scholarly journals Identification of Phytaspase Interactors via the Proximity-Dependent Biotin-Based Identification Approach

2021 ◽  
Vol 22 (23) ◽  
pp. 13123
Author(s):  
Anastasia D. Teplova ◽  
Marina V. Serebryakova ◽  
Raisa A. Galiullina ◽  
Nina V. Chichkova ◽  
Andrey B. Vartapetian
Keyword(s):  
Serine Proteases ◽  
Proteolytic Enzymes ◽  
Regulatory Protein ◽  
Direct Interaction ◽  
Hydrolytic Activity ◽  
Post Translational Modification ◽  
Enhanced Expression ◽  
Identification Approach ◽  
Regulatory Functions

Proteolytic enzymes are instrumental in various aspects of plant development, including senescence. This may be due not only to their digestive activity, which enables protein utilization, but also to fulfilling regulatory functions. Indeed, for the largest family of plant serine proteases, subtilisin-like proteases (subtilases), several members of which have been implicated in leaf and plant senescence, both non-specific proteolysis and regulatory protein processing have been documented. Here, we strived to identify the protein partners of phytaspase, a plant subtilase involved in stress-induced programmed cell death that possesses a characteristic aspartate-specific hydrolytic activity and unusual localization dynamics. A proximity-dependent biotin identification approach in Nicotiana benthamiana leaves producing phytaspase fused to a non-specific biotin ligase TurboID was employed. Although the TurboID moiety appeared to be unstable in the apoplast environment, several intracellular candidate protein interactors of phytaspase were identified. These were mainly, though not exclusively, represented by soluble residents of the endoplasmic reticulum, namely endoplasmin, BiP, and calreticulin-3. For calreticultin-3, whose gene is characterized by an enhanced expression in senescing leaves, direct interaction with phytaspase was confirmed in an in vitro binding assay using purified proteins. In addition, an apparent alteration of post-translational modification of calreticultin-3 in phytaspase-overproducing plant cells was observed.

Agonist-induced association of tropomyosin with protein kinase Cα in colonic smooth muscle

2005 ◽  
Vol 288 (2) ◽  
pp. G268-G276 ◽  
Author(s):  
Sita Somara ◽  
Haiyan Pang ◽  
Khalil N. Bitar
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Thin Filament ◽  
Regulatory Protein ◽  
Direct Interaction ◽  
Muscle Cells ◽  
Smooth Muscle Contraction ◽  
Particulate Fraction ◽  
Calcium Dependent

Smooth muscle contraction regulated by myosin light chain phosphorylation is also regulated at the thin-filament level. Tropomyosin, a thin-filament regulatory protein, regulates contraction by modulating actin-myosin interactions. Present investigation shows that acetylcholine induces PKC-mediated and calcium-dependent phosphorylation of tropomyosin in colonic smooth muscle cells. Our data also shows that acetylcholine induces a significant and sustained increase in PKC-mediated association of tropomyosin with PKCα in the particulate fraction of colonic smooth muscle cells. Immunoblotting studies revealed that in colonic smooth muscle cells, there is no significant change in the amount of tropomyosin or actin in particulate fraction in response to acetylcholine, indicating that the increased association of tropomyosin with PKCα in the particulate fraction may be due to acetylcholine-induced translocation of PKCα to the particulate fraction. To investigate whether the association of PKCα with tropomyosin was due to a direct interaction, we performed in vitro direct binding assay. Tropomyosin cDNA amplified from colonic smooth muscle mRNA was expressed as GST-tropomyosin fusion protein. In vitro binding experiments using GST-tropomyosin and recombinant PKCα indicated direct interaction of tropomyosin with PKCα. PKC-mediated phosphorylation of tropomyosin and direct interaction of PKCα with tropomyosin suggest that tropomyosin could be a substrate for PKC. Phosphorylation of tropomyosin may aid in holding the slided tropomyosin away from myosin binding sites on actin, resulting in actomyosin interaction and sustained contraction.

scholarly journals Production of proteolytic enzymes in mast cells, fibroblasts, vascular smooth muscle and endothelial cells cultivated under normoxic or hypoxic conditions

2010 ◽  
pp. 711-719 ◽  
Author(s):  
H Maxová ◽  
L Bačáková ◽  
V Lisá ◽  
J Novotná ◽  
H Tomášová ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Mast Cells ◽  
Vascular Smooth Muscle ◽  
Serine Proteases ◽  
Proteolytic Enzymes ◽  
Cell Types ◽  
In Vitro Production ◽  
Hypoxic Conditions

Matrix metalloproteinases (MMPs) is a family of proteolytic enzymes involved in remodeling of extracellular matrix. Although proteolytic enzymes are produced by many cell types, mast cells seem to be more important than other types in remodeling of pulmonary arteries during hypoxia. Therefore, we tested in vitro production of MMPs and serine proteases in four cell types (mast cells, fibroblasts, vascular smooth muscle cells and endothelial cells) cultivated for 48 h under normoxic or hypoxic (3 % O2) conditions. MMP-13 was visualized by immunohistochemistry, MMP-2 and MMP-9 were detected by zymography in cell lysates. Enzymatic activities (MMPs, tryptase and chymase) were estimated in the cultivation media. Hypoxia had a minimal effect on total MMP activity in the cultivation media of all types of cells, but immunofluorescence revealed higher intensity of MMP-13 in the cells exposed to hypoxia except of fibroblasts. Tryptase activity was three times higher and chymase activity twice higher in mast cells cultivated in hypoxia than in those cultured in normoxia. Among all cell types studied here, mast cells are the most abundant source of proteolytic enzymes under normoxic and hypoxic conditions. Moreover, in these cells hypoxia increases the production of both specific serine proteases tryptase and chymase, which can act as MMPs activators.

scholarly journals Serine Protease HtrA1 Associates with Microtubules and Inhibits Cell Migration

2009 ◽  
Vol 29 (15) ◽  
pp. 4177-4187 ◽  
Author(s):  
Jeremy Chien ◽  
Takayo Ota ◽  
Giovanni Aletti ◽  
Ravi Shridhar ◽  
Mariarosaria Boccellino ◽  
...  
Keyword(s):  
High Temperature ◽  
Heat Shock ◽  
Cell Motility ◽  
Serine Proteases ◽  
Microtubule Assembly ◽  
Unfolded Proteins ◽  
Microtubule Stability ◽  
Enhanced Expression

ABSTRACT HtrA1 belongs to a family of serine proteases found in organisms ranging from bacteria to humans. Bacterial HtrA1 (DegP) is a heat shock-induced protein that behaves as a chaperone at low temperature and as a protease at high temperature to help remove unfolded proteins during heat shock. In contrast to bacterial HtrA1, little is known about the function of human HtrA1. Here, we report the first evidence that human HtrA1 is a microtubule-associated protein and modulates microtubule stability and cell motility. Intracellular HtrA1 is localized to microtubules in a PDZ (PSD95, Dlg, ZO1) domain-dependent, nocodazole-sensitive manner. During microtubule assembly, intracellular HtrA associates with centrosomes and newly polymerized microtubules. In vitro, purified HtrA1 promotes microtubule assembly. Moreover, HtrA1 cosediments and copurifies with microtubules. Purified HtrA1 associates with purified α- and β-tubulins, and immunoprecipitation of endogenous HtrA1 results in coprecipitation of α-, β-, and γ-tubulins. Finally, downregulation of HtrA1 promotes cell motility, whereas enhanced expression of HtrA1 attenuates cell motility. These results offer an original identification of HtrA1 as a microtubule-associated protein and provide initial mechanistic insights into the role of HtrA1 in theregulation of cell motility by modulating microtubule stability.

scholarly journals Novel Translational Control through an Iron-Responsive Element by Interaction of Multifunctional Protein YB-1 and IRP2

2002 ◽  
Vol 22 (18) ◽  
pp. 6375-6383 ◽  
Author(s):  
Megumi Ashizuka ◽  
Takao Fukuda ◽  
Takanori Nakamura ◽  
Kanemitsu Shirasuna ◽  
Kazuhiro Iwai ◽  
...  
Keyword(s):  
Translational Control ◽  
Ribosomal Proteins ◽  
Translational Regulation ◽  
Regulatory Protein ◽  
Direct Interaction ◽  
In Vitro Translation ◽  
High Concentration ◽  
Iron Responsive Element ◽  
Responsive Element

ABSTRACT The eukaryotic Y-box-binding protein YB-1 functions in various biological processes, including DNA repair, cell proliferation, and transcriptional and translational controls. To gain further insight into how human YB-1 plays its role in pleiotropic functions, we here used two-hybrid screenings to identify partners of this protein; the results showed that YB-1 itself, iron-regulatory protein 2 (IRP2), and five ribosomal proteins each served as partners to YB-1. We then examined the biological effect of the interaction of YB-1 and IRP2 on translational regulation. Both in vitro binding and coimmunoprecipitation assays showed the direct interaction of YB-1 and IRP2 in the presence of a high concentration of iron. RNA gel shift assays showed that YB-1 reduced the formation of the IRP2-mRNA complex when the iron-responsive element of the ferritin mRNA 5′ untranslated region (UTR) was used as a probe. By using an in vitro translation assay using luciferase mRNA ligated to the ferritin mRNA 5′UTR as a reporter construct, we showed that both YB-1 and IRP2 inhibited the translation of the mRNA. However, coadministration of YB-1 and IRP2 proteins abrogated the inhibition of protein synthesis by each protein. An In vivo coimmunoprecipitation assay showed that IRP2 bound to YB-1 in the presence of iron and a proteasome inhibitor. The direct interaction of YB-1 and IRP2 provides the first evidence of the involvement of YB-1 in the translational regulation of an iron-related protein.

scholarly journals SpoIVB and CtpB Are Both Forespore Signals in the Activation of the Sporulation Transcription Factor σK in Bacillus subtilis

2007 ◽  
Vol 189 (16) ◽  
pp. 6021-6027 ◽  
Author(s):  
Nathalie Campo ◽  
David Z. Rudner
Keyword(s):  
Transcription Factor ◽  
Bacillus Subtilis ◽  
Mother Cell ◽  
Serine Proteases ◽  
Regulatory Protein ◽  
Signal Transduction Pathway ◽  
Proteolytic Activation ◽  
Proper Timing

ABSTRACT The proteolytic activation of the mother cell transcription factor pro-σK is controlled by a signal transduction pathway during sporulation in the bacterium Bacillus subtilis. The pro-σK processing enzyme SpoIVFB, a membrane-embedded metalloprotease, is held inactive by two other integral membrane proteins, SpoIVFA and BofA, in the mother cell membrane that surrounds the forespore. Two signaling serine proteases, SpoIVB and CtpB, trigger pro-σK processing by cleaving the regulatory protein SpoIVFA. The SpoIVB signal is absolutely required to activate pro-σK processing and is derived from the forespore compartment. CtpB is necessary for the proper timing of σK activation and was thought to be a mother cell signal. Here, we show that the ctpB gene is expressed in both the mother cell and forespore compartments but that synthesis in the forespore under the control of σG is both necessary and sufficient for the proper timing of pro-σK processing. We further show that SpoIVB cleaves CtpB in vitro and in vivo but that this cleavage does not appear to be necessary for CtpB activation. Thus, both signaling proteins are made in the forespore and independently target the same regulatory protein.

Proteolysis on the body surface of pyrethroid-sensitive and resistant Varroa destructor

2013 ◽  
Vol 58 (1) ◽  
Author(s):  
Aneta Strachecka ◽  
Grzegorz Borsuk ◽  
Krzysztof Olszewski ◽  
Jerzy Paleolog ◽  
Zbigniew Lipiński
Keyword(s):  
Protease Inhibitor ◽  
Proteolytic Activity ◽  
Body Surface ◽  
Serine Proteases ◽  
Protein Concentration ◽  
Proteolytic Enzymes ◽  
Electrophoretic Analysis ◽  
The Body ◽  
Activity Levels

AbstractThe aim of this work was to determine the activity of proteases and protease inhibitors sampled from the body surface of tau-fluvalinate-sensitive and resistant V. destructor. Proteins were isolated from the tau-fluvalinate-sensitive and resistant mites, while mites untreated with tau-fluvalinate constituted the control. Subsequently, the following methodology was applied: protein concentration assay by the Lowry method — as modified by Schacterle and Pollack; assay of proteolytic activity in relation to various substrates (gelatine, haemoglobin, ovoalbumin, albumin, cytochrome C, casein) by the modified Anson method; identification of proteolytic activity in relation to diagnostic inhibitors of proteolytic enzymes (pepstatin A, PMSF, iodoacetamide, o-phenantrolin), using the Lee and Lin method; identification of acidic, neutral and basic protease activities by means of the modified Anson method; electrophoretic analysis of proteins in a polyacrylamide gel for protease detection with the Laemmli method and for protease inhibitor detection with the Felicioli method. The highest value of protein concentration was found in the tau-fluvalinate-sensitive V. destructor, while the highest activity levels of acidic, neutral and alkaline proteases were observed in the tau-fluvalinate-resistant mites. Aspartic, serine, thiolic and metallic proteases were found in the drug-resistant and drug-sensitive Varroa mites. The control samples were found to contain aspartic and serine proteases. In an acidic and alkaline environment, the results revealed a complete loss of inhibitor activities in the in vitro analyses and electrophoresis. Serine protease inhibitor activities (at pH 7.0) were high, especially in the group of tau-fluvalinate-resistant mites.

scholarly journals A direct interaction between a glutamine-rich activator and the N terminus of TFIIB can mediate transcriptional activation in vivo.

1995 ◽  
Vol 15 (4) ◽  
pp. 2311-2320 ◽  
Author(s):  
J Colgan ◽  
H Ashali ◽  
J L Manley
Keyword(s):  
Zinc Finger ◽  
Transcriptional Activation ◽  
Regulatory Protein ◽  
Direct Interaction ◽  
Mutant Form ◽  
Transcription Factor Iib ◽  
Protein Binding Assay ◽  
N Terminus

Studies examining the mechanism by which transcriptional activators function have suggested that the general transcription factor IIB (TFIIB) can be a target for certain regulatory proteins. For example, we showed previously that expression of a mutant form of TFIIB can specifically inhibit activation in vivo mediated by the strong, glutamine-rich activator protein GAL4-ftzQ. Using transient cotransfection assays, we have defined the regions in both GAL4-ftzQ and TFIIB that are required for activity in vivo and provide evidence that a potential zinc finger structure at the N terminus of TFIIB is necessary for the observed functional interaction between the two proteins. Using a protein binding assay, we have demonstrated that GAL4-ftzQ can specifically interact with TFIIB in vitro. This interaction requires the same regions in both molecules necessary for function in vivo and is reduced or eliminated by mutations predicted to disrupt the zinc finger in TFIIB. These results support the idea that a direct interaction between a regulatory protein and TFIIB can be important for transcriptional activation in vivo and, combined with previous data of others, suggest that different activators can function by contacting distinct regions of TFIIB.

scholarly journals A Root-Knot Nematode Secretory Peptide Functions as a Ligand for a Plant Transcription Factor

2006 ◽  
Vol 19 (5) ◽  
pp. 463-470 ◽  
Author(s):  
Guozhong Huang ◽  
Ruihua Dong ◽  
Rex Allen ◽  
Eric L. Davis ◽  
Thomas J. Baum ◽  
...  
Keyword(s):  
Regulatory Protein ◽  
Direct Interaction ◽  
Giant Cells ◽  
Root Knot Nematode ◽  
Root Cells ◽  
Host Interaction ◽  
Nematode Parasitism ◽  
Parasitism Genes

Parasitism genes expressed in the esophageal gland cells of root-knot nematodes encode proteins that are secreted into host root cells to transform the recipient cells into enlarged multinucleate feeding cells called giant-cells. Expression of a root-knot nematode parasitism gene which encodes a novel 13-amino-acid secretory peptide in plant tissues stimulated root growth. Two SCARECROW-like transcription factors of the GRAS protein family were identified as the putative targets for this bioactive nematode peptide in yeast two-hybrid analyses and confirmed by in vitro and in vivo coimmunoprecipitations. This discovery is the first demonstration of a direct interaction of a nematode-secreted parasitism peptide with a plant-regulatory protein, which may represent an early signaling event in the root-knot nematode-host interaction.

Anti-Diabetic Activity of a Mineraloid Isolate, in vitro and in Genetically Diabetic Mice

2011 ◽  
Vol 81 (1) ◽  
pp. 34-42 ◽  
Author(s):  
Joel Deneau ◽  
Taufeeq Ahmed ◽  
Roger Blotsky ◽  
Krzysztof Bojanowski
Keyword(s):  
Dna Microarrays ◽  
Human Fibroblasts ◽  
Diabetic Animal ◽  
In Vitro Tests ◽  
Diabetic Mice ◽  
Fossil Material ◽  
Expression Of Genes ◽  
Enhanced Expression ◽  
Genetically Diabetic Mice

Type II diabetes is a metabolic disease mediated through multiple molecular pathways. Here, we report anti-diabetic effect of a standardized isolate from a fossil material - a mineraloid leonardite - in in vitro tests and in genetically diabetic mice. The mineraloid isolate stimulated mitochondrial metabolism in human fibroblasts and this stimulation correlated with enhanced expression of genes coding for mitochondrial proteins such as ATP synthases and ribosomal protein precursors, as measured by DNA microarrays. In the diabetic animal model, consumption of the Totala isolate resulted in decreased weight gain, blood glucose, and glycated hemoglobin. To our best knowledge, this is the first description ever of a fossil material having anti-diabetic activity in pre-clinical models.

Defibrotide Inhibits Platelet Activation by Cathepsin G Released From Stimulated Polymorphonuclear Leukocytes

1992 ◽  
Vol 67 (06) ◽  
pp. 660-664 ◽  
Author(s):  
Virgilio Evangelista ◽  
Paola Piccardoni ◽  
Giovanni de Gaetano ◽  
Chiara Cerletti
Keyword(s):  
Platelet Activation ◽  
Polymorphonuclear Leukocytes ◽  
Direct Interaction ◽  
Cathepsin G ◽  
In Vivo Test ◽  
Cell Functions ◽  
Test Models ◽  
Antithrombotic Effects

SummaryDefibrotide is a polydeoxyribonucleotide with antithrombotic effects in experimental animal models. Most of the actions of this drug have been observed in in vivo test models but no effects have been reported in in vitro systems. In this paper we demonstrate that defibrotide interferes with polymorphonuclear leukocyte-induced human platelet activation in vitro. This effect was not related to any direct interaction with polymorphonuclear leukocytes or platelets, but was due to the inhibition of cathepsin G, the main biochemical mediator of this cell-cell cooperation. Since cathepsin G not only induces platelet activation but also affects some endothelial cell functions, the anticathepsin G activity of defibrotide could help to explain the antithrombotic effect of this drug.

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