Arginine Sensor Research Articles

DNA module platform for developing colorimetric aptamer sensors.

Here we present a DNA module platform for developing simple aptamer sensors based on a microarray format combined with secondary structure prediction in silico. The platform comprises four parts: (i) aptamer, (ii) joint module, (iii) terminal stem, and (iv) a DNAzyme that catalyzes a redox reaction controlled by a structural change induced by aptamer/target binding. First, we developed a joint module, capable of sensing a conformational change in the aptamer region, that was linked to the signal transmission activity of a DNAzyme as the reporter in a concentration-dependent manner with the AMP aptamer. This module design was then used to generate an arginine sensor simply by replacing the AMP aptamer region with a previously reported arginine aptamer. Using this DNA module platform, we were also able to customize a microarray containing >10,000 sequences designed by in silico secondary structure prediction and successfully identify a new aptamer against patulin. Our results show that the DNA module platform can be used to readily devise sensors based on known aptamers as well as create new aptamer sensors by array-based screening.

The CASTOR Proteins Are Arginine Sensors for the mTORC1 Pathway

Amino acids signal to the mTOR complex I (mTORC1) growth pathway through the Rag GTPases. Multiple distinct complexes regulate the Rags, including GATOR1, a GTPase activating protein (GAP), and GATOR2, a positive regulator of unknown molecular function. Arginine stimulation of cells activates mTORC1, but how it is sensed is not well understood. Recently, SLC38A9 was identified as a putative lysosomal arginine sensor required for arginine to activate mTORC1 but how arginine deprivation represses mTORC1 is unknown. Here, we show that CASTOR1, a previously uncharacterized protein, interacts with GATOR2 and is required for arginine deprivation to inhibit mTORC1. CASTOR1 homodimerizes and can also heterodimerize with the related protein, CASTOR2. Arginine disrupts the CASTOR1-GATOR2 complex by binding to CASTOR1 with a dissociation constant of ~30 μM, and its arginine-binding capacity is required for arginine to activate mTORC1 in cells. Collectively, these results establish CASTOR1 as an arginine sensor for the mTORC1 pathway.

CASTORing New Light on Amino Acid Sensing

The activation state of mTORC1, a master regulator of cell growth, is particularly sensitive to changes in the intracellular levels of the amino acid arginine, but the sensing mechanisms are poorly understood. In this issue of Cell, Chantranupong et al. identify CASTOR1 as a direct arginine sensor that acts through the GATOR2 complex to regulate mTORC1.

Plumbagin as colorimetric and ratiometric sensor for arginine

A simple and selective naphthoquinone based fluorescent probe involving plumbagin (Pn=1,4-naphthoquinone) which is a naturally occurring phytochemical is reported for sensing of arginine in aqueous medium. The sensing probe Pn upon binding to Arg, induced a visible color change from light yellow to pink. The 1:1 Pn/Arg complex was characterized by Job's plot, ESI-MS and DFT calculations. This naked eye colorimetric sensing is insensitive to other amino acids namely Asp, Glu, Thr, Lys, His, Cys, Met, Ser and Phe. In the absence of arginine, excited state intramolecular proton transfer (ESIPT) occurs and when arginine is added, intermolecular proton transfer from Pn to arginine takes place resulting in colorimetric and ratiometric response.

Metabolism. Lysosomal amino acid transporter SLC38A9 signals arginine sufficiency to mTORC1.

The mechanistic target of rapamycin complex 1 (mTORC1) protein kinase is a master growth regulator that responds to multiple environmental cues. Amino acids stimulate, in a Rag-, Ragulator-, and vacuolar adenosine triphosphatase-dependent fashion, the translocation of mTORC1 to the lysosomal surface, where it interacts with its activator Rheb. Here, we identify SLC38A9, an uncharacterized protein with sequence similarity to amino acid transporters, as a lysosomal transmembrane protein that interacts with the Rag guanosine triphosphatases (GTPases) and Ragulator in an amino acid-sensitive fashion. SLC38A9 transports arginine with a high Michaelis constant, and loss of SLC38A9 represses mTORC1 activation by amino acids, particularly arginine. Overexpression of SLC38A9 or just its Ragulator-binding domain makes mTORC1 signaling insensitive to amino acid starvation but not to Rag activity. Thus, SLC38A9 functions upstream of the Rag GTPases and is an excellent candidate for being an arginine sensor for the mTORC1 pathway.

Arginine Wettability and Impedance Dual-Signal Response by Aldehyde Calix[4]arene Self-Assembled Monolayers.

A new aldehyde calix[4]arene (C4AH) was synthesized efficiently to give a yield of 90 % and was used to form self-assembled monolayers (SAMS) by means of click chemistry to give a highly selective wettability and impedance sensor for arginine (Arg). The success of the C4AH functional interface and Arg-responsive events were confirmed by contact-angle (CA) measurements and electrochemical impedance spectroscopy (EIS). Furthermore, the C4AH functional interface can be used as wettability-responsive cycles at least eight times. This type of sensor operates on the principle that Arg has a high pKa value of 12.48, and is able to form strong hydrogen bonds with aldehyde selectivity, which results in a significant CA change. The proposed wettability-sensing devices display a remarkable specificity, intuitiveness, and convenience, which should be suitable for the production of arginine recognition chips that can be used in the fields of biology and medical science.

Filamin A mutations cause periventricular heterotopia with Ehlers-Danlos syndrome

To define the clinical, radiologic, and genetic features of periventricular heterotopia (PH) with Ehlers-Danlos syndrome (EDS). Exonic sequencing and single stranded conformational polymorphism (SSCP) analysis was performed on affected individuals. Linkage analysis using microsatellite markers on the X-chromosome was performed on a single pedigree. Western blotting evaluated for loss of filamin A (FLNA) protein and Southern blotting assessed for any potential chromosome rearrangement in this region. The authors report two familial cases and nine additional sporadic cases of the EDS-variant form of PH, which is characterized by nodular brain heterotopia, joint hypermobility, and development of aortic dilatation in early adulthood. MRI typically demonstrated bilateral nodular PH, indistinguishable from PH due to FLNA mutations. Exonic sequencing or SSCP analyses of FLNA revealed a 2762 delG single base pair deletion in one affected female. Another affected female harbored a C116 single point mutation, resulting in an A39G change. A third affected female had a 4147 delG single base pair deletion. One pedigree with no detectable exonic mutation demonstrated positive linkage to the FLNA locus Xq28, an affected individual in this family also had no detectable FLNA protein, but no chromosomal rearrangement was detected. These results suggest that the Ehlers-Danlos variant of periventricular heterotopia (PH), in part, represents an overlapping syndrome with X-linked dominant PH due to filamin A mutations.

Swapping functional specificity of a MADS box protein: residues required for Arg80 regulation of arginine metabolism.

Arg80 and Mcm1, two members of the MADS box family of DNA-binding proteins, regulate the metabolism of arginine in association with Arg81, the arginine sensor. In spite of the high degree of sequence conservation between the MADS box domains of the Arg80 and Mcm1 proteins (56 of 81 amino acids), these domains are not interchangeable. To determine which amino acids define the specificity of Arg80, we swapped the amino acids in each secondary-structure element of the Arg80 MADS box domain with the corresponding amino acids of Mcm1 and assayed the ability of these chimeras to regulate arginine-metabolic genes in place of the wild-type Arg80. Also performed was the converse experiment in which each variant residue in the Mcm1 MADS box domain was swapped with the corresponding residue of Arg80 in the context of an Arg80-Mcm1 fusion protein. We show that multiple regions of Arg80 are important for its function. Interestingly, the residues which have important roles in determining the specificity of Arg80 are not those which could contact the DNA but are residues that are likely to be involved in protein interactions. Many of these residues are clustered on one side of the protein, which could serve as an interface for interaction with Arg81 or Mcm1. This interface is distinct from the region used by the Mcm1 and human serum response factor MADS box proteins to interact with their cofactors. It is possible that this alternative interface is used by other MADS box proteins to interact with their cofactors.

A chiral sensor for arginine and lysine

[structure: see text] We provide access to a new class of C1- or C2-symmetrical host molecules 1 and 2 based on a spirobisindane skeleton. Whereas 1 is selective for short, rigid diamines, 2 prefers longer alpha,omega-dications. Of all the amino acid methyl esters, only those of lysine and arginine with the correct distance between their cationic groups form strong 1:1 complexes in DMSO with 2. NMR titrations reveal high association constants as well as discrimination between the enantiomers of lysine and arginine.

BasT, a membrane-bound transducer protein for amino acid detection in Halobacterium salinarum.

Halophilic archaea, such as eubacteria, use methyl-accepting chemotaxis proteins (MCPs) to sense their environment. We show here that BasT is a halobacterial transducer protein (Htp) responsible for chemotaxis towards five attractant amino acids. The C-terminus of the protein exhibits the highly conserved regions that are diagnostic for MCPs: the signalling domain for communication with the histidine kinase and the methylation sites that interact with the methylation/demethylation enzymes for adaptation. Hydropathy analysis predicts an enterobacterial-type transducer protein topology for BasT, with an extracellular putative ligand-binding domain flanked by two transmembrane helices and a cytoplasmic domain. BasT-inactivated mutant cells are missing a membrane protein radiolabelled with L-[methyl-3H]-methionine in wild-type cells, confirming that BasT is methylatable and membrane bound. Behavioural analysis of the basT mutant cells by capillary and chemical-in-plug assays demonstrates complete loss of chemotactic responses towards five (leucine, isoleucine, valine, methionine and cysteine) of the six attractant amino acids for Halobacterium salinarum, whereas they still respond to arginine. The volatile methyl group production assays also corroborate these findings and confirm that BasT signalling induces methyl group turnover. Our data identify BasT as the chemotaxis transducer protein for the branched chain amino acids leucine, isoleucine and valine as well as for methionine and cysteine. Thus, BasT and the arginine sensor Car cover the entire spectrum of chemotactic responses towards attractant amino acids in H. salinarum.

Effect of inhibition of gluconeogenesis on arginine-induced insulin secretion

It is well known that several amino acids, such as arginine, are potent stimuli for insulin and glucagon secretion from the pancreas. Recently, vagal arginine sensors, which modulate arginine-induced pancreatic hormone secretion, have been reported to exist in the liver. The present investigation was designed to evaluate the role played by gluconeogenesis in this hepatic influence. To this end, we studied the effects of an intraperitoneal injection of 3-mercaptopicolinic acid (3-MPA), a gluconeogenic inhibitor, on the pancreatic hormonal response induced by intraperitoneal administration of arginine (1 g/kg body mass) to hepatic vagotomized and sham vagotomized rats. Fifteen min following the injection of arginine, the increases in glucose and insulin concentrations were significantly lower in rats with an inhibited gluconeogenesis than in rats with an intact capacity for gluconeogenesis. There were no effects of the hepatic vagotomy on the arginine-induced hormonal responses either with or without the 3-MPA injection. The results suggest that gluconeogenesis is implicated in the hepatic modulation of arginine-induced pancreatic hormone secretion.

Modulation of insulin secretion by hepatic vagotomy in cirrhotic rats

There is evidence that glucose sensors and arginine sensors are present in the hepato-portal system and exert a reflex regulation of the pancreatic neuroendocrine system in normal rats. To investigate the function of these sensor systems in liver cirrhosis, we examined the effect of hepatic vagotomy on plasma glucose and insulin concentrations after intraperitoneal (IP) injection of glucose or l-arginine in carbon tetrachloride (CCl 4)-induced cirrhotic rats. After IP glucose injection, hepatic vagotomy decreased plasma insulin with elevation of plasma glucose in control rats; however, in cirrhotic rats, this procedure did not affect either plasma glucose or insulin concentrations. After IP arginine injection, hepatic vagotomy increased plasma insulin and reduced plasma glucose in control rats, although in cirrhotic rats, this procedure did not affect plasma glucose but did increase plasma insulin concentrations. These results suggest that function of the glucose sensor in CCl 4-induced cirrhotic rats is disturbed, although that of the arginine sensor is retained.

Glycine sensors in the hepato-portal system and their reflex effects on pancreatic efferents in the rat

We earlier reported the existence of vagal arginine, alanine, and leucine sensors in the liver which modulate amino acid-induced pancreatic hormone secretion. To determine the possible existence of glycine sensors in the liver and their reflex effects, afferent discharges from the hepatic branch and efferent discharges from the pancreatic branch of the vagus nerve were recorded. Intraportal administration of 0.1, 1, and 10 mM of l-glycine solution (0.1 ml) depressed the afferent discharge rate of the hepatic branch of the nerve in a dose-dependent manner, and increased the efferent discharge rate of the pancreatic vagal branch. The results suggest the existence of glycine sensors in the hepato-portal system that exert reflex regulation on the pancreatic vagus nerve activity.

Modulation of Arginine-Induced Insulin and Glucagon Secretion by the Hepatic Vagus Nerve in the Rat: Effects of Celiac Vagotomy and Administration of Atropine

We hypothesized the existence of vagal arginine sensors in the liver which modulate arginine-induced pancreatic hormone secretion. The present study was carried out to examine the efferent pathways and receptor mechanisms from arginine sensors using selective vagotomies and autonomic drugs on the secretion of insulin and glucagon after ip injection of L-arginine (1 g/kg BW) in rats in an unanesthetized and unrestrained state. Hepatic vagotomy (sectioning of the hepatic branch of the vagus nerve) enhanced both plasma insulin and glucagon concentrations after ip arginine more than those in sham-vagotomized (control) rats. The effect of hepatic vagotomy was blocked by adding celiac vagotomy (sectioning of the celiac branches of the vagus nerve) or by previous administration of atropine methyl nitrate (10 mg/kg BW), but not by phentolamine (1 mg/kg BW) or propranolol (2 mg/kg BW). Celiac vagotomy alone did not affect the plasma insulin concentration; however, it reduced the plasma glucagon concentration after ip arginine compared to that in sham-vagotomized rats. Administration of atropine alone did not affect plasma insulin or glucagon concentrations after ip arginine. These results suggest that celiac branches of the vagus nerve act as efferent pathways to the pancreas through a muscarinic receptor mechanism in the hepatic arginine sensor-mediated pancreatic neuroendocrine system. The physiological role of these hepatic sensors may be to prevent arginine-induced exaggerated pancreatic hormone secretion and maintain blood glucose homeostasis.

Amino acid sensors sensitive to alanine and leucine exist in the hepato-portal system in the rat

We reported the existence of vagal arginine sensors in the liver which modulate arginine-induced pancreatic hormone secretion. The present study was carried out to examine the possible existence of other amino acid sensors such as l-alanine and l-leucine in the hepato-portal system in rats using an electrophysiological approach. Afferent discharges were recorded from fine filaments dissected from the peripheral cut end of the hepatic branch of the vagus nerve. Administration of 0.1, 1, and 10 mM l-alanine and l-leucine solution (0.1 ml) into the portal vein caused an increase in the discharge rate of hepatic vagal afferents in a dose-dependent manner. The results suggest the existence of vagal amino acid sensors which are sensitive to alanine and leucine in the hepato-portal system.

Hyper-Response of Insulin Release to Arginine in Streptozotocin-Induced Diabetic Rats with Hepatic Vagotomy.

Vagal glucose receptor and arginine sensor have been found to exist in the liver of normal rats. The present study was designed to examine whether these sensory receptor systems function in streptozotocin-induced mildly diabetic rats. One week after streptozotocin injection (35 mg/kg body wt. iv), hepatic vagotomy and installation of a jugular vein catheter were performed. Three days after the surgery, intraperitoneal glucose (1 g/kg body wt.) or arginine (1 g/kg body wt.) stimulation was done with the rat in an unanesthetized and unrestrained state. Hepatic vagotomy significantly reduced glucose-induced insulin release with an increase in plasma glucose in control and diabetic rats. The rate of reduction of plasma insulin release was similar in the two groups. Hepatic vagotomy also significantly enhanced arginine-induced insulin release in both groups, but in control rats there was no change in the response to plasma glucose, while in diabetic rats there was a significant reduction. The rate of enhancement of plasma insulin release by hepatic vagotomy was significantly greater in diabetic than in control rats. The results indicate that streptozotocin-induced mildly diabetic rats have hyperfunction of the arginine sensor and normofunction of the glucose receptor and modulate insulin release through these receptor systems in the liver.

Liver arginine sensors regulate insulin and glucagon secretion after intraperitoneal arginine

Liver arginine sensors regulate insulin and glucagon secretion after intraperitoneal arginine

Arginine sensors in the hepato-portal system and their reflex effects on pancreatic efferents in the rat

To investigate the possible existence of arginine sensors in the liver and their reflex effects, afferent discharges from the hepatic branch of the vagus nerve and efferent discharges from the pancreatic branches of the splanchnic and vagus nerves were recorded. Intraportal injection of arginine increased afferent discharge rate of the hepatic branch of the vagus nerve, slightly depressed the efferent discharge rate of the pancreatic vagal branch, and increased the efferent discharge rate of the pancreatic splanchnic branch. The results suggest the existence of arginine sensors in the hepato-portal system that exert reflex regulation of the pancreatic neuroendocrine system.