance. Skin-derived precursors (SKPs) may be good candidates for use in cell-based therapy as the only neural stem cells which can be isolated from an accessible tissue, skin. Herein, we presented a simple protocol to enrich neural SKPs by monolayer adherent cultivation to prove the efficacy of this method. To enrich neural SKPs from dermal cell populations, we have found that a monolayer adherent cultivation helps to increase the numbers of neural precursor cells. Indeed, we have cultured dermal cells as monolayer under serum-supplemented (control) and serum-supplemented culture, followed by serum free cultivation (test) and compared. Finally, protein markers of SKPs were assessed and compared in both experimental groups and differentiation potential was evaluated in enriched culture. The cells of enriched culture concurrently expressed fibronectin, vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors as compared to control culture. In addition, they possessed a multipotential capacity to differentiate into neurogenic, glial, adipogenic, osteogenic and skeletal myogenic cell lineages. It was concluded that serum-free adherent culture reinforced by growth factors have been shown to be effective on proliferation of skin-derived neural precursor cells (skin-NPCs) and drive their selective and rapid expansion.

Cucumber metal transport protein MTP8 confers increased tolerance to manganese when expressed in yeast and Arabidopsis thaliana

PubMed Central

Migocka, Magdalena; Papierniak, Anna; Maciaszczyk-Dziubińska, Ewa; Poździk, Piotr; Posyniak, Ewelina; Garbiec, Arnold; Filleur, Sophie

2014-01-01

Cation diffusion facilitator (CDF) proteins are ubiquitous divalent cation transporters that have been proved to be essential for metal homeostasis and tolerance in Archaebacteria, Bacteria, and Eukaryota. In plants, CDFs are designated as metal tolerance proteins (MTPs). Due to the lack of genomic resources, studies on MTPs in other plants, including cultivated crops, are lacking. Here, the identification and organization of genes encoding members of the MTP family in cucumber are described. The first functional characterization of a cucumber gene encoding a member of the Mn-CDF subgroup of CDF proteins, designated as CsMTP8 based on the highest homology to plant MTP8, is also presented. The expression of CsMTP8 in Saccharomyces cerevisiae led to increased Mn accumulation in yeast cells and fully restored the growth of mutants hypersensitive to Mn in Mn excess. Similarly, the overexpression of CsMTP8 in Arabidopsis thaliana enhanced plant tolerance to high Mn in nutrition media as well as the accumulation of Mn in plant tissues. When fused to green fluorescent protein (GFP), CsMTP8 localized to the vacuolar membranes in yeast cells and to Arabidopsis protoplasts. In cucumber, CsMTP8 was expressed almost exclusively in roots, and the level of gene transcript was markedly up-regulated or reduced under elevated Mn or Mn deficiency, respectively. Taken together, the results suggest that CsMTP8 is an Mn transporter localized in the vacuolar membrane, which participates in the maintenance of Mn homeostasis in cucumber root cells. PMID:25039075

Myelin-mediated inhibition of oligodendrocyte precursor differentiation can be overcome by pharmacological modulation of Fyn-RhoA and protein kinase C signalling

PubMed Central

Baer, Alexandra S.; Syed, Yasir A.; Kang, Sung Ung; Mitteregger, Dieter; Vig, Raluca; ffrench-Constant, Charles; Franklin, Robin J. M.; Altmann, Friedrich; Lubec, Gert

2009-01-01

Failure of oligodendrocyte precursor cell (OPC) differentiation contributes significantly to failed myelin sheath regeneration (remyelination) in chronic demyelinating diseases. Although the reasons for this failure are not completely understood, several lines of evidence point to factors present following demyelination that specifically inhibit differentiation of cells capable of generating remyelinating oligodendrocytes. We have previously demonstrated that myelin debris generated by demyelination inhibits remyelination by inhibiting OPC differentiation and that the inhibitory effects are associated with myelin proteins. In the present study, we narrow down the spectrum of potential protein candidates by proteomic analysis of inhibitory protein fractions prepared by CM and HighQ column chromatography followed by BN/SDS/SDS–PAGE gel separation using Nano-HPLC-ESI-Q-TOF mass spectrometry. We show that the inhibitory effects on OPC differentiation mediated by myelin are regulated by Fyn-RhoA-ROCK signalling as well as by modulation of protein kinase C (PKC) signalling. We demonstrate that pharmacological or siRNA-mediated inhibition of RhoA-ROCK-II and/or PKC signalling can induce OPC differentiation in the presence of myelin. Our results, which provide a mechanistic link between myelin, a mediator of OPC differentiation inhibition associated with demyelinating pathologies and specific signalling pathways amenable to pharmacological manipulation, are therefore of significant potential value for future strategies aimed at enhancing CNS remyelination. PMID:19208690

Numb endocytic adapter proteins regulate the transport and processing of the amyloid precursor protein in an isoform-dependent manner: implications for Alzheimer disease pathogenesis.

PubMed

Kyriazis, George A; Wei, Zelan; Vandermey, Miriam; Jo, Dong-Gyu; Xin, Ouyang; Mattson, Mark P; Chan, Sic L

2008-09-12

Central to the pathogenesis of Alzheimer disease is the aberrant processing of the amyloid precursor protein (APP) to generate amyloid beta-peptide (Abeta), the principle component of amyloid plaques. The cell fate determinant Numb is a phosphotyrosine binding domain (PTB)-containing endocytic adapter protein that interacts with the carboxyl-terminal domain of APP. The physiological relevance of this interaction is unknown. Mammals produce four alternatively spliced variants of Numb that differ in the length of their PTB and proline-rich region. In the current study, we determined the influence of the four human Numb isoforms on the intracellular trafficking and processing of APP. Stable expression of Numb isoforms that differ in the PTB but not in the proline-rich region results in marked differences in the sorting of APP to the recycling and degradative pathways. Neural cells expressing Numb isoforms that lack the insert in the PTB (short PTB (SPTB)) exhibited marked accumulation of APP in Rab5A-labeled early endosomal and recycling compartments, whereas those expressing isoforms with the insertion in the PTB (long PTB (LPTB)) exhibited reduced amounts of cellular APP and its proteolytic derivatives relative to parental control cells. Neither the activities of the beta- and gamma-secretases nor the expression of APP mRNA were significantly different in the stably transfected cells, suggesting that the differential effects of the Numb proteins on APP metabolism is likely to be secondary to altered APP trafficking. In addition, the expression of SPTB-Numb increases at the expense of LPTB-Numb in neuronal cultures subjected to stress, suggesting a role for Numb in stress-induced Abeta production. Taken together, these results suggest distinct roles for the human Numb isoforms in APP metabolism and may provide a novel potential link between altered Numb isoform expression and increased Abeta generation.

Phosphorylation of FE65 Ser610 by serum- and glucocorticoid-induced kinase 1 modulates Alzheimer's disease amyloid precursor protein processing

PubMed Central

Chow, Wan Ning Vanessa; Ngo, Jacky Chi Ki; Li, Wen; Chen, Yu Wai; Tam, Ka Ming Vincent; Chan, Ho Yin Edwin; Miller, Christopher C.J.; Lau, Kwok-Fai

2015-01-01

Alzheimer's disease (AD) is a fatal neurodegenerative disease affecting 36 million people worldwide. Genetic and biochemical research indicate that the excessive generation of amyloid-β peptide (Aβ) from amyloid precursor protein (APP), is a major part of AD pathogenesis. FE65 is a brain-enriched adaptor protein that binds to APP. However, the role of FE65 in APP processing and the mechanisms that regulate binding of FE65 to APP are not fully understood. In the present study, we show that serum- and glucocorticoid-induced kinase 1 (SGK1) phosphorylates FE65 on Ser610 and that this phosphorylation attenuates FE65 binding to APP. We also show that FE65 promotes amyloidogenic processing of APP and that FE65 Ser610 phosphorylation inhibits this effect. Furthermore, we found that the effect of FE65 Ser610 phosphorylation on APP processing is linked to a role of FE65 in metabolic turnover of APP via the proteasome. Thus FE65 influences APP degradation via the proteasome and phosphorylation of FE65 Ser610 by SGK1 regulates binding of FE65 to APP, APP turnover and processing. PMID:26188042

The Psen1-L166P-knock-in mutation leads to amyloid deposition in human wild-type amyloid precursor protein YAC transgenic mice

PubMed Central

Vidal, Ruben; Sammeta, Neeraja; Garringer, Holly J.; Sambamurti, Kumar; Miravalle, Leticia; Lamb, Bruce T.; Ghetti, Bernardino

2012-01-01

Genetically engineered mice have been generated to model cerebral β-amyloidosis, one of the hallmarks of Alzheimer disease (AD) pathology, based on the overexpression of a mutated cDNA of the amyloid-β precursor protein (AβPP) or by knock-in of the murine Aβpp gene alone or with presenilin1 mutations. Here we describe the generation and initial characterization of a new mouse line based on the presence of 2 copies of the human genomic region encoding the wild-type AβPP and the L166P presenilin 1 mutation. At ∼6 mo of age, double-mutant mice develop amyloid pathology, with signs of neuritic dystrophy, intracellular Aβ accumulation, and glial inflammation, an increase in AβPP C-terminal fragments, and an 8 times increase in Aβ42 levels with a 40% decrease in Aβ40 levels, leading to a significant increase (14 times) of Aβ42/Aβ40 ratios, with minimal effects on presenilin or the Notch1 pathway in the brain. We conclude that in mice, neither mutations in AβPP nor overexpression of an AβPP isoform are a prerequisite for Aβ pathology. This model will allow the study of AD pathogenesis and testing of therapeutic strategies in a more relevant environment without experimental artifacts due to the overexpression of a single-mutant AβPP isoform using exogenous promoters.—Vidal, R., Sammeta, N., Garringer, H. J., Sambamurti, K., Miravalle, L., Lamb B. T., Ghetti, B. The Psen1-L166P-knock-in mutation leads to amyloid deposition in human wild-type amyloid precursor protein YAC transgenic mice. PMID:22459153

Lost region in amyloid precursor protein (APP) through TALEN-mediated genome editing alters mitochondrial morphology.

PubMed

Wang, Yajie; Wu, Fengyi; Pan, Haining; Zheng, Wenzhong; Feng, Chi; Wang, Yunfu; Deng, Zixin; Wang, Lianrong; Luo, Jie; Chen, Shi

2016-02-29

Alzheimer's disease (AD) is characterized by amyloid-β (Aβ) deposition in the brain. Aβ plaques are produced through sequential β/γ cleavage of amyloid precursor protein (APP), of which there are three main APP isoforms: APP695, APP751 and APP770. KPI-APPs (APP751 and APP770) are known to be elevated in AD, but the reason remains unclear. Transcription activator-like (TAL) effector nucleases (TALENs) induce mutations with high efficiency at specific genomic loci, and it is thus possible to knock out specific regions using TALENs. In this study, we designed and expressed TALENs specific for the C-terminus of APP in HeLa cells, in which KPI-APPs are predominantly expressed. The KPI-APP mutants lack a 12-aa region that encompasses a 5-aa trans-membrane (TM) region and 7-aa juxta-membrane (JM) region. The mutated KPI-APPs exhibited decreased mitochondrial localization. In addition, mitochondrial morphology was altered, resulting in an increase in spherical mitochondria in the mutant cells through the disruption of the balance between fission and fusion. Mitochondrial dysfunction, including decreased ATP levels, disrupted mitochondrial membrane potential, increased ROS generation and impaired mitochondrial dehydrogenase activity, was also found. These results suggest that specific regions of KPI-APPs are important for mitochondrial localization and function.

Tonoplast Na+/H+ Antiport Activity and Its Energization by the Vacuolar H+-ATPase in the Halophytic Plant Mesembryanthemum crystallinum L.

PubMed Central

Barkla, B. J.; Zingarelli, L.; Blumwald, E.; Smith, JAC.

1995-01-01

Tonoplast vesicles were isolated from leaf mesophyll tissue of the inducible Crassulacean acid metabolism plant Mesembryanthemum crystallinum to investigate the mechanism of vacuolar Na+ accumulation in this halophilic species. In 8-week-old plants exposed to 200 mM NaCl for 2 weeks, tonoplast H+-ATPase activity was approximately doubled compared with control plants of the same age, as determined by rates of both ATP hydrolysis and ATP-dependent H+ transport. Evidence was also obtained for the presence of an electroneutral Na+/H+ antiporter at the tonoplast that is constitutively expressed, since extravesicular Na+ was able to dissipate a pre-existing transmembrane pH gradient. Initial rates of H+ efflux showed saturation kinetics with respect to extravesicular Na+ concentration and were 2.1-fold higher from vesicles of salt-treated plants compared with the controls. Na+-dependent H+ efflux also showed a high selectivity for Na+ over K+, was insensitive to the transmembrane electrical potential difference, and was more than 50% inhibited by 200 [mu]M N-amidino-3,5-diamino-6-chloropyrazinecarboxamide hydrochloride. The close correlation between increased Na+/H+ antiport and H+-ATPase activities in response to salt treatment suggests that accumulation of the very high concentrations of vacuolar Na+ found in M. crystallinum is energized by the H+ electrochemical gradient across the tonoplast. PMID:12228611

Tonoplast Na+/H+ Antiport Activity and Its Energization by the Vacuolar H+-ATPase in the Halophytic Plant Mesembryanthemum crystallinum L.

PubMed

Barkla, B. J.; Zingarelli, L.; Blumwald, E.; Smith, JAC.

1995-10-01

Tonoplast vesicles were isolated from leaf mesophyll tissue of the inducible Crassulacean acid metabolism plant Mesembryanthemum crystallinum to investigate the mechanism of vacuolar Na+ accumulation in this halophilic species. In 8-week-old plants exposed to 200 mM NaCl for 2 weeks, tonoplast H+-ATPase activity was approximately doubled compared with control plants of the same age, as determined by rates of both ATP hydrolysis and ATP-dependent H+ transport. Evidence was also obtained for the presence of an electroneutral Na+/H+ antiporter at the tonoplast that is constitutively expressed, since extravesicular Na+ was able to dissipate a pre-existing transmembrane pH gradient. Initial rates of H+ efflux showed saturation kinetics with respect to extravesicular Na+ concentration and were 2.1-fold higher from vesicles of salt-treated plants compared with the controls. Na+-dependent H+ efflux also showed a high selectivity for Na+ over K+, was insensitive to the transmembrane electrical potential difference, and was more than 50% inhibited by 200 [mu]M N-amidino-3,5-diamino-6-chloropyrazinecarboxamide hydrochloride. The close correlation between increased Na+/H+ antiport and H+-ATPase activities in response to salt treatment suggests that accumulation of the very high concentrations of vacuolar Na+ found in M. crystallinum is energized by the H+ electrochemical gradient across the tonoplast.

Hypoperfusion induces overexpression of beta-amyloid precursor protein mRNA in a focal ischemic rodent model.

PubMed

Shi, J; Yang, S H; Stubley, L; Day, A L; Simpkins, J W

2000-01-17

Silent stroke is one of the risk factors of dementia. In the present study, we used a novel focal ischemic animal model to investigate the effects of comparatively small changes of cerebral blood flow (CBF) on the expression of beta-amyloid precursor protein (APP) mRNA. Focal ischemia was achieved by introducing a 4-0 monofilament to the bifurcation of anterior and middle cerebral arteries. Brain samples were harvested from ischemic core and penumbra of cortices at 1, 4 and 7 days following ischemia. The expression of APP mRNA was assessed by RT-PCR. The CBF was decreased to 50% for 1 day after stroke and recovered to 90% at the fourth day after stroke. The changes of CBF were accompanied by an increase in the expression of APP mRNA. APP mRNA increased to 208% and 152% in the penumbra and core ischemic regions, respectively, on the fourth day after MCAO and remained high through the seventh day of ischemia. This study suggests brain hypoperfusion enhances APP mRNA expression and may contribute to the progression of cognitive impairment after silent stroke.

HMGCS2 promotes autophagic degradation of the amyloid-β precursor protein through ketone body-mediated mechanisms.

PubMed

Hu, Li-Tian; Zhu, Bing-Lin; Lai, Yu-Jie; Long, Yan; Zha, Jing-Si; Hu, Xiao-Tong; Zhang, John H; Chen, Guo-Jun

2017-04-29

HMGCS2 (mitochondrial 3-hydroxy-3-methylglutaryl-COA synthase 2) is a control enzyme in ketogenesis. The mitochondrial localization and interaction with APP (β-amyloid precursor protein) suggest that HMGCS2 may play a role in the pathophysiology of AD (Alzheimer's disease). Here we report that overexpression of HMGCS2 decreased levels of APP and related CTFs (carboxy-terminal fragments), which was largely prevented by an autophagic inhibitor chloroquine. In addition, HMGCS2 enhancement of autophagic marker LC3II was diminished by rapamycin, an inhibitor of mechanistic target of rapamycin. Moreover, deprivation of EBSS (Earle's Balanced Salt Solution) significantly augmented the effect of HMGCS2 on LC3II, while acetoacetate reversed the reduction of LC3II, APP and CTFs which was induced by HMGCS2 knockdown. In the presence of acetoacetate, rapamycin failed to induce further increase of LC3II, which mimicked the effect of HMGCS2 overexpression. Finally, HMGCS2 enhanced the antioxidant response. Collectively, HMGCS2 shares with ketone bodies common features in autophagic clearance of APP and CTFs, suggesting that ketone bodies play an important role in HMGCS2 regulation of the autophagy. Copyright © 2017 Elsevier Inc. All rights reserved.

Positive evolutionary selection of an HD motif on Alzheimer precursor protein orthologues suggests a functional role.

PubMed

Miklós, István; Zádori, Zoltán

2012-02-01

HD amino acid duplex has been found in the active center of many different enzymes. The dyad plays remarkably different roles in their catalytic processes that usually involve metal coordination. An HD motif is positioned directly on the amyloid beta fragment (Aβ) and on the carboxy-terminal region of the extracellular domain (CAED) of the human amyloid precursor protein (APP) and a taxonomically well defined group of APP orthologues (APPOs). In human Aβ HD is part of a presumed, RGD-like integrin-binding motif RHD; however, neither RHD nor RXD demonstrates reasonable conservation in APPOs. The sequences of CAEDs and the position of the HD are not particularly conserved either, yet we show with a novel statistical method using evolutionary modeling that the presence of HD on CAEDs cannot be the result of neutral evolutionary forces (p<0.0001). The motif is positively selected along the evolutionary process in the majority of APPOs, despite the fact that HD motif is underrepresented in the proteomes of all species of the animal kingdom. Position migration can be explained by high probability occurrence of multiple copies of HD on intermediate sequences, from which only one is kept by selective evolutionary forces, in a similar way as in the case of the "transcription binding site turnover." CAED of all APP orthologues and homologues are predicted to bind metal ions including Amyloid-like protein 1 (APLP1) and Amyloid-like protein 2 (APLP2). Our results suggest that HDs on the CAEDs are most probably key components of metal-binding domains, which facilitate and/or regulate inter- or intra-molecular interactions in a metal ion-dependent or metal ion concentration-dependent manner. The involvement of naturally occurring mutations of HD (Tottori (D7N) and English (H6R) mutations) in early onset Alzheimer's disease gives additional support to our finding that HD has an evolutionary preserved function on APPOs.

Positive Evolutionary Selection of an HD Motif on Alzheimer Precursor Protein Orthologues Suggests a Functional Role

PubMed Central

Miklós, István; Zádori, Zoltán

2012-01-01

HD amino acid duplex has been found in the active center of many different enzymes. The dyad plays remarkably different roles in their catalytic processes that usually involve metal coordination. An HD motif is positioned directly on the amyloid beta fragment (Aβ) and on the carboxy-terminal region of the extracellular domain (CAED) of the human amyloid precursor protein (APP) and a taxonomically well defined group of APP orthologues (APPOs). In human Aβ HD is part of a presumed, RGD-like integrin-binding motif RHD; however, neither RHD nor RXD demonstrates reasonable conservation in APPOs. The sequences of CAEDs and the position of the HD are not particularly conserved either, yet we show with a novel statistical method using evolutionary modeling that the presence of HD on CAEDs cannot be the result of neutral evolutionary forces (p<0.0001). The motif is positively selected along the evolutionary process in the majority of APPOs, despite the fact that HD motif is underrepresented in the proteomes of all species of the animal kingdom. Position migration can be explained by high probability occurrence of multiple copies of HD on intermediate sequences, from which only one is kept by selective evolutionary forces, in a similar way as in the case of the “transcription binding site turnover.” CAED of all APP orthologues and homologues are predicted to bind metal ions including Amyloid-like protein 1 (APLP1) and Amyloid-like protein 2 (APLP2). Our results suggest that HDs on the CAEDs are most probably key components of metal-binding domains, which facilitate and/or regulate inter- or intra-molecular interactions in a metal ion-dependent or metal ion concentration-dependent manner. The involvement of naturally occurring mutations of HD (Tottori (D7N) and English (H6R) mutations) in early onset Alzheimer's disease gives additional support to our finding that HD has an evolutionary preserved function on APPOs. PMID:22319430

An affinity-directed protein missile system for targeted proteolysis.

PubMed

Fulcher, Luke J; Macartney, Thomas; Bozatzi, Polyxeni; Hornberger, Annika; Rojas-Fernandez, Alejandro; Sapkota, Gopal P

2016-10-01

The von Hippel-Lindau (VHL) protein serves to recruit the hypoxia-inducible factor alpha (HIF1α) protein under normoxia to the CUL2 E3 ubiquitin ligase for its ubiquitylation and degradation through the proteasome. In this report, we modify VHL to engineer an affinity-directed protein missile (AdPROM) system to direct specific endogenous target proteins for proteolysis in mammalian cells. The proteolytic AdPROM construct harbours a cameloid anti-green fluorescence protein (aGFP) nanobody that is fused to VHL for either constitutive or tetracycline-inducible expression. For target proteins, we exploit CRISPR/Cas9 to rapidly generate human kidney HEK293 and U2OS osteosarcoma homozygous knock-in cells harbouring GFP tags at the VPS34 (vacuolar protein sorting 34) and protein associated with SMAD1 (PAWS1, aka FAM83G) loci, respectively. Using these cells, we demonstrate that the expression of the VHL-aGFP AdPROM system results in near-complete degradation of the endogenous GFP-VPS34 and PAWS1-GFP proteins through the proteasome. Additionally, we show that Tet-inducible destruction of GFP-VPS34 results in the degradation of its associated partner, UVRAG, and reduction in levels of cellular phosphatidylinositol 3-phosphate. © 2016 The Authors.

An affinity-directed protein missile system for targeted proteolysis

PubMed Central

Fulcher, Luke J.; Macartney, Thomas; Bozatzi, Polyxeni; Hornberger, Annika; Rojas-Fernandez, Alejandro

2016-01-01

The von Hippel–Lindau (VHL) protein serves to recruit the hypoxia-inducible factor alpha (HIF1α) protein under normoxia to the CUL2 E3 ubiquitin ligase for its ubiquitylation and degradation through the proteasome. In this report, we modify VHL to engineer an affinity-directed protein missile (AdPROM) system to direct specific endogenous target proteins for proteolysis in mammalian cells. The proteolytic AdPROM construct harbours a cameloid anti-green fluorescence protein (aGFP) nanobody that is fused to VHL for either constitutive or tetracycline-inducible expression. For target proteins, we exploit CRISPR/Cas9 to rapidly generate human kidney HEK293 and U2OS osteosarcoma homozygous knock-in cells harbouring GFP tags at the VPS34 (vacuolar protein sorting 34) and protein associated with SMAD1 (PAWS1, aka FAM83G) loci, respectively. Using these cells, we demonstrate that the expression of the VHL-aGFP AdPROM system results in near-complete degradation of the endogenous GFP-VPS34 and PAWS1-GFP proteins through the proteasome. Additionally, we show that Tet-inducible destruction of GFP-VPS34 results in the degradation of its associated partner, UVRAG, and reduction in levels of cellular phosphatidylinositol 3-phosphate. PMID:27784791

Cleavage sites within the poliovirus capsid protein precursors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Larsen, G.R.; Anderson, C.W.; Dorner, A.J.

1982-01-01

Partial amino-terminal sequence analysis was performed on radiolabeled poliovirus capsid proteins VP1, VP2, and VP3. A computer-assisted comparison of the amino acid sequences obtained with that predicted by the nucleotide sequence of the poliovirus genome allows assignment of the amino terminus of each capsid protein to a unique position within the virus polyprotein. Sequence analysis of trypsin-digested VP4, which has a blocked amino terminus, demonstrates that VP4 is encoded at or very near to the amino terminus of the polyprotein. The gene order of the capsid proteins is VP4-VP2-VP3-VP1. Cleavage of VP0 to VP4 and VP2 is shown to occurmore » between asparagine and serine, whereas the cleavages that separate VP2/VP3 and VP3/VP1 occur between glutamine and glycine residues. This finding supports the hypothesis that the cleavage of VP0, which occurs during virion morphogenesis, is distinct from the cleavages that separate functional regions of the polyprotein.« less

Electron tomographic characterization of a vacuolar reticulum and of six vesicle types that occupy different cytoplasmic domains in the apex of tip-growing Chara rhizoids.

PubMed

Limbach, Christoph; Staehelin, L Andrew; Sievers, Andreas; Braun, Markus

2008-04-01

We provide a 3D ultrastructural analysis of the membrane systems involved in tip growth of rhizoids of the green alga Chara. Electron tomography of cells preserved by high-pressure freeze fixation has enabled us to distinguish six different types of vesicles in the apical cytoplasm where the tip growth machinery is accommodated. The vesicle types are: dark and light secretory vesicles, plasma membrane-associated clathrin-coated vesicles (PM-CCVs), Spitzenkoerper-associated clathrin-coated vesicles (Sp-CCVs) and coated vesicles (Sp-CVs), and microvesicles. Each of these vesicle types exhibits a distinct distribution pattern, which provides insights into their possible function for tip growth. The PM-CCVs are confined to the cytoplasm adjacent to the apical plasma membrane. Within this space they are arranged in clusters often surrounding tubular plasma membrane invaginations from which CCVs bud. This suggests that endocytosis and membrane recycling are locally confined to specialized apical endocytosis sites. In contrast, exocytosis of secretory vesicles occurs over the entire membrane area of the apical dome. The Sp-CCVs and the Sp-CVs are associated with the aggregate of endoplasmic reticulum membranes in the center of the growth-organizing Spitzenkoerper complex. Here, Sp-CCVs are seen to bud from undefined tubular membranes. The subapical region of rhizoids contains a vacuolar reticulum that extends along the longitudinal cell axis and consists of large, vesicle-like segments interconnected by thin tubular domains. The tubular domains are encompassed by thin filamentous structures resembling dynamin spirals which could drive peristaltic movements of the vacuolar reticulum similar to those observed in fungal hyphae. The vacuolar reticulum appears to serve as a lytic compartment into which multivesicular bodies deliver their internal vesicles for molecular recycling and degradation.

Anti-amyloid precursor protein antibodies inhibit amyloid-β production by steric hindrance

PubMed Central

Thomas, Rhian S.; Liddell, J. Eryl; Kidd, Emma J.

2015-01-01

Summary Cleavage of amyloid precursor protein (APP) by β- and γ-secretases results in the production of amyloid-β (Aβ) in Alzheimer’s disease (AD). We raised two monoclonal antibodies, 2B3 and 2B12, that recognise the β-secretase cleavage site on APP but not Aβ. We hypothesised that these antibodies would reduce Aβ levels via steric hindrance of β-secretase. Both antibodies decreased extracellular Aβ levels from astrocytoma cells, but 2B3 was more potent than 2B12. Levels of soluble sAPPα from the non-amyloidogenic α-secretase pathway and intracellular APP were not affected by either antibody nor were there any effects on cell viability. 2B3 exhibited a higher affinity for APP than 2B12 and its epitope appeared to span the cleavage site while 2B12 bound slightly upstream. Both of these factors probably contribute to its greater effect on Aβ levels. After 60 minutes incubation at pH 4.0, most 2B3 and 2B12 remained bound to their antigen, suggesting that the antibodies will remain bound to APP in the acidic endosomes where β-secretase cleavage probably occurs. Only 2B3 and 2B12, but not control antibodies, inhibited the cleavage of sAPPα by β-secretase in a cell-free assay where effects of antibody internalisation and intracellular degradation were excluded. 2B3 virtually abolished this cleavage. In addition, levels of C-terminal APP fragments, βCTF, generated following β-secretase cleavage, were significantly reduced in cells after incubation with 2B3. These results strongly suggest that anti-cleavage site antibodies can generically reduce Aβ levels via inhibition of β-secretase by steric hindrance and may provide a novel alternative therapy for AD. PMID:21122073

Rrp5p, Noc1p and Noc2p form a protein module which is part of early large ribosomal subunit precursors in S. cerevisiae

PubMed Central

Hierlmeier, Thomas; Merl, Juliane; Sauert, Martina; Perez-Fernandez, Jorge; Schultz, Patrick; Bruckmann, Astrid; Hamperl, Stephan; Ohmayer, Uli; Rachel, Reinhard; Jacob, Anja; Hergert, Kristin; Deutzmann, Rainer; Griesenbeck, Joachim; Hurt, Ed; Milkereit, Philipp; Baßler, Jochen; Tschochner, Herbert

2013-01-01

Eukaryotic ribosome biogenesis requires more than 150 auxiliary proteins, which transiently interact with pre-ribosomal particles. Previous studies suggest that several of these biogenesis factors function together as modules. Using a heterologous expression system, we show that the large ribosomal subunit (LSU) biogenesis factor Noc1p of Saccharomyces cerevisiae can simultaneously interact with the LSU biogenesis factor Noc2p and Rrp5p, a factor required for biogenesis of the large and the small ribosomal subunit. Proteome analysis of RNA polymerase-I-associated chromatin and chromatin immunopurification experiments indicated that all members of this protein module and a specific set of LSU biogenesis factors are co-transcriptionally recruited to nascent ribosomal RNA (rRNA) precursors in yeast cells. Further ex vivo analyses showed that all module members predominantly interact with early pre-LSU particles after the initial pre-rRNA processing events have occurred. In yeast strains depleted of Noc1p, Noc2p or Rrp5p, levels of the major LSU pre-rRNAs decreased and the respective other module members were associated with accumulating aberrant rRNA fragments. Therefore, we conclude that the module exhibits several binding interfaces with pre-ribosomes. Taken together, our results suggest a co- and post-transcriptional role of the yeast Rrp5p–Noc1p–Noc2p module in the structural organization of early LSU precursors protecting them from non-productive RNase activity. PMID:23209026

Role of the Cellular Prion Protein in Oligodendrocyte Precursor Cell Proliferation and Differentiation in the Developing and Adult Mouse CNS

PubMed Central

Bribián, Ana; Gavín, Rosalina; Reina, Manuel; García-Verdugo, José Manuel; Torres, Juan María; de Castro, Fernando; del Río, José Antonio

2012-01-01

There are numerous studies describing the signaling mechanisms that mediate oligodendrocyte precursor cell (OPC) proliferation and differentiation, although the contribution of the cellular prion protein (PrPc) to this process remains unclear. PrPc is a glycosyl-phosphatidylinositol (GPI)-anchored glycoprotein involved in diverse cellular processes during the development and maturation of the mammalian central nervous system (CNS). Here we describe how PrPc influences oligodendrocyte proliferation in the developing and adult CNS. OPCs that lack PrPc proliferate more vigorously at the expense of a delay in differentiation, which correlates with changes in the expression of oligodendrocyte lineage markers. In addition, numerous NG2-positive cells were observed in cortical regions of adult PrPc knockout mice, although no significant changes in myelination can be seen, probably due to the death of surplus cells. PMID:22529900

ERG2 and ERG24 Are Required for Normal Vacuolar Physiology as Well as Candida albicans Pathogenicity in a Murine Model of Disseminated but Not Vaginal Candidiasis

PubMed Central

Luna-Tapia, Arturo; Peters, Brian M.; Eberle, Karen E.; Kerns, Morgan E.; Foster, Timothy P.; Marrero, Luis; Noverr, Mairi C.; Fidel, Paul L.

2015-01-01

Several important classes of antifungal agents, including the azoles, act by blocking ergosterol biosynthesis. It was recently reported that the azoles cause massive disruption of the fungal vacuole in the prevalent human pathogen Candida albicans. This is significant because normal vacuolar function is required to support C. albicans pathogenicity. This study examined the impact of the morpholine antifungals, which inhibit later steps of ergosterol biosynthesis, on C. albicans vacuolar integrity. It was found that overexpression of either the ERG2 or ERG24 gene, encoding C-8 sterol isomerase or C-14 sterol reductase, respectively, suppressed C. albicans sensitivity to the morpholines. In addition, both erg2Δ/Δ and erg24Δ/Δ mutants were hypersensitive to the morpholines. These data are consistent with the antifungal activity of the morpholines depending upon the simultaneous inhibition of both Erg2p and Erg24p. The vacuoles within both erg2Δ/Δ and erg24Δ/Δ C. albicans strains exhibited an aberrant morphology and accumulated large quantities of the weak base quinacrine, indicating enhanced vacuolar acidification compared with that of control strains. Both erg mutants exhibited significant defects in polarized hyphal growth and were avirulent in a mouse model of disseminated candidiasis. Surprisingly, in a mouse model of vaginal candidiasis, both mutants colonized mice at high levels and induced a pathogenic response similar to that with the controls. Thus, while targeting Erg2p or Erg24p alone could provide a potentially efficacious therapy for disseminated candidiasis, it may not be an effective strategy to treat vaginal infections. The potential value of drugs targeting these enzymes as adjunctive therapies is discussed. PMID:26231054

A Python Analytical Pipeline to Identify Prohormone Precursors and Predict Prohormone Cleavage Sites

PubMed Central

Southey, Bruce R.; Sweedler, Jonathan V.; Rodriguez-Zas, Sandra L.

2008-01-01

Neuropeptides and hormones are signaling molecules that support cell–cell communication in the central nervous system. Experimentally characterizing neuropeptides requires significant efforts because of the complex and variable processing of prohormone precursor proteins into neuropeptides and hormones. We demonstrate the power and flexibility of the Python language to develop components of an bioinformatic analytical pipeline to identify precursors from genomic data and to predict cleavage as these precursors are en route to the final bioactive peptides. We identified 75 precursors in the rhesus genome, predicted cleavage sites using support vector machines and compared the rhesus predictions to putative assignments based on homology to human sequences. The correct classification rate of cleavage using the support vector machines was over 97% for both human and rhesus data sets. The functionality of Python has been important to develop and maintain NeuroPred (http://neuroproteomics.scs.uiuc.edu/neuropred.html), a user-centered web application for the neuroscience community that provides cleavage site prediction from a wide range of models, precision and accuracy statistics, post-translational modifications, and the molecular mass of potential peptides. The combined results illustrate the suitability of the Python language to implement an all-inclusive bioinformatics approach to predict neuropeptides that encompasses a large number of interdependent steps, from scanning genomes for precursor genes to identification of potential bioactive neuropeptides. PMID:19169350

Amyloid Precursor Protein (APP) May Act as a Substrate and a Recognition Unit for CRL4CRBN and Stub1 E3 Ligases Facilitating Ubiquitination of Proteins Involved in Presynaptic Functions and Neurodegeneration.

PubMed

Del Prete, Dolores; Rice, Richard C; Rajadhyaksha, Anjali M; D'Adamio, Luciano

2016-08-12

The amyloid precursor protein (APP), whose mutations cause Alzheimer disease, plays an important in vivo role and facilitates transmitter release. Because the APP cytosolic region (ACR) is essential for these functions, we have characterized its brain interactome. We found that the ACR interacts with proteins that regulate the ubiquitin-proteasome system, predominantly with the E3 ubiquitin-protein ligases Stub1, which binds the NH2 terminus of the ACR, and CRL4(CRBN), which is formed by Cul4a/b, Ddb1, and Crbn, and interacts with the COOH terminus of the ACR via Crbn. APP shares essential functions with APP-like protein-2 (APLP2) but not APP-like protein-1 (APLP1). Noteworthy, APLP2, but not APLP1, interacts with Stub1 and CRL4(CRBN), pointing to a functional pathway shared only by APP and APLP2. In vitro ubiquitination/ubiquitome analysis indicates that these E3 ligases are enzymatically active and ubiquitinate the ACR residues Lys(649/650/651/676/688) Deletion of Crbn reduces ubiquitination of Lys(676) suggesting that Lys(676) is physiologically ubiquitinated by CRL4(CRBN) The ACR facilitated in vitro ubiquitination of presynaptic proteins that regulate exocytosis, suggesting a mechanism by which APP tunes transmitter release. Other dementia-related proteins, namely Tau and apoE, interact with and are ubiquitinated via the ACR in vitro This, and the evidence that CRBN and CUL4B are linked to intellectual disability, prompts us to hypothesize a pathogenic mechanism, in which APP acts as a modulator of E3 ubiquitin-protein ligase(s), shared by distinct neuronal disorders. The well described accumulation of ubiquitinated protein inclusions in neurodegenerative diseases and the link between the ubiquitin-proteasome system and neurodegeneration make this concept plausible. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Vacuolar Localization of Endoproteinases EP(1) and EP(2) in Barley Mesophyll Cells.

PubMed

Thayer, S S; Huffaker, R C

1984-05-01

The localization of two previously characterized endoproteinases (EP(1) and EP(2)) that comprise more than 95% of the protease activity in primary Hordeum vulgare L. var Numar leaves was determined. Intact vacuoles released from washed mesophyll protoplasts by gentle osmotic shock and increase in pH, were purified by flotation through a four-step Ficoll gradient. These vacuoles contained endoproteinases that rapidly degraded purified barley ribulose-1,5-bisphosphate carboxylase (RuBPCase) substrate. Breakdown products and extent of digestion of RuBPCase were determined using 12% polyacrylamide-sodium dodecyl sulfate gels. Coomassie brilliant blue- or silver-stained gels were scanned, and the peaks were integrated to provide quantitative information. The characteristics of the vacuolar endoproteinases (e.g. sensitivity to various inhibitors and activators, and the molecular weights of the breakdown products, i.e. peptide maps) closely resembled those of purified EP(1) and partially purified EP(2). It is therefore concluded that EP(1) and EP(2) are localized in the vacuoles of mesophyll cells.

Molecular Basis of ADP Inhibition of Vacuolar (V)-type ATPase/Synthase*

PubMed Central

Kishikawa, Jun-ichi; Nakanishi, Atsuko; Furuike, Shou; Tamakoshi, Masatada; Yokoyama, Ken

2014-01-01

Reduction of ATP hydrolysis activity of vacuolar-type ATPase/synthase (V0V1) as a result of ADP inhibition occurs as part of the normal mechanism of V0V1 of Thermus thermophilus but not V0V1 of Enterococcus hirae or eukaryotes. To investigate the molecular basis for this difference, domain-swapped chimeric V1 consisting of both T. thermophilus and E. hirae enzymes were generated, and their function was analyzed. The data showed that the interaction between the nucleotide binding and C-terminal domains of the catalytic A subunit from E. hirae V1 is central to increasing binding affinity of the chimeric V1 for phosphate, resulting in reduction of the ADP inhibition. These findings together with a comparison of the crystal structures of T. thermophilus V1 with E. hirae V1 strongly suggest that the A subunit adopts a conformation in T. thermophilus V1 different from that in E. hirae V1. This key difference results in ADP inhibition of T. thermophilus V1 by abolishing the binding affinity for phosphate during ATP hydrolysis. PMID:24247239

Role of membrane contact sites in protein import into mitochondria

PubMed Central

Horvath, Susanne E; Rampelt, Heike; Oeljeklaus, Silke; Warscheid, Bettina; van der Laan, Martin; Pfanner, Nikolaus

2015-01-01

Mitochondria import more than 1,000 different proteins from the cytosol. The proteins are synthesized as precursors on cytosolic ribosomes and are translocated by protein transport machineries of the mitochondrial membranes. Five main pathways for protein import into mitochondria have been identified. Most pathways use the translocase of the outer mitochondrial membrane (TOM) as the entry gate into mitochondria. Depending on specific signals contained in the precursors, the proteins are subsequently transferred to different intramitochondrial translocases. In this article, we discuss the connection between protein import and mitochondrial membrane architecture. Mitochondria possess two membranes. It is a long-standing question how contact sites between outer and inner membranes are formed and which role the contact sites play in the translocation of precursor proteins. A major translocation contact site is formed between the TOM complex and the presequence translocase of the inner membrane (TIM23 complex), promoting transfer of presequence-carrying preproteins to the mitochondrial inner membrane and matrix. Recent findings led to the identification of contact sites that involve the mitochondrial contact site and cristae organizing system (MICOS) of the inner membrane. MICOS plays a dual role. It is crucial for maintaining the inner membrane cristae architecture and forms contacts sites to the outer membrane that promote translocation of precursor proteins into the intermembrane space and outer membrane of mitochondria. The view is emerging that the mitochondrial protein translocases do not function as independent units, but are embedded in a network of interactions with machineries that control mitochondrial activity and architecture. PMID:25514890

Crystal Structure of Poliovirus 3CD Protein: Virally Encoded Protease and Precursor to the RNA-Dependent RNA Polymerase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marcotte,L.; Wass, A.; Gohara, D.

2007-01-01

Poliovirus 3CD is a multifunctional protein that serves as a precursor to the protease 3Cpro and the viral polymerase 3Dpol and also plays a role in the control of viral replication. Although 3CD is a fully functional protease, it lacks polymerase activity. We have solved the crystal structures of 3CD at a 3.4- Angstroms resolution and the G64S fidelity mutant of 3Dpol at a 3.0- Angstroms resolution. In the 3CD structure, the 3C and 3D domains are joined by a poorly ordered polypeptide linker, possibly to facilitate its cleavage, in an arrangement that precludes intramolecular proteolysis. The polymerase active sitemore » is intact in both the 3CD and the 3Dpol G64S structures, despite the disruption of a network proposed to position key residues in the active site. Therefore, changes in molecular flexibility may be responsible for the differences in fidelity and polymerase activities. Extensive packing contacts between symmetry-related 3CD molecules and the approach of the 3C domain's N terminus to the VPg binding site suggest how 3Dpol makes biologically relevant interactions with the 3C, 3CD, and 3BCD proteins that control the uridylylation of VPg during the initiation of viral replication. Indeed, mutations designed to disrupt these interfaces have pronounced effects on the uridylylation reaction in vitro.« less

Proton pump inhibitors as anti vacuolar-ATPases drugs: a novel anticancer strategy.

PubMed

Spugnini, Enrico P; Citro, Gennaro; Fais, Stefano

2010-05-08

The vacuolar ATPases are ATP-dependent proton pumps whose functions include the acidification of intracellular compartments and the extrusion of protons through the cell cytoplasmic membrane. These pumps play a pivotal role in the regulation of cell pH in normal cells and, to a much greater extent, in tumor cells. In fact, the glucose metabolism in hypoxic conditions by the neoplasms leads to an intercellular pH drift towards acidity. The acid microenvironment is modulated through the over-expression of H+ transporters that are also involved in tumor progression, invasiveness, distant spread and chemoresistance. Several strategies to block/downmodulate the efficiency of these transporters are currently being investigated. Among them, proton pump inhibitors have shown to successfully block the H+ transporters in vitro and in vivo, leading to apoptotic death. Furthermore, their action seems to synergize with conventional chemotherapy protocols, leading to chemosensitization and reversal of chemoresistance. Aim of this article is to critically revise the current knowledge of this cellular machinery and to summarize the therapeutic strategies developed to counter this mechanism.

Differential expression of vacuolar and defective cell wall invertase genes in roots and seeds of metalliferous and non-metalliferous populations of Rumex dentatus under copper stress.

PubMed

Xu, Zhong-Rui; Cai, Shen-Wen; Huang, Wu-Xing; Liu, Rong-Xiang; Xiong, Zhi-Ting

2018-01-01

Acid invertase activities in roots and young seeds of a metalliferous population (MP) of Rumex dentatus were previously observed to be significantly higher than those of a non-metalliferous population (NMP) under Cu stress. To date, no acid invertase gene has been cloned from R. dentatus. Here, we isolated four full-length cDNAs from the two populations of R. dentatus, presumably encoding cell wall (RdnCIN1 and RdmCIN1 from the NMP and MP, respectively) and vacuolar invertases (RdnVIN1 and RdmVIN1 from the NMP and MP, respectively). Unexpectedly, RdnCIN1 and RdmCIN1 most likely encode special defective invertases with highly attenuated sucrose-hydrolyzing capacity. The transcript levels of RdmCIN1 were significantly higher than those of RdnCIN1 in roots and young seeds under Cu stress, whereas under control conditions, the former was initially lower than the latter. Unexpected high correlations were observed between the transcript levels of RdnCIN1 and RdmCIN1 and the activity of cell wall invertase, even though RdnCIN1 and RdmCIN1 do not encode catalytically active invertases. Similarly, the transcript levels of RdmVIN1 in roots and young seeds were increased under Cu stress, whereas those of RdnVIN1 were decreased. The high correlations between the transcript levels of RdnVIN1 and RdmVIN1 and the activity of vacuolar invertase indicate that RdnVIN1 and RdmVIN1 might control distinct vacuolar invertase activities in the two populations. Moreover, a possible indirect role for acid invertases in Cu tolerance, mediated by generating a range of sugars used as nutrients and signaling molecules, is discussed. Copyright © 2017 Elsevier Inc. All rights reserved.

K+ channel openers prevent global ischemia-induced expression of c-fos, c-jun, heat shock protein, and amyloid beta-protein precursor genes and neuronal death in rat hippocampus.

PubMed Central

Heurteaux, C; Bertaina, V; Widmann, C; Lazdunski, M

1993-01-01

Transient global forebrain ischemia induces in rat brain a large increase of expression of the immediate early genes c-fos and c-jun and of the mRNAs for the 70-kDa heat-shock protein and for the form of the amyloid beta-protein precursor including the Kunitz-type protease-inhibitor domain. At 24 hr after ischemia, this increased expression is particularly observed in regions that are vulnerable to the deleterious effects of ischemia, such as pyramidal cells of the CA1 field in the hippocampus. In an attempt to find conditions which prevent the deleterious effects of ischemia, representatives of three different classes of K+ channel openers, (-)-cromakalim, nicorandil, and pinacidil, were administered both before ischemia and during the reperfusion period. This treatment totally blocked the ischemia-induced expression of the different genes. In addition it markedly protected neuronal cells against degeneration. The mechanism of the neuroprotective effects involves the opening of ATP-sensitive K+ channels since glipizide, a specific blocker of that type of channel, abolished the beneficial effects of K+ channel openers. The various classes of K+ channel openers seem to deserve attention as potential drugs for cerebral ischemia. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8415718

An abundantly expressed mucin-like protein from Toxocara canis infective larvae: the precursor of the larval surface coat glycoproteins.

PubMed Central

Gems, D; Maizels, R M

1996-01-01

Evasion of host immunity by Toxocara canis infective larvae is mediated by the nematode surface coat, which is shed in response to binding by host antibody molecules or effector cells. The major constituent of the coat is the TES-120 glycoprotein series. We have isolated a 730-bp cDNA from the gene encoding the apoprotein precursor of TES-120. The mRNA is absent from T. canis adults but hyperabundant in larvae, making up approximately 10% of total mRNA, and is trans-spliced with the nematode 5' leader sequence SL1. It encodes a 15.8-kDa protein (after signal peptide removal) containing a typical mucin domain: 86 amino acid residues, 72.1% of which are Ser or Thr, organized into an array of heptameric repeats, interspersed with proline residues. At the C-terminal end of the putative protein are two 36-amino acid repeats containing six Cys residues, in a motif that can also be identified in several genes in Caenorhabditis elegans. Although TES-120 displays size and charge heterogeneity, there is a single copy gene and a homogeneous size of mRNA. The association of overexpression of some membrane-associated mucins with immunosuppression and tumor metastasis suggests a possible model for the role of the surface coat in immune evasion by parasitic nematodes. Images Fig. 1 Fig. 4 PMID:8643687

Meat flavor precursors and factors influencing flavor precursors--A systematic review.

PubMed

Khan, Muhammad Issa; Jo, Cheorun; Tariq, Muhammad Rizwan

2015-12-01

Flavor is the sensory impression sensed by taste and smell buds and is a leading factor determining the meat quality and purchasing decision of the consumer. Meat flavor is characteristic of volatiles produced as a result of reactions of non-volatile components that are induced thermally. The water soluble compounds having low molecular weight and meat lipids are important precursors of cooked meat flavor. The Maillard reaction, lipid oxidation, and vitamin degradation are leading reactions during cooking which develop meat flavor from uncooked meat with little aroma and bloody taste. The pre-slaughter and postmortem factors like animal breed, sex, age, feed, aging and cooking conditions contribute to flavor development of cooked meat. The objective of this review is to highlight the flavor chemistry, meat flavor precursors and factors affecting meat flavor precursors. Copyright © 2015 Elsevier Ltd. All rights reserved.

Molecular basis of autosomal dominant neurohypophyseal diabetes insipidus. Cellular toxicity caused by the accumulation of mutant vasopressin precursors within the endoplasmic reticulum.

PubMed Central

Ito, M; Jameson, J L; Ito, M

1997-01-01

Mutations in the arginine vasopressin (AVP) gene cause autosomal dominant familial neurohypophyseal diabetes insipidus (FNDI). The dominant inheritance pattern has been postulated to reflect neuronal toxicity of the mutant proteins, but the mechanism for such cytotoxicity is unknown. In this study, wild-type or several different mutant AVP genes were stably expressed in neuro2A neuroblastoma cells. When cells were treated with valproic acid to induce neuronal differentiation, each of the mutants caused reduced viability. Metabolic labeling revealed diminished intracellular trafficking of mutant AVP precursors and confirmed inefficient secretion of immunoreactive AVP. Immunofluorescence studies demonstrated marked accumulation of mutant AVP precursors within the endoplasmic reticulum. These studies suggest that the cellular toxicity in FNDI may be caused by the intracellular accumulation of mutant precursor proteins. PMID:9109434

SomethiNG 2 talk about-Transcriptional regulation in embryonic and adult oligodendrocyte precursors.

PubMed

Küspert, Melanie; Wegner, Michael

2016-05-01

Glial cells that express the chondroitin sulfate proteoglycan NG2 represent an inherently heterogeneous population. These so-called NG2-glia are present during development and in the adult CNS, where they are referred to as embryonic oligodendrocyte precursors and adult NG2-glia, respectively. They give rise to myelinating oligodendrocytes at all times of life. Over the years much has been learnt about the transcriptional network in embryonic oligodendrocyte precursors, and several transcription factors from the HLH, HMG-domain, zinc finger and homeodomain protein families have been identified as main constituents. Much less is known about the corresponding network in adult NG2-glia. Here we summarize and discuss current knowledge on functions of each of these transcription factor families in NG2-glia, and where possible compare transcriptional regulation in embryonic oligodendrocyte precursors and adult NG2-glia. This article is part of a Special Issue entitled SI:NG2-glia (Invited only). Copyright © 2015 Elsevier B.V. All rights reserved.

Vacuolar zinc transporter Zrc1 is required for detoxification of excess intracellular zinc in the human fungal pathogen Cryptococcus neoformans.

PubMed

Cho, Minsu; Hu, Guanggan; Caza, Mélissa; Horianopoulos, Linda C; Kronstad, James W; Jung, Won Hee

2018-01-01

Zinc is an important transition metal in all living organisms and is required for numerous biological processes. However, excess zinc can also be toxic to cells and cause cellular stress. In the model fungus Saccharomyces cerevisiae, a vacuolar zinc transporter, Zrc1, plays important roles in the storage and detoxification of excess intracellular zinc to protect the cell. In this study, we identified an ortholog of the S. cerevisiae ZRC1 gene in the human fungal pathogen Cryptococcus neoformans. Zrc1 was localized in the vacuolar membrane in C. neoformans, and a mutant lacking ZRC1 showed significant growth defects under high-zinc conditions. These results suggested a role for Zrc1 in zinc detoxification. However, contrary to our expectation, the expression of Zrc1 was induced in cells grown in zinc-limited conditions and decreased upon the addition of zinc. These expression patterns were similar to those of Zip1, the high-affinity zinc transporter in the plasma membrane of C. neoformans. Furthermore, we used the zrc1 mutant in a murine model of cryptococcosis to examine whether a mammalian host could inhibit the survival of C. neoformans using zinc toxicity. We found that the mutant showed no difference in virulence compared with the wildtype strain. This result suggests that Zrc1-mediated zinc detoxification is not required for the virulence of C. neoformans, and imply that zinc toxicity may not be an important aspect of the host immune response to the fungus.

Phenol Oxidase Mediated Protein Cross-Linking

DTIC Science & Technology

1989-06-26

proteins and Fasciola eggshell precursor proteins have their tyrosine residues oxidized to DOPA prior to secretion. We have been trying to determine...reproducibly with "Arnow’s Stain" although Fasciola DOPA protein kindly supplied by Herb Waite stains reproducibly in our hands. NOVEL SILVER STAINING OF

Clinical features of avian vacuolar myelinopathy in American coots

USGS Publications Warehouse

Larsen, R.S.; Nutter, F.B.; Augspurger, T.; Rocke, T.E.; Tomlinson, L.; Thomas, N.J.; Stoskopf, M.K.

2002-01-01

Objectivea??To characterize clinical features of avian vacuolar myelinopathy (AVM) in American coots. Designa??Case-control study. Animalsa??26 AVM-affected American coots and 12 unaffected coots. Proceduresa??Complete physical, neurologic, hematologic, and plasma biochemical evaluations were performed. Affected coots received supportive care. All coots died or were euthanatized, and AVM status was confirmed via histopathologic findings. Resultsa??3 severely affected coots were euthanatized immediately after examination. Seventeen affected coots were found dead within 7 days of admission, but 5 affected coots survived > 21 days and had signs of clinical recovery. Abnormal physical examination findings appeared to be related to general debilitation. Ataxia (88%), decreased withdrawal reflexes (88%), proprioceptive deficits (81%), decreased vent responses (69%), beak or tongue weakness (42%), and head tremors (31%), as well as absent pupillary light responses (46%), anisocoria (15%), apparent blindness (4%), nystagmus (4%), and strabismus (4%) were detected. Few gross abnormalities were detected at necropsy, but histologically, all AVM-affected coots had severe vacuolation of white matter of the brain. None of the control coots had vacuolation. Conclusions and Clinical Relevancea??Although there was considerable variability in form and severity of clinical neurologic abnormalities, clinical signs common in AVM-affected birds were identified. Clinical recovery of some AVM-affected coots can occur when supportive care is administered. Until the etiology is identified, caution should be exercised when rehabilitating and releasing coots thought to be affected by AVM.

Knowns and unknowns of plasma membrane protein degradation in plants.

PubMed

Liu, Chuanliang; Shen, Wenjin; Yang, Chao; Zeng, Lizhang; Gao, Caiji

2018-07-01

Plasma membrane (PM) not only creates a physical barrier to enclose the intracellular compartments but also mediates the direct communication between plants and the ever-changing environment. A tight control of PM protein homeostasis by selective degradation is thus crucial for proper plant development and plant-environment interactions. Accumulated evidences have shown that a number of plant PM proteins undergo clathrin-dependent or membrane microdomain-associated endocytic routes to vacuole for degradation in a cargo-ubiquitination dependent or independent manner. Besides, several trans-acting determinants involved in the regulation of endocytosis, recycling and multivesicular body-mediated vacuolar sorting have been identified in plants. More interestingly, recent findings have uncovered the participation of selective autophagy in PM protein turnover in plants. Although great progresses have been made to identify the PM proteins that undergo dynamic changes in subcellular localizations and to explore the factors that control the membrane protein trafficking, several questions remain to be answered regarding the molecular mechanisms of PM protein degradation in plants. In this short review article, we briefly summarize recent progress in our understanding of the internalization, sorting and degradation of plant PM proteins. More specifically, we focus on discussing the elusive aspects underlying the pathways of PM protein degradation in plants. Copyright © 2018 Elsevier B.V. All rights reserved.

A strategy for targeting recombinant proteins to protein storage vacuoles by fusion to Brassica napus napin in napin-depleted seeds.

PubMed

Hegedus, Dwayne D; Baron, Marcus; Labbe, Natalie; Coutu, Cathy; Lydiate, Derek; Lui, Helen; Rozwadowski, Kevin

2014-03-01

Seeds are capable of accumulating high levels of seed storage proteins (SSP), as well as heterologous proteins under certain conditions. Arabidopsis thaliana was used to develop a strategy to deplete seeds of an endogenous SSP and then replenish them with the same protein fused to a heterologous protein. In several other studies, competition with endogenous SSP for space and metabolic resources was shown to affect the accumulation of recombinant proteins in seeds. We used RNAi to reduce the expression of the five napin genes and deplete the seeds of this SSP. Targeting a recombinant protein to a vacuole or structure within the seed where it can be protected from cytosolic proteases can also promote its accumulation. To achieve this, a synthetic Brassica napus napin gene (Bn napin) was designed that was both impervious to the A. thaliana napin (At napin) RNAi construct and permitted fusion to a heterologous protein, in this case green fluorescent protein (GFP). GFP was placed in several strategic locations within Bn napin with consideration to maintaining structure, processing sites and possible vacuolar targeting signals. In transgenic A. thaliana plants, GFP was strongly localized to the seed protein storage vacuole in all Bn napin fusion configurations tested, but not when expressed alone. This SSP depletion-replenishment strategy outlined here would be applicable to expression of recombinant proteins in industrial crops that generally have large repertoires of endogenous SSP genes. Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.

Amyloid precursor protein modulates Nav1.6 sodium channel currents through a Go-coupled JNK pathway.

PubMed

Li, Shao; Wang, Xi; Ma, Quan-Hong; Yang, Wu-Lin; Zhang, Xiao-Gang; Dawe, Gavin S; Xiao, Zhi-Cheng

2016-12-23

Amyloid precursor protein (APP), commonly associated with Alzheimer's disease, also marks axonal degeneration. In the recent studies, we demonstrated that APP aggregated at nodes of Ranvier (NORs) in myelinated central nervous system (CNS) axons and interacted with Nav1.6. However, the physiological function of APP remains unknown. In this study, we described reduced sodium current densities in APP knockout hippocampal neurons. Coexpression of APP or its intracellular domains containing a VTPEER motif with Na v 1.6 sodium channels in Xenopus oocytes resulted in an increase in peak sodium currents, which was enhanced by constitutively active Go mutant and blocked by a dominant negative mutant. JNK and CDK5 inhibitor attenuated increases in Nav1.6 sodium currents induced by overexpression of APP. Nav1.6 sodium currents were increased by APPT668E (mutant Thr to Glu) and decreased by T668A (mutant Thr to ALa) mutant, respectively. The cell surface expression of Nav1.6 sodium channels in the white matter of spinal cord and the spinal conduction velocity is decreased in APP, p35 and JNK3 knockout mice. Therefore, APP modulates Nav1.6 sodium channels through a Go-coupled JNK pathway, which is dependent on phosphorylation of APP at Thr668.

Amyloid precursor protein modulates Nav1.6 sodium channel currents through a Go-coupled JNK pathway

PubMed Central

Li, Shao; Wang, Xi; Ma, Quan-Hong; Yang, Wu-lin; Zhang, Xiao-Gang; Dawe, Gavin S.; Xiao, Zhi-Cheng

2016-01-01

Amyloid precursor protein (APP), commonly associated with Alzheimer’s disease, also marks axonal degeneration. In the recent studies, we demonstrated that APP aggregated at nodes of Ranvier (NORs) in myelinated central nervous system (CNS) axons and interacted with Nav1.6. However, the physiological function of APP remains unknown. In this study, we described reduced sodium current densities in APP knockout hippocampal neurons. Coexpression of APP or its intracellular domains containing a VTPEER motif with Nav1.6 sodium channels in Xenopus oocytes resulted in an increase in peak sodium currents, which was enhanced by constitutively active Go mutant and blocked by a dominant negative mutant. JNK and CDK5 inhibitor attenuated increases in Nav1.6 sodium currents induced by overexpression of APP. Nav1.6 sodium currents were increased by APPT668E (mutant Thr to Glu) and decreased by T668A (mutant Thr to ALa) mutant, respectively. The cell surface expression of Nav1.6 sodium channels in the white matter of spinal cord and the spinal conduction velocity is decreased in APP, p35 and JNK3 knockout mice. Therefore, APP modulates Nav1.6 sodium channels through a Go-coupled JNK pathway, which is dependent on phosphorylation of APP at Thr668. PMID:28008944

The Kunitz-protease inhibitor domain in amyloid precursor protein reduces cellular mitochondrial enzymes expression and function.

PubMed

Chua, Li-Min; Lim, Mei-Li; Wong, Boon-Seng

2013-08-09

Mitochondrial dysfunction is a prominent feature of Alzheimer's disease (AD) and this can be contributed by aberrant metabolic enzyme function. But, the mechanism causing this enzymatic impairment is unclear. Amyloid precursor protein (APP) is known to be alternatively spliced to produce three major isoforms in the brain (APP695, APP751, APP770). Both APP770 and APP751 contain the Kunitz Protease Inhibitory (KPI) domain, but the former also contain an extra OX-2 domain. APP695 on the other hand, lacks both domains. In AD, up-regulation of the KPI-containing APP isoforms has been reported. But the functional contribution of this elevation is unclear. In the present study, we have expressed and compared the effect of the non-KPI containing APP695 and the KPI-containing APP751 on mitochondrial function. We found that the KPI-containing APP751 significantly decreased the expression of three major mitochondrial metabolic enzymes; citrate synthase, succinate dehydrogenase and cytochrome c oxidase (COX IV). This reduction lowers the NAD(+)/NADH ratio, COX IV activity and mitochondrial membrane potential. Overall, this study demonstrated that up-regulation of the KPI-containing APP isoforms is likely to contribute to the impairment of metabolic enzymes and mitochondrial function in AD. Copyright © 2013 Elsevier Inc. All rights reserved.

Evolution of vacuolar proton pyrophosphatase domains and volutin granules: clues into the early evolutionary origin of the acidocalcisome

PubMed Central

2011-01-01

Background Volutin granules appear to be universally distributed and are morphologically and chemically identical to acidocalcisomes, which are electron-dense granular organelles rich in calcium and phosphate, whose functions include storage of phosphorus and various metal ions, metabolism of polyphosphate, maintenance of intracellular pH, osmoregulation and calcium homeostasis. Prokaryotes are thought to differ from eukaryotes in that they lack membrane-bounded organelles. However, it has been demonstrated that as in acidocalcisomes, the calcium and polyphosphate-rich intracellular "volutin granules (polyphosphate bodies)" in two bacterial species, Agrobacterium tumefaciens, and Rhodospirillum rubrum, are membrane bound and that the vacuolar proton-translocating pyrophosphatases (V-H+PPases) are present in their surrounding membranes. Volutin granules and acidocalcisomes have been found in organisms as diverse as bacteria and humans. Results Here, we show volutin granules also occur in Archaea and are, therefore, present in the three superkingdoms of life (Archaea, Bacteria and Eukarya). Molecular analyses of V-H+PPase pumps, which acidify the acidocalcisome lumen and are diagnostic proteins of the organelle, also reveal the presence of this enzyme in all three superkingdoms suggesting it is ancient and universal. Since V-H+PPase sequences contained limited phylogenetic signal to fully resolve the ancestral nodes of the tree, we investigated the divergence of protein domains in the V-H+PPase molecules. Using Protein family (Pfam) database, we found a domain in the protein, PF03030. The domain is shared by 31 species in Eukarya, 231 in Bacteria, and 17 in Archaea. The universal distribution of the V-H+PPase PF03030 domain, which is associated with the V-H+PPase function, suggests the domain and the enzyme were already present in the Last Universal Common Ancestor (LUCA). Conclusion The importance of the V-H+PPase function and the evolutionary dynamics of these

Glycogen Synthase Kinase 3 Inhibition Promotes Lysosomal Biogenesis and Autophagic Degradation of the Amyloid-β Precursor Protein

PubMed Central

Parr, Callum; Carzaniga, Raffaela; Gentleman, Steve M.; Van Leuven, Fred; Walter, Jochen

2012-01-01

Alzheimer's disease (AD) has been associated with altered activity of glycogen synthase kinase 3 (GSK3) isozymes, which are proposed to contribute to both neurofibrillary tangles and amyloid plaque formation. However, the molecular basis by which GSK3 affects the formation of Aβ remains unknown. Our aim was to identify the underlying mechanisms of GSK3-dependent effects on the processing of amyloid precursor protein (APP). For this purpose, N2a cells stably expressing APP carrying the Swedish mutation were treated with specific GSK3 inhibitors or transfected with GSK3α/β short interfering RNA. We show that inhibition of GSK3 leads to decreased expression of APP by enhancing its degradation via an increase in the number of lysosomes. This induction of the lysosomal/autophagy pathway was associated with nuclear translocation of transcription factor EB (TFEB), a master regulator of lysosomal biogenesis. Our data indicate that GSK3 inhibition reduces Aβ through an increase of the degradation of APP and its carboxy-terminal fragment (CTF) by activation of the lysosomal/autophagy pathway. These results suggest that an increased propensity toward autophagic/lysosomal alterations in AD patients could have consequences for neuronal function. PMID:22927642

Enhanced salt stress tolerance of rice plants expressing a vacuolar H+-ATPase subunit c1 (SaVHAc1) gene from the halophyte grass Spartina alterniflora Löisel

USDA-ARS?s Scientific Manuscript database

The physiological role of a vacuolar ATPase subunit c1 (SaVHAc1) from a halophyte grass Spartina alterniflora was studied through its expression in rice. The SaVHAc1– expressing plants showed enhanced tolerance to salt stress than the wild-type plants, mainly through adjustments in early stage and p...

Synthesis and assembly of retrovirus Gag precursors into immature capsids in vitro.

PubMed Central

Sakalian, M; Parker, S D; Weldon, R A; Hunter, E

1996-01-01

The assembly of retroviral particles is mediated by the product of the gag gene; no other retroviral gene products are necessary for this process. While most retroviruses assemble their capsids at the plasma membrane, viruses of the type D class preassemble immature capsids within the cytoplasm of infected cells. This has allowed us to determine whether immature capsids of the prototypical type D retrovirus, Mason-Pfizer monkey virus (M-PMV), can assemble in a cell-free protein synthesis system. We report here that assembly of M-PMV Gag precursor proteins can occur in this in vitro system. Synthesized particles sediment in isopycnic gradients to the appropriate density and in thin-section electron micrographs have a size and appearance consistent with those of immature retrovirus capsids. The in vitro system described in this report appears to faithfully mimic the process of assembly which occurs in the host cell cytoplasm, since M-PMV gag mutants defective in in vivo assembly also fail to assemble in vitro. Likewise, the Gag precursor proteins of retroviruses that undergo type C morphogenesis, Rous sarcoma virus and human immunodeficiency virus, which do not preassemble capsids in vivo, fail to assemble particles in this system. Additionally, we demonstrate, with the use of anti-Gag antibodies, that this cell-free system can be utilized for analysis in vitro of potential inhibitors of retrovirus assembly. PMID:8648705

Disruption of the vacuolar-type H+-ATPase complex in liver causes MTORC1-independent accumulation of autophagic vacuoles and lysosomes.

PubMed

Kissing, Sandra; Rudnik, Sönke; Damme, Markus; Lüllmann-Rauch, Renate; Ichihara, Atsuhiro; Kornak, Uwe; Eskelinen, Eeva-Liisa; Jabs, Sabrina; Heeren, Jörg; De Brabander, Jef K; Haas, Albert; Saftig, Paul

2017-04-03

The vacuolar-type H + -translocating ATPase (v-H + -ATPase) has been implicated in the amino acid-dependent activation of the mechanistic target of rapamycin complex 1 (MTORC1), an important regulator of macroautophagy. To reveal the mechanistic links between the v-H + -ATPase and MTORC1, we destablilized v-H + -ATPase complexes in mouse liver cells by induced deletion of the essential chaperone ATP6AP2. ATP6AP2-mutants are characterized by massive accumulation of endocytic and autophagic vacuoles in hepatocytes. This cellular phenotype was not caused by a block in endocytic maturation or an impaired acidification. However, the degradation of LC3-II in the knockout hepatocytes appeared to be reduced. When v-H + -ATPase levels were decreased, we observed lysosome association of MTOR and normal signaling of MTORC1 despite an increase in autophagic marker proteins. To better understand why MTORC1 can be active when v-H + -ATPase is depleted, the activation of MTORC1 was analyzed in ATP6AP2-deficient fibroblasts. In these cells, very little amino acid-elicited activation of MTORC1 was observed. In contrast, insulin did induce MTORC1 activation, which still required intracellular amino acid stores. These results suggest that in vivo the regulation of macroautophagy depends not only on v-H + -ATPase-mediated regulation of MTORC1.

The yeast protein kinase Sch9 adjusts V-ATPase assembly/disassembly to control pH homeostasis and longevity in response to glucose availability.

PubMed

Wilms, Tobias; Swinnen, Erwin; Eskes, Elja; Dolz-Edo, Laura; Uwineza, Alice; Van Essche, Ruben; Rosseels, Joëlle; Zabrocki, Piotr; Cameroni, Elisabetta; Franssens, Vanessa; De Virgilio, Claudio; Smits, Gertien J; Winderickx, Joris

2017-06-01

The conserved protein kinase Sch9 is a central player in the nutrient-induced signaling network in yeast, although only few of its direct substrates are known. We now provide evidence that Sch9 controls the vacuolar proton pump (V-ATPase) to maintain cellular pH homeostasis and ageing. A synthetic sick phenotype arises when deletion of SCH9 is combined with a dysfunctional V-ATPase, and the lack of Sch9 has a significant impact on cytosolic pH (pHc) homeostasis. Sch9 physically interacts with, and influences glucose-dependent assembly/disassembly of the V-ATPase, thereby integrating input from TORC1. Moreover, we show that the role of Sch9 in regulating ageing is tightly connected with V-ATPase activity and vacuolar acidity. As both Sch9 and the V-ATPase are highly conserved in higher eukaryotes, it will be interesting to further clarify their cooperative action on the cellular processes that influence growth and ageing.

The Candida albicans stress response gene Stomatin-Like Protein 3 is implicated in ROS-induced apoptotic-like death of yeast phase cells

PubMed Central

Salcedo, Eugenia C.

2018-01-01

The ubiquitous presence of SPFH (Stomatin, Prohibitin, Flotillin, HflK/HflC) proteins in all domains of life suggests that their function would be conserved. However, SPFH functions are diverse with organism-specific attributes. SPFH proteins play critical roles in physiological processes such as mechanosensation and respiration. Here, we characterize the stomatin ORF19.7296/SLP3 in the opportunistic human pathogen Candida albicans. Consistent with the localization of stomatin proteins, a Slp3p-Yfp fusion protein formed visible puncta along the plasma membrane. We also visualized Slp3p within the vacuolar lumen. Slp3p primary sequence analyses identified four putative S-palmitoylation sites, which may facilitate membrane localization and are conserved features of stomatins. Plasma membrane insertion sequences are present in mammalian and nematode SPFH proteins, but are absent in Slp3p. Strikingly, Slp3p was present in yeast cells, but was absent in hyphal cells, thus categorizing it as a yeast-phase specific protein. Slp3p membrane fluorescence significantly increased in response to cellular stress caused by plasma membrane, cell wall, oxidative, or osmotic perturbants, implicating SLP3 as a general stress-response gene. A slp3Δ/Δ homozygous null mutant had no detected phenotype when slp3Δ/Δ mutants were grown in the presence of a variety of stress agents. Also, we did not observe a defect in ion accumulation, filamentation, endocytosis, vacuolar structure and function, cell wall structure, or cytoskeletal structure. However, SLP3 over-expression triggered apoptotic-like death following prolonged exposure to oxidative stress or when cells were induced to form hyphae. Our findings reveal the cellular localization of Slp3p, and for the first time associate Slp3p function with the oxidative stress response. PMID:29389961

Polyimide Precursor Solid Residuum

NASA Technical Reports Server (NTRS)

Weiser, Erik S. (Inventor); St.Clair, Terry L. (Inventor); Echigo, Yoshiaki (Inventor); Kaneshiro, Hisayasu (Inventor)

2001-01-01

A polyimide precursor solid residuum is an admixture of an aromatic dianhydride or derivative thereof and an aromatic diamine or derivative thereof plus a complexing agent, which is complexed with the admixture by hydrogen bonding. The polyimide precursor solid residuum is effectively employed in the preparation of polyimide foam and the fabrication of polyimide foam structures.

Alteration of complex sphingolipid composition and its physiological significance in yeast Saccharomyces cerevisiae lacking vacuolar ATPase.

PubMed

Tani, Motohiro; Toume, Moeko

2015-12-01

In the yeast Saccharomyces cerevisiae, complex sphingolipids have three types of polar head group and five types of ceramide; however, the physiological significance of the structural diversity is not fully understood. Here, we report that deletion of vacuolar H+-ATPase (V-ATPase) in yeast causes dramatic alteration of the complex sphingolipid composition, which includes decreases in hydroxylation at the C-4 position of long-chain bases and the C-2 position of fatty acids in the ceramide moiety, decreases in inositol phosphorylceramide (IPC) levels, and increases in mannosylinositol phosphorylceramide (MIPC) and mannosyldiinositol phosphorylceramide [M(IP)2C] levels. V-ATPase-deleted cells exhibited slow growth at pH 7.2, whereas the increase in MIPC levels was significantly enhanced when V-ATPase-deleted cells were incubated at pH 7.2. The protein expression levels of MIPC and M(IP)2C synthases were significantly increased in V-ATPase-deleted cells incubated at pH 7.2. Loss of MIPC synthesis or an increase in the hydroxylation level of the ceramide moiety of sphingolipids on overexpression of Scs7 and Sur2 sphingolipid hydroxylases enhanced the growth defect of V-ATPase-deleted cells at pH 7.2. On the contrary, the growth rate of V-ATPase-deleted cells was moderately increased on the deletion of SCS7 and SUR2. In addition, supersensitivities to Ca2+, Zn2+ and H2O2, which are typical phenotypes of V-ATPase-deleted cells, were enhanced by the loss of MIPC synthesis. These results indicate the possibility that alteration of the complex sphingolipid composition is an adaptation mechanism for a defect of V-ATPase.

ERG2 and ERG24 Are Required for Normal Vacuolar Physiology as Well as Candida albicans Pathogenicity in a Murine Model of Disseminated but Not Vaginal Candidiasis.

PubMed

Luna-Tapia, Arturo; Peters, Brian M; Eberle, Karen E; Kerns, Morgan E; Foster, Timothy P; Marrero, Luis; Noverr, Mairi C; Fidel, Paul L; Palmer, Glen E

2015-10-01

Several important classes of antifungal agents, including the azoles, act by blocking ergosterol biosynthesis. It was recently reported that the azoles cause massive disruption of the fungal vacuole in the prevalent human pathogen Candida albicans. This is significant because normal vacuolar function is required to support C. albicans pathogenicity. This study examined the impact of the morpholine antifungals, which inhibit later steps of ergosterol biosynthesis, on C. albicans vacuolar integrity. It was found that overexpression of either the ERG2 or ERG24 gene, encoding C-8 sterol isomerase or C-14 sterol reductase, respectively, suppressed C. albicans sensitivity to the morpholines. In addition, both erg2Δ/Δ and erg24Δ/Δ mutants were hypersensitive to the morpholines. These data are consistent with the antifungal activity of the morpholines depending upon the simultaneous inhibition of both Erg2p and Erg24p. The vacuoles within both erg2Δ/Δ and erg24Δ/Δ C. albicans strains exhibited an aberrant morphology and accumulated large quantities of the weak base quinacrine, indicating enhanced vacuolar acidification compared with that of control strains. Both erg mutants exhibited significant defects in polarized hyphal growth and were avirulent in a mouse model of disseminated candidiasis. Surprisingly, in a mouse model of vaginal candidiasis, both mutants colonized mice at high levels and induced a pathogenic response similar to that with the controls. Thus, while targeting Erg2p or Erg24p alone could provide a potentially efficacious therapy for disseminated candidiasis, it may not be an effective strategy to treat vaginal infections. The potential value of drugs targeting these enzymes as adjunctive therapies is discussed. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Plant 14-3-3 proteins assist ion channels and pumps.

PubMed

de Boer, A H

2002-08-01

Turgor pressure is a cellular parameter, important for a range of physiological processes in plants, like cell elongation, gas exchange and gravitropic/phototropic bending. Regulation of turgor pressure involves ion and water transport at the expense of metabolic energy (ATP). The primary pump in the plasma membrane (the H(+)-ATPase) is a key player in turgor regulation since it provides the driving force for ion uptake, followed by water influx through osmosis. Using the phytotoxin fusicoccin (a well-known activator of the ATPase) as a tool, 14-3-3 proteins were identified as regulators of the H(+)-ATPase. Since fusicoccin has a dramatic effect on K(+) accumulation and cellular respiration as well, we studied whether 14-3-3 proteins play a role in the regulation of the mitochondrial F(0)F(1)-ATP synthase and ion channels in the vacuolar and plasma membranes. Besides the plasma membrane H(+)-ATPase, we have identified thus far at least four other transport proteins that are regulated by 14-3-3 proteins. The mechanism of regulation will be described and the possibility that 14-3-3 proteins act as coordinators of ion transporters with varied but interdependent functions will be discussed.

Fetzima (levomilnacipran), a drug for major depressive disorder as a dual inhibitor for human serotonin transporters and beta-site amyloid precursor protein cleaving enzyme-1.

PubMed

Rizvi, Syed Mohd Danish; Shaikh, Sibhghatulla; Khan, Mahiuddin; Biswas, Deboshree; Hameed, Nida; Shakil, Shazi

2014-01-01

Pharmacological management of Major Depressive Disorder includes the use of serotonin reuptake inhibitors which targets serotonin transporters (SERT) to increase the synaptic concentrations of serotonin. Beta-site amyloid precursor protein cleaving enzyme-1 (BACE-1) is responsible for amyloid β plaque formation. Hence it is an interesting target for Alzheimer's disease (AD) therapy. This study describes molecular interactions of a new Food and Drug Administration approved antidepressant drug named 'Fetzima' with BACE-1 and SERT. Fetzima is chemically known as levomilnacipran. The study has explored a possible link between the treatment of Depression and AD. 'Autodock 4.2' was used for docking study. The free energy of binding (ΔG) values for 'levomilnacipran-SERT' interaction and 'levomilnacipran-BACE1' interaction were found to be -7.47 and -8.25 kcal/mol, respectively. Levomilnacipran was found to interact with S438, known to be the most important amino acid residue of serotonin binding site of SERT during 'levomilnacipran-SERT' interaction. In the case of 'levomilnacipran-BACE1' interaction, levomilnacipran interacted with two very crucial aspartic acid residues of BACE-1, namely, D32 and D228. These residues are accountable for the cleavage of amyloid precursor protein and the subsequent formation of amyloid β plaques in AD brain. Hence, Fetzima (levomilnacipran) might act as a potent dual inhibitor of SERT and BACE-1 and expected to form the basis of a future dual therapy against depression and AD. It is an established fact that development of AD is associated with Major Depressive Disorder. Therefore, the design of new BACE-1 inhibitors based on antidepressant drug scaffolds would be particularly beneficial.

Amyloid Precursor Protein Haploinsufficiency Preferentially Mediates Brain Iron Accumulation in Mice Transgenic for The Huntington's Disease Mutation.

PubMed

Berggren, Kiersten; Agrawal, Sonal; Fox, Julia A; Hildenbrand, Justin; Nelson, Ryan; Bush, Ashley I; Fox, Jonathan H

2017-01-01

Huntington's disease (HD) is an autosomal dominant disorder caused by a CAG expansion in the huntingtin gene that results in expression of mutant huntingtin protein. Iron accumulates in HD brain neurons. Amyloid precursor protein (APP) promotes neuronal iron export. However, the role of APP in brain iron accumulation in HD is unclear. To determine the effects of APP insufficiency on HD in YAC128 mice. We crossed APP hemizygous mice (APP+/-) with YAC128 mice that are transgenic (Tg) for human mutant huntingtin (hmHTT) to generate APP+/+ hmHTT-/-, APP+/- hmHTT-/-, APP+/+ hmHTT+/- and APP+/- hmHTT+/- progeny. Mice were evaluated for behavioral, biochemical and neuropathology HD outcomes at 2-12 months of age. APP heterozygosity decreased cortical APP 25% and 60% in non-Tg and Tg mice, respectively. Cerebral and striatal iron levels were increased by APP knockdown in Tg mice only. Nest-building behavior was decreased in Tg mice; APP knockdown decreased nest building in non-Tg but not Tg mice. Rota-rod endurance was decreased in Tg mice. APP+/- hHTT+/- mice demonstrated additional decreases in rota-rod endurance from 4-10 months of age. Tg mice had smaller striatal volumes and fewer striatal neurons but were not affected by APP knockdown. APP heterozygosity results in greater decreases of cortical APP in Tg versus non-Tg mice. Mutant huntingtin transgenic mice develop brain iron accumulation as a result of greater suppression of APP levels. Elevated brain iron in Tg mice was associated with a decline in motor endurance consistent with a disease promoting effect of iron in the YAC128 model of human HD.

Maintaining protein homeostasis: early and late endosomal dual recycling for the maintenance of intracellular pools of the plasma membrane protein Chs3

PubMed Central

Arcones, Irene; Sacristán, Carlos; Roncero, Cesar

2016-01-01

The major chitin synthase activity in yeast cells, Chs3, has become a paradigm in the study of the intracellular traffic of transmembrane proteins due to its tightly regulated trafficking. This includes an efficient mechanism for the maintenance of an extensive reservoir of Chs3 at the trans-Golgi network/EE, which allows for the timely delivery of the protein to the plasma membrane. Here we show that this intracellular reservoir of Chs3 is maintained not only by its efficient AP-1–mediated recycling, but also by recycling through the retromer complex, which interacts with Chs3 at a defined region in its N-terminal cytosolic domain. Moreover, the N-terminal ubiquitination of Chs3 at the plasma membrane by Rsp5/Art4 distinctly labels the protein and regulates its retromer-mediated recycling by enabling Chs3 to be recognized by the ESCRT machinery and degraded in the vacuole. Therefore the combined action of two independent but redundant endocytic recycling mechanisms, together with distinct labels for vacuolar degradation, determines the final fate of the intracellular traffic of the Chs3 protein, allowing yeast cells to regulate morphogenesis, depending on environmental constraints. PMID:27798229