scholarly journals Identification of Binding Proteins for TSC22D1 Family Proteins Using Mass Spectrometry

2021 ◽  
Vol 22 (20) ◽  
pp. 10913
Author(s):  
Ryouta Kamimura ◽  
Daisuke Uchida ◽  
Shin-ichiro Kanno ◽  
Ryo Shiraishi ◽  
Toshiki Hyodo ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Amino Acids ◽  
Binding Proteins ◽  
Guanine Nucleotide ◽  
Binding Assays ◽  
Guanine Nucleotide Binding Protein ◽  
Cell Systems ◽  
Guanine Nucleotide Binding

TSC-22 (TGF-β stimulated clone-22) has been reported to induce differentiation, growth inhibition, and apoptosis in various cells. TSC-22 is a member of a family in which many proteins are produced from four different family genes. TSC-22 (corresponding to TSC22D1-2) is composed of 144 amino acids translated from a short variant mRNA of the TSC22D1 gene. In this study, we attempted to determine the intracellular localizations of the TSC22D1 family proteins (TSC22D1-1, TSC-22 (TSC22D1-2), and TSC22(86) (TSC22D1-3)) and identify the binding proteins for TSC22D1 family proteins by mass spectrometry. We determined that TSC22D1-1 was mostly localized in the nucleus, TSC-22 (TSC22D1-2) was localized in the cytoplasm, mainly in the mitochondria and translocated from the cytoplasm to the nucleus after DNA damage, and TSC22(86) (TSC22D1-3) was localized in both the cytoplasm and nucleus. We identified multiple candidates of binding proteins for TSC22D1 family proteins in in vitro pull-down assays and in vivo binding assays. Histone H1 bound to TSC-22 (TSC22D1-2) or TSC22(86) (TSC22D1-3) in the nucleus. Guanine nucleotide-binding protein-like 3 (GNL3), which is also known as nucleostemin, bound to TSC-22 (TSC22D1-2) in the nucleus. Further investigation of the interaction of the candidate binding proteins with TSC22D1 family proteins would clarify the biological roles of TSC22D1 family proteins in several cell systems.

scholarly journals Multiplatform Physiologic and Metabolic Phenotyping Reveals Microbial Toxicity

mSystems ◽  
2018 ◽  
Vol 3 (6) ◽  
Author(s):  
Jingwei Cai ◽  
Robert G. Nichols ◽  
Imhoi Koo ◽  
Zachary A. Kalikow ◽  
Limin Zhang ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Amino Acids ◽  
Flow Cytometry ◽  
Free Radical ◽  
Gut Microbiota ◽  
Structure And Function ◽  
Metabolic Phenotyping ◽  
And Function

ABSTRACTThe gut microbiota is susceptible to modulation by environmental stimuli and therefore can serve as a biological sensor. Recent evidence suggests that xenobiotics can disrupt the interaction between the microbiota and host. Here, we describe an approach that combinesin vitromicrobial incubation (isolated cecal contents from mice), flow cytometry, and mass spectrometry- and1H nuclear magnetic resonance (NMR)-based metabolomics to evaluate xenobiotic-induced microbial toxicity. Tempol, a stabilized free radical scavenger known to remodel the microbial community structure and functionin vivo, was studied to assess its direct effect on the gut microbiota. The microbiota was isolated from mouse cecum and was exposed to tempol for 4 h under strict anaerobic conditions. The flow cytometry data suggested that short-term tempol exposure to the microbiota is associated with disrupted membrane physiology as well as compromised metabolic activity. Mass spectrometry and NMR metabolomics revealed that tempol exposure significantly disrupted microbial metabolic activity, specifically indicated by changes in short-chain fatty acids, branched-chain amino acids, amino acids, nucleotides, glucose, and oligosaccharides. In addition, a mouse study with tempol (5 days gavage) showed similar microbial physiologic and metabolic changes, indicating that thein vitroapproach reflectedin vivoconditions. Our results, through evaluation of microbial viability, physiology, and metabolism and a comparison ofin vitroandin vivoexposures with tempol, suggest that physiologic and metabolic phenotyping can provide unique insight into gut microbiota toxicity.IMPORTANCEThe gut microbiota is modulated physiologically, compositionally, and metabolically by xenobiotics, potentially causing metabolic consequences to the host. We recently reported that tempol, a stabilized free radical nitroxide, can exert beneficial effects on the host through modulation of the microbiome community structure and function. Here, we investigated a multiplatform phenotyping approach that combines high-throughput global metabolomics with flow cytometry to evaluate the direct effect of tempol on the microbiota. This approach may be useful in deciphering how other xenobiotics directly influence the microbiota.

Tonic inhibition of renal response to vasopressin by a pertussis toxin substrate

1988 ◽  
Vol 255 (6) ◽  
pp. F1107-F1115
Author(s):  
W. B. Jeffries ◽  
R. Fallet ◽  
G. D. Gong ◽  
P. Van Dreal ◽  
W. A. Pettinger
Keyword(s):  
Camp Accumulation ◽  
Guanine Nucleotide Binding Protein ◽  
Renal Response ◽  
Collecting Tubule ◽  
Adenylate Cyclase Stimulation ◽  
V2 Receptor ◽  
Guanine Nucleotide Binding

The putative role of the inhibitory guanine nucleotide binding protein (Gi) in modulating the renal response to vasopressin was investigated using islet activating protein (IAP). IAP treatment in rats in vivo abolished the capacity of alpha 2-adrenoceptors to reverse vasopressin-induced adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in microdissected cortical collecting tubule (CCT) segments. IAP pretreatment also caused a marked upward shift in the dose-response curve of vasopressin (10(-10) to 10(-4) M)-induced cAMP accumulation. Augmentation of the response to vasopressin in rat CCT was dependent on the in vivo dose of IAP and paralleled the loss in alpha 2-adrenoceptor responsiveness. In the isolated perfused kidney the antinatriuretic and antidiuretic effects of the V2-receptor agonist desamino-8-D-arginine vasopressin (DDAVP) (1 pM) were enhanced following IAP pretreatment. alpha 2-Adrenoceptor stimulation (30 nM epinephrine) inhibited the renal effects of DDAVP (1 pM) in kidneys from control but not IAP-pretreated rats. Interestingly, IAP pretreatment alone caused increased urine flow rate and enhanced excretion of sodium and chloride without affecting potassium excretion or renal hemodynamics in vitro. Our results suggest that an IAP substrate, probably Gi, 1) is required for signal transduction by renal alpha 2-adrenoceptors, 2) may tonically modulate the response to vasopressin in the CCT but not of parathyroid hormone in the proximal convoluted tubule, and 3) participates in renal water and electrolyte reabsorption independent of exogenous adenylate cyclase stimulation.

scholarly journals Gαi1and Gαi3regulate macrophage polarization by forming a complex containing CD14 and Gab1

2015 ◽  
Vol 112 (15) ◽  
pp. 4731-4736 ◽  
Author(s):  
Xianjing Li ◽  
Duowei Wang ◽  
Zhen Chen ◽  
Ermei Lu ◽  
Zhuo Wang ◽  
...  
Keyword(s):  
Binding Protein ◽  
Macrophage Polarization ◽  
Growth Factor Receptor ◽  
Guanine Nucleotide Binding Protein ◽  
Akt Activation ◽  
Proteasome Pathway ◽  
Guanine Nucleotide Binding ◽  
Underlying Mechanisms

Heterotrimeric G proteins have been implicated in Toll-like receptor 4 (TLR4) signaling in macrophages and endothelial cells. However, whether guanine nucleotide-binding protein G(i) subunit alpha-1 and alpha-3 (Gαi1/3) are required for LPS responses remains unclear, and if so, the underlying mechanisms need to be studied. In this study, we demonstrated that, in response to LPS, Gαi1/3form complexes containing the pattern recognition receptor (PRR) CD14 and growth factor receptor binding 2 (Grb2)-associated binding protein (Gab1), which are required for activation of PI3K-Akt signaling. Gαi1/3deficiency decreased LPS-induced TLR4 endocytosis, which was associated with decreased phosphorylation of IFN regulatory factor 3 (IRF3). Gαi1/3knockdown in bone marrow-derived macrophage cells (Gαi1/3KD BMDMs) exhibited an M2-like phenotype with significantly suppressed production of TNF-α, IL-6, IL-12, and NO in response to LPS. The altered polarization coincided with decreased Akt activation. Further, Gαi1/3deficiency caused LPS tolerance in mice. In vitro studies revealed that, in LPS-tolerant macrophages, Gαi1/3were down-regulated partially by the proteasome pathway. Collectively, the present findings demonstrated that Gαi1/3can interact with CD14/Gab1, which modulates macrophage polarization in vitro and in vivo.

scholarly journals The Yeast Trimeric Guanine Nucleotide-Binding Protein α Subunit, Gpa2p, Controls the Meiosis-Specific Kinase Ime2p Activity in Response to Nutrients

1999 ◽  
Vol 19 (9) ◽  
pp. 6110-6119 ◽  
Author(s):  
Mariel Donzeau ◽  
Wolfhard Bandlow
Keyword(s):  
Kinase Activity ◽  
Binding Protein ◽  
Nucleotide Binding ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding Protein ◽  
Α Subunit ◽  
Guanine Nucleotide Binding ◽  
Diploid Cells ◽  
Nucleotide Binding Protein

ABSTRACT Saccharomyces cerevisiae Gpa2p, the α subunit of a heterotrimeric guanine nucleotide-binding protein (G protein), is involved in the regulation of vegetative growth and pseudohyphal development. Here we report that Gpa2p also controls sporulation by interacting with the regulatory domain of Ime2p (Sme1p), a protein kinase essential for entrance of meiosis and sporulation. Protein-protein interactions between Gpa2p and Ime2p depend on the GTP-bound state of Gpa2p and correlate with down-regulation of Ime2p kinase activity in vitro. Overexpression of Ime2p inhibits pseudohyphal development and enables diploid cells to sporulate even in the presence of glucose or nitrogen. In contrast, overexpression of Gpa2p in cells simultaneously overproducing Ime2p results in a drastic reduction of sporulation efficiency, demonstrating an inhibitory effect of Gpa2p on Ime2p function. Furthermore, deletion of GPA2 accelerates sporulation on low-nitrogen medium. These observations are consistent with the following model. In glucose-containing medium, diploid cells do not sporulate because Ime2p is inactive or expressed at low levels. Upon starvation, expression of Gpa2p and Ime2p is induced but sporulation is prevented as long as nitrogen is present in the medium. The negative control of Ime2p kinase activity is exerted at least in part through the activated form of Gpa2p and is released as soon as nutrients are exhausted. This model attributes a switch function to Gpa2p in the meiosis-pseudohyphal growth decision.

scholarly journals The Caulobacter crescentus CgtA Protein Displays Unusual Guanine Nucleotide Binding and Exchange Properties

1999 ◽  
Vol 181 (18) ◽  
pp. 5825-5832 ◽  
Author(s):  
Bin Lin ◽  
Kelly L. Covalle ◽  
Janine R. Maddock
Keyword(s):  
Rate Constant ◽  
Binding Proteins ◽  
Caulobacter Crescentus ◽  
Nucleotide Binding ◽  
Guanine Nucleotide ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Guanine Nucleotide Binding ◽  
Exchange Properties

ABSTRACT The Caulobacter crescentus CgtA protein is a member of the Obg-GTP1 subfamily of monomeric GTP-binding proteins. In vitro, CgtA specifically bound GTP and GDP but not GMP or ATP. CgtA bound GTP and GDP with moderate affinity at 30°C and displayed equilibrium binding constants of 1.2 and 0.5 μM, respectively, in the presence of Mg2+. In the absence of Mg2+, the affinity of CgtA for GTP and GDP was reduced 59- and 6-fold, respectively.N-Methyl-3′-O-anthranoyl (mant)–guanine nucleotide analogs were used to quantify GDP and GTP exchange. Spontaneous dissociation of both GDP and GTP in the presence of 5 to 12 mM Mg2+ was extremely rapid (kd = 1.4 and 1.5 s−1, respectively), 103- to 105-fold faster than that of the well-characterized eukaryotic Ras-like GTP-binding proteins. The dissociation rate constant of GDP increased sevenfold in the absence of Mg2+. Finally, there was a low inherent GTPase activity with a single-turnover rate constant of 5.0 × 10−4s−1 corresponding to a half-life of hydrolysis of 23 min. These data clearly demonstrate that the guanine nucleotide binding and exchange properties of CgtA are different from those of the well-characterized Ras-like GTP-binding proteins. Furthermore, these data are consistent with a model whereby the nucleotide occupancy of CgtA is controlled by the intracellular levels of guanine nucleotides.

scholarly journals Opioid peptides promote cholera-toxin-catalysed ADP-ribosylation of the inhibitory guanine-nucleotide-binding protein (Gi) in membranes of neuroblastoma x glioma hybrid cells

1988 ◽  
Vol 252 (2) ◽  
pp. 369-373 ◽  
Author(s):  
G Milligan ◽  
F R McKenzie
Keyword(s):  
Cholera Toxin ◽  
G Protein ◽  
Pertussis Toxin ◽  
Opioid Peptides ◽  
Hybrid Cells ◽  
Guanine Nucleotide Binding Protein ◽  
Adp Ribosylation ◽  
Guanine Nucleotide Binding

NG108-15 neuroblastoma x glioma hybrid cells express a major 45 kDa substrate for cholera toxin and a 40 kDa substrate(s) for pertussis toxin when ADP-ribosylation is performed in the presence of GTP. In the absence of exogenous GTP, however, cholera toxin was shown to catalyse incorporation of radioactivity into a 40 kDa protein as well as into the 45 kDa polypeptide. In membranes of cells which had been pretreated in vivo with pertussis toxin, the 40 kDa band was no longer a substrate for either pertussis or cholera toxin in vitro, whereas in membranes from cholera-toxin-pretreated cells the 40 kDa band was still a substrate for fresh cholera toxin in vitro and for pertussis toxin. In this cell line, opioid peptides have been shown to inhibit adenylate cyclase exclusively by interacting with Gi (inhibitory G-protein) and with no other pertussis-toxin-sensitive G-protein. Opioid agonists, but not antagonists, promoted the cholera-toxin-catalysed ADP-ribosylation of the 40 kDa polypeptide, hence demonstrating that this cholera-toxin substrate was indeed the alpha-subunit of Gi. These results demonstrate that Gi can be a substrate for either cholera or pertussis toxin under appropriate conditions.

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