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1.
T A Yacoubian S R Slone A J Harrington S Hamamichi J M Schieltz K A Caldwell G A Caldwell D G Standaert 《Cell death & disease》2010,1(1):e2
14-3-3 proteins are important negative regulators of cell death pathways. Recent studies have revealed alterations in 14-3-3s in Parkinson''s disease (PD) and the ability of 14-3-3s to interact with α-synuclein (α-syn), a protein central to PD pathophysiology. In a transgenic α-syn mouse model, we found reduced expression of 14-3-3θ, -ɛ, and -γ. These same isoforms prevent α-syn inclusion formation in an H4 neuroglioma cell model. Using dopaminergic cell lines stably overexpressing each 14-3-3 isoform, we found that overexpression of 14-3-3θ, -ɛ, or -γ led to resistance to both rotenone and 1-methyl-4-phenylpyridinium, whereas other isoforms were not protective against both toxins. Inhibition of a single protective isoform, 14-3-3θ, by shRNA did not increase vulnerability to neurotoxic injury, but toxicity was enhanced by broad-based inhibition of 14-3-3 action with the peptide inhibitor difopein. Using a transgenic C. elegans model of PD, we confirmed the ability of both human 14-3-3θ and a C. elegans 14-3-3 homologue (ftt-2) to protect dopaminergic neurons from α-syn toxicity. Collectively, these data show a strong neuroprotective effect of enhanced 14-3-3 expression – particularly of the 14-3-3θ, -ɛ, and -γ isoforms – in multiple cellular and animal models of PD, and point to the potential value of these proteins in the development of neuroprotective therapies for human PD. 相似文献
2.
Organelles serve as biochemical reactors in the cell, and often display characteristic scaling trends with cell size, suggesting mechanisms that coordinate their sizes. In this study, we measure the vacuole-cell size scaling trends in budding yeast using optical microscopy and a novel, to our knowledge, image analysis algorithm. Vacuole volume and surface area both show characteristic scaling trends with respect to cell size that are consistent among different strains. Rapamycin treatment was found to increase vacuole-cell size scaling trends for both volume and surface area. Unexpectedly, these increases did not depend on macroautophagy, as similar increases in vacuole size were observed in the autophagy deficient mutants atg1Δ and atg5Δ. Rather, rapamycin appears to act on vacuole size by inhibiting retrograde membrane trafficking, as the atg18Δ mutant, which is defective in retrograde trafficking, shows similar vacuole size scaling to rapamycin-treated cells and is itself insensitive to rapamycin treatment. Disruption of anterograde membrane trafficking in the apl5Δ mutant leads to complementary changes in vacuole size scaling. These quantitative results lead to a simple model for vacuole size scaling based on proportionality between cell growth rates and vacuole growth rates. 相似文献
3.
4.
Background
In plants, 14-3-3 proteins are encoded by a large multigene family and are involved in signaling pathways to regulate plant development and protection from stress. Although twelve Populus 14-3-3s were identified based on the Populus trichocarpa genome V1.1 in a previous study, no systematic analysis including genome organization, gene structure, duplication relationship, evolutionary analysis and expression compendium has been conducted in Populus based on the latest P. trichocarpa genome V3.0.Principal Findings
Here, a comprehensive analysis of Populus 14-3-3 family is presented. Two new 14-3-3 genes were identified based on the latest P. trichocarpa genome. In P. trichocarpa, fourteen 14-3-3 genes were grouped into ε and non-ε group. Exon-intron organizations of Populus 14-3-3s are highly conserved within the same group. Genomic organization analysis indicated that purifying selection plays a pivotal role in the retention and maintenance of Populus 14-3-3 family. Protein conformational analysis indicated that Populus 14-3-3 consists of a bundle of nine α-helices (α1-α9); the first four are essential for formation of the dimer, while α3, α5, α7, and α9 form a conserved peptide-binding groove. In addition, α1, α3, α5, α7, and α9 were evolving at a lower rate, while α2, α4, and α6 were evolving at a relatively faster rate. Microarray analyses showed that most Populus 14-3-3s are differentially expressed across tissues and upon exposure to various stresses.Conclusions
The gene structures and their coding protein structures of Populus 14-3-3s are highly conserved among group members, suggesting that members of the same group might also have conserved functions. Microarray and qRT-PCR analyses showed that most Populus 14-3-3s were differentially expressed in various tissues and were induced by various stresses. Our investigation provided a better understanding of the complexity of the 14-3-3 gene family in poplars. 相似文献5.
?yvind Halskau Jr. Ming Ying Anne Baumann Rune Kleppe David Rodriguez-Larrea Bj?rg Alm?s Jan Haavik Aurora Martinez 《The Journal of biological chemistry》2009,284(47):32758-32769
Tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of catecholamines, is activated by phosphorylation-dependent binding to 14-3-3 proteins. The N-terminal domain of TH is also involved in interaction with lipid membranes. We investigated the binding of the N-terminal domain to its different partners, both in the unphosphorylated (TH-(1–43)) and Ser19-phosphorylated (THp-(1–43)) states by surface plasmon resonance. THp-(1–43) showed high affinity for 14-3-3 proteins (Kd ∼ 0.5 μm for 14-3-3γ and -ζ and 7 μm for 14-3-3η). The domains also bind to negatively charged membranes with intermediate affinity (concentration at half-maximal binding S0.5 = 25–58 μm (TH-(1–43)) and S0.5 = 135–475 μm (THp-(1–43)), depending on phospholipid composition) and concomitant formation of helical structure. 14-3-3γ showed a preferential binding to membranes, compared with 14-3-3ζ, both in chromaffin granules and with liposomes at neutral pH. The affinity of 14-3-3γ for negatively charged membranes (S0.5 = 1–9 μm) is much higher than the affinity of TH for the same membranes, compatible with the formation of a ternary complex between Ser19-phosphorylated TH, 14-3-3γ, and membranes. Our results shed light on interaction mechanisms that might be relevant for the modulation of the distribution of TH in the cytoplasm and membrane fractions and regulation of l-DOPA and dopamine synthesis. 相似文献
6.
Long Nam Nguyen Gabriele Vargas Cesar Giang Thi Thu Le David L. Silver Leonardo Nimrichter Joshua D. Nosanchuk 《PLoS pathogens》2012,8(8)
We have recently observed that a fatty acid auxotrophic mutant (fatty acid synthase, Fas2Δ/Δ) of the emerging human pathogenic yeast Candida parapsilosis dies after incubation in various media including serum. In the present study we describe the mechanism for cell death induced by serum and glucose containing media. We show that Fas2Δ/Δ yeast cells are profoundly susceptible to glucose leading us to propose that yeast cells lacking fatty acids exhibit uncontrolled metabolism in response to glucose. We demonstrate that incubation of Fas2Δ/Δ yeast cells with serum leads to cell death, and this process can be prevented with inhibition of protein or DNA synthesis, indicating that newly synthesized cellular components are detrimental to the mutant cells. Furthermore, we have found that cell death is mediated by mitochondria. Suppression of electron transport enzymes using inhibitors such as cyanide or azide prevents ROS overproduction and Fas2Δ/Δ yeast cell death. Additionally, deletion of mitochondrial DNA, which encodes several subunits for enzymes of the electron transport chain, significantly reduces serum-induced Fas2Δ/Δ yeast cell death. Therefore, our results show that serum and glucose media induce Fas2Δ/Δ yeast cell death by triggering unbalanced metabolism, which is regulated by mitochondria. To our knowledge, this is the first study to critically define a link between cytosolic fatty acid synthesis and mitochondrial function in response to serum stress in C. parapsilosis. 相似文献
7.
de Medina-Redondo M Arnáiz-Pita Y Clavaud C Fontaine T del Rey F Latgé JP Vázquez de Aldana CR 《PloS one》2010,5(11):e14046
Background
The formation of the cell wall in Schizosaccharomyces pombe requires the coordinated activity of enzymes involved in the biosynthesis and modification of β-glucans. The β(1,3)-glucan synthase complex synthesizes linear β(1,3)-glucans, which remain unorganized until they are cross-linked to other β(1,3)-glucans and other cell wall components. Transferases of the GH72 family play important roles in cell wall assembly and its rearrangement in Saccharomyces cerevisiae and Aspergillus fumigatus. Four genes encoding β(1,3)-glucanosyl-transferases -gas1+, gas2+, gas4+ and gas5+- are present in S. pombe, although their function has not been analyzed.Methodology/Principal Findings
Here, we report the characterization of the catalytic activity of gas1p, gas2p and gas5p together with studies directed to understand their function during vegetative growth. From the functional point of view, gas1p is essential for cell integrity and viability during vegetative growth, since gas1Δ mutants can only grow in osmotically supported media, while gas2p and gas5p play a minor role in cell wall construction. From the biochemical point of view, all of them display β(1,3)-glucanosyl-transferase activity, although they differ in their specificity for substrate length, cleavage point and product size. In light of all the above, together with the differences in expression profiles during the life cycle, the S. pombe GH72 proteins may accomplish complementary, non-overlapping functions in fission yeast.Conclusions/Significance
We conclude that β(1,3)-glucanosyl-transferase activity is essential for viability in fission yeast, being required to maintain cell integrity during vegetative growth. 相似文献8.
9.
Ivan Kadurin Anita Alvarez-Laviada Shu Fun Josephine Ng Ryan Walker-Gray Marianna D'Arco Michael G. Fadel Wendy S. Pratt Annette C. Dolphin 《The Journal of biological chemistry》2012,287(40):33554-33566
The accessory α2δ subunits of voltage-gated calcium channels are membrane-anchored proteins, which are highly glycosylated, possess multiple disulfide bonds, and are post-translationally cleaved into α2 and δ. All α2δ subunits have a C-terminal hydrophobic, potentially trans-membrane domain and were described as type I transmembrane proteins, but we found evidence that they can be glycosylphosphatidylinositol-anchored. To probe further the function of membrane anchoring in α2δ subunits, we have now examined the properties of α2δ-1 constructs truncated at their putative glycosylphosphatidylinositol anchor site, located before the C-terminal hydrophobic domain (α2δ-1ΔC-term). We find that the majority of α2δ-1ΔC-term is soluble and secreted into the medium, but unexpectedly, some of the protein remains associated with detergent-resistant membranes, also termed lipid rafts, and is extrinsically bound to the plasma membrane. Furthermore, heterologous co-expression of α2δ-1ΔC-term with CaV2.1/β1b results in a substantial enhancement of the calcium channel currents, albeit less than that produced by wild-type α2δ-1. These results call into question the role of membrane anchoring of α2δ subunits for calcium current enhancement. 相似文献
10.
Roderick A. M. Williams Jeremy C. Mottram Graham H. Coombs 《The Journal of biological chemistry》2013,288(5):3678-3690
Macroautophagy in Leishmania, which is important for the cellular remodeling required during differentiation, relies upon the hydrolytic activity of two ATG4 cysteine peptidases (ATG4.1 and ATG4.2). We have investigated the individual contributions of each ATG4 to Leishmania major by generating individual gene deletion mutants (Δatg4.1 and Δatg4.2); double mutants could not be generated, indicating that ATG4 activity is required for parasite viability. Both mutants were viable as promastigotes and infected macrophages in vitro and mice, but Δatg4.2 survived poorly irrespective of infection with promastigotes or amastigotes, whereas this was the case only when promastigotes of Δatg4.1 were used. Promastigotes of Δatg4.2 but not Δatg4.1 were more susceptible than wild type promastigotes to starvation and oxidative stresses, which correlated with increased reactive oxygen species levels and oxidatively damaged proteins in the cells as well as impaired mitochondrial function. The antioxidant N-acetylcysteine reversed this phenotype, reducing both basal and induced autophagy and restoring mitochondrial function, indicating a relationship between reactive oxygen species levels and autophagy. Deletion of ATG4.2 had a more dramatic effect upon autophagy than did deletion of ATG4.1. This phenotype is consistent with a reduced efficiency in the autophagic process in Δatg4.2, possibly due to ATG4.2 having a key role in removal of ATG8 from mature autophagosomes and thus facilitating delivery to the lysosomal network. These findings show that there is a level of functional redundancy between the two ATG4s, and that ATG4.2 appears to be the more important. Moreover, the low infectivity of Δatg4.2 demonstrates that autophagy is important for the virulence of the parasite. 相似文献
11.
N van Oosterwijk J Knol L Dijkhuizen R van der Geize BW Dijkstra 《The Journal of biological chemistry》2012,287(37):30975-30983
3-Ketosteroid Δ4-(5α)-dehydrogenases (Δ4-(5α)-KSTDs) are enzymes that introduce a double bond between the C4 and C5 atoms of 3-keto-(5α)-steroids. Here we show that the ro05698 gene from Rhodococcus jostii RHA1 codes for a flavoprotein with Δ4-(5α)-KSTD activity. The 1.6 Å resolution crystal structure of the enzyme revealed three conserved residues (Tyr-319, Tyr-466, and Ser-468) in a pocket near the isoalloxazine ring system of the FAD co-factor. Site-directed mutagenesis of these residues confirmed that they are absolutely essential for catalytic activity. A crystal structure with bound product 4-androstene-3,17-dione showed that Ser-468 is in a position in which it can serve as the base abstracting the 4β-proton from the C4 atom of the substrate. Ser-468 is assisted by Tyr-319, which possibly is involved in shuttling the proton to the solvent. Tyr-466 is at hydrogen bonding distance to the C3 oxygen atom of the substrate and can stabilize the keto-enol intermediate occurring during the reaction. Finally, the FAD N5 atom is in a position to be able to abstract the 5α-hydrogen of the substrate as a hydride ion. These features fully explain the reaction catalyzed by Δ4-(5α)-KSTDs. 相似文献
12.
Hydrolysis of Casein-Derived Peptides αS1-Casein(f1-9) and β-Casein(f193-209) by Lactobacillus helveticus Peptidase Deletion Mutants Indicates the Presence of a Previously Undetected Endopeptidase
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Jeffrey E. Christensen Jeffery R. Broadbent James L. Steele 《Applied microbiology》2003,69(2):1283-1286
Peptides derived from hydrolysis of αS1-casein(f1-9) [αS1-CN(f1-9)] and β-CN(f193-209) with cell extracts of Lactobacillus helveticus CNRZ32 and single-peptidase mutants (ΔpepC, ΔpepE, ΔpepN, ΔpepO, and ΔpepX) were isolated by using reverse-phase high-performance liquid chromatography and were characterized by mass spectrometry. The peptides identified suggest that there was activity of an endopeptidase, distinct from previously identified endopeptidases (PepE and PepO), with specificity for peptide bonds C terminal to Pro residues. Identification of hydrolysis products derived from a carboxyl-blocked form of β-CN(f193-209) confirmed that the peptides were derived from the activity of an endopeptidase. 相似文献
13.
Involvement of Acyl Coenzyme A Oxidase Isozymes in Biotransformation of Methyl Ricinoleate into γ-Decalactone by Yarrowia lipolytica
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Yves Wach Cline Laroche Karin Bergmark Charlotte Mller-Andersen Mario Aguedo Marie-Thrse Le Dall Huijie Wang Jean-Marc Nicaud Jean-Marc Belin 《Applied microbiology》2000,66(3):1233-1236
We reported previously on the function of acyl coenzyme A (acyl-CoA) oxidase isozymes in the yeast Yarrowia lipolytica by investigating strains disrupted in one or several acyl-CoA oxidase-encoding genes (POX1 through POX5) (H. Wang et al., J. Bacteriol. 181:5140–5148, 1999). Here, these mutants were studied for lactone production. Monodisrupted strains produced similar levels of lactone as the wild-type strain (50 mg/liter) except for Δpox3, which produced 220 mg of γ-decalactone per liter after 24 h. The Δpox2 Δpox3 double-disrupted strain, although slightly affected in growth, produced about 150 mg of lactone per liter, indicating that Aox2p was not essential for the biotransformation. The Δpox2 Δpox3 Δpox5 triple-disrupted strain produced and consumed lactone very slowly. On the contrary, the Δpox2 Δpox3 Δpox4 Δpox5 multidisrupted strain did not grow or biotransform methyl ricinoleate into γ-decalactone, demonstrating that Aox4p is essential for the biotransformation. 相似文献
14.
Zhang Y Liu L Wu X An X Stubbe J Huang M 《The Journal of biological chemistry》2011,286(48):41499-41509
The β2 subunit of class Ia ribonucleotide reductase (RNR) contains a diferric tyrosyl radical cofactor (Fe2III-Tyr•) that is essential for nucleotide reduction. The β2 subunit of Saccharomyces cerevisiae is a heterodimer of Rnr2 (β) and Rnr4 (β′). Although only β is capable of iron binding and Tyr• formation, cells lacking β′ are either dead or exhibit extremely low Tyr• levels and RNR activity depending on genetic backgrounds. Here, we present evidence supporting the model that β′ is required for iron loading and Tyr• formation in β in vivo via a pathway that is likely dependent on the cytosolic monothiol glutaredoxins Grx3/Grx4 and the Fe-S cluster protein Dre2. rnr4 mutants are defective in iron loading into nascent β and are hypersensitive to iron depletion and the Tyr•-reducing agent hydroxyurea. Transient induction of β′ in a GalRNR4 strain leads to a concomitant increase in iron loading and Tyr• levels in β. Tyr• can also be rapidly generated using endogenous iron when permeabilized Δrnr4 spheroplasts are supplemented with recombinant β′ and is inhibited by adding an iron chelator prior to, but not after, β′ supplementation. The growth defects of rnr4 mutants are enhanced by deficiencies in grx3/grx4 and dre2. Moreover, depletion of Dre2 in GalDRE2 cells leads to a decrease in both Tyr• levels and ββ′ activity. This result, in combination with previous findings that a low level of Grx3/4 impairs RNR function, strongly suggests that Grx3/4 and Dre2 serve in the assembly of the deferric Tyr• cofactor in RNR. 相似文献
15.
The p53 family member p63 has been shown to be critical for growth, proliferation and chemosensitivity. Here we demonstrate that the c-Abl tyrosine kinase phosphorylates the widely expressed ΔNp63α isoform and identify multiple sites by mass spectrometry in vitro and in vivo. Phopshorylation by c-Abl results in greater protein stability of both ectopically expressed and endogenous ΔNp63α. c-Abl phosphorylation of ΔNp63α induces its binding to Yes-associated protein (YAP) and silencing of YAP by siRNA reduces the c-Abl-induced increase of ΔNp63α levels. We further show that cisplatin induces c-Abl phosphorylation of ΔNp63α and its binding to YAP. Overexpression of ΔNp63α, but not the c-Abl phosphosites mutant, protects cells from cisplatin treatment. Finally, we demonstrate the rescue of p63 siRNA-mediated loss of viability with p63siRNA insensitive construct of ΔNp63α but not the phosphosites mutant. These results demonstrate that c-Abl phosphorylation of ΔNp63α regulates its protein stability, by inducing binding of YAP, and is critical for cell viability. 相似文献
16.
Emma F. Barry Fernando A. Felquer Jason A. Powell Lisa Biggs Frank C. Stomski Andrea Urbani Hayley Ramshaw Peter Hoffmann Matthew C. Wilce Michele A. Grimbaldeston Angel F. Lopez Mark A. Guthridge 《The Journal of biological chemistry》2009,284(18):12080-12090
Integrated cascades of protein tyrosine and serine/threonine
phosphorylation play essential roles in transducing signals in response to
growth factors and cytokines. How adaptor or scaffold proteins assemble
signaling complexes through both phosphotyrosine and phosphoserine/threonine
residues to regulate specific signaling pathways and biological responses is
unclear. We show in multiple cell types that endogenous 14-3-3ζ is
phosphorylated on Tyr179 in response to granulocyte macrophage
colony-stimulating factor. Importantly, 14-3-3ζ can function as an
intermolecular bridge that couples to phosphoserine residues and also directly
binds the SH2 domain of Shc via Tyr179. The assembly of these
14-3-3:Shc scaffolds is specifically required for the recruitment of a
phosphatidylinositol 3-kinase signaling complex and the regulation of CTL-EN
cell survival in response to cytokine. The biological significance of these
findings was further demonstrated using primary bone marrow-derived mast cells
from 14-3-3ζ-/- mice. We show that cytokine was able to
promote Akt phosphorylation and viability of primary mast cells derived from
14-3-3ζ-/- mice when reconstituted with wild type
14-3-3ζ, but the Akt phosphorylation and survival response was reduced in
cells reconstituted with the Y179F mutant. Together, these results show that
14-3-3:Shc scaffolds can act as multivalent signaling nodes for the
integration of both phosphoserine/threonine and phosphotyrosine pathways to
regulate specific cellular responses.The ability of a cell to respond to extrinsic stimuli critically hinges on
its ability to regulate specific intracellular protein-protein interactions in
a reversible manner. Such signals are relayed within the cell through the
assembly of signaling complexes that are built using protein scaffolds. One
important mechanism by which this occurs is via the binding of Src homology 2
(SH2)5 or
phosphotyrosine-binding (PTB) domains to phosphotyrosine residues
(1,
2). Importantly, the ability of
individual SH2 or PTB domains to recognize specific phosphotyrosine motifs in
different proteins enables the assembly of purpose-built signaling complexes
that promote signaling via specific pathways
(3). In some cases, signaling
proteins not only contain more than one SH2 and/or PTB domain but are also
themselves tyrosine-phosphorylated, leading to a network of
phosphotyrosine-mediated protein-protein interactions.Although less well studied, phosphoserine/threonine-binding proteins are
also important for the assembly of signaling complexes. For example, the
14-3-3 family of proteins is able to bind phosphoserine/threonine residues in
a sequence-specific context (RSX(S/T)XP and
RXXX(S/T)XP, where (S/T) is phosphoserine/threonine)
(4,
5). The 14-3-3 proteins have
been proposed to function as “modifiers” or
“sequestrators”; however, because of their dimeric structure, they
have also been proposed to function as “adaptor” or
“scaffold” proteins through their ability to bring together two
serine/threonine phosphorylated proteins
(4–7).
Additionally, a number of phosphoserine/threonine-binding modules such as
tryptophan-tryptophan (WW), Forkhead-associated (FHA), Polo box (PBD), and
BRCA1 C-terminal (BRCT) domains have been shown to interact with
phosphoserine/threonine residues within a sequence-specific context and have
also been proposed to be important for the assembly of multi-protein signaling
complexes (8).The genes/cassettes encoding each phosphotyrosine- and
phosphoserine/threonine-binding protein/module arose as a separate
evolutionary event, and the DNA encoding these modules has been subject to
frequent duplication and shuffling. For example, the 14-3-3 family of proteins
is ubiquitously expressed in mammalian tissues and is composed of seven
different isoforms, each encoded by a separate gene
(6). In addition, duplication
and shuffling of SH2, PTB, WW, FHA, PBD, and BCRT cassettes has led to their
wide distribution among signaling proteins. Yet, despite the frequent
duplication and shuffling of the DNA encoding these domains throughout
evolution, proteins that contain both a phosphotyrosine-binding cassette
(e.g. SH2 or PTB) and a phosphoserine/threonine-binding cassette
(e.g. 14-3-3, WW, FHA, PBD, and BCRT) have not been identified. This
is perhaps surprising given the highly integrated nature of phosphotyrosine
and phosphoserine/threonine signaling and would suggest that alternative
strategies to regulate integration are at play.We show here that 14-3-3ζ is tyrosine-phosphorylated, enabling it to
interact with Shc and provide a scaffold for the assembly of signaling
complexes via both phosphoserine/threonine and phosphotyrosine residues. Our
results show that Tyr179 of 14-3-3ζ directly binds to the SH2
domain of Shc and that this interaction is critical for the assembly of a
phosphatidylinositol (PI) 3-kinase signaling complex in response to
granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation.
Moreover, we show that Tyr179 of 14-3-3ζ is necessary and
sufficient for the ability of GM-CSF to regulate PI 3-kinase and cell survival
in the CTL-EN line. Furthermore, reconstitution of primary mast cells derived
from 14-3-3ζ-/- mice with wild type (wt) or mutant
14-3-3ζ demonstrated an important role for Tyr179 in
cytokine-mediated Akt phosphorylation and cell survival. These multivalent
14-3-3:Shc scaffolds provide a novel mechanism by which
phosphoserine/threonine and phosphotyrosine pathways can be integrated for the
regulation of specific cellular responses. 相似文献
17.
Okamoto Y Shirakashi Y Ihara M Urushitani M Oono M Kawamoto Y Yamashita H Shimohama S Kato S Hirano A Tomimoto H Ito H Takahashi R 《PloS one》2011,6(5):e20427
Background and Purpose
Cu/Zn superoxide dismutase (SOD1) is a major component of Lewy body-like hyaline inclusion (LBHI) found in the postmortem tissue of SOD1-linked familial amyotrophic lateral sclerosis (FALS) patients. In our recent studies, 14-3-3 proteins have been found in the ubiquitinated inclusions inside the anterior horn cells of spinal cords with sporadic amyotrophic lateral sclerosis (ALS). To further investigate the role of 14-3-3 proteins in ALS, we performed immunohistochemical analysis of 14-3-3 proteins and compared their distributions with those of SOD1 in FALS patients and SOD1-overexpressing mice.Methods
We examined the postmortem brains and the spinal cords of three FALS cases (A4V SOD1 mutant). Transgenic mice expressing the G93A mutant human SOD1 (mutant SOD1-Tg mice), transgenic mice expressing the wild-type human SOD1 (wild-type SOD1-Tg mice), and non-Tg wild-type mice were also subjected to the immunohistochemical analysis.Results
In all the FALS patients, LBHIs were observed in the cytoplasm of the anterior horn cells, and these inclusions were immunopositive intensely for pan 14-3-3, 14-3-3β, and 14-3-3γ. In the mutant SOD1-Tg mice, a high degree of immunoreactivity for misfolded SOD1 (C4F6) was observed in the cytoplasm, with an even greater degree of immunoreactivity present in the cytoplasmic aggregates of the anterior horn cells in the lumbar spinal cord. Furthermore, we have found increased 14-3-3β and 14-3-3γ immunoreactivities in the mutant SOD1-Tg mice. Double immunofluorescent staining showed that C4F6 and 14-3-3 proteins were partially co-localized in the spinal cord with FALS and the mutant SOD1-Tg mice. In comparison, the wild-type SOD1-Tg and non-Tg wild-type mice showed no or faint immunoreactivity for C4F6 and 14-3-3 proteins (pan 14-3-3, 14-3-3β, and 14-3-3γ) in any neuronal compartments.Discussion
These results suggest that 14-3-3 proteins may be associated with the formation of SOD1-containing inclusions, in FALS patients and the mutant SOD1-Tg mice. 相似文献18.
Autophagy targets cytoplasmic cargo to a lytic compartment for degradation. Autophagy-related (Atg) proteins, including the transmembrane protein Atg9, are involved in different steps of autophagy in yeast and mammalian cells. Functional classification of core Atg proteins in plants has not been clearly confirmed, partly because of the limited availability of reliable assays for monitoring autophagic flux. By using proUBQ10-GFP-ATG8a as an autophagic marker, we showed that autophagic flux is reduced but not completely compromised in Arabidopsis thaliana atg9 mutants. In contrast, we confirmed full inhibition of auto-phagic flux in atg7 and that the difference in autophagy was consistent with the differences in mutant phenotypes such as hypersensitivity to nutrient stress and selective autophagy. Autophagic flux is also reduced by an inhibitor of phosphatidylinositol kinase. Our data indicated that atg9 is phenotypically distinct from atg7 and atg2 in Arabidopsis, and we proposed that ATG9 and phosphatidylinositol kinase activity contribute to efficient autophagy in Arabidopsis. 相似文献
19.
Background
Autophagy is a basic cellular homeostatic process important to cell fate decisions under conditions of stress. Dysregulation of autophagy impacts numerous human diseases including cancer and chronic obstructive lung disease. This study investigates the role of autophagy in idiopathic pulmonary fibrosis.Methods
Human lung tissues from patients with IPF were analyzed for autophagy markers and modulating proteins using western blotting, confocal microscopy and transmission electron microscopy. To study the effects of TGF-β1 on autophagy, human lung fibroblasts were monitored by fluorescence microscopy and western blotting. In vivo experiments were done using the bleomycin-induced fibrosis mouse model.Results
Lung tissues from IPF patients demonstrate evidence of decreased autophagic activity as assessed by LC3, p62 protein expression and immunofluorescence, and numbers of autophagosomes. TGF-β1 inhibits autophagy in fibroblasts in vitro at least in part via activation of mTORC1; expression of TIGAR is also increased in response to TGF-β1. In the bleomycin model of pulmonary fibrosis, rapamycin treatment is antifibrotic, and rapamycin also decreases expression of á-smooth muscle actin and fibronectin by fibroblasts in vitro. Inhibition of key regulators of autophagy, LC3 and beclin-1, leads to the opposite effect on fibroblast expression of á-smooth muscle actin and fibronectin.Conclusion
Autophagy is not induced in pulmonary fibrosis despite activation of pathways known to promote autophagy. Impairment of autophagy by TGF-β1 may represent a mechanism for the promotion of fibrogenesis in IPF. 相似文献20.
John H. Northrop 《The Journal of general physiology》1964,48(1):73-78
Lambda coli phage is not inactivated by chymotrypsin, trypsin, or ficin. T2 phage is slowly inactivated by high concentrations of (α-, β-, γ-, or Δ-chymotrypsin, but not by trypsin or ficin. P1 phage is slowly inactivated by α-, β-, or γ-chymotrypsin, or ficin, more rapidly by Δ-chymotrypsin, and much more rapidly by trypsin. Crystalline egg albumin, crystalline serum albumin, E. coli nucleoprotein, and yeast nucleoprotein are hydrolyzed slowly by α-chymotrypsin. Yeast nucleoprotein, like P1 phage, is hydrolyzed more rapidly by Δ-chymotrypsin than by α-chymotrypsin, but not by trypsin or ficin. Neither phages nor native proteins were attacked by papain, carboxypeptidase, deoxyribonuclease, or ribonuclease. 相似文献