Suppressive role of regucalcin in protein output in liver cells

Introduction Regucalcin was discovered in 1978 as a novel calcium-binding protein that does not contain the EF-hand motif of the calcium-binding domain, differing from calmodulin and other calcium-related proteins. The name ‘regucalcin’ was proposed for this calcium-binding protein which regulates various Ca2+-dependant or Ca2+/calmodulin-dependent enzyme activations. Regucalcin and its gene (rgn) are identified in over 15 species, comprising the regucalcin family. Regucalcin has been demonstrated to play a multifuncti-onal role in cell regulation including the depression of apoptotic cell dea-th and cell proliferation, induced through various signalling factors in many cell types in vitro. Cytoplasmic regucalcin translocates to the nucleus and suppresses the synthesis of nuclear DNA and RNA. Moreover, regucalcin has been shown to have a suppressive effect on protein synthesis, a stimulatory effect on protein degradation, suppresses aminoacyltRNA synthetase and activates proteases. This review discusses the suppressive role of regucalcin in protein output in liver cells. Conclusion Regucalcin may play an important role in regulating protein turnover by suppressing protein synthesis and stimulating protein degradation in normal and proliferating liver cells. Introduction


Introduction Discussion
The author has referenced some of its own studies in this review.These referenced studies have been conducted in accordance with the Declaration of Helsinki (1964) and the protocols of these studies have been approved by the relevant ethics committees related to the institution in which they were performed.All human subjects, in these referenced studies, gave informed consent to participate in these studies.

RGN suppresses nuclear DNA and RNA synthesis
Nuclear localisation of RGN Calmodulin stimulates nuclear DNA synthesis in liver cells 17 , and the effect of calmodulin is mediated through α-adrenergic stimulation 18,19 .RGN has been shown to be localised in the nuclei of rat liver cells 20,21 .Endogenous RGN has been shown to bind calmodulin 22,23 .Nuclear RGN translocation was not appreciably changed in the presence of adenosine 5'-triphosphate (ATP; 2 mM), guanosine 5'-triphosphate (GTP, 2 mM) and calcium chloride (0.1 mM), suggesting that its translocation is not mediated through the nuclear localisation signal 23 .ATP and GTP are required for the nuclear import of proteins that are localised in the nuclei.It was observed that ATP or GTP did not regulate the translocation of RGN into the nuclei of rat liver cells.The nuclear localisation of RGN was found to be independent of Ca 2+ 23 .

Physiology & Biochemistry
Regucalcin is a calcium-binding protein that does not contain the EF-hand motif as the calcium-binding domain, which is found in several calcium-binding proteins [1][2][3] .The name 'regucalcin' was proposed for this calcium-binding protein which suppresses the activity of various enzymes that are activated by Ca 2+ or Ca 2+ /calmodulin 2,3 .The rgn gene is localised on X chromosome and consists of seven exons and six introns 4,5 .RGN and its gene are identified in over 15 species, together comprising the 'regucalcin' family; the gene species are found to be highly conserved in vertebrate species [6][7][8] .RGN gene expression is regulated through various transcription factors (including AP-1, NF1-A1, RGPR-p117, β-catenin, SP1 and others), identified as enhancer and suppressor factors.Gene expression is also regulated by hormonal stimulation and physiological state 8,[9][10][11][12][13][14] .RGN has been demonstrated to play a multi-functional role in the regulation of various tissues and types of cells 3,[15][16][17][18] .RGN plays a crucial role in the maintenance of intracellular Ca 2+ levels through the activation of various Ca 2+ pump enzymes 15 .Cytoplasmic RGN is translocated into the nucleus and has been shown to suppress nuclear Ca 2+ -dependent protein kinase and protein phosphatase activities, Ca 2+activated DNA fragmentation, and D-NA and RNA synthesis 15,16 , in various types of cells.RGN has been proposed to play a pivotal role as a suppressor protein in various signal transductions to maintain cell homeostasis for stimuli 15,16 .
RGN has been shown to have a suppressive effect on protein synthesis, protein output in cells and a stimulatory effect on protein degradation.In this review, we have Nuclear protein transport is blocked in the presence of the lectin wheat germ agglutinin (WGA) 24 .Nuclear RGN translocation was not appreciably changed by the presence of WGA in the reaction mixture 23 .These findings suggest that the nuclear translocation of RGN is not related to the nuclear localisation signal that is responsible for selection of intranuclear active transport.The molecular weight of RGN is ~33 kDa 6 .RGN may be passively transported to the nucleus through nuclear pores in the liver cells.
Using the Far-Western blot analysis, RGN has been shown to bind proteins in isolated rat liver nuclei 25 .The results from this analysis showed the existence of protein components that bind to RGN in the nuclei isolated from rat liver cells 25 .Using the Western blot analysis for RGN, along with a DNA cellulose-binding assay, RGN has also been demonstrated to bind DNA 25 .
Using immunocytochemical analysis, RGN has also been shown to localise in the nuclei of cloned, normal rat kidney, proximal, tubular epithelial NRK52E cells 26 .The nuclear localisation of RGN is enhanced through the hormonal signalling process that involves the activation of protein kinase C in vitro 26 .

RGN suppresses nuclear enzyme activity
Isolated rat liver nuclei showed DNA endonuclease activity upon activation of Ca 2+ , resulting in extensive DNA hydrolysis 27 .Incubation of isolated rat liver nuclei with ATP, NAD + and submicromolar Ca 2+ concentrations resulted in extensive DNA hydrolysis.ATP stimulated a calmodulindependent nuclear Ca 2+ uptake system which apparently mediated endonuclease activation.The characteristics of this endonuclease activity indicate that it may be responsible for the Ca 2+ -dependent fragmentation of DNA involved in programmed cell death (apoptosis) and in certain forms of chemically-induced cell killing 27,28 .RGN has been shown to have a suppressive effect on Ca 2+ -activated DNA fragmentation in isolated rat liver nuclei 29 .The presence of RGN (0.5-2.0 μM) completely depressed the activation of liver nuclear DNA fragmentation when 10 μM Ca 2+ was added 29 .It was also shown that RGN could bind to nuclear proteins in the absence or presence of 1.0 mM Ca 2+ 30 .Hence, RGN may have a direct suppressive effect on DNA endonuclease in liver nuclei.Several studies have shown that Ca 2+ plays an important role in the regulation of nuclear functions 31,32 .A sustained increase in cytosolic Ca 2+ levels precedes the activation of DNA fragmentation that is involved in programmed cell death (apoptosis) and in certain forms of chemically induced cell killing 27,28 .Hence, RGN may suppress apoptosis, induced through the activation of DNA fragmentation.
GTPase Ran (ras-related nuclear protein) is required for protein export from the nucleus and protein import into the nucleus 33 .GTPase was found in the nuclei isolated from rat liver cells 34 .Liver nuclear GTPase activity was increased after calcium addition in the enzyme reaction mixture; this increase was not seen in the presence of trifluoperazine, an antagonist of calmodulin 34 .The presence of exogenous RGN (0.5 μM) in the enzyme reaction mixture caused an inhibitory effect on GTPase activity in the rat liver nuclei 34 .This effect was also seen in the presence of ethyleneglycol bis (2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), a chelator of Ca 2+ .The inhibitory effect of RGN on liver nuclear GTPase activity was independent of Ca 2+ /calmodulin in the nucleus.RGN has been shown to have an inhibitory effect on the activation of enzymes by Ca 2+ /calmodulin due to its ability to bind Ca 2+ and/or calmodulin [35][36][37][38] .RGN may directly inhibit GTPase activity in the liver nucleus.
RGN may be able to regulate the process of signal transduction from the cytoplasm to the nucleus in liver cells.This process is mediated through various protein kinases.The role of RGN in the regulation of Ca 2+ -dependent protein kinase and protein tyrosine phosphatase activities, in isolated liver nuclei, has been examined 21,23,39 .Nuclear Ca 2+dependent protein kinase and protein tyrosine phosphatase activities were increased in the presence of anti-RGN monoclonal antibodies in the enzyme reaction mixture, and these increases were completely depressed after addition of RGN 23 .Endogenous RGN has been demonstrated to have a suppressive effect on the enhancement of protein kinase activity with a proliferation of liver cells 40 .Protein kinase activity is enhanced in the cytosol and nucleus of regenerating rat liver cells with proliferating cells in vivo 40 .RGN has been shown to have a suppressive effect on tyrosine kinase, protein kinase C and Ca 2+ /calmodulin-dependent protein kinase, in the cytoplasm and nuclei of regenerating rat liver 35,36,40 .
Protein phosphatase activity towards phosphotyrosine, phosphoserine and phosphothreonine, is found in the rat liver nuclei 21 .The addition of RGN in the enzyme reaction mixture caused a significant decrease in phosphatase activity towards phosphotyrosine, phosphoserine and phosphothreonine 21 .Nuclear protein phosphatase activity was elevated in the presence of anti-RGN monoclonal antibodies (25 ng/mL and 50 ng/mL of reaction mixture) 21 .This elevation was completely depressed after addition of RGN.Hence, endogenous RGN may regulate the activity of various protein phosphatases in rat liver nuclei.
RGN suppresses nuclear DNA and RNA synthesis RGN has been shown to have an inhibitory effect on DNA synthesis activity in the nuclei of normal rat liver 41  present in the liver nucleus 41 .Liver nuclear DNA synthesis activity was increased in the presence of EGTA in the reaction mixture, suggesting that Ca 2+ suppresses DNA synthesis activity in the nucleus.The effect of RGN in suppressing nuclear DNA synthesis may not be related to Ca 2+ in the liver nucleus.
Liver nuclear DNA synthesis has been shown to be stimulated in regenerating rat liver in vivo 41 .Nuclear DNA synthesis was markedly increased one day after hepatectomy, and this increase was also seen at three days after hepatecctomy 41 .Nuclear DNA synthesis was enhanced in the presence of EGTA, a Ca 2+ chelator, in the incubation mixture 41 .The presence of Ca 2+ (1.0-25 μM) caused a significant decrease in the nuclear DNA synthesis of normal rat liver.RGN (0.25 μM and 0.5 μM) caused an inhibition of nuclear DNA synthesis in normal rat liver cells 41 .This inhibition was also seen in the presence of Ca 2+ .The inhibitory effect of RGN was remarkable in regenerating rat liver nuclei in comparison with that of normal rat liver nuclei 42 .Thus, RGN has been shown to have suppressive effects on nuclear DNA synthesis in regenerating rat liver with proliferating cells.
RGN has been also shown to have a suppressive effect on DNA synthesis activity in the nuclei isolated from rat renal cortex 43 .The addition of RGN (0.1-0.5 μM) in the reaction mixture, had an inhibitory effect on nuclear DNA synthesis activity, independent of calcium addition 44 .The presence of anti-RGN monoclonal antibodies (10-50 ng/ml) in the reaction mixture caused a significant increase in nuclear DNA synthesis activity 44 .This increase was completely depressed in the presence of RGN (0.5 μM).Endogenous RGN has been found to have a suppressive effect on DNA synthesis in the nuclei of the rat renal cortex 44 .
RGN has been shown to have suppressive effects on RNA synthesis in the nuclei isolated from normal rat liver in vitro 44 and regenerating rat liver in vivo 45 .Ca 2+ has a stimulatory effect on RNA synthesis in the liver nucleus 46,47 .This effect may be partly mediated through Ca 2+ -dependent protein kinases 46,47 .The stimulatory effect of Ca 2+ on nuclear RNA synthesis activity was completely depressed in the presence of RGN 44,45 .The addition of Ca 2+ with higher concentrations has been reported to have an inhibitory effect on nuclear RNA synthesis in rat liver cells 46 .It has been shown that inactivation of RNA polymerase III transcription is calcium dependent 47 .The effect of RGN in decreasing nuclear RNA synthesis activity in normal rat liver was not seen in the presence of α-amanitin, an inhibitor of RNA polymerase II and III 45 , suggesting that the suppressive effect of RGN is partly due to its inhibitory action on RNA polymerase II and III 44,45 .RGN has been shown to inhibit Ca 2+dependent protein kinases in the rat liver nucleus 40 .Presumably, the effect of RGN in decreasing RNA synthesis activity in the liver nucleus is partly involved in its inhibitory action on the activities of both RNA polymerase II and III and Ca 2+ -dependent protein kinases.
As described above, RGN has been found to have a suppressive effect on liver nuclear DNA and RNA synthesis [45][46] .This effect of RGN may partly influence protein output in liver cells.

RGN suppresses protein synthesis
RGN has been shown to have a regulatory effect on protein synthesis and protein degradation, suggesting that RGN plays a role in the regulation of protein turnover in cells.Protein synthesis is depressed in a variety of eukaryotic cell types exposed to conditions depleting Ca 2+ but not Mg 2+ 48 .It has also been proposed that hormones (vasopressin and α-adrenergic agonist), which have been known to mobilise sequestered Ca 2+ within liver cells, inhibit amino acid incorporation by influencing a Ca 2+ requirement associated with protein synthesis 49 .Moreover, vasopressin inhibits the rate of protein synthesis in isolated hepatocytes partially depleted of Ca 2+ 50 .These investigations propose the hypothesis that a sequestered pool of intracellular Ca 2+ is required for the maintenance of high rates of protein synthesis in liver cells.On the other hand, vasopressin and α-adrenergic agonist cause an increase in the intracellular free Ca 2+ concentration of hepatocytes that are not depleted of Ca 2+ 51,52 .Whether the increase in intracellular Ca 2+ influences hepatic protein synthesis, is still undefined, however, it may be important to clarify the effect of Ca 2+ addition on hepatic protein synthesis in vitro.
Ca 2+ , among various metals, can uniquely inhibit in vitro protein synthesis using the 5500 g supernatant fraction (the microsomes and cytosol) of the rat liver homogenate 53 .This inhibition was seen after addition of 1.0 μM Ca 2+ .Addition of Ca 2+ caused a remarkable decrease in the activity of aminoacyl (leucyl)-tRNA synthetase, which is a rate-limiting enzyme in the hepatic cytosol that influences protein synthesis during translation 53 .Ca 2+ may directly inhibit hepatic protein synthesis in a subcellular fraction of liver cells.Ca 2+ is required for protein synthesis in hepatocytes exposed to conditions depleting the cation 53 .Calmodulin, which can amplify the effects of Ca 2+ on enzymes, did not have an appreciable effect on in vitro protein synthesis using the 5500 g supernatant fraction of the liver homogenate in the presence of Ca 2+ (10 μM) 53 .The activity of aminoacyl-tRNA synthetase in the hepatic cytosol was not altered through calmodulin 54 .The mechanism by which Ca 2+ is required in the protein synthesis of hepatocytes depleted of Ca 2+ , may be complex.
The role of RGN in the regulation of in vitro protein synthesis, using the 5500 g supernatant fraction of the rat liver homogenate, has been previously investigated 53  a remarkable inhibition of hepatic protein synthesis in vitro 53 .RGN could not reverse the Ca 2+ -induced inhibition of protein synthesis 53 although RGN has been shown to reverse the effect of Ca 2+ on many enzymes in liver cells.Since RGN can bind to liver cytosolic proteins and its binding is slightly enhanced with the co-existence of 0.1 μM Ca 2+ 50 , Ca 2+binding RGN and/or Ca 2+ -free RGN may be able to inhibit hepatic protein synthesis.In fact, the presence of RGN (1 μM and 2 μM) could fairly decrease hepatic protein synthesis, which was found to be reduced after addition of 10 μM Ca 2+ 53 .RGN may play a role in the regulation of protein synthesis, independent of Ca 2+ , in liver cells.
RGN has been shown to inhibit hepatic aminoacyl-tRNA synthetase activity 53 .The inhibitory effect of RGN was seen in the presence of Ca 2+ (10 μM).The inhibitory effect of RGN on hepatic protein synthesis may be partly based on a remarkable decrease of aminoacyl-tRNA synthetase activity.Since iodinated RGN can bind the proteins in the hepatic cytosol, RGN may bind aminoacyl (leucyl)-tRNA synthetase 30 .
The role of endogenous RGN on protein synthesis has been examined using anti-RGN monoclonal antibodies 54 .The presence of anti-RGN monoclonal antibodies in the reaction mixture caused a significant increase in protein synthesis and [ 3 H] leucyl-tRNA synthetase activity in normal rat liver.These increases were completely depressed after addition of exogenous RGN (1.0 μM).Liver cytosol contained approximately 16 μg of RGN per 1 mg of cytosolic protein; the reaction mixture contained about 0.17-0.19μM of endogenous RGN, because cytosolic protein in the range of 360-390 μg was added to a mixture of 1 mL.Hence, endogenous RGN may have a suppressive effect on protein synthesis in liver cells.
Hepatic protein synthesis has been shown to be enhanced in regenerating rat liver, which induces a proliferation of liver cells after partial hepatectomy in vivo 54 .This enhancement was remarkable at 24 h and 48 h after partial hepatectomy.Hepatic protein synthesis in regenerating liver was further enhanced in the presence of anti-RGN monoclonal antibodies in the reaction mixture.Endogenous RGN may play a suppressive role in the enhancement of protein synthesis in regenerating liver.

RGN stimulates protein degradation
Evidence for the role of Ca 2+ -activated proteases (calpains), which induce protein degradation, is implicated in signal transduction 55 .Two neutral Ca 2+ -requiring proteases, differing in molecular size, have been isolated from the rabbit liver cytosol 56 .Both are recovered as inactive proenzymes that can be converted to the active forms using high (0.1-1.0 mM) concentrations of Ca 2+ in the absence of a substrate or using low (1-5 μM) concentrations of Ca 2+ in the presence of a protein substrate 56 .The activated proteases required only 1-5 μM Ca 2+ for maximal activity 57 .
RGN has been shown to stimulate protein degradation in the liver cytosol 57 .The addition of RGN (0.25-2.0 μM) into the enzyme reaction mixture has been found to induce a remarkable increase of neutral protease activity in the presence of 5.0 μM Ca 2+ 57 .The effect of RGN was seen at 0.25 μM.RGN may activate Ca 2+ -requiring proteases in the rat liver cytosol.The effect of RGN in increasing liver cytosolic protease activity was also seen in the absence of Ca 2+ 57 .This increase was remarkable as compared with that of Ca 2+ addition.RGN may activate Ca 2+ -not requiring neutral protease in the rat liver cytosol.RGN has a reversible effect on the activation of various enzymes by Ca 2+ and/or calmodulin [35][36][37] .The finding that RGN can activate neutral proteases in the rat liver cytosol in the presence or absence of Ca 2+ , was novel.The activating effect of RGN on liver cytosolic protease activity was not seen in the presence of anti-RGN antiserum in the enzyme reaction mixture 57 , suggesting a role of endogenous RGN in the activation of cytosolic protease.
The activating effect of RGN on neutral proteases in the liver cytosol has been already characterised 58 .Leupeptin is a potent inhibitor of SH-protease.The RGN-increased protease activity was inhibited in the presence of leupeptin in the enzyme reaction mixture 58 .RGN may activate neutral cysteinyl-protease in the liver cytosol.The effect of RGN in increasing proteolytic activity in the rat liver cytosol was not depressed in the presence of diisopropylfluorophosphate (DFP), an inhibitor of serine-protease, although DFP alone had an inhibitory effect on the proteolytic activity 58 .RGN did not act on serine-proteases in the liver cytosol.The effect of RGN was also seen in the presence of a chelator of metal ions, suggesting that RGN does not activate metal-related proteases in the liver cytosol.
The proteolytic activity in the liver cytosol was markedly increased after addition of dithiothreitol (DTT), a protecting reagent for the thiol (SH) group, in the enzyme reaction mixture, and this increase was completely inhibited in the presence of N-ethylmaleimide (NEM), an SH group-modifying agent 58 .RGN was found to increase proteolytic activity in the presence of DTT in the liver cytosol although it was completely inhibited in the presence of NEM 58 .RGN may act on the SH group of cysteinyl-proteases in the liver cytosol.
All authors contributed to the concep on, design, and prepara on of the manuscript, as well as read and approved the fi nal manuscript.
All authors abide by the Associa on for Medical Ethics (AME) ethical rules of disclosure.
Licensee OA Publishing London 2013.Creative Commons Attribution Licence (CC-BY) F : Yamaguchi M. Suppressive role of regucalcin in protein output in liver cells.OA Biotechnology 2013 Jan 31;2(1):6.μM)-increased proteolytic activity in the rat liver cytosol was inhibited in the presence of NEM, indicating that neutral proteases (including calpains), which are activated by Ca 2+ , exist in the liver cytoplasm 58 .
The activity of calpains, which are thiol proteases 60 , increases in hepatocytes following addition of ATP 61 .The proteolytic activity in liver cytosol was decreased after the addition of calpastatin, a specific inhibitory of calpains, indicating the existence of calpains in the cytosol 58 .RGN-increased proteolytic activity was depressed in the presence of calpastatin.Hence, RGN may be an activator for calpains.m-calpain isolated from rabbit skeletal muscle was activated by RGN, independent of Ca 2+ .This activation was inhibited after the addition of NEM 58 .RGN acts on the SH group in m-calpain.Presumably, RGN may be able to activate both m-calpains and μ-calpains.
The role of RGN in the regulation of neutral proteolytic activity in rat kidney cortex cytosol has also been demonstrated 62,63 .RGN had an activating effect on the neutral proteolytic activity in the kidney cortex cytosol 62 .This increase was depressed in the presence of anti-RGN monoclonal antibodies, supporting the view that endogenous RGN plays a role as an activator of proteases in the renal cortex cytosol 62 .The activating effect of RGN on proteases is seen with concentrations of 0.01-0.25 μM 62 .The concentration of RGN present in rat kidney tissue has been found to be ~5.3 μM.Hence, it can be inferred that RGN plays a physiological role in the activation of thiol proteases in renal cortex cells.
The effect of RGN on proteolytic activity was not altered in the presence of calcium chloride (0.01 and 1.0 mM) or EGTA (1.0 mM), indicating that the effect of RGN was independent of Ca 2+ .RGN may be responsible for the activation of both calpains and other proteases in the kidney cortex cytosol.RGN has also been shown to activate SH proteases in the rat renal cortex cytosol.Presumably, RGN acts on the SH-groups of proteases in the rat renal cortex cytosol.
RGN uniquely activates thiol proteases, independent of Ca 2+ , in the liver cytosol, whereas it has no effect on serine proteases and metalloproteases 57,58 .Similar effects of RGN were also found in the renal cortex cytosol 63 .RGN may be an activator of thiol proteases in liver and kidney cells.RGN also plays a role as an activator in other tissues that express RGN.
Calpains are ubiquitous, non-lysosomal, calcium-dependent proteases that may play important roles in Ca 2+ -mediated intracellular processes 55,56,61 .The ability of calpain to alter the limited proteolysis and the activity or function of numerous cytoskeletal proteins, protein kinases, receptors and transcription factors, suggests the involvement of these proteases in various Ca 2+ -regulated cellular functions 60 .Calpains may also play an integral role in modulating the activity of protein kinase C, a key protein in many signal transduction processes 64 since they convert the native Ca 2+ /phospholipids-dependent kinase to a soluble form that does not require Ca 2+ or phospholipids for activity.RGN can increase the activity of thiol proteases, including calpains, in rat liver and renal cortex cytosols.RGN may play a pivotal role in the regulation of cellular functions related to Ca 2+ , mediated through thiol proteases.Presumably, RGN plays an important role in the regulation of signal transduction involved with proteases.
As summarised in Figure 1, RGN may play a role as a suppressor in the enhancement of cytoplasm protein levels by inhibiting protein synthesis and activating protein degradation in normal and prolifer-

Conclusion
RGN plays a pivotal role in cell regulation as a suppressor protein in signal transduction for various stimuli in various types of cells.RGN has been shown to have a suppressive effect on cell proliferation and apoptosis that are mediated through various hormones, cytokines and other factors.Moreover, RGN has been demonstrated to suppress protein synthesis and stimulate proteolysis in cells.The suppressive effect of RGN on protein synthesis is mediated through the inhibition of transcriptional and translational processes.The stimulatory effect of RGN on proteolysis is mediated through the activation of thiol protease.The suppressive effect of RGN on protein output in cells is revealed in normal and proliferating liver cells.RGN may play a role as a regulatory factor in protein turnover in cells.Specifically, RGN may have a suppressive effect on the overexpression of protein output in proliferating cells to maintain cell homeostasis.Hence, downregulation of RGN expression may suppress the development of carcinogenesis that enhances cell protein output.Future research should aim at targeting the expression of the RGN gene for the suppression of carcinogenesis.

Abbreviations list
presented our findings concerning these properties in relation to the regulation of aminoacyl-tRNA synthetase and proteases in liver cells.Licensee OA Publishing London 2013.Creative Commons Attribution Licence (CC-BY) F : Yamaguchi M. Suppressive role of regucalcin in protein output in liver cells.OA Biotechnology 2013 Jan 31;2(1):6.
. The inhibitory effect of RGN was seen in the presence of EGTA, a chelator of Ca 2+ , in the reaction mixture.Ca 2+ is Licensee OA Publishing London 2013.Creative Commons Attribution Licence (CC-BY) F : Yamaguchi M. Suppressive role of regucalcin in protein output in liver cells.OA Biotechnology 2013 Jan 31;2(1):6.

Figure 1 :
Figure 1:RGN has a suppressive effect on protein output by depressing protein synthesis and stimulating protein degradation in normal and proliferating liver cells.This effect may be based on the suppression of nuclear DNA and RNA synthesis and aminoacyl t-RNA synthetase, a rate-limited enzyme involved in the translation of proteins.RGN also stimulates proteolysis that is mediated through the activation of thiol proteases.RGN may regulate protein turnover in the cells.Also, RGN may play a pathophysiological role in the suppression of the overexpression of protein output in proliferating cells.