Regucalcin : new development in biotechnology

Regucalcin, which is a suppressor protein in cell signalling, was discovered in 1978. The regucalcin gene is localized on the X chromosome and is identified in over 15 species consisting of the regucalcin family. Regucalcin is highly conserved in vertebrate species throughout evolution. Regucalcin is found to play a multifunctional role in cell regulation. Regucalcin is trans located into the nucleus and regulates nuclear function in cells. Overexpression of endogenous regucalcin in cells has been demonstrated to suppress cell proliferation and apoptotic cell death. The analysis of proteome and differential gene expression has shown a unique suppression of regucalcin expression among many proteins in the development of carcinogenesis. Clinical studies of regucalcin for disease and the application of regucalcin in clinical fields are discussed.


Editorial
OA Biotechnology has been launched with this issue.The author, an Editorin-Chief, here contributes an editorial concerning the new development in biotechnology of the calcium signal-ling-related protein regucalcin (RGN) that was discovered by the author.
Calcium ions (Ca 2+ ) play a pivotal role as second messengers in the regulation of many cell functions.Ca 2+ regulates muscle contraction, neurotransmission, hormone secretion, cell mitosis, apoptotic cell death, and gene expression.Calcium signal is transmitted to intracellular responses, which are amplified through calmodulin and protein kinase C [1][2][3] .Calcium signalling is important in the regulation of cell metabolism.RGN was discovered in 1978 as a novel calcium-binding protein that does not contain the EF-hand motif, which is a calcium-binding domain [4][5][6][7][8][9][10] .The name RGN was proposed for this calcium-binding protein, which regulates various Ca 2+ -or Ca 2+ /calmodulin-dependent enzyme activations in liver cells [7][8][9] .In addition, the protein named as senescence marker protein-30 (SMP30), which is identical to RGN, was also reported after the discovery of RGN 11,12 .The gene encoding RGN (rgn) is localized on the X chromosome, and the organization of rgn consists of 7 exons and 6 introns [13][14][15] .RGN and its gene are identified in over 15 species consisting of RGN family 16 .
The molecular weight of rat RGN is estimated as 33,388 Da, consisting of 299 amino acid residues, and the isoelectric point of RGN is 5.20 [4][5][6]10 . Theapparent association constant for Ca 2+ of RGN is found to be 4.19 × 10 5 M −1 by equilibrium dialysis, and there are 6.52 high-affinity sites per molecule of protein 5 .Extrapolation of the regression line to infinite Ca 2+ concentration indicated a maximal Ca 2+ -binding of 2.28 × 10 −4 mol of Ca 2+ per gram of protein.Calmodulin exists as a monomer with a molecular weight of 17,000 and contains 4 Ca 2+binding sites 1 .The α-helical content of RGN in Ca 2+ -free buffer is estimated to be 34%, and the presence of 1.0 mM Ca 2+ decreased this by 4.5%.Ca 2+ binding to RGN has been shown to induce conformational changes using circular dichroism spectroscopy. Tis binding loosens the structural conformation of RGN.The α-helical content of calmodulin is 30%; this content is increased after Ca 2+ binding, and its conformation is then tightened 1 . Bnding of Ca 2+ to calmodulin increases hydrophobicity.This represents the mechanism that calmodulin activates its target proteins.However, RGN reverses the activation of many enzymes by Ca 2+ /calmodulin 9 .The role of RGN may be different from that of calmodulin in cells.
RGN shows a hydrophobic character as a molecule.There is a hydrophobic sequence in both the N-terminal and C-terminal regions of the RGN molecules 10 .The most common EF-hand is composed of the helix-loop-helix-domain. RGN has a relatively high content of glycine and much lower amounts of glutamic acid, valine, aspartic acid and lysine 10 .RGN contains approximately 20% (mol%) glutamic (16 residues) and aspartic acids (24 residues) and approximately 17% amide residues (lysine, histidine and arginine) and, thus, a high proportion of charged residues 10 .Acidic amino acids comprise approximately one-fifth of the RGN molecule.These amino acids may be related to Ca 2+ binding.The result of crystal structure with X-ray diffraction data shows that human RGN contains the metal site bound with either a Ca 2+ or a Zn 2+ atom, suggesting that the Ca 2+bound form may be physiologically relevant for stressed cells with an elevated Ca 2+ level 17 , supporting our finding for RGN as a calcium-binding protein.
RGN is highly conserved in vertebrate species throughout evolution.Various transcription factors (including AP-1, NF1-A1, RGPR-p117, β-catenine and others) have been shown to enhance transcription activity of rgn expression that is mediated through the mechanism of calcium and other signalling 16,18,19 .Other transcriptional factors may be found in future studies.This will bring more exploration of the regulatory mechanism of rgn expression.The expression of RGN mRNA is regulated through many hormonal factors (including calcium, calcitonin, insulin, oestrogen, aldosterone, dexamethasone, transforming growth factor-β, tumour necrosis factor-α, lipopolysaccharide and other factors) in vivo and in vitro.RGN may participate in a physiological aspect that is related to these hormonal and cytokine effects.
There is growing evidence that RGN plays a multifunctional role in cell regulation through a mechanism related to the processes of Ca 2+ -dependent and -independent signalling in many cell types [20][21][22] .RGN is greatly expressed in the liver and kidney cortex and is also found in other tissues including brain, heart, bone, breast, lung, prostate and other tissues 23,24 .The role of RGN in the regulation of liver and kidney cell function is investigated in detail.RGN plays a pivotal role in maintaining intracellular calcium homeostasis due to activating Ca 2+ pump enzymes and suppressing calcium signalling from the cytoplasm to the nucleus in proliferation cells 20,21 .RGN is translocated into the nucleus of liver cells and regulates nuclear function 20,21 .RGN has a suppressive effect on the activities of Ca 2+ -dependent and -independent protein kinases and protein phosphatases in the cytoplasm and nucleus of cells and nuclear deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) syntheses 20,21 .RGN regulates protein turnover by suppressing protein synthesis and stimulating protein degradation 20,21 .Overexpression of endogenous RGN in cells has been demonstrated to suppress cell proliferation and apoptotic cell death that is induced by various signalling factors 20,21 .RGN is the first time finding for a protein molecule that acts as a suppressor protein in cell signaling 21 , although it is well known that many proteins enhance cell signal transduction so far.From these findings, it is proposed that RGN plays a pivotal role in maintaining cell homeostasis for intracellular signalling stimulation 20,21 .
***Thus, RGN may play an important role as a regulatory protein in the regulation of a living body.The pathophysiological role of RGN in vivo has been found.A deficiency of RGN induces an accumulation of liver lipid and also causes a decrease in Lascorbic acid (vitamin C) in mice 25,26 , although this may not be significant because vitamin C is not synthesized in humans.Overexpression of endogenous RGN induces osteoporosis and hyperlipidemia in RGN transgenic rats, indicating the pathophysiological role of RGN in metabolic disorders [27][28][29] .
RGN has been proposed to be a target molecule in metabolic disorders and disease.The expression of rgn in the liver and kidney is suppressed in various pathophysiological states including carcinogenesis, diabetes, hypertensive state or drugs that induce liver and kidney disorders.Liver RGN is markedly released into the serum of rats with liver injury, suggesting a role as a specific biomarker of chronic liver disease with hepatitis.The analysis for proteome and differential gene expression has shown a unique suppression of RGN expression among many proteins in the development of carcinogenesis and kidney damage.Moreover, rgn expression is down-regulated in the development of carcinogenesis in liver tissues, sug-gesting that its suppression induces promotion of tumour cells.The suppression of rgn expression may reveal a pathophysiological role.
***The gene therapy that targets rgn may be useful as a therapeutic tool for disease with the attenuation of rgn expression 30 .The development of drugs that modulate the RGN molecule may have a clinical significance in the restoration of metabolic disorders implicated to RGN.Clinical studies of RGN for disease and its application in clinical fields will be expected.