The CBL-interacting protein kinase CIPK26 is a novel interactor of Arabidopsis NADPH oxidase AtRbohF that negatively modulates its ROS-producing activity in a heterologous expression system

2012 ◽  
Vol 153 (2) ◽  
pp. 191-195 ◽  
Author(s):  
S. Kimura ◽  
T. Kawarazaki ◽  
H. Nibori ◽  
M. Michikawa ◽  
A. Imai ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nadph Oxidase ◽  
Heterologous Expression ◽  
Expression System ◽  
Heterologous Expression System ◽  
Interacting Protein

scholarly journals Reconstitution of β-adrenergic regulation of CaV1.2: Rad-dependent and Rad-independent protein kinase A mechanisms

2020 ◽  
Author(s):  
Moshe Katz ◽  
Suraj Subramaniam ◽  
Orna Chomsky-Hecht ◽  
Vladimir Tsemakhovich ◽  
Anouar Belkacemi ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Heterologous Expression ◽  
Protein Kinase A ◽  
Calcium Influx ◽  
Expression System ◽  
Β Subunit ◽  
Heterologous Expression System ◽  
Adrenergic Regulation ◽  
Adrenergic Modulation ◽  
Kinase A

AbstractIntroductionCardiac L-type voltage-gated CaV1.2 channels are crucial in physiological regulation of cardiac excitation-contraction coupling. Adrenergic modulation of CaV1.2 starts with activation of β-adrenergic receptors (AR) and culminates in protein kinase A (PKA) - induced increase of calcium influx through CaV1.2 channels. To date, this cascade has never been fully reconstituted in heterologous systems; even partial reconstitution proved challenging and controversial. A recent study identified Rad, a calcium channel inhibitory protein, as an essential component of the adrenergic signaling cascade. We corroborated this finding, further characterized, and fully reconstituted, the complete β-AR CaV1.2 modulation cascade in a heterologous expression system.ObjectiveOur primary goal was to heterologously reconstitute the complete β-adrenergic cascade, and to investigate the role of Rad and additional molecular determinants in adrenergic regulation of cardiac CaV1.2.Methods and ResultsWe utilized the Xenopus oocyte heterologous expression system. We expressed CaV1.2 channel subunits, without or with Rad and β1-AR or β2-AR. To activate PKA, we injected cyclic AMP (cAMP) into the oocytes, or extracellularly applied isoproterenol (Iso) to stimulate β-AR. Whole-cell Ba2+ currents served as readout. We find and distinguish between two distinct pathways of PKA modulation of CaV1.2: Rad-dependent (~80% of total) and Rad-independent. We separate the two mechanisms by showing distinct requirements for the cytosolic N- and distal C- termini of α1C and for the CaVβ subunit. Finally, for the first time, we reconstitute the complete pathway using agonist activation of either β1-AR or β2-AR. The reconstituted system reproduces the known features of β-AR regulation in cardiomyocytes, such as a >2-fold increase in CaV1.2 current, a hyperpolarizing shift in activation curve, and a high constitutive activity of β2-AR.ConclusionsThe adrenergic modulation of CaV1.2 is composed of two distinct pathways, Rad-independent and Rad-dependent. The latter contributes most of the β-AR-induced enhancement of CaV1.2 activity, crucially depends on CaVβ subunit, and is differently regulated by β1-AR and β2-AR. The reconstitution of the full β-AR cascade provides the means to address central unresolved issues related to roles of auxiliary proteins in the cascade, CaV1.2 isoforms, and will help to develop therapies for catecholamine-induced cardiac pathologies.

scholarly journals Characterization of Leader Processing Shows That Partially Processed Mersacidin Is Activated by AprE After Export

2021 ◽  
Vol 12 ◽  
Author(s):  
Jakob H. Viel ◽  
Amanda Y. van Tilburg ◽  
Oscar P. Kuipers
Keyword(s):  
Antimicrobial Activity ◽  
Synthetic Biology ◽  
Heterologous Expression ◽  
Expression System ◽  
Heterologous Expression System ◽  
Proteolytic Activation ◽  
E Coli ◽  
Gram Positive Bacteria ◽  
Precursor Peptide ◽  
Potential Applications

The ribosomally synthesized and post-translationally modified peptide mersacidin is a class II lanthipeptide with good activity against Gram-positive bacteria. The intramolecular lanthionine rings, that give mersacidin its stability and antimicrobial activity, are specific structures with potential applications in synthetic biology. To add the mersacidin modification enzymes to the synthetic biology toolbox, a heterologous expression system for mersacidin in Escherichia coli has recently been developed. While this system was able to produce fully modified mersacidin precursor peptide that could be activated by Bacillus amyloliquefaciens supernatant and showed that mersacidin was activated in an additional proteolytic step after transportation out of the cell, it lacked a mechanism for clean and straightforward leader processing. Here, the protease responsible for activating mersacidin was identified and heterologously produced in E. coli, improving the previously reported heterologous expression system. By screening multiple proteases, the stringency of proteolytic activity directly next to a very small lanthionine ring is demonstrated, and the full two-step proteolytic activation of mersacidin was elucidated. Additionally, the effect of partial leader processing on diffusion and antimicrobial activity is assessed, shedding light on the function of two-step leader processing.

scholarly journals TheNitrosopumilus maritimusCdvB, but Not FtsZ, Assembles into Polymers

Archaea ◽  
2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
Kian-Hong Ng ◽  
Vinayaka Srinivas ◽  
Ramanujam Srinivasan ◽  
Mohan Balasubramanian
Keyword(s):  
Cell Division ◽  
Heterologous Expression ◽  
Fission Yeast ◽  
Mammalian Cells ◽  
Expression System ◽  
Related Protein ◽  
Heterologous Expression System

Euryarchaeota and Crenarchaeota are two major phyla of archaea which use distinct molecular apparatuses for cell division. Euryarchaea make use of the tubulin-related protein FtsZ, while Crenarchaea, which appear to lack functional FtsZ, employ the Cdv (cell division) components to divide. Ammonia oxidizing archaeon (AOA)Nitrosopumilus maritimusbelongs to another archaeal phylum, the Thaumarchaeota, which has both FtsZ and Cdv genes in the genome. Here, we used a heterologous expression system to characterize FtsZ and Cdv proteins fromN. maritimusby investigating the ability of these proteins to form polymers. We show that one of the Cdv proteins inN. maritimus, the CdvB (Nmar_0816), is capable of forming stable polymers when expressed in fission yeast. TheN. maritimusCdvB is also capable of assembling into filaments in mammalian cells. However,N. maritimusFtsZ does not assemble into polymers in our system. The ability of CdvB, but not FtsZ, to polymerize is consistent with a recent finding showing that several Cdv proteins, but not FtsZ, localize to the mid-cell site in the dividingN. maritimus. Thus, we propose that it is Cdv proteins, rather than FtsZ, that function as the cell division apparatus inN. maritimus.

scholarly journals Assembly of Methyl Coenzyme M Reductase in the Methanogenic ArchaeonMethanococcus maripaludis

2018 ◽  
Vol 200 (7) ◽  
Author(s):  
Zhe Lyu ◽  
Chau-Wen Chou ◽  
Hao Shi ◽  
Liangliang Wang ◽  
Robel Ghebreab ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Methane Production ◽  
Active Sites ◽  
Net Primary Production ◽  
Expression System ◽  
Anaerobic Oxidation Of Methane ◽  
Coenzyme M ◽  
Heterologous Expression System ◽  
Content Type ◽  
Methyl Coenzyme M Reductase

ABSTRACTMethyl coenzyme M reductase (MCR) is a complex enzyme that catalyzes the final step in biological methanogenesis. To better understand its assembly, the recombinant MCR from the thermophileMethanothermococcus okinawensis(rMCRok) was expressed in the mesophileMethanococcus maripaludis. The rMCRokwas posttranslationally modified correctly and contained McrD and the unique nickel tetrapyrrole coenzyme F430. Subunits of the nativeM. maripaludis(MCRmar) were largely absent, suggesting that the recombinant enzyme was formed by an assembly of cotranscribed subunits. Strong support for this hypothesis was obtained by expressing a chimeric operon comprising the His-taggedmcrAfromM. maripaludisand themcrBDCGfromM. okinawensisinM. maripaludis. The His-tagged purified rMCR then contained theM. maripaludisMcrA and theM. okinawensisMcrBDG. The present study prompted us to form a working model for MCR assembly, which can be further tested by the heterologous expression system established here.IMPORTANCEApproximately 1.6% of the net primary production of plants, algae, and cyanobacteria are processed by biological methane production in anoxic environments. This accounts for about 74% of the total global methane production, up to 25% of which is consumed by anaerobic oxidation of methane (AOM). Methyl coenzyme M reductase (MCR) is the key enzyme in both methanogenesis and AOM. MCR is assembled as a dimer of two heterotrimers, where posttranslational modifications and F430cofactors are embedded in the active sites. However, this complex assembly process remains unknown. Here, we established a heterologous expression system for MCR to learn how MCR is assembled.

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