scholarly journals In vivo and in vitro stimulatory effect of mating pheromone on the endogenous protein phosphorylation in Rhodosporidium toruloides.

1982 ◽  
Vol 46 (8) ◽  
pp. 2179-2181 ◽  
Author(s):  
Tokichi MIYAKAWA ◽  
Hiroyuki MARUYAMA ◽  
Eiko TSUCHIYA ◽  
Sakuzo FUKUI
Keyword(s):  
Protein Phosphorylation ◽  
Rhodosporidium Toruloides ◽  
Mating Pheromone ◽  
Endogenous Protein

scholarly journals DNAJB6b is Downregulated in Synucleinopathies

2021 ◽  
pp. 1-13
Author(s):  
Jonas Folke ◽  
Sertan Arkan ◽  
Isak Martinsson ◽  
Susana Aznar ◽  
Gunnar Gouras ◽  
...  
Keyword(s):  
Neurodegenerative Diseases ◽  
Substantia Nigra Pars Compacta ◽  
Endogenous Protein ◽  
Highly Sensitive ◽  
Brain Material ◽  
Health And Disease ◽  
Isoform B

Background: α-synuclein (α-syn) aggregation contributes to the progression of multiple neurodegenerative diseases. We recently found that the isoform b of the co-chaperone DNAJB6 is a strong suppressor of a-syn aggregation in vivo and in vitro. However, nothing is known about the role of the endogenous isoform b of DNAJB6 (DNAJB6b) in health and disease, due to lack of specific antibodies. Objective: Here we generated a novel anti-DNAJB6b antibody to analyze the localization and expression this isoform in cells, in tissue and in clinical material. Methods: To address this we used immunocytochemistry, immunohistochemistry, as well as a novel quantitative DNAJB6 specific ELISA method. Results: The endogenous protein is mainly expressed in the cytoplasm and in neurites in vitro, where it is found more in dendrites than in axons. We further verified in vivo that DNAJB6b is expressed in the dopaminergic neurons of the substantia nigra pars compacta (SNpc), which is a neuronal subpopulation highly sensitive to α-syn aggregation, that degenerate to a large extend in patients with Parkinson’s disease (PD) and multiple system atrophy (MSA). When we analyzed the expression levels of DNAJB6b in brain material from PD and MSA patients, we found a downregulation of DNAJB6b by use of ELISA based quantification. Interestingly, this was also true when analyzing tissue from patients with progressive supranuclear palsy, a taupathic atypical parkinsonian disorder. However, the total level of DNAJB6 was upregulated in these three diseases, which may indicate an upregulation of the other major isoform of DNAJB6, DNAJB6a. Conclusion: This study shows that DNAJB6b is downregulated in several different neurodegenerative diseases, which makes it an interesting target to further investigate in relation to amyloid protein aggregation and disease progression.

Microtubule-entrained kinase activities associated with the cortical cytoskeleton during cytokinesis

1997 ◽  
Vol 110 (12) ◽  
pp. 1373-1386 ◽  
Author(s):  
G.R. Walker ◽  
C.B. Shuster ◽  
D.R. Burgess
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Protein Phosphorylation ◽  
Immunoblot Analysis ◽  
Negative Regulator ◽  
Mitogen Activated Protein Kinase ◽  
Mitotic Apparatus ◽  
Cortical Cytoskeleton

Research over the past few years has demonstrated the central role of protein phosphorylation in regulating mitosis and the cell cycle. However, little is known about how the mechanisms regulating the entry into mitosis contribute to the positional and temporal regulation of the actomyosin-based contractile ring formed during cytokinesis. Recent studies implicate p34cdc2 as a negative regulator of myosin II activity, suggesting a link between the mitotic cycle and cytokinesis. In an effort to study the relationship between protein phosphorylation and cytokinesis, we examined the in vivo and in vitro phosphorylation of actin-associated cortical cytoskeletal (CSK) proteins in an isolated model of the sea urchin egg cortex. Examination of cortices derived from eggs or zygotes labeled with 32P-orthophosphate reveals a number of cortex-associated phosphorylated proteins, including polypeptides of 20, 43 and 66 kDa. These three major phosphoproteins are also detected when isolated cortices are incubated with [32P]ATP in vitro, suggesting that the kinases that phosphorylate these substrates are also specifically associated with the cortex. The kinase activities in vivo and in vitro are stimulated by fertilization and display cell cycle-dependent activities. Gel autophosphorylation assays, kinase assays and immunoblot analysis reveal the presence of p34cdc2 as well as members of the mitogen-activated protein kinase family, whose activities in the CSK peak at cell division. Nocodazole, which inhibits microtubule formation and thus blocks cytokinesis, significantly delays the time of peak cortical protein phosphorylation as well as the peak in whole-cell histone H1 kinase activity. These results suggest that a key element regulating cortical contraction during cytokinesis is the timing of protein kinase activities associated with the cortical cytoskeleton that is in turn regulated by the mitotic apparatus.

scholarly journals Glycosyl-phosphatidylinositol anchor attachment in a yeast in vitro system

1997 ◽  
Vol 328 (2) ◽  
pp. 669-675 ◽  
Author(s):  
L. Tamara DOERING ◽  
Randy SCHEKMAN
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fusion Proteins ◽  
Attachment Site ◽  
Gpi Anchor ◽  
Mating Pheromone ◽  
Peptide Substrates ◽  
In Vitro System ◽  
Glycosyl Phosphatidylinositol

The yeast mating pheromone precursor prepro-alpha factor was fused to C-terminal signals for glycosyl-phosphatidylinositol (GPI) anchor attachment, based on the sequence of the Saccharomyces cerevisiae protein Gas1p. Maturation of fusion proteins expressed in vivo required the presence of both a functional GPI attachment site and the synthesis of GPI precursors. Constructs were translated in vitro for use in cell-free studies of glycolipid attachment. The radiolabelled polypeptides were post-translationally translocated into yeast microsomes, where at least one third of the molecules received a GPI anchor. This approach offers distinct advantages over anchor attachment reactions that require co-translational translocation of secretory peptide substrates.

scholarly journals Biochemical studies of the excitable membrane of Paramecium tetraurelia VI. Endogenous protein substrates for in vitro and in vivo phosphorylation in cilia and ciliary membranes.

1981 ◽  
Vol 91 (1) ◽  
pp. 167-174 ◽  
Author(s):  
R M Lewis ◽  
D L Nelson
Keyword(s):  
Protein Kinase ◽  
Cyclic Gmp ◽  
Excitable Membrane ◽  
Protein Kinase Activity ◽  
Paramecium Tetraurelia ◽  
Phosphoprotein Phosphatase ◽  
Endogenous Protein ◽  
Endogenous Substrates

The endogenous protein kinases of isolated Paramecium tetraurelia cilia phosphorylated approximately 30 ciliary polypeptides in vitro. Labeling with [gamma-32P]ATP was not proportional to the amount of each protein in cilia; some minor polypeptides (e.g., 67,000 and 180,000 mol wt) were more heavily labeled than some major polypeptides. Certain of the endogenous substrates for protein kinase were localized in the ciliary membrane (130,000, 86,000, 67,000, and 45,000 mol wt); others were found in axonemes or in both fractions. With cilia from bacterized cultures in the undefined Cerophyl medium, the labeling of specific endogenous phosphate acceptors was altered by pH, cyclic AMP, and cyclic GMP, but the labeling pattern was not affected by the presence of Na+ or K+ (15 mM), Ba++ (5 mM), Ca++ (10(-5) or 10(-4) M), or EGTA. Very similar results were obtained with cilia from cells grown axenically in a semidefined medium; the molecular weights and the extent of phosphorylation of the phosphopolypeptides were comparable to those of cilia from bacterized Cerophyl cultures, although no significant cyclic nucleotide effects were observed in the axenic cilia. Most of the phosphopolypeptides labeled in vitro also turned over rapidly in vitro. The phosphoprotein phosphatase responsible for turnover was partially inhibited by 5 mM NaF. The pattern of ciliary polypeptides labeled in vivo was similar to that observed in the in vitro experiments, although the relative intensities of labeling differed. Six behavioral mutants of Paramecium, known to have defects in the excitable membrane that regulates the ciliary beat, showed normal patterns of ciliary protein phosphorylation in vitro, with and without added cyclic nucleotides, at both pH 6.0 and pH 8.0. The mutants also had apparently normal phosphoprotein phosphatase. The Paranoiac A mutant, however, showed a reduction in cyclic GMP-stimulated protein kinase activity.

In Vitro and in Vivo Neutralization of Murine Activated Protein C

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3364-3364
Author(s):  
Laurent Burnier ◽  
Jose A. Fernandez ◽  
John H. Griffin
Keyword(s):  
Active Site ◽  
Protein C ◽  
Activated Protein C ◽  
Activity Level ◽  
Protective Effects ◽  
Wild Type ◽  
Amidolytic Activity ◽  
Endogenous Protein

Abstract Abstract 3364 Activated Protein C (APC) is a circulating serine protease with two major roles to maintain homeostasis. APC acts via multiple receptors, including protease-activated receptor 1, to exert anti-apoptotic and vascular integrity protective effects. A number of protective effects of pharmacologic APC are reported in the literature, with beneficial effects in kidney, brain and irradiation-induced pathologies. The functional protections of the endogenous protein C systems are challenging to study. A better understanding of its mechanisms at different cellular levels and in different tissues is needed to enable evaluation of its further usage in humans. To that end, new tools should be considered to increase our knowledge. To help evaluate the endogenous murine protein C system and to be able to neutralize pharmacologic APC, we have made and characterized a novel rat monoclonal anti-mouse protein C antibody, SPC-54, that almost completely ablates in vitro and in vivo APC activity. In solid phase binding assays, the Kd of SPC-54 for APC was about 8 nM. In biochemical assays, SPC-54 inhibited amidolytic activity of wild-type murine APC by > 95%. SPC-54 was similarly a potent inhibitor (> 90%) of the amidolytic activity of the 5A-APC mutant. IC50 value for wild-type APC and the 5A-APC mutant were comparable. SPC-54 was pre-incubated with APC, followed by the addition of a 20 fold molar excess of biotinylated FPR-chloromethylketone, quantification of biotinylation of APC was readily made by SDS-PAGE and Western blots using infrared-coupled streptavidin. SPC-54 blocked successfully active site titration of APC using this biotinylated active site titrant. These and other experiments suggest that the SPC-54 epitope is located in the vicinity of the active site, such that it blocks different small substrates from reaching the active site. When we performed thrombin generation assays using mouse platelet-poor plasma to check whether SPC-54 was a potent blocker of APC activity in plasma, we showed that SPC-54 neutralized almost completely exogenous APC anticoagulant activity in a dose-dependent manner. Using native polyacrylamide gel migration, Western immunoblotting and immuno-precipitation with protein G-agarose, we confirmed that SPC-54 was bound to protein C in plasma after infusing mice with SPC-54 (10 mg/kg). Moreover, using a modified ELISA that is capable to quantify the pool of activatable protein C, the plasma protein C activity level was considerably decreased (> 80%) in mice after a single injection of SPC-54 (10 mg/kg), and that this effect of neutralizing circulating protein C was sustained for at least 7 days. For in vivo proof of concept, we performed murine tissue factor-induced thromboembolism experiments. Results showed a severe decrease in survival of mice that were pre-infused with SPC-54 when compared to control (survival time of 7 min vs. 42.5 min respectively, P = 0.0016). Moreover, blood perfusion in lungs of mice infused with SPC-54 (10 mg/kg) was dramatically impaired (decrease of 54%, P < 0.0001) as revealed by infrared quantification of Evans Blue dye as marker of vascular perfusion. We also used endotoxemia murine models to assess effects of SPC-54. SPC-54 decreased survival after endotoxin challenge (25 mg/kg, LD50 dose) in mice infused with SPC-54 (10 mg/kg) at 7 hours after LPS. Mortality was 100% after 36 h in the SPC-54 group, whereas controls, which received either boiled SPC-54 antibodies or PBS vehicle, showed a mortality of about 50% (P < 0.001). In summary, SPC-54 is a potent rat monoclonal antibody that neutralizes murine APC activities in vitro and in vivo. Its characteristic ability to dampen the endogenous protein C/APC system is of value to understand better the role of the endogenous protein C system in murine injury models and also to neutralize pharmacologic murine APC. Disclosures: No relevant conflicts of interest to declare.

Protein phosphorylation at tyrosine is induced by the v-erbB gene product in vivo and in vitro

Cell ◽  
1985 ◽  
Vol 40 (3) ◽  
pp. 609-618 ◽  
Author(s):  
Thomas Gilmore ◽  
Jeffrey E. Declue ◽  
G.Steven Martin
Keyword(s):  
Protein Phosphorylation ◽  
Gene Product

scholarly journals Multiple genes coding for precursors of rhodotorucine A, a farnesyl peptide mating pheromone of the basidiomycetous yeast Rhodosporidium toruloides.

1989 ◽  
Vol 9 (8) ◽  
pp. 3491-3498 ◽  
Author(s):  
R Akada ◽  
K Minomi ◽  
J Kai ◽  
I Yamashita ◽  
T Miyakawa ◽  
...  
Keyword(s):  
Tandem Repeats ◽  
Rhodosporidium Toruloides ◽  
Basidiomycetous Yeast ◽  
Peptide Sequence ◽  
In Vitro Translation ◽  
Mating Pheromone ◽  
S1 Nuclease ◽  
Amino Terminal ◽  
Carboxy Terminal

Haploid cells of mating type A of the basidiomycetous yeast Rhodosporidium toruloides secrete a mating pheromone, rhodotorucine A, which is an undecapeptide containing S-farnesyl cysteine at its carboxy terminus. To analyze the processing and secretion pathway of rhodotorucine A, we isolated both genomic and complementary DNAs encoding the peptide moiety. We identified three distinct genes, RHA1, RHA2, and RHA3, encoding four, five, and three copies of the pheromone peptide, respectively. Complementary DNA clones were classified into two types. One type was homologous to RHA1, and the other type was homologous to RHA2. Transcription start sites were identified by primer extension and S1 nuclease protection, from which the site of the initiator methionine was verified. A primary precursor of rhodotorucine A was detected as a 7-kilodalton protein by immunoprecipitation of in vitro translation products. On the basis of these results, we propose similar three-precursor structures of rhodotorucine A, each containing the amino-terminal peptide sequence Met-Val-Ala. The precursors contain three, four, or five tandem repeats of the pheromone peptide, each separated by a spacer peptide, Thr-Val-Ser(Ala)-Lys, and each precursor has the carboxy-terminal sequence Thr-Val-Ala. This structure suggests that primary precursors of rhodotorucine A do not contain canonical signal sequences.

Inhibition of Ty1 transposition by mating pheromones in Saccharomyces cerevisiae

1991 ◽  
Vol 11 (5) ◽  
pp. 2736-2743
Author(s):  
H Xu ◽  
J D Boeke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Yeast Cells ◽  
Mating Pheromone ◽  
Yeast Saccharomyces Cerevisiae ◽  
Transposition Frequency ◽  
Mating Pheromones ◽  
Retroviral Replication ◽  
Viruslike Particles

The Ty1 elements in the yeast Saccharomyces cerevisiae are a family of retrotransposons which transpose via a process similar to that of retroviral replication. We report here that the Ty1 transposition process can be blocked posttranscriptionally by treatment of cells with mating pheromones. When haploid yeast cells are treated with appropriate mating pheromones, the transposition frequency of a marked Ty1 element driven by the GAL1 promoter is greatly diminished. Ty1 viruslike particles (VLPs), the putative intermediates for transposition, can be isolated from mating pheromone-treated cells. These VLPs accumulate to normal levels but are aberrant in that they produce very few reverse transcripts of Ty1 RNA both in vivo and in vitro and contain subnormal amounts of p90-TYB and related proteins. In addition, a TYA phosphoprotein product accumulates in treated cells, and some species of TYB proteins have decreased stability. We also show that decreased transposition in mating pheromone-treated cells is not a consequence of simply blocking cell division, since Ty1 transposes at a nearly normal rate in yeast cells arrested in G2 by the drug nocodazole.

scholarly journals Inhibition of Ty1 transposition by mating pheromones in Saccharomyces cerevisiae.

1991 ◽  
Vol 11 (5) ◽  
pp. 2736-2743 ◽  
Author(s):  
H Xu ◽  
J D Boeke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Yeast Cells ◽  
Mating Pheromone ◽  
Yeast Saccharomyces Cerevisiae ◽  
Transposition Frequency ◽  
Mating Pheromones ◽  
Retroviral Replication ◽  
Viruslike Particles

The Ty1 elements in the yeast Saccharomyces cerevisiae are a family of retrotransposons which transpose via a process similar to that of retroviral replication. We report here that the Ty1 transposition process can be blocked posttranscriptionally by treatment of cells with mating pheromones. When haploid yeast cells are treated with appropriate mating pheromones, the transposition frequency of a marked Ty1 element driven by the GAL1 promoter is greatly diminished. Ty1 viruslike particles (VLPs), the putative intermediates for transposition, can be isolated from mating pheromone-treated cells. These VLPs accumulate to normal levels but are aberrant in that they produce very few reverse transcripts of Ty1 RNA both in vivo and in vitro and contain subnormal amounts of p90-TYB and related proteins. In addition, a TYA phosphoprotein product accumulates in treated cells, and some species of TYB proteins have decreased stability. We also show that decreased transposition in mating pheromone-treated cells is not a consequence of simply blocking cell division, since Ty1 transposes at a nearly normal rate in yeast cells arrested in G2 by the drug nocodazole.

Role for ICAT in β-catenin-dependent nuclear signaling and cadherin functions

2004 ◽  
Vol 286 (4) ◽  
pp. C747-C756 ◽  
Author(s):  
Cara J. Gottardi ◽  
Barry M. Gumbiner
Keyword(s):  
Cultured Cells ◽  
Mdck Cells ◽  
Primary Role ◽  
Protein Levels ◽  
Nuclear Signaling ◽  
Endogenous Protein ◽  
Hepatocyte Growth Factor Treatment

Inhibitor of β-catenin and TCF-4 (ICAT) is a 9-kDa polypeptide that inhibits β-catenin nuclear signaling by binding β-catenin and competing its interaction with the transcription factor TCF (T cell factor), but basic characterization of the endogenous protein and degree to which it alters other β-catenin functions is less well understood. At the subcellular level, we show that ICAT localizes to both cytoplasmic and nuclear compartments. In intestinal tissue, ICAT is upregulated in the mature, nondividing enterocyte population lining intestinal villi and is absent in the β-catenin/TCF signaling-active crypt region, suggesting that its protein levels may be inversely related with β-catenin signaling activity. However, ICAT protein levels are not altered by activation or inhibition of Wnt signaling in cultured cells, suggesting that ICAT expression is not a direct target of the Wnt/β-catenin pathway. In cells where β-catenin levels are elevated by Wnt, a fraction of this β-catenin pool is associated with ICAT, suggesting that ICAT may buffer the cell from increased levels of β-catenin. Distinct from TCF and cadherin, ICAT does not protect the soluble pool of β-catenin from degradation by the adenomatous polyposis coli containing “destruction complex.” Although ICAT inhibits β-catenin binding to the cadherin as well as TCF in vitro, stable overexpression of ICAT in Madin-Darby canine kidney (MDCK) epithelial cells shows no obvious alterations in the cadherin complex, suggesting that the ability of ICAT to inhibit β-catenin binding to the cadherin may be restricted in vivo. MDCK cells overexpressing ICAT do, however, exhibit enhanced cell scattering on hepatocyte growth factor treatment, suggesting a possible role in the regulation of dynamic rather than steady-state cell-cell adhesions. These findings confirm ICAT's primary role in β-catenin signaling inhibition and further suggest that ICAT may have consequences for cadherin-based adhesive function in certain circumstances, implying a broader role than previously described.

Sign in / Sign up

Export Citation Format

Share Document