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Aims Vimentin citrullination, the calcium (Ca2+ )-dependent peptidylarginine deiminase (PAD)-mediated conversion of an arginine residue of vimentin to a citrulline residue, has emerged as a pathophysiological outcome of autoimmune diseases and neurodegeneration. However, the roles, functions, and expression of citrullinated vimentin have not yet been elucidated because available antibodies are limited. Methods We developed mouse monoclonal IgG1 and IgM specific for vimentin citrullinated at position R440 or R450 and applied Western blotting, immunohistochemistry, and immunofluorescent staining to investigate the pathogenesis of prion diseases in animal models, in patients with prion diseases, and in vitro. Results Vimentin was found to be highly citrullinated at R440 and R450, and these citrullinated forms were mainly expressed in reactive astrocytes in the brain tissues of scrapie-infected mice. Full-length and cleaved forms of citrullinated vimentin were found in the cerebral cortices of sporadic Creutzfeldt-Jakob disease (sCJD) patients. The distribution of citrullinated vimentin was mainly confirmed in vimentin-, GFAP-, and YKL-40-positive reactive astrocytes. Biochemically, citrullination promoted resistance to the caspase-3- and caspase-9-mediated fragmentation of vimentin. Additionally, citrullination led to increased cytoplasmic and integral membrane/organelle vimentin enrichment, which indicated changes in the intrinsic solubility and distribution of vimentin. Conclusions Our observations suggest that citrullinated vimentin acts as a specific indicator for the reactive state of astrocytes under abnormal neurological conditions. In addition, these novel antibodies will be helpful for studying the role of citrullinated vimentin in the pathogenesis of human disorders.

Peptidylarginine deiminases (PADs) are posttranslational modification enzymes that citrullinate (deiminate) protein arginine residues in a calcium-dependent manner, yielding citrulline residues. Enzymatic citrullination abolishes positive charges of native protein molecules, inevitably causing significant alterations in their structure and function. Previously, we reported the abnormal accumulation of citrullinated proteins and an increase of PAD2 content in hippocampi of patients with Alzheimer disease. In this study, we investigated PAD expression by using dibutyryl cAMP (dbcAMP) in human astrocytoma U-251MG cells. Under normal culture conditions, PAD2 and PAD3 mRNA expression is detectable with quantitative PCR in U-251MG cells. The addition of dbcAMP in a dose-dependent manner significantly increased this mRNA expression and protein levels. Moreover, PAD enzyme activity also increased significantly and dose-dependently. Furthermore, the expression of PAD2 and PAD3 mRNA was inhibited by the cAMP-dependent PKA inhibitor KT5720, suggesting that such expression of dbcAMP-induced PAD2 and PAD3 mRNA is mediated by the cAMP-PKA signaling pathway in U-251MG cells. This is the first report to document the PAD2 and PAD3 mRNA expression induced by dbcAMP and to attribute the induction of these genes to mediation by the cAMP-PKA signaling pathway in U-251MG cells. © 2016 Wiley Periodicals, Inc.

Prion infection leads to neuronal cell death, glial cell activation, and the accumulation of misfolded prion proteins. However, the altered cellular environments in animals with prion diseases are poorly understood. In the central nervous system, cells connect the cytoplasm of adjacent cells via connexin (Cx)-assembled gap junction channels to allow the direct exchange of small molecules, including ions, neurotransmitters, and signaling molecules, which regulate the activities of the connected cells. Here, we investigate the role of Cx43 in the pathogenesis of prion diseases. Upregulated Cx43 expression, which was dependent on c-Jun N-Terminal Kinase (JNK)/c-Jun signaling cascades, was found in prion-affected brain tissues and hippocampal neuronal cells. Scrapie infection-induced Cx43 formed aggregated plaques within the cytoplasmic compartments at the cell-cell interfaces. The ethidium bromide (EtBr) uptake assay and scrape-loading dye transfer assay demonstrated that increased Cx43 has functional consequences for the activity of Cx43 hemichannels. Interestingly, blockade of PrPSc accumulation reduced Cx43 expression through the inhibition of JNK signaling, indicating that PrPSc accumulation may be directly involved in JNK activation-mediated Cx43 upregulation. Overall, our findings describe a scrapie infection-mediated novel regulatory signaling pathway of Cx43 expression and may suggest a role for Cx43 in the pathogenesis of prion diseases.

Calsenilin modulates A-type potassium channels, regulates presenilin-mediated γ-secretase activity, and represses prodynorphin and c-fos genes expression. RhoA is involved in various cellular functions including proliferation, differentiation, migration, transcription, and regulation of the actin cytoskeleton. Although recent studies demonstrate that calsenilin can directly interact with RhoA and that RhoA inactivation is essential for neuritogenesis, it is uncertain whether there is a link between calsenilin and RhoA-regulated neuritogenesis. Here, we investigated the role of calsenilin in RhoA-regulated neuritogenesis using in vitro and in vivo systems. We found that calsenilin induced RhoA inactivation, which accompanied RhoA phosphorylation and the reduced phosphorylation levels of LIM kinase (LIMK) and cofilin. Interestingly, PC12 cells overexpressing either full-length (FL) or the caspase 3-derived C-terminal fragment (CTF) of calsenilin significantly inactivated RhoA through its interaction with RhoA and p190 Rho GTPase-activating protein (p190RhoGAP). In addition, cells expressing FL and the CTF of calsenilin had increased neurite outgrowth compared to cells expressing the N-terminal fragment (NTF) of calsenilin or vector alone. Moreover, Tat-C3 and Y27632 treatment significantly increased the percentage of neurite-bearing cells, neurite length, and the number of neurites in cells. Finally, calsenilin deficiency in the brains of calsenilin-knockout mice significantly interfered with RhoA inactivation. These findings suggest that calsenilin contributes to neuritogenesis through RhoA inactivation.

Hepatic stellate cells (HSCs) play pivotal roles in hepatic fibrosis as they synthesize glial fibrillary acidic protein (GFAP), which is increased in activated HSCs. GFAP-expressing HSCs and myofibroblasts accumulate in and around hepatic fibrosis lesions. Peptidylarginine deiminase 2 (PAD2) is responsible for the citrullination of GFAP (cit-GFAP). However, the involvement of PAD2 and cit-GFAP in hepatic fibrosis remains unclear. To determine the expression of PAD2 and cit-GFAP in hepatic fibrosis, C57BL/6 mice underwent bile duct ligation (BDL) or a sham operation. In BDL livers, the expression of PAD2 and its enzyme activity were significantly increased compared with controls. In addition, PAD2-postitive cells were rarely observed in only the portal vein and the small bile duct in sham-operated livers, whereas an increased number of PAD2-positive cells were detected in the bile duct and Glisson's sheath in BDL livers. Interestingly, PAD2 was colocalized with α-SMA-positive cells and CK19-positive cells in BDL livers, indicating upregulated PAD2 in activated HSCs and portal fibroblasts of the livers of BDL mice. We also found that citrullinated proteins were highly accumulated in the livers of BDL mice compared with controls. Moreover, the expression level of GFAP and the amount of cit-GFAP were higher in BDL livers than in control livers. In correlation with PAD2 localization, cit-GFAP was observed in α-SMA-positive and CK19-positive cells in the livers of BDL mice. These results suggest that the increased expression and activation of PAD2 along with increased citrullinated proteins, specifically cit-GFAP, may play important roles in the pathogenesis of hepatic fibrosis.

Peptidylarginine deiminases (PADs) are posttranslational modification enzymes that convert protein arginine to citrulline residues in a calcium ion-dependent manner. Previously, we reported the abnormal accumulation of citrullinated proteins and the increase in the amount of PAD2 in hippocampi from Alzheimer's disease (AD) patients. Moreover, glial fibrillary acidic protein (GFAP), an astrocyte-specific marker protein, and vimentin were identified as citrullinated proteins by using two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. To clarify the substrate specificity of PADs against GFAP, we prepared recombinant human (rh)PAD1, rhPAD2, rhPAD3, rhPAD4, and rhGFAP. After incubation of rhGFAP with rhPAD1, rhPAD2, rhPAD3, and rhPAD4, citrullinated (cit-)rhGFAP was detected by Western blotting. The citrullination of rhGFAP by rhPAD2 was unique, specific, and time dependent; additionally, rhPAD1 slightly citrullinated rhGFAP. We then generated eight anti-cit-rhGFAP monoclonal antibodies, CTGF-125, -128, -129, -1212, -1213, -1221, -122R, and -1224R, which reacted specifically with cit-rhGFAP. Two of those eight monoclonal antibodies, CTGF-122R and -1224R, reacted with both cit-rhGFAP and rhGFAP in Western blots. By using the CTGF-1221 antibody and a tandem mass spectrometer, we identified the two independent citrullination sites (R270Cit and R416Cit) of cit-rhGFAP. Immunohistochemical analysis with CTGF-1221 antibody revealed cit-GFAP staining in the hippocampus of AD brain, and the cit-GFAP-positive cells appeared to be astrocyte-like cells. These collective results strongly suggest that PAD2 is responsible for the citrullination of GFAP in the progression of AD and that the monoclonal antibody CTGF-1221, reacting with cit-GFAP at R270Cit and R416Cit, is useful for immunohistochemical investigation of AD brains.

Cl-amidine, which is a small-molecule inhibitor of PAD, has therapeutic potential for inflammation-mediated diseases. However, little is known regarding the manner by which PAD inhibition by Cl-amidine regulates inflammatory conditions. Here, we investigated the effects of PAD inhibition by Cl-amidine on the functioning of DCs, which are pivotal immune cells that mediate inflammatory diseases. When DC maturation was induced by TLR agonists, reduced cytokine levels (IL-6, IL-1β, and IL-12p70) were observed in Cl-amidine-treated DCs. Cl-amidine-treated, LPS-activated DCs exhibited alterations in their mature and functional statuses with up-regulated antigen uptake, down-regulated CD80, and MHC molecules. In addition, Cl-amidine-treated DCs dysregulated peptide-MHC class formations. Interestingly, the decreased cytokines were independent of MAPK/NF-κB signaling pathways and transcription levels, indicating that PAD inhibition by Cl-amidine may be involved in post-transcriptional steps of cytokine production. Transmission electron microscopy revealed morphotypical changes with reduced dendrites in the Cl-amidine-treated DCs, along with altered cellular compartments, including fragmented ERs and the formation of foamy vesicles. Furthermore, in vitro and in vivo Cl-amidine treatments impaired the proliferation of nai¨ve CD4+ and CD8+ T cells. Overall, our findings suggest that Cl-amidine has therapeutic potential for treating inflammation-mediated diseases.

The conversion of peptidylarginine into peptidylcitrulline by calcium-dependent peptidylarginine deiminases (PADs) has been implicated in the pathogenesis of a number of diseases, identifying PADs as therapeutic targets for various diseases. The PAD inhibitor Cl-amidine ameliorates the disease course, severity, and clinical manifestation in multiple disease models, and it also modulates dendritic cell (DC) functions such as cytokine production, antigen presentation, and T cell proliferation. The beneficial effects of Cl-amidine make it an attractive compound for PAD-targeting therapeutic strategies in inflammatory diseases. Here, we found that Cl-amidine inhibited nitric oxide (NO) generation in a time- and dose-dependent manner in maturing DCs activated by lipopolysaccharide (LPS). This suppression of NO generation was independent of changes in NO synthase (NOS) enzyme activity levels but was instead dependent on changes in inducible NO synthase (iNOS) transcription and expression levels. Several upstream signaling pathways for iNOS expression, including the mitogen-activated protein kinase, nuclear factor-κB p65 (NF-κB p65), and hypoxia-inducible factor 1 pathways, were not affected by Cl-amidine. By contrast, the LPS-induced signal transducer and the activator of transcription (STAT) phosphorylation and activator protein-1 (AP-1) transcriptional activities (c-Fos, JunD, and phosphorylated c-Jun) were decreased in Cl-amidine-treated DCs. Inhibition of Janus kinase/STAT signaling dramatically suppressed iNOS expression and NO production, whereas AP-1 inhibition had no effect. These results indicate that Cl-amidine-inhibited STAT activation may suppress iNOS expression. Additionally, we found mildly reduced cyclooxygenase-2 expression and prostaglandin E2 production in Cl-amidine-treated DCs. Our findings indicate that Cl-amidine acts as a novel suppressor of iNOS expression, suggesting that Cl-amidine has the potential to ameliorate the effects of excessive iNOS/NO-linked immune responses.

The failure of Mycobacterium bovis BCG as a TB vaccine against TB reactivation suggests that latency-associated proteins should be included in alternative TB vaccine development. Further, antigens known to generate protective immunity against the strong Th1 stimulatory response to reactivated TB should be included in novel vaccine design. Recent studies have emphasized the importance of Rpfs from Mycobacterium tuberculosis in the reactivation process and cellular immunity. However, little is known about how RpfB mediates protective immunity against M. tuberculosis. Here, we investigated the functional roles and signaling mechanisms of RpfB in DCs and its implications in the development of T cell immunity. DCs treated with RpfB displayed features of mature and functional status, with elevated expression of cell surface molecules (CD80, CD86, and MHC class I and II) and proinflammatory cytokine production (TNF-α, IL-1β, IL-6, and IL-12p70). Activation of DCs was mediated by direct binding of RpfB to TLR4, followed by MyD88/TRIF-dependent signaling to MAPKs and NF-κB signaling pathways. Specifically, we found that the RpfB G5 domain is the most important part in RpfB binding to TLR4. RpfB-treated DCs effectively polarized naïve CD4+ and CD8+ T cells to secrete IFN-γ and IL-2. Importantly, RpfB induced the expansion of memory CD4+/CD8+CD44highCD62Llow T cells in the spleen of M. tuberculosis-infected mice. Our data suggest that RpfB regulates innate immunity and activates adaptive immunity through TLR4, a finding that may help in the design of more effective vaccines.

The post-translational citrullination (deimination) process is mediated by peptidylarginine deiminases (PADs), which convert peptidylarginine into peptidylcitrulline in the presence of high calcium concentrations. Over the past decade, PADs and protein citrullination have been commonly implicated as abnormal pathological features in neurodegeneration and inflammatory responses associated with diseases such as multiple sclerosis, Alzheimer disease and rheumatoid arthritis. Based on this evidence, we investigated the roles of PADs and citrullination in the pathogenesis of prion diseases. Prion diseases (also known as transmissible spongiform encephalopathies) are fatal neurodegenerative diseases that are pathologically well characterized as the accumulation of disease-associated misfolded prion proteins, spongiform changes, glial cell activation and neuronal loss. We previously demonstrated that the upregulation of PAD2, mainly found in reactive astrocytes of infected brains, leads to excessive citrullination, which is correlated with disease progression. Further, we demonstrated that various cytoskeletal and energy metabolism-associated proteins are particularly vulnerable to citrullination. Our recent in vivo and in vitro studies elicited altered functions of enolase as the result of citrullination; these altered functions included reduced enzyme activity, increased protease sensitivity and enhanced plasminogen-binding affinity. These findings suggest that PAD2 and citrullinated proteins may play a key role in the brain pathology of prion diseases. By extension, we believe that abnormal increases in protein citrullination may be strong evidence of neurodegeneration.

Peptidylarginine deiminases (PADs) are posttranslational modification enzymes that citrullinate (deiminate) protein arginine residues in a calcium-dependent manner, yielding citrulline residues. Enzymatic citrullination abolishes positive charges of native protein molecules, inevitably causing significant alterations in their structure and function. Previously, we reported the abnormal accumulation of citrullinated proteins and an increase of PAD2 content in hippocampi of patients with Alzheimer disease. In this study, we investigated PAD expression by using dibutyryl cAMP (dbcAMP) in human astrocytoma U-251MG cells. Under normal culture conditions, PAD2 and PAD3 mRNA expression is detectable with quantitative PCR in U-251MG cells. The addition of dbcAMP in a dose-dependent manner significantly increased this mRNA expression and protein levels. Moreover, PAD enzyme activity also increased significantly and dose-dependently. Furthermore, the expression of PAD2 and PAD3 mRNA was inhibited by the cAMP-dependent PKA inhibitor KT5720, suggesting that such expression of dbcAMP-induced PAD2 and PAD3 mRNA is mediated by the cAMP-PKA signaling pathway in U-251MG cells. This is the first report to document the PAD2 and PAD3 mRNA expression induced by dbcAMP and to attribute the induction of these genes to mediation by the cAMP-PKA signaling pathway in U-251MG cells. © 2016 Wiley Periodicals, Inc.

The citrullination of enolase by PAD (peptidylarginine deiminase) has emerged as an important post-translational modification in human disorders; however, the physiological function of citrullination remains unknown. In the present study, we report that citrullination diversely regulates the biological functions of ENO1 (α-enolase) and NSE (neuron-specific enolase). We developed three mouse IgG1 monoclonal antibodies with specificity to the following: (i) citrullination of Arg9 of ENO1 [ENO1Cit9; anti-CE1 (citrullinated enolase 1) antibody]; (ii) citrullination of Arg9 in ENO1 and NSE (ENO1Cit9/NSECit9; anti-CE1/2 antibody); and (iii) citrullination of Arg429 of NSE (NSECit429; anti-CE2 antibody). Regardless of the total protein expression level, the levels of ENO1Cit9 and NSECit429 were elevated, and their immunoreactivities were also increased in cortical neuronal cells or around blood vessels in the frontal cortex of patients with sporadic Creutzfeldt-Jakob disease and Alzheimer's disease compared with controls. In a time- and dose-dependent manner, PAD negatively regulated enolase activity via citrullination, and enolase in diseased patients was more inactive than in controls. Interestingly, the citrullination of enolase effectively promoted its proteolytic degradation by Ca2+-dependent calpain-1, and leupeptin (calpain inhibitor I) abrogated this degradation. Surprisingly, using an affinity assay, the citrullination of enolase enhanced its plasminogen-binding affinity, which was blocked by the lysine analogue ϵ-aminocaproic acid. These findings suggest that PAD-mediated citrullination regulates the diverse physiological activities of enolase and that CE may be a candidate diagnostic/prognostic factor for degenerative diseases.

Peptidylarginine deiminases (PADs)-mediated post-translational citrullination processes play key roles in protein functions and structural stability through the conversion of arginine to citrulline in the presence of excessive calcium concentrations. In brain, PAD2 is abundantly expressed and can be involved in citrullination in disease. Recently, we have reported pathological characterization of PAD2 and citrullinated proteins in scrapie-infected mice, but the implication of protein citrullination in the pathophysiology in human prion disease is not clear. In the present study, we explored the molecular and biological involvement of PAD2 and the pathogenesis of citrullinated proteins in frontal cortex of patients with sporadic Creutzfeldt-Jakob disease (sCJD). We found increased expression of PAD2 in reactive astrocytes that also contained increased levels of citrullinated proteins. In addition, PAD activity was significantly elevated in patients with sCJD compared to controls. From two-dimensional gel electrophoresis and MALDI-TOF mass analysis, we found various citrullinated candidates, including cytoskeletal and energy metabolism-associated proteins such as vimentin, glial fibrillary acidic protein, enolase, and phosphoglycerate kinase. Based on these findings, our investigations suggest that PAD2 activation and aberrant citrullinated proteins could play a role in pathogenesis and have value as a marker for the postmortem classification of neurodegenerative diseases.

Peptidylarginine deiminases (PADs), which are a group of posttranslational modification enzymes, are involved in protein citrullination (deimination) by the conversion of peptidylarginine to peptidylcitrulline in a calcium concentration-dependent manner. Among the PADs, PAD2 is widely distributed in various tissues and is the only type that is expressed in brain. To elucidate the involvement of protein citrullination by PAD2 in the pathogenesis of brain-specific prion diseases, we examined the profiles of citrullinated proteins using the brains of scrapie-infected mice as a prion disease model. We found that, compared with controls, increased levels of citrullinated proteins of various molecular weights were detected in different brain sections of scrapie-infected mice. In support of this data, expression levels of PAD2 protein as well as its enzyme activity were significantly increased in brain sections of scrapie-infected mice, including hippocampus, brain stem, and striatum. Additionally, the expression levels of PAD2 mRNA were increased during scrapie infection. Moreover, PAD2 immunoreactivity was increased in scrapie-infected brains, with staining detected primarily in reactive astrocytes. Using two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry, various citrullinated proteins were identified in the brains of scrapie-infected mice, including glial fibrillary acidic protein, myelin basic protein, enolases, and aldolases. This study suggests that accumulated citrullinated proteins and abnormal activation of PAD2 may function in the pathogenesis of prion diseases and serve as potential therapeutic targets.

Nitration of tyrosine residues is taken as evidence for intracellular formation of peroxynitrite. Cytochrome c (cyt c) can be nitrated by peroxynitrite and nitrated cyt c has been observed in cells and tissues under stress conditions. Here we studied the biochemical properties of nitrated cyt c in order to understand its potential roles in nitrative stress. Nitration of cyt c resulted in disruption of the heme-methionine bond and rapid binding to cyanide. Equilibrium unfolding by guanidine hydrochloride showed that cyt c was slightly destabilized upon nitration but the unfolding transition of nitrated cyt c was highly cooperative indicating that the overall folding was largely preserved. Nitrated cyt c could not be reduced by superoxide and did not support electron transfer between ascorbate and cyt c oxidase. Nitration of cyt c resulted in a tremendous increase in peroxidase activity so that nitrated cyt c rapidly oxidized dihydrodichlorofluorescein even in the presence of a high concentration of glutathione. Enhanced peroxidase activity of nitrated cyt c was responsible for H2O2-induced oxidation of phospholipid membranes and H2O2/NO2--mediated nitration of other proteins. These results suggest that nitration of cyt c by peroxynitrite may exacerbate oxidative damage to mitochondrial proteins and membranes.

PrPSc is formed from a normal glycosylphosphatidylinositol (GPI)-anchored prion protein (PrPC) by a posttranslational modification. Most GPI-anchored proteins have been shown to be cleaved by GPI phospholipases. Recently, GPI-phospholipase D (GPI-PLD) was shown to be a strictly specific enzyme for GPI anchors. To investigate the involvement of GPI-PLD in the processes of neurodegeneration in prion diseases, we examined the mRNA and protein expression levels of GPI-PLD in the brains of a prion animal model (scrapie), and in both the brains and cerebrospinal fluids (CSF) of sporadic and familial Creutzfeldt-Jakob disease (CJD) patients. We found that compared with controls, the expression of GPI-PLD was dramatically down-regulated in the brains of scrapie-infected mice, especially in the caveolin-enriched membrane fractions. Interestingly, the observed decrease in GPI-PLD expression levels began at the same time that PrPSc began to accumulate in the infected brains and this decrease was also observed in both the brain and CSF of CJD patients; however, no differences in expression were observed in either the brains or CSF specimens from Alzheimer's disease patients. Taken together, these results suggest that the down-regulation of GPI-PLD protein may be involved in prion propagation in the brains of prion diseases.

Peptidylarginine deiminases (PADs) are known to be directly affected by Ca2+ homeostasis and irreversibly catalyze the posttranslational deimination (or citrullination) of peptidylarginine to peptidylcitrulline in a Ca2+-dependent manner. Over the past decade, Ca2+-dependent peptidylarginine deiminases (PADs) and deiminated proteins have emerged as key molecules in neurodegeneration and inflammatory responses associated with diseases such as multiple sclerosis, Alzheimer disease, prion disease, and rheumatoid arthritis. Prion diseases (also known as transmissible spongiform encephalopathies) are well characterized by various neurological symptoms and common histopathological features, including spongiform degeneration, reactive gliosis, neuronal loss, and the accumulation of disease-associated misfolded prion proteins (termed PrPSc) in the central nervous system. Although current knowledge of the molecular and pathological features of PAD enzymes is still limited in neurodegenerative diseases, we previously demonstrated that the upregulation of PAD2 and abnormal accumulation of deiminated proteins, which are correlated with disease progression, were found in prion disease-affected human and mouse brains. Proteomic analysis and electron immunogold labeling further revealed that a variety of deiminated proteins with increased PAD2 in infected brains were widely distributed in different subcellular compartments in neuronal and glial cells. Our recent in vivo and in vitro studies elicited altered functions of enolase as the result of citrullination; these altered functions included reduced enzyme activity, increased protease sensitivity, and enhanced plasminogen-binding affinity. Further investigation of deiminated proteins and their potential physiological and pathogenic functions in prion diseases will be useful for the development of diagnostic tools as well as potential pathogenic targets in prion diseases.

Presenilin 1 (PS1) is a component of the γ-secretase complex that cleaves a variety of type I membrane proteins, including the β-amyloid precursor protein (β-APP), Notch, and neuronal (N)- and epithelial (E)-cadherins. N-cadherin is an essential adhesion molecule that forms a complex with, and is cleaved by, PS1/γ-secretase and β-catenin in the plasma membrane. The purpose of this study was to determine whether calsenilin, a presenilin-interacting protein, has a functional role in PS1/γ-secretase-mediated N-cadherin e-cleavage using Western blot analysis, RT-PCR, immunoprecipitation, subcellular fractionation, biotinylation, and a luciferase reporter assay in SH-SY5Y neuroblastoma cells. Here, we demonstrate that the expression of calsenilin leads to a disruption of PS1/γ-secretase-mediated e-cleavage of N-cadherin, which results in the significant accumulation of N-cadherin C-terminal fragment 1 (Ncad/CTF1), the reduction of cytoplasmic Ncad/CTF2 release, and a deceleration of PS1-CTF delivery to the cell surface. Interestingly, we also found that the expression of calsenilin is associated with the redistribution of β-catenin from the cell surface to a cytoplasmic pool, as well as with the negative regulation of genes that are targets of T-cell factor/β-catenin nuclear signaling. Taken together, our findings suggest that calsenilin is a novel negative regulator of N-cadherin processing that plays an important role in β-catenin signaling.

Peptidylarginine deiminase (PAD) and citrullinated proteins have emerged as key molecules in various human diseases, but detailed subcellular localizations of PAD2 and citrullinated proteins are poorly mapped in brain under normal and pathologic conditions. We performed subcellular fractionation and electron microscopic analysis using brains of normal and scrapie-infected mice. Peptidylarginine deiminase 2 was abundantly present in cytosol and weakly in microsomal and mitochondrial fractions and expression in these fractions was higher in brains of scrapie-infected mice. Despite relatively low PAD2 expression, in microsomal and mitochondrial fractions, citrullinated proteins were present at high levels in these fractions in scrapie-infected brains. Surprisingly, increased PAD2 expression and accumulated citrullinated proteins were also found in nuclear fractions in scrapie-infected brains. By electron microscopy, PAD2 and citrullinated proteins in scrapie-infected brains were widely distributed in most cellular compartments including mitochondria, endoplasmic reticulum, glial filaments, nuclei, and Golgi apparatus in astrocytes and hippocampal neurons. Taken together, we report for the first time the nuclear localization of PAD2 and the detailed subcellular localization of PAD2 and of citrullinated proteins in scrapie-infected brains. Our findings suggest that different subcellular compartmentalization of PAD2 and citrullinated proteins may have different physiological roles in normal and neurodegenerative conditions.

Peptidylarginine deiminases (PADs), which are a group of posttranslational modification enzymes, are involved in protein citrullination (deimination) by the conversion of peptidylarginine to peptidylcitrulline in a calcium concentration-dependent manner. Among the PADs, PAD2 is widely distributed in various tissues and is the only type that is expressed in brain. To elucidate the involvement of protein citrullination by PAD2 in the pathogenesis of brain-specific prion diseases, we examined the profiles of citrullinated proteins using the brains of scrapie-infected mice as a prion disease model. We found that, compared with controls, increased levels of citrullinated proteins of various molecular weights were detected in different brain sections of scrapie-infected mice. In support of this data, expression levels of PAD2 protein as well as its enzyme activity were significantly increased in brain sections of scrapie-infected mice, including hippocampus, brain stem, and striatum. Additionally, the expression levels of PAD2 mRNA were increased during scrapie infection. Moreover, PAD2 immunoreactivity was increased in scrapie-infected brains, with staining detected primarily in reactive astrocytes. Using two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry, various citrullinated proteins were identified in the brains of scrapie-infected mice, including glial fibrillary acidic protein, myelin basic protein, enolases, and aldolases. This study suggests that accumulated citrullinated proteins and abnormal activation of PAD2 may function in the pathogenesis of prion diseases and serve as potential therapeutic targets.

In Magnetic resonance imaging(MRI), imaging a moving organ such as the heart requires a trigger so that successive scans can be synchronized. In the case of cardiac imaging, the QRS complex of ECG is used as a trigger signal for MRI scan. But, gradient and RF(radio frequency) artifacts which are caused to static and dynamic field in MRI scanner cause interference in the ECG. Also, the signal shape of theses artifacts can be similar to the QRS-complex, causing possible misinterpretation during patient monitoring and false gating of the MRI. In case of using general FIR or IIR band-pass filters for minimizing the artifacts, artifact-reduction-ratio is not excellent. So, an adaptive real-time digital filter is proposed for reduction of noise by gradient and RF(radio frequency) artifacts. The proposed filter for MRI-Gating is based on the noise-canceller with NLMS(Normalized Least Mean Square) algorithm. The reference signals of the adaptive noise canceller are a combination of the noisy three channel ECG signals. Various tests were done on normal volunteers in various scenarios[SE(spin echo), FSE(fast spin echo), TR(transition time), TX-Gain, RX-gain, Spin-echo-degree, soon]. The noise canceller’s performance was measured offline, simulating real-time processing by point-by-point operations. In conclusions, the proposed method showed the acceptable quality of ECG signal with sufficient SNR for gating the MRI and possibility of real time implementation

The multiple post-translational modifications of proteins display specific gain- or loss-of-function under normal and abnormal conditions. These modifications are precisely regulated by post-translational modification enzymes. The altered molecular status perturbs the pattern of gene expression and decides on a direction to signal transduction cascades as well as intrinsic properties of the proteins. Ultimately, it strictly maintains intracellular environment or results in disease manifestations. Recently, it has become that enzyme-dependent modification of arginine residue to citrulline exerts an important role in the induction of autoimmunity including rheumatoid arthritis, multiple sclerosis, and cancer. The modification of arginine residue to citrulline on proteins is called "citrullination" or "deimination" and is regulated by the calcium-dependent enzyme peptidylarginine deiminase (PAD). Now many effective PAD inhibitors (for example, Cl-amidine) have developed that ameliorates disease phenotypes. In this review, we discuss crucial roles of PAD enzyme and citrullination, the effectiveness of PAD inhibitors, and the implication in pathology.