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Ziqing Hei - One of the best experts on this subject based on the ideXlab platform.
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Connexin 32 deficiency protects the liver against ischemia reperfusion injury
European Journal of Pharmacology, 2020Co-Authors: Weifeng Yao, Dongdong Yuan, Fei Huang, Chaojin Chen, Huixin Chen, Yiqian Liu, Jun Cai, Ziqing HeiAbstract:Hepatic ischemia/reperfusion (I/R) injury is a common complication in the clinical setting. Our previous study has shown that Connexin 32 (Cx32) plays a major role in renal I/R injury; however, the role of Cx32 in hepatic I/R injury remains unknown. Liver tissue and serum samples from patients undergoing orthotopic liver transplantation (OLT) were used to evaluate the function of Cx32 in OLT post-reperfusion injury. Then, partial hepatic ischemia was established in global Cx32 knockout mice and wild-type mice followed by reperfusion. Hepatic injury markers were examined. Cx32 small interfering RNA and the p53 inhibitor, pifithrin-α, tenovin-1 were used to examine the relationship between Cx32 and the p53/puma pathways in the BRL-3A and murine primary hepatocytes hypoxia/reoxygenation (H/R) model. Corresponding to liver damage, Cx32 was significantly induced both during OLT in human patients and partial hepatic I/R in mice. Cx32 KO mice exhibited less liver injury than controls. Cx32 deficiency significantly suppressed the p53/puma pathways and hepatocyte apoptosis. Similar results were observed in the BRL-3A and murine primary hepatocytes H/R model. Propofol protected against OLT post-reperfusion injury and hepatocyte apoptosis by inhibiting Cx32. In conclusion Cx32 is a novel regulator of hepatic I/R injury through the modulation of hepatocyte apoptosis and damage, largely via the p53/puma signaling pathway.
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Connexin 32 deficiency protects the liver against ischemia/reperfusion injury.
European journal of pharmacology, 2020Co-Authors: Weifeng Yao, Dongdong Yuan, Fei Huang, Chaojin Chen, Huixin Chen, Yiqian Liu, Jun Cai, Ziqing HeiAbstract:Hepatic ischemia/reperfusion (I/R) injury is a common complication in the clinical setting. Our previous study has shown that Connexin 32 (Cx32) plays a major role in renal I/R injury; however, the role of Cx32 in hepatic I/R injury remains unknown. Liver tissue and serum samples from patients undergoing orthotopic liver transplantation (OLT) were used to evaluate the function of Cx32 in OLT post-reperfusion injury. Then, partial hepatic ischemia was established in global Cx32 knockout mice and wild-type mice followed by reperfusion. Hepatic injury markers were examined. Cx32 small interfering RNA and the p53 inhibitor, pifithrin-α, tenovin-1 were used to examine the relationship between Cx32 and the p53/puma pathways in the BRL-3A and murine primary hepatocytes hypoxia/reoxygenation (H/R) model. Corresponding to liver damage, Cx32 was significantly induced both during OLT in human patients and partial hepatic I/R in mice. Cx32 KO mice exhibited less liver injury than controls. Cx32 deficiency significantly suppressed the p53/puma pathways and hepatocyte apoptosis. Similar results were observed in the BRL-3A and murine primary hepatocytes H/R model. Propofol protected against OLT post-reperfusion injury and hepatocyte apoptosis by inhibiting Cx32. In conclusion Cx32 is a novel regulator of hepatic I/R injury through the modulation of hepatocyte apoptosis and damage, largely via the p53/puma signaling pathway.
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Effects of Connexin 32-Mediated Lung Inflammation Resolution During Liver Ischemia Reperfusion.
Digestive diseases and sciences, 2020Co-Authors: Zheng Zhang, Weifeng Yao, Dongdong Yuan, Fei Huang, Yue Liu, Gangjian Luo, Ziqing HeiAbstract:Background Hepatic ischemia reperfusion (HIR) leads to a lung inflammatory response and subsequent pulmonary barrier dysfunction. The gap junction communication protein Connexin 32 (Cx32), which is widely expressed in the lungs, participates in intercellular signaling. This study determined whether the communication protein Cx32 could affect pulmonary inflammation caused by HIR.
Antonio Villalobo - One of the best experts on this subject based on the ideXlab platform.
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Phosphorylation of Connexin-32 by protein kinase C prevents its proteolysis by mu-calpain and m-calpain.
The Journal of biological chemistry, 1993Co-Authors: Maribel Elvira, Juan Diez, Kevin K. W. Wang, Antonio VillaloboAbstract:Abstract Isolated Connexin-32s from rat and mouse liver are proteolyzed in vitro by the intracellular Ca(2+)-dependent neutral proteases, mu-calpain and m-calpain, producing a major fragment of 26 kDa. Connexin-26 is not proteolyzed by calpain. Calpain cleaves Connexin-32 at its C-terminal end as shown by 125I-calmodulin binding experiments. Connexin-32, but not Connexin-26, is phosphorylated by both protein kinase A and protein kinase C in serine residues and the sites of phosphorylation by both kinases remain in the major 26-kDa fragment resulting from calpain proteolysis. Phosphorylation of Connexin-32 by protein kinase C, but not by protein kinase A, prevents the proteolytic attack of mu-calpain and m-calpain. Phosphorylation of Connexin-32 by protein kinase A and protein kinase C does not prevent its proteolysis by papain, alpha-chymotrypsin, proteinase K, and trypsin.
Terry L. Dowd - One of the best experts on this subject based on the ideXlab platform.
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structural studies of n terminal mutants of Connexin 26 and Connexin 32 using 1 h nmr spectroscopy
Archives of Biochemistry and Biophysics, 2016Co-Authors: Y. Batir, Thaddeus A. Bargiello, Terry L. DowdAbstract:Alterations in gap junctions underlie the etiologies of syndromic deafness (KID) and Charcot-Marie Tooth disease (CMTX). Functional gap junctions are composed of Connexin molecules with N-termini containing a flexible turn around G12, inserting the N-termini into the channel pore allowing voltage gating. The loss of this turn correlates with loss of Connexin 32 (Cx32) function by impaired trafficking to the cell membrane. Using (1)H NMR we show the N-terminus of a syndromic deafness mutation Cx26G12R, producing "leaky channels", contains a turn around G12 which is less structured and more flexible than wild-type. In contrast, the N-terminal structure of the same mutation in Cx32 chimera, Cx32*43E1G12R shows a larger constricted turn and no membrane current expression but forms membrane inserted hemichannels. Their function was rescued by formation of heteromeric channels with wild type subunits. We suggest the inflexible Cx32G12R N-terminus blocks ion conduction in homomeric channels and this channel block is relieved by incorporation of wild type subunits. In contrast, the increased open probability of Cx26G12R hemichannels is likely due to the addition of positive charge in the channel pore changing pore electrostatics and impairing hemichannel regulation by Ca(2+). These results provide mechanistic information on aberrant channel activity observed in disease.
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NMR and structural data for Connexin 32 and Connexin 26 N-terminal peptides.
Data in brief, 2016Co-Authors: Y. Batir, Thaddeus A. Bargiello, Terry L. DowdAbstract:In this article we present 1H and 13C chemical shift assignments, secondary structural propensity data and normalized temperature coefficient data for N-terminal peptides of Connexin 26 (Cx26), Cx26G12R and Cx32G12R mutants seen in syndromic deafness and Charcot Marie Tooth Disease respectively, published in “Structural Studies of N-Terminal Mutants of Connexin 26 and Connexin 32 Using 1H NMR Spectroscopy” (Y. Batir, T.A. Bargiello, T.L. Dowd, 2016) [1]. The mutation G12R affects the structure of both Cx26 and Cx32 peptides differently. We present data from secondary structure propensity chemical shift analysis which calculates a secondary structure propensity (SSP) score for both disordered or folded peptides and proteins using the difference between the 13C secondary chemical shifts of the Cα and Cβ protons. This data supplements the calculated NMR structures from NOESY data [1]. We present and compare the SSP data for the Cx26 vs Cx26G12R peptides and the Cx32 and Cx32G12R peptides. In addition, we present plots of temperature coefficients obtained for Cx26, Cx26G12R and Cx32G12R peptides collected previously [1] and normalized to their random coil temperature coefficients, “Random coil 1H chemical shifts obtained as a function of temperature and trifluoroethanol concentration for the peptide series GGXGG” (G. Merutka, H.J. Dyson, P.E. Wright, 1995) [2]. Reductions in these normalized temperature coefficients are directly observable for residues in different segments of the peptide and this data informs on solvent accessibility of the NH protons and NH protons which may be more constrained due to the formation of H bonds.
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Structural studies of N-terminal mutants of Connexin 32 using 1H NMR spectroscopy
Archives of biochemistry and biophysics, 2012Co-Authors: B.d. Kalmatsky, Thaddeus A. Bargiello, Y. Batir, Terry L. DowdAbstract:The amino terminus of gap junction proteins, Connexins, plays a fundamental role in voltage gating and ion permeation. We have previously shown with 1H NMR that the structure of the N-terminus of functional Connexin molecules contains a flexible turn around G12 (Arch. Biochem. Biophys.490:9,2009) allowing the N-terminus to form a portion of the channel pore near the cytoplasmic entrance. The mutants of nonfunctional Connexin molecules G12S and G12Y were found to prevent this turn. Previous functional studies of loci at which Cx32 mutations cause a peripheral neuropathy, Charcot-Marie-Tooth disease, have shown that G12S is not plasma membrane inserted. Presently, we solve the structure of nonfunctional Connexin 32 mutants W3D and Y7D which do not appear to be membrane inserted. Using 2D 1H NMR, we report that similar to G12S and G12Y, alterations in hydrophobic sidechain interactions disrupt (Y7D) or constrain (W3D) the flexible turn around G12. The alteration in the open turn around residue 12, observed in all nonfunctional mutants G12S, G12Y, W3D and Y7D correlates with loss of function. We propose that loss of the open turn causes the N-terminus to extend out of the channel pore and this misfolding may target mutants for destruction in the endoplasmic reticulum.
Rudolf Martini - One of the best experts on this subject based on the ideXlab platform.
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Macrophage‐Related Demyelination In Peripheral Nerves Of Mice Deficient In The Gap Junction Protein Connexin 32
Journal of the Peripheral Nervous System, 2002Co-Authors: Igor Kobsar, Mathias Mäurer, Thomas Ott, Rudolf MartiniAbstract:Mice deficient in the gap junction protein Connexin 32 (Cx32) develop a slowly progressing demyelinating neuropathy, with enlarged periaxonal collars, abnormal non-compacted myelin domains and axonal sprouts. These mice serve as a model for the X-linked form of inherited demyelin- ating neuropathies in humans. Based on our previous findings that macrophages are involved in demyelination in other myelin mutants (i.e. mice heterozygously deficient in P0), we considered the possibility that macrophages might be also mediators of demyelination in Cx32-deficient mice. Indeed, we detected an age-related increase in the number of macrophages in demyelinating nerves of Cx32-deficient mice. In addition, immunoelectron microscopy revealed macrophages in an apposition to degenerating myelin reminiscent of a macrophage-mediated demyelinating neuropathy. We conclude that involvement of macrophages might be a widespread phenomenon in genetically-determined demyelination.
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Macrophage-related demyelination in peripheral nerves of mice deficient in the gap junction protein Connexin 32.
Neuroscience letters, 2002Co-Authors: Igor Kobsar, Mathias Mäurer, Thomas Ott, Rudolf MartiniAbstract:Mice deficient in the gap junction protein Connexin 32 (Cx32) develop a slowly progressing demyelinating neuropathy, with enlarged periaxonal collars, abnormal non-compacted myelin domains and axonal sprouts. These mice serve as a model for the X-linked form of inherited demyelinating neuropathies in humans. Based on our previous findings that macrophages are involved in demyelination in other myelin mutants (i.e. mice heterozygously deficient in P0), we considered the possibility that macrophages might be also mediators of demyelination in Cx32-deficient mice. Indeed, we detected an age-related increase in the number of macrophages in demyelinating nerves of Cx32-deficient mice. In addition, immunoelectron microscopy revealed macrophages in an apposition to degenerating myelin reminiscent of a macrophage-mediated demyelinating neuropathy. We conclude that involvement of macrophages might be a widespread phenomenon in genetically-determined demyelination.
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Accelerated demyelination of peripheral nerves in mice deficient in Connexin 32 and protein zero
Journal of neuroscience research, 1998Co-Authors: Dirk H H Neuberg, Rudolf Martini, Melitta Schachner, Stefano Carenini, Ueli SuterAbstract:Mutant mice that lack either protein zero (P0) or Connexin 32 (Cx32) were generated previously to investigate the function of these myelin proteins in peripheral nerves and to assess the value of these mice as animal models for hereditary human peripheral neuropathies. Mice that are completely devoid of P0 expression (P00/0) show a complex phenotype that is characterized by hypomyelination, compromised myelin compaction, and degeneration of myelin and axons early in life. In contrast, young mouse mutants that have retained one wild-type allele of the P0 gene (P0+/0) reveal morphologically normal myelin but start to develop signs of demyelination and remyelination at 4 months of age. A similar late-onset myelin deficiency was observed in Cx32-deficient mice (Cx320/0). We have now generated mice deficient for Cx32 and P0. In animals that lack both proteins (Cx320/0/P00/0), the phenotype is morphologically identical to mice that solely lack P0. Animals that lack Cx32 and carry one functional P0 allele (Cx320/0/P0+/0) revealed demyelination and remyelination as evidenced by thin myelin and Schwann cell onion bulb formation already at the age of 4 weeks, a time point when no pathology was observed in the single mutants. These morphological deficits were also more prominent in 4-month-old Cx320/0/P0+/0 animals compared to the single mutants. Our data support the view that Cx32 and P0 are crucial molecules for the maintenance of myelin. Furthermore, the function of Cx32 in the peripheral nervous system appears to be largely dispensable when myelin compaction is impaired. J. Neurosci. Res. 53:542–550, 1998. © 1998 Wiley-Liss, Inc.
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structural abnormalities and deficient maintenance of peripheral nerve myelin in mice lacking the gap junction protein Connexin 32
The Journal of Neuroscience, 1997Co-Authors: Patrizia Anzini, Klaus Willecke, Dirk H H Neuberg, Melitta Schachner, Eric Nelles, Jurgen Zielasek, Klaus V Toyka, Ueli Suter, Rudolf MartiniAbstract:Mutations affecting the Connexin 32 (Cx32) gene are associated with the X-linked form of the hereditary peripheral neuropathy Charcot–Marie–Tooth disease (CMTX). We show that Cx32-deficient mice develop a late-onset progressive peripheral neuropathy with abnormalities comparable to those associated with CMTX, thus providing proof of the critical role of Cx32 in the maintenance of peripheral nerve myelin and an animal model for CMTX. Frequently observed features include abnormally thin myelin sheaths, cellular onion bulb formation reflecting myelin degeneration-induced Schwann cell proliferation, and enlarged periaxonal collars while nerve conductance properties are altered only slightly. These observations are consistent with earlier hypotheses suggesting a function of Cx32 as a channel-forming protein that facilitates the communication between the abaxonal and adaxonal aspects of Schwann cell cytoplasm.
Thaddeus A. Bargiello - One of the best experts on this subject based on the ideXlab platform.
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structural studies of n terminal mutants of Connexin 26 and Connexin 32 using 1 h nmr spectroscopy
Archives of Biochemistry and Biophysics, 2016Co-Authors: Y. Batir, Thaddeus A. Bargiello, Terry L. DowdAbstract:Alterations in gap junctions underlie the etiologies of syndromic deafness (KID) and Charcot-Marie Tooth disease (CMTX). Functional gap junctions are composed of Connexin molecules with N-termini containing a flexible turn around G12, inserting the N-termini into the channel pore allowing voltage gating. The loss of this turn correlates with loss of Connexin 32 (Cx32) function by impaired trafficking to the cell membrane. Using (1)H NMR we show the N-terminus of a syndromic deafness mutation Cx26G12R, producing "leaky channels", contains a turn around G12 which is less structured and more flexible than wild-type. In contrast, the N-terminal structure of the same mutation in Cx32 chimera, Cx32*43E1G12R shows a larger constricted turn and no membrane current expression but forms membrane inserted hemichannels. Their function was rescued by formation of heteromeric channels with wild type subunits. We suggest the inflexible Cx32G12R N-terminus blocks ion conduction in homomeric channels and this channel block is relieved by incorporation of wild type subunits. In contrast, the increased open probability of Cx26G12R hemichannels is likely due to the addition of positive charge in the channel pore changing pore electrostatics and impairing hemichannel regulation by Ca(2+). These results provide mechanistic information on aberrant channel activity observed in disease.
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NMR and structural data for Connexin 32 and Connexin 26 N-terminal peptides.
Data in brief, 2016Co-Authors: Y. Batir, Thaddeus A. Bargiello, Terry L. DowdAbstract:In this article we present 1H and 13C chemical shift assignments, secondary structural propensity data and normalized temperature coefficient data for N-terminal peptides of Connexin 26 (Cx26), Cx26G12R and Cx32G12R mutants seen in syndromic deafness and Charcot Marie Tooth Disease respectively, published in “Structural Studies of N-Terminal Mutants of Connexin 26 and Connexin 32 Using 1H NMR Spectroscopy” (Y. Batir, T.A. Bargiello, T.L. Dowd, 2016) [1]. The mutation G12R affects the structure of both Cx26 and Cx32 peptides differently. We present data from secondary structure propensity chemical shift analysis which calculates a secondary structure propensity (SSP) score for both disordered or folded peptides and proteins using the difference between the 13C secondary chemical shifts of the Cα and Cβ protons. This data supplements the calculated NMR structures from NOESY data [1]. We present and compare the SSP data for the Cx26 vs Cx26G12R peptides and the Cx32 and Cx32G12R peptides. In addition, we present plots of temperature coefficients obtained for Cx26, Cx26G12R and Cx32G12R peptides collected previously [1] and normalized to their random coil temperature coefficients, “Random coil 1H chemical shifts obtained as a function of temperature and trifluoroethanol concentration for the peptide series GGXGG” (G. Merutka, H.J. Dyson, P.E. Wright, 1995) [2]. Reductions in these normalized temperature coefficients are directly observable for residues in different segments of the peptide and this data informs on solvent accessibility of the NH protons and NH protons which may be more constrained due to the formation of H bonds.
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Structural studies of N-terminal mutants of Connexin 32 using 1H NMR spectroscopy
Archives of biochemistry and biophysics, 2012Co-Authors: B.d. Kalmatsky, Thaddeus A. Bargiello, Y. Batir, Terry L. DowdAbstract:The amino terminus of gap junction proteins, Connexins, plays a fundamental role in voltage gating and ion permeation. We have previously shown with 1H NMR that the structure of the N-terminus of functional Connexin molecules contains a flexible turn around G12 (Arch. Biochem. Biophys.490:9,2009) allowing the N-terminus to form a portion of the channel pore near the cytoplasmic entrance. The mutants of nonfunctional Connexin molecules G12S and G12Y were found to prevent this turn. Previous functional studies of loci at which Cx32 mutations cause a peripheral neuropathy, Charcot-Marie-Tooth disease, have shown that G12S is not plasma membrane inserted. Presently, we solve the structure of nonfunctional Connexin 32 mutants W3D and Y7D which do not appear to be membrane inserted. Using 2D 1H NMR, we report that similar to G12S and G12Y, alterations in hydrophobic sidechain interactions disrupt (Y7D) or constrain (W3D) the flexible turn around G12. The alteration in the open turn around residue 12, observed in all nonfunctional mutants G12S, G12Y, W3D and Y7D correlates with loss of function. We propose that loss of the open turn causes the N-terminus to extend out of the channel pore and this misfolding may target mutants for destruction in the endoplasmic reticulum.
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Connexin 32 increases the proliferative response of schwann cells to neuregulin 1 nrg1
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Mona M Freidin, Michael V. L. Bennett, Thaddeus A. Bargiello, Samantha Asche, Charles K AbramsAbstract:Connexin 32 (Cx32), a gap junction protein, is found within the para-nodal region and Schmidt-Lanterman incisures of myelinating Schwann cells (SCs). In developing and regenerating peripheral nerves, pro-myelinating SCs express Cx32 mRNA and protein in conjunction with the expression of myelin specific genes. Neuregulin-1 (Nrg1), a member of the neuregulin family of growth factors, controls SC proliferation and differentiation depending on the cellular environment and the particular stage of SC maturation. Primary cultures of purified SCs from newborn mouse sciatic nerve were used to characterize both the role of Nrg1 in the expression of Cx32 and, conversely, the role of Cx32 in SC responsiveness to Nrg1. Glial growth factor 2, an isoform of Nrg1, up-regulated Cx32 in both proliferating and non-proliferating SCs. However, SCs from Cx32-KO mice exhibited a significantly smaller mitogenic response to glial growth factor 2. Electrical coupling between Cx32-KO SCs did not differ from that between WT SCs, indicating the presence of other Connexins. These results suggest a link between Cx32 expression and Nrg1 regulation of SC proliferation that does not involve Cx32-mediated intercellular communication.
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Changes in permeability caused by Connexin 32 mutations underlie X-linked Charcot-Marie-Tooth disease.
Neuron, 1997Co-Authors: Michael V. L. Bennett, E. Brady Trexler, Vytautas Verselis, Thaddeus A. BargielloAbstract:The relationship between the loss of Connexin 32 function and clinical manifestations of X-linked Charcot-Marie-Tooth (CMTX) disease is unknown. Here, we report that eight of nine CMTX mutations investigated form channels with measurable electrical conductance. Single-channel studies of two mutations demonstrate reduced junctional permeability caused by a decrease in either pore size (S26L) or open channel probability (M34T) that favors residency in a low-conductance substate. Permeation of second messengers such as cAMP through reflexive gap junctions between adjacent cytoplasmic loops of myelinating Schwann cells is likely to be reduced or absent in these channels. We propose that CMTX mutations impair the transduction of signals arising from normal glial-neuronal interactions and thereby cause demyelination and axonal degeneration.