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Stephen G. Young - One of the best experts on this subject based on the ideXlab platform.
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Deficiency of Isoprenylcysteine Carboxyl Methyltransferase (ICMT) Leads to Progressive Loss of Photoreceptor Function
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2016Co-Authors: Jeffrey R. Christiansen, Martin O. Bergo, Stephen G. Young, Saravanan Kolandaivelu, Nachiket Devdatta Pendse, Visvanathan RamamurthyAbstract:Retinal neurons use multiple strategies to fine-tune visual signal transduction, including post-translational modifications of proteins, such as addition of an isoprenyl lipid to a carboxyl-terminal cysteine in proteins that terminate with a “ CAAX motif.” We previously showed that RAS converting enzyme 1 (RCE1)-mediated processing of isoprenylated proteins is required for photoreceptor maintenance and function. However, it is not yet known whether the requirement for the RCE1-mediated protein processing is related to the absence of the endoproteolytic processing step, the absence of the subsequent methylation step by isoprenylcysteine methyltransferase (ICMT), or both. To approach this issue and to understand the significance of protein methylation, we generated mice lacking Icmt expression in the retina. In the absence of Icmt expression, rod and cone light-mediated responses diminished progressively. Lack of ICMT-mediated methylation led to defective association of isoprenylated transducin and cone phosphodiesterase 6 (PDE6α′) with photoreceptor membranes and resulted in decreased levels of transducin, PDE6α′, and cone G-protein coupled receptor kinase-1 (GRK1). In contrast to our earlier findings with retina-specific RCE1 knock-out mice, rod PDE6 in Icmt -deficient mice trafficked normally to the photoreceptor outer segment, suggesting that the failure to remove the − AAX is responsible for blocking the movement of PDE6 to the outer segment. Our findings demonstrate that carboxyl methylation of isoprenylated proteins is crucial for maintenance of photoreceptor function. SIGNIFICANCE STATEMENT In this report, we show that an absence of isoprenylcysteine methyltransferase-mediated protein methylation leads to progressive loss of vision. Photoreceptors also degenerate, although at a slower pace than the rate of visual loss. The reduction in photoresponses is due to defective association of crucial players in phototransduction cascade. Unlike the situation with RCE1 deficiency, where both methylation and removal of − AAX were affected, the transport of isoprenylated proteins in isoprenylcysteine methyltransferase-deficient retinas was not dependent on methylation. This finding implies that the retention of the − AAX in PDE6 catalytic subunits in RCE1 −/− mice is responsible for impeding their transport to the rod photoreceptor outer segment. In conclusion, lack of methylation of isoprenylcysteines leads to age-dependent photoreceptor dysfunction.
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RCE1 deficiency accelerates the development of K-RAS-induced myeloproliferative disease.
Blood, 2006Co-Authors: Annika M. Wahlstrom, Stephen G. Young, Briony A. Cutts, Christin Karlsson, Karin M. E. Andersson, Meng Liu, Anna-karin M. Sjogren, Birgitta Swolin, Martin O. BergoAbstract:The RAS proteins undergo farnesylation of a carboxyl-terminal cysteine (the "C" of the carboxyl-terminal CaaX motif). After farnesylation, the 3 amino acids downstream from the farnesyl cysteine (the -aaX of the CaaX motif) are released by RAS-converting enzyme 1 (RCE1). We previously showed that inactivation of RCE1 in mouse fibroblasts mislocalizes RAS proteins away from the plasma membrane and inhibits RAS transformation. Therefore, we hypothesized that the inactivation of RCE1 might inhibit RAS transformation in vivo. To test this hypothesis, we used Cre/loxP recombination techniques to simultaneously inactivate RCE1 and activate a latent oncogenic K-RAS allele in hematopoietic cells in mice. Normally, activation of the oncogenic K-RAS allele in hematopoietic cells leads to rapidly progressing and lethal myeloproliferative disease. Contrary to our hypothesis, the inactivation of RCE1 actually increased peripheral leukocytosis, increased the release of immature hematopoietic cells into the circulation and the infiltration of cells into liver and spleen, and caused mice to die more rapidly. Moreover, in the absence of RCE1, splenocytes and bone marrow cells expressing oncogenic K-RAS yielded more and larger colonies when grown in methylcellulose. We conclude that the inactivation of RCE1 worsens the myeloproliferative disease caused by oncogenic K-RAS.
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Endoproteolytic Processing of RhoA by RCE1 Is Required for the Cleavage of RhoA by Yersinia enterocolitica Outer Protein T
Infection and immunity, 2006Co-Authors: Florian Fueller, Martin O. Bergo, Stephen G. Young, Klaus Aktories, Gudula SchmidtAbstract:The bacterial toxin Yersinia outer protein T (YopT) is a cysteine protease that cleaves Rho GTPases immediately upstream of a carboxyl-terminal isoprenylcysteine. By clipping off the lipid anchor, YopT releases Rho GTPases from membranes, resulting in rounding up of mammalian cells in culture. The proteolytic activity of YopT depends on the isoprenylation of the cysteine within the carboxyl-terminal CaaX motif, a reaction carried out by geranylgeranyltransferase type I. The CaaX motif (where "a" indicates aliphatic amino acids) of Rho proteins undergoes two additional processing steps: endoproteolytic removal of the last three amino acids (i.e., -aaX) by RCE1 (Ras-converting enzyme 1) and methylation of the geranylgeranylcysteine by Icmt (isoprenylcysteine carboxyl methyltransferase). In in vitro experiments, RhoA retaining -aaX cannot be cleaved by YopT. Nothing is known, however, about the influence of RCE1-mediated removal of -aaX on the activity of YopT in living cells. We hypothesized that RCE1-deficient mouse fibroblasts, in which the geranylgeranylated Rho proteins are not endoproteolytically processed, would be resistant to YopT. Indeed, this was the case. Microinjection of recombinant YopT into RCE1-deficient fibroblasts had no impact on the subcellular localization of RhoA and no impact on cell morphology. To determine if carboxyl methylation is also required for YopT action, we microinjected YopT into Icmt-deficient fibroblasts. In contrast to the results with RCE1-deficient cells, YopT cleaved RhoA and caused rounding up of the Icmt-deficient cells. Our data demonstrate that RCE1-mediated removal of -aaX from isoprenylated Rho GTPases is required for the proteolytic activity of YopT in living cells, whereas carboxyl methylation by Icmt is not.
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on the physiological importance of endoproteolysis of caax proteins heart specific RCE1 knockout mice develop a lethal cardiomyopathy
Journal of Biological Chemistry, 2004Co-Authors: Martin O. Bergo, Bryant J. Gavino, James C. Otto, Patrick J. Casey, Patricia Ambroziak, Hsiao D. Lieu, Quinn M. Walker, Stephen G. YoungAbstract:Abstract Proteins terminating with a CAAX motif, such as the Ras proteins and the nuclear lamins, undergo post-translational modification of a C-terminal cysteine with an isoprenyl lipid via a process called protein prenylation. After prenylation, the last three residues of CAAX proteins are clipped off by RCE1, an integral membrane endoprotease of the endoplasmic reticulum. Prenylation is crucial to the function of many CAAX proteins, but the physiologic significance of endoproteolytic processing has remained obscure. To address this issue, we used Cre/loxP recombination techniques to create mice lacking RCE1 in the heart, an organ where RCE1 is expressed at particularly high levels. The hearts from heart-specific RCE1 knockout mice manifested reduced levels of both the RCE1 mRNA and CAAX endoprotease activity, and the hearts manifested an accumulation of CAAX protein substrates. The heart-specific RCE1 knockout mice initially appeared healthy but died starting at 3-5 months of age. By 10 months of age, ∼70% of the mice had died. Pathological studies revealed that the heart-specific RCE1 knockout mice had a dilated cardiomyopathy. By contrast, liver-specific RCE1 knockout mice appeared healthy, had normal transaminase levels, and had normal liver histology. These studies indicate that the endoproteolytic processing of CAAX proteins is essential for cardiac function but is less important for the liver.
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On the Physiological Importance of Endoproteolysis of CAAX Proteins HEART-SPECIFIC RCE1 KNOCKOUT MICE DEVELOP A LETHAL CARDIOMYOPATHY
The Journal of biological chemistry, 2003Co-Authors: Martin O. Bergo, Bryant J. Gavino, James C. Otto, Patrick J. Casey, Patricia Ambroziak, Hsiao D. Lieu, Quinn M. Walker, Stephen G. YoungAbstract:Proteins terminating with a CAAX motif, such as the Ras proteins and the nuclear lamins, undergo post-translational modification of a C-terminal cysteine with an isoprenyl lipid via a process called protein prenylation. After prenylation, the last three residues of CAAX proteins are clipped off by RCE1, an integral membrane endoprotease of the endoplasmic reticulum. Prenylation is crucial to the function of many CAAX proteins, but the physiologic significance of endoproteolytic processing has remained obscure. To address this issue, we used Cre/loxP recombination techniques to create mice lacking RCE1 in the heart, an organ where RCE1 is expressed at particularly high levels. The hearts from heart-specific RCE1 knockout mice manifested reduced levels of both the RCE1 mRNA and CAAX endoprotease activity, and the hearts manifested an accumulation of CAAX protein substrates. The heart-specific RCE1 knockout mice initially appeared healthy but died starting at 3-5 months of age. By 10 months of age, approximately 70% of the mice had died. Pathological studies revealed that the heart-specific RCE1 knockout mice had a dilated cardiomyopathy. By contrast, liver-specific RCE1 knockout mice appeared healthy, had normal transaminase levels, and had normal liver histology. These studies indicate that the endoproteolytic processing of CAAX proteins is essential for cardiac function but is less important for the liver.
Martin O. Bergo - One of the best experts on this subject based on the ideXlab platform.
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Targeting RAS-converting enzyme 1 overcomes senescence and improves progeria-like phenotypes of ZMPSTE24 deficiency.
Aging cell, 2020Co-Authors: Haidong Yao, Xue Chen, Muhammad Kashif, Ting Wang, Mohamed X. Ibrahim, Elin Tüksammel, Gwladys Revêchon, Maria J. Eriksson, Clotilde Wiel, Martin O. BergoAbstract:Several progeroid disorders are caused by deficiency in the endoprotease ZMPSTE24 which leads to accumulation of prelamin A at the nuclear envelope. ZMPSTE24 cleaves prelamin A twice: at the third carboxyl-terminal amino acid following farnesylation of a -CSIM motif; and 15 residues upstream to produce mature lamin A. The carboxyl-terminal cleavage can also be performed by RAS-converting enzyme 1 (RCE1) but little is known about the importance of this cleavage for the ability of prelamin A to cause disease. Here, we found that knockout of RCE1 delayed senescence and increased proliferation of ZMPSTE24-deficient fibroblasts from a patient with non-classical Hutchinson-Gilford progeria syndrome (HGPS), but did not influence proliferation of classical LMNA-mutant HGPS cells. Knockout of RCE1 in Zmpste24-deficient mice at postnatal week 4-5 increased body weight and doubled the median survival time. The absence of RCE1 in Zmpste24-deficient fibroblasts did not influence nuclear shape but reduced an interaction between prelamin A and AKT which activated AKT-mTOR signaling and was required for the increased proliferation. Prelamin A levels increased in RCE1-deficient cells due to a slower turnover rate but its localization at the nuclear rim was unaffected. These results strengthen the idea that the presence of misshapen nuclei does not prevent phenotype improvement and suggest that targeting RCE1 might be useful for treating the rare progeroid disorders associated with ZMPSTE24 deficiency.
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Deficiency of Isoprenylcysteine Carboxyl Methyltransferase (ICMT) Leads to Progressive Loss of Photoreceptor Function
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2016Co-Authors: Jeffrey R. Christiansen, Martin O. Bergo, Stephen G. Young, Saravanan Kolandaivelu, Nachiket Devdatta Pendse, Visvanathan RamamurthyAbstract:Retinal neurons use multiple strategies to fine-tune visual signal transduction, including post-translational modifications of proteins, such as addition of an isoprenyl lipid to a carboxyl-terminal cysteine in proteins that terminate with a “ CAAX motif.” We previously showed that RAS converting enzyme 1 (RCE1)-mediated processing of isoprenylated proteins is required for photoreceptor maintenance and function. However, it is not yet known whether the requirement for the RCE1-mediated protein processing is related to the absence of the endoproteolytic processing step, the absence of the subsequent methylation step by isoprenylcysteine methyltransferase (ICMT), or both. To approach this issue and to understand the significance of protein methylation, we generated mice lacking Icmt expression in the retina. In the absence of Icmt expression, rod and cone light-mediated responses diminished progressively. Lack of ICMT-mediated methylation led to defective association of isoprenylated transducin and cone phosphodiesterase 6 (PDE6α′) with photoreceptor membranes and resulted in decreased levels of transducin, PDE6α′, and cone G-protein coupled receptor kinase-1 (GRK1). In contrast to our earlier findings with retina-specific RCE1 knock-out mice, rod PDE6 in Icmt -deficient mice trafficked normally to the photoreceptor outer segment, suggesting that the failure to remove the − AAX is responsible for blocking the movement of PDE6 to the outer segment. Our findings demonstrate that carboxyl methylation of isoprenylated proteins is crucial for maintenance of photoreceptor function. SIGNIFICANCE STATEMENT In this report, we show that an absence of isoprenylcysteine methyltransferase-mediated protein methylation leads to progressive loss of vision. Photoreceptors also degenerate, although at a slower pace than the rate of visual loss. The reduction in photoresponses is due to defective association of crucial players in phototransduction cascade. Unlike the situation with RCE1 deficiency, where both methylation and removal of − AAX were affected, the transport of isoprenylated proteins in isoprenylcysteine methyltransferase-deficient retinas was not dependent on methylation. This finding implies that the retention of the − AAX in PDE6 catalytic subunits in RCE1 −/− mice is responsible for impeding their transport to the rod photoreceptor outer segment. In conclusion, lack of methylation of isoprenylcysteines leads to age-dependent photoreceptor dysfunction.
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RAS-converting enzyme 1-mediated endoproteolysis is required for trafficking of rod phosphodiesterase 6 to photoreceptor outer segments
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Jeffrey R. Christiansen, Martin O. Bergo, Saravanan Kolandaivelu, Visvanathan RamamurthyAbstract:Prenylation is the posttranslational modification of a carboxyl-terminal cysteine residue of proteins that terminate with a CAAX motif. Following prenylation, the last three amino acids are cleaved off by the endoprotease, RAS-converting enzyme 1 (RCE1), and the prenylcysteine residue is methylated. Although it is clear that prenylation increases membrane affinity of CAAX proteins, less is known about the importance of the postprenylation processing steps. RCE1 function has been studied in a variety of tissues but not in neuronal cells. To approach this issue, we generated mice lacking RCE1 in the retina. Retinal development proceeded normally in the absence of RCE1, but photoreceptor cells failed to respond to light and subsequently degenerated in a rapid fashion. In contrast, the inner nuclear and ganglion cell layers were unaffected. We found that the multimeric rod phosphodiesterase 6 (PDE6), a prenylated protein and RCE1 substrate, was unable to be transported to the outer segments in RCE1-deficient photoreceptor cells. PDE6 present in the inner segment of RCE1-deficient photoreceptor cells was assembled and functional. Synthesis and transport of transducin, and rhodopsin kinase 1 (GRK1), also prenylated substrates of RCE1, was unaffected by RCE1 deficiency. We conclude that RCE1 is essential for the intracellular trafficking of PDE6 and survival of photoreceptor cells.
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RCE1 deficiency accelerates the development of K-RAS-induced myeloproliferative disease.
Blood, 2006Co-Authors: Annika M. Wahlstrom, Stephen G. Young, Briony A. Cutts, Christin Karlsson, Karin M. E. Andersson, Meng Liu, Anna-karin M. Sjogren, Birgitta Swolin, Martin O. BergoAbstract:The RAS proteins undergo farnesylation of a carboxyl-terminal cysteine (the "C" of the carboxyl-terminal CaaX motif). After farnesylation, the 3 amino acids downstream from the farnesyl cysteine (the -aaX of the CaaX motif) are released by RAS-converting enzyme 1 (RCE1). We previously showed that inactivation of RCE1 in mouse fibroblasts mislocalizes RAS proteins away from the plasma membrane and inhibits RAS transformation. Therefore, we hypothesized that the inactivation of RCE1 might inhibit RAS transformation in vivo. To test this hypothesis, we used Cre/loxP recombination techniques to simultaneously inactivate RCE1 and activate a latent oncogenic K-RAS allele in hematopoietic cells in mice. Normally, activation of the oncogenic K-RAS allele in hematopoietic cells leads to rapidly progressing and lethal myeloproliferative disease. Contrary to our hypothesis, the inactivation of RCE1 actually increased peripheral leukocytosis, increased the release of immature hematopoietic cells into the circulation and the infiltration of cells into liver and spleen, and caused mice to die more rapidly. Moreover, in the absence of RCE1, splenocytes and bone marrow cells expressing oncogenic K-RAS yielded more and larger colonies when grown in methylcellulose. We conclude that the inactivation of RCE1 worsens the myeloproliferative disease caused by oncogenic K-RAS.
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Endoproteolytic Processing of RhoA by RCE1 Is Required for the Cleavage of RhoA by Yersinia enterocolitica Outer Protein T
Infection and immunity, 2006Co-Authors: Florian Fueller, Martin O. Bergo, Stephen G. Young, Klaus Aktories, Gudula SchmidtAbstract:The bacterial toxin Yersinia outer protein T (YopT) is a cysteine protease that cleaves Rho GTPases immediately upstream of a carboxyl-terminal isoprenylcysteine. By clipping off the lipid anchor, YopT releases Rho GTPases from membranes, resulting in rounding up of mammalian cells in culture. The proteolytic activity of YopT depends on the isoprenylation of the cysteine within the carboxyl-terminal CaaX motif, a reaction carried out by geranylgeranyltransferase type I. The CaaX motif (where "a" indicates aliphatic amino acids) of Rho proteins undergoes two additional processing steps: endoproteolytic removal of the last three amino acids (i.e., -aaX) by RCE1 (Ras-converting enzyme 1) and methylation of the geranylgeranylcysteine by Icmt (isoprenylcysteine carboxyl methyltransferase). In in vitro experiments, RhoA retaining -aaX cannot be cleaved by YopT. Nothing is known, however, about the influence of RCE1-mediated removal of -aaX on the activity of YopT in living cells. We hypothesized that RCE1-deficient mouse fibroblasts, in which the geranylgeranylated Rho proteins are not endoproteolytically processed, would be resistant to YopT. Indeed, this was the case. Microinjection of recombinant YopT into RCE1-deficient fibroblasts had no impact on the subcellular localization of RhoA and no impact on cell morphology. To determine if carboxyl methylation is also required for YopT action, we microinjected YopT into Icmt-deficient fibroblasts. In contrast to the results with RCE1-deficient cells, YopT cleaved RhoA and caused rounding up of the Icmt-deficient cells. Our data demonstrate that RCE1-mediated removal of -aaX from isoprenylated Rho GTPases is required for the proteolytic activity of YopT in living cells, whereas carboxyl methylation by Icmt is not.
Patrick J. Casey - One of the best experts on this subject based on the ideXlab platform.
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on the physiological importance of endoproteolysis of caax proteins heart specific RCE1 knockout mice develop a lethal cardiomyopathy
Journal of Biological Chemistry, 2004Co-Authors: Martin O. Bergo, Bryant J. Gavino, James C. Otto, Patrick J. Casey, Patricia Ambroziak, Hsiao D. Lieu, Quinn M. Walker, Stephen G. YoungAbstract:Abstract Proteins terminating with a CAAX motif, such as the Ras proteins and the nuclear lamins, undergo post-translational modification of a C-terminal cysteine with an isoprenyl lipid via a process called protein prenylation. After prenylation, the last three residues of CAAX proteins are clipped off by RCE1, an integral membrane endoprotease of the endoplasmic reticulum. Prenylation is crucial to the function of many CAAX proteins, but the physiologic significance of endoproteolytic processing has remained obscure. To address this issue, we used Cre/loxP recombination techniques to create mice lacking RCE1 in the heart, an organ where RCE1 is expressed at particularly high levels. The hearts from heart-specific RCE1 knockout mice manifested reduced levels of both the RCE1 mRNA and CAAX endoprotease activity, and the hearts manifested an accumulation of CAAX protein substrates. The heart-specific RCE1 knockout mice initially appeared healthy but died starting at 3-5 months of age. By 10 months of age, ∼70% of the mice had died. Pathological studies revealed that the heart-specific RCE1 knockout mice had a dilated cardiomyopathy. By contrast, liver-specific RCE1 knockout mice appeared healthy, had normal transaminase levels, and had normal liver histology. These studies indicate that the endoproteolytic processing of CAAX proteins is essential for cardiac function but is less important for the liver.
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On the Physiological Importance of Endoproteolysis of CAAX Proteins HEART-SPECIFIC RCE1 KNOCKOUT MICE DEVELOP A LETHAL CARDIOMYOPATHY
The Journal of biological chemistry, 2003Co-Authors: Martin O. Bergo, Bryant J. Gavino, James C. Otto, Patrick J. Casey, Patricia Ambroziak, Hsiao D. Lieu, Quinn M. Walker, Stephen G. YoungAbstract:Proteins terminating with a CAAX motif, such as the Ras proteins and the nuclear lamins, undergo post-translational modification of a C-terminal cysteine with an isoprenyl lipid via a process called protein prenylation. After prenylation, the last three residues of CAAX proteins are clipped off by RCE1, an integral membrane endoprotease of the endoplasmic reticulum. Prenylation is crucial to the function of many CAAX proteins, but the physiologic significance of endoproteolytic processing has remained obscure. To address this issue, we used Cre/loxP recombination techniques to create mice lacking RCE1 in the heart, an organ where RCE1 is expressed at particularly high levels. The hearts from heart-specific RCE1 knockout mice manifested reduced levels of both the RCE1 mRNA and CAAX endoprotease activity, and the hearts manifested an accumulation of CAAX protein substrates. The heart-specific RCE1 knockout mice initially appeared healthy but died starting at 3-5 months of age. By 10 months of age, approximately 70% of the mice had died. Pathological studies revealed that the heart-specific RCE1 knockout mice had a dilated cardiomyopathy. By contrast, liver-specific RCE1 knockout mice appeared healthy, had normal transaminase levels, and had normal liver histology. These studies indicate that the endoproteolytic processing of CAAX proteins is essential for cardiac function but is less important for the liver.
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Absence of the CAAX Endoprotease RCE1: Effects on Cell Growth and Transformation
Molecular and cellular biology, 2002Co-Authors: Martin O. Bergo, James C. Otto, Edward Kim, Patrick J. Casey, Patricia Ambroziak, Cria Gregory, Amanda George, Hiroki Nagase, Allan Balmain, Stephen G. YoungAbstract:After isoprenylation, the Ras proteins and other CAAX proteins undergo two additional enzymatic modifications—endoproteolytic release of the last three amino acids of the protein by the protease RCE1 and methylation of the carboxyl-terminal isoprenylcysteine by the methyltransferase Icmt. This postisoprenylation processing is thought to be important for the association of Ras proteins with membranes. Blocking postisoprenylation processing, by inhibiting RCE1, has been suggested as a potential approach for retarding cell growth and blocking cellular transformation. The objective of this study was to develop a cell culture system for addressing these issues. We generated mice with a conditional RCE1 allele (RCE1flox) and produced RCE1flox/flox fibroblasts. Cre-mediated excision of RCE1 (thereby producing RCE1Δ/Δ fibroblasts) eliminated Ras endoproteolytic processing and methylation and caused a partial mislocalization of truncated K-Ras and H-Ras fusion proteins within cells. RCE1Δ/Δ fibroblasts grew more slowly than RCE1flox/flox fibroblasts. The excision of RCE1 also reduced Ras-induced transformation, as judged by the growth of colonies in soft agar. The excision of RCE1 from a RCE1flox/flox skin carcinoma cell line also significantly retarded the growth of cells, and this effect was exaggerated by cotreatment of the cells with a farnesyltransferase inhibitor. These studies support the idea that interference with postisoprenylation processing retards cell growth, limits Ras-induced transformation, and sensitizes tumor cells to a farnesyltransferase inhibitor.
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Non-peptidic, non-prenylic inhibitors of the prenyl protein-specific protease RCE1.
Bioorganic & medicinal chemistry letters, 2001Co-Authors: Martin Schlitzer, Ann M. Winter-vann, Patrick J. CaseyAbstract:Several compounds designed as bisubstrate analogues of protein farnesyltransferase inhibited the prenyl protein-specific protease RCE1, qualifying them as lead structures for a novel class of non-peptidic, non-prenylic inhibitors of this protease.
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The C-terminal Polylysine Region and Methylation of K-Ras Are Critical for the Interaction between K-Ras and Microtubules
The Journal of biological chemistry, 2000Co-Authors: Zhui Chen, Martin O. Bergo, Stephen G. Young, James C. Otto, Patrick J. CaseyAbstract:After synthesis in the cytosol, Ras proteins must be targeted to the inner leaflet of the plasma membrane for biological activity. This targeting requires a series of C-terminal posttranslational modifications initiated by the addition of an isoprenoid lipid in a process termed prenylation. A search for factors involved in the intracellular trafficking of Ras has identified a specific and prenylation-dependent interaction between tubulin/microtubules and K-Ras. In this study, we examined the structural requirements for this interaction between K-Ras and microtubules. By using a series of chimeras in which regions of the C terminus of K-Ras were replaced with those of Ha-Ras and vice versa, we found that the polylysine region of K-Ras located immediately upstream of the prenylation site is required for binding of K-Ras to microtubules. Studies in intact cells confirmed the importance of the K-Ras polylysine region for microtubule binding, as deletion or replacement of this region resulted in loss of paclitaxel-induced mislocalization of a fluorescent K-Ras fusion protein. The additional modifications in the prenyl protein processing pathway also affected the interaction of K-Ras with microtubules. Removal of the three C-terminal amino acids of farnesylated K-Ras with the specific endoprotease RCE1p abolished its binding to microtubules. Interestingly, however, methylation of the C-terminal prenylcysteine restored binding. Consistent with these results, localization of the fluorescent K-Ras fusion protein remained paclitaxel-sensitive in cells lacking RCE1, whereas no paclitaxel effect was observed in cells lacking the methyltransferase. These studies show that the polylysine region of K-Ras is critical for its interaction with microtubules and provide the first evidence for a functional consequence of Ras C-terminal proteolysis and methylation.
James C. Otto - One of the best experts on this subject based on the ideXlab platform.
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on the physiological importance of endoproteolysis of caax proteins heart specific RCE1 knockout mice develop a lethal cardiomyopathy
Journal of Biological Chemistry, 2004Co-Authors: Martin O. Bergo, Bryant J. Gavino, James C. Otto, Patrick J. Casey, Patricia Ambroziak, Hsiao D. Lieu, Quinn M. Walker, Stephen G. YoungAbstract:Abstract Proteins terminating with a CAAX motif, such as the Ras proteins and the nuclear lamins, undergo post-translational modification of a C-terminal cysteine with an isoprenyl lipid via a process called protein prenylation. After prenylation, the last three residues of CAAX proteins are clipped off by RCE1, an integral membrane endoprotease of the endoplasmic reticulum. Prenylation is crucial to the function of many CAAX proteins, but the physiologic significance of endoproteolytic processing has remained obscure. To address this issue, we used Cre/loxP recombination techniques to create mice lacking RCE1 in the heart, an organ where RCE1 is expressed at particularly high levels. The hearts from heart-specific RCE1 knockout mice manifested reduced levels of both the RCE1 mRNA and CAAX endoprotease activity, and the hearts manifested an accumulation of CAAX protein substrates. The heart-specific RCE1 knockout mice initially appeared healthy but died starting at 3-5 months of age. By 10 months of age, ∼70% of the mice had died. Pathological studies revealed that the heart-specific RCE1 knockout mice had a dilated cardiomyopathy. By contrast, liver-specific RCE1 knockout mice appeared healthy, had normal transaminase levels, and had normal liver histology. These studies indicate that the endoproteolytic processing of CAAX proteins is essential for cardiac function but is less important for the liver.
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On the Physiological Importance of Endoproteolysis of CAAX Proteins HEART-SPECIFIC RCE1 KNOCKOUT MICE DEVELOP A LETHAL CARDIOMYOPATHY
The Journal of biological chemistry, 2003Co-Authors: Martin O. Bergo, Bryant J. Gavino, James C. Otto, Patrick J. Casey, Patricia Ambroziak, Hsiao D. Lieu, Quinn M. Walker, Stephen G. YoungAbstract:Proteins terminating with a CAAX motif, such as the Ras proteins and the nuclear lamins, undergo post-translational modification of a C-terminal cysteine with an isoprenyl lipid via a process called protein prenylation. After prenylation, the last three residues of CAAX proteins are clipped off by RCE1, an integral membrane endoprotease of the endoplasmic reticulum. Prenylation is crucial to the function of many CAAX proteins, but the physiologic significance of endoproteolytic processing has remained obscure. To address this issue, we used Cre/loxP recombination techniques to create mice lacking RCE1 in the heart, an organ where RCE1 is expressed at particularly high levels. The hearts from heart-specific RCE1 knockout mice manifested reduced levels of both the RCE1 mRNA and CAAX endoprotease activity, and the hearts manifested an accumulation of CAAX protein substrates. The heart-specific RCE1 knockout mice initially appeared healthy but died starting at 3-5 months of age. By 10 months of age, approximately 70% of the mice had died. Pathological studies revealed that the heart-specific RCE1 knockout mice had a dilated cardiomyopathy. By contrast, liver-specific RCE1 knockout mice appeared healthy, had normal transaminase levels, and had normal liver histology. These studies indicate that the endoproteolytic processing of CAAX proteins is essential for cardiac function but is less important for the liver.
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Absence of the CAAX Endoprotease RCE1: Effects on Cell Growth and Transformation
Molecular and cellular biology, 2002Co-Authors: Martin O. Bergo, James C. Otto, Edward Kim, Patrick J. Casey, Patricia Ambroziak, Cria Gregory, Amanda George, Hiroki Nagase, Allan Balmain, Stephen G. YoungAbstract:After isoprenylation, the Ras proteins and other CAAX proteins undergo two additional enzymatic modifications—endoproteolytic release of the last three amino acids of the protein by the protease RCE1 and methylation of the carboxyl-terminal isoprenylcysteine by the methyltransferase Icmt. This postisoprenylation processing is thought to be important for the association of Ras proteins with membranes. Blocking postisoprenylation processing, by inhibiting RCE1, has been suggested as a potential approach for retarding cell growth and blocking cellular transformation. The objective of this study was to develop a cell culture system for addressing these issues. We generated mice with a conditional RCE1 allele (RCE1flox) and produced RCE1flox/flox fibroblasts. Cre-mediated excision of RCE1 (thereby producing RCE1Δ/Δ fibroblasts) eliminated Ras endoproteolytic processing and methylation and caused a partial mislocalization of truncated K-Ras and H-Ras fusion proteins within cells. RCE1Δ/Δ fibroblasts grew more slowly than RCE1flox/flox fibroblasts. The excision of RCE1 also reduced Ras-induced transformation, as judged by the growth of colonies in soft agar. The excision of RCE1 from a RCE1flox/flox skin carcinoma cell line also significantly retarded the growth of cells, and this effect was exaggerated by cotreatment of the cells with a farnesyltransferase inhibitor. These studies support the idea that interference with postisoprenylation processing retards cell growth, limits Ras-induced transformation, and sensitizes tumor cells to a farnesyltransferase inhibitor.
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The C-terminal Polylysine Region and Methylation of K-Ras Are Critical for the Interaction between K-Ras and Microtubules
The Journal of biological chemistry, 2000Co-Authors: Zhui Chen, Martin O. Bergo, Stephen G. Young, James C. Otto, Patrick J. CaseyAbstract:After synthesis in the cytosol, Ras proteins must be targeted to the inner leaflet of the plasma membrane for biological activity. This targeting requires a series of C-terminal posttranslational modifications initiated by the addition of an isoprenoid lipid in a process termed prenylation. A search for factors involved in the intracellular trafficking of Ras has identified a specific and prenylation-dependent interaction between tubulin/microtubules and K-Ras. In this study, we examined the structural requirements for this interaction between K-Ras and microtubules. By using a series of chimeras in which regions of the C terminus of K-Ras were replaced with those of Ha-Ras and vice versa, we found that the polylysine region of K-Ras located immediately upstream of the prenylation site is required for binding of K-Ras to microtubules. Studies in intact cells confirmed the importance of the K-Ras polylysine region for microtubule binding, as deletion or replacement of this region resulted in loss of paclitaxel-induced mislocalization of a fluorescent K-Ras fusion protein. The additional modifications in the prenyl protein processing pathway also affected the interaction of K-Ras with microtubules. Removal of the three C-terminal amino acids of farnesylated K-Ras with the specific endoprotease RCE1p abolished its binding to microtubules. Interestingly, however, methylation of the C-terminal prenylcysteine restored binding. Consistent with these results, localization of the fluorescent K-Ras fusion protein remained paclitaxel-sensitive in cells lacking RCE1, whereas no paclitaxel effect was observed in cells lacking the methyltransferase. These studies show that the polylysine region of K-Ras is critical for its interaction with microtubules and provide the first evidence for a functional consequence of Ras C-terminal proteolysis and methylation.
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Isoprenylcysteine Carboxyl Methyltransferase Deficiency in Mice
The Journal of biological chemistry, 2000Co-Authors: Martin O. Bergo, Gordon K. Leung, James C. Otto, Patrick J. Casey, Patricia Ambroziak, Anita Quintal Gomes, Miguel C. Seabra, Stephen G. YoungAbstract:After isoprenylation, Ras and other CAAX proteins undergo endoproteolytic processing by RCE1 and methylation of the isoprenylcysteine by Icmt (isoprenylcysteine carboxyl methyltransferase). We reported previously that RCE1-deficient mice died during late gestation or soon after birth. We hypothesized that Icmt deficiency might cause a milder phenotype, in part because of reports suggesting the existence of more than one activity for methylating isoprenylated proteins. To address this hypothesis and also to address the issue of other methyltransferase activities, we generated Icmt-deficient mice. Contrary to our expectation, Icmt deficiency caused a more severe phenotype than RCE1 deficiency, with virtually all of the knockout embryos (Icmt-/-) dying by mid-gestation. An analysis of chimeric mice produced from Icmt-/- embryonic stem cells showed that the Icmt-/- cells retained the capacity to contribute to some tissues (e.g. skeletal muscle) but not to others (e.g. brain). Lysates from Icmt-/- embryos lacked the ability to methylate either recombinant K-Ras or small molecule substrates (e.g. N-acetyl-S-geranylgeranyl-l-cysteine). In addition, Icmt-/- cells lacked the ability to methylate Rab proteins. Thus, Icmt appears to be the only enzyme participating in the carboxyl methylation of isoprenylated proteins.
Patricia Ambroziak - One of the best experts on this subject based on the ideXlab platform.
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on the physiological importance of endoproteolysis of caax proteins heart specific RCE1 knockout mice develop a lethal cardiomyopathy
Journal of Biological Chemistry, 2004Co-Authors: Martin O. Bergo, Bryant J. Gavino, James C. Otto, Patrick J. Casey, Patricia Ambroziak, Hsiao D. Lieu, Quinn M. Walker, Stephen G. YoungAbstract:Abstract Proteins terminating with a CAAX motif, such as the Ras proteins and the nuclear lamins, undergo post-translational modification of a C-terminal cysteine with an isoprenyl lipid via a process called protein prenylation. After prenylation, the last three residues of CAAX proteins are clipped off by RCE1, an integral membrane endoprotease of the endoplasmic reticulum. Prenylation is crucial to the function of many CAAX proteins, but the physiologic significance of endoproteolytic processing has remained obscure. To address this issue, we used Cre/loxP recombination techniques to create mice lacking RCE1 in the heart, an organ where RCE1 is expressed at particularly high levels. The hearts from heart-specific RCE1 knockout mice manifested reduced levels of both the RCE1 mRNA and CAAX endoprotease activity, and the hearts manifested an accumulation of CAAX protein substrates. The heart-specific RCE1 knockout mice initially appeared healthy but died starting at 3-5 months of age. By 10 months of age, ∼70% of the mice had died. Pathological studies revealed that the heart-specific RCE1 knockout mice had a dilated cardiomyopathy. By contrast, liver-specific RCE1 knockout mice appeared healthy, had normal transaminase levels, and had normal liver histology. These studies indicate that the endoproteolytic processing of CAAX proteins is essential for cardiac function but is less important for the liver.
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On the Physiological Importance of Endoproteolysis of CAAX Proteins HEART-SPECIFIC RCE1 KNOCKOUT MICE DEVELOP A LETHAL CARDIOMYOPATHY
The Journal of biological chemistry, 2003Co-Authors: Martin O. Bergo, Bryant J. Gavino, James C. Otto, Patrick J. Casey, Patricia Ambroziak, Hsiao D. Lieu, Quinn M. Walker, Stephen G. YoungAbstract:Proteins terminating with a CAAX motif, such as the Ras proteins and the nuclear lamins, undergo post-translational modification of a C-terminal cysteine with an isoprenyl lipid via a process called protein prenylation. After prenylation, the last three residues of CAAX proteins are clipped off by RCE1, an integral membrane endoprotease of the endoplasmic reticulum. Prenylation is crucial to the function of many CAAX proteins, but the physiologic significance of endoproteolytic processing has remained obscure. To address this issue, we used Cre/loxP recombination techniques to create mice lacking RCE1 in the heart, an organ where RCE1 is expressed at particularly high levels. The hearts from heart-specific RCE1 knockout mice manifested reduced levels of both the RCE1 mRNA and CAAX endoprotease activity, and the hearts manifested an accumulation of CAAX protein substrates. The heart-specific RCE1 knockout mice initially appeared healthy but died starting at 3-5 months of age. By 10 months of age, approximately 70% of the mice had died. Pathological studies revealed that the heart-specific RCE1 knockout mice had a dilated cardiomyopathy. By contrast, liver-specific RCE1 knockout mice appeared healthy, had normal transaminase levels, and had normal liver histology. These studies indicate that the endoproteolytic processing of CAAX proteins is essential for cardiac function but is less important for the liver.
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Absence of the CAAX Endoprotease RCE1: Effects on Cell Growth and Transformation
Molecular and cellular biology, 2002Co-Authors: Martin O. Bergo, James C. Otto, Edward Kim, Patrick J. Casey, Patricia Ambroziak, Cria Gregory, Amanda George, Hiroki Nagase, Allan Balmain, Stephen G. YoungAbstract:After isoprenylation, the Ras proteins and other CAAX proteins undergo two additional enzymatic modifications—endoproteolytic release of the last three amino acids of the protein by the protease RCE1 and methylation of the carboxyl-terminal isoprenylcysteine by the methyltransferase Icmt. This postisoprenylation processing is thought to be important for the association of Ras proteins with membranes. Blocking postisoprenylation processing, by inhibiting RCE1, has been suggested as a potential approach for retarding cell growth and blocking cellular transformation. The objective of this study was to develop a cell culture system for addressing these issues. We generated mice with a conditional RCE1 allele (RCE1flox) and produced RCE1flox/flox fibroblasts. Cre-mediated excision of RCE1 (thereby producing RCE1Δ/Δ fibroblasts) eliminated Ras endoproteolytic processing and methylation and caused a partial mislocalization of truncated K-Ras and H-Ras fusion proteins within cells. RCE1Δ/Δ fibroblasts grew more slowly than RCE1flox/flox fibroblasts. The excision of RCE1 also reduced Ras-induced transformation, as judged by the growth of colonies in soft agar. The excision of RCE1 from a RCE1flox/flox skin carcinoma cell line also significantly retarded the growth of cells, and this effect was exaggerated by cotreatment of the cells with a farnesyltransferase inhibitor. These studies support the idea that interference with postisoprenylation processing retards cell growth, limits Ras-induced transformation, and sensitizes tumor cells to a farnesyltransferase inhibitor.
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Isoprenylcysteine Carboxyl Methyltransferase Deficiency in Mice
The Journal of biological chemistry, 2000Co-Authors: Martin O. Bergo, Gordon K. Leung, James C. Otto, Patrick J. Casey, Patricia Ambroziak, Anita Quintal Gomes, Miguel C. Seabra, Stephen G. YoungAbstract:After isoprenylation, Ras and other CAAX proteins undergo endoproteolytic processing by RCE1 and methylation of the isoprenylcysteine by Icmt (isoprenylcysteine carboxyl methyltransferase). We reported previously that RCE1-deficient mice died during late gestation or soon after birth. We hypothesized that Icmt deficiency might cause a milder phenotype, in part because of reports suggesting the existence of more than one activity for methylating isoprenylated proteins. To address this hypothesis and also to address the issue of other methyltransferase activities, we generated Icmt-deficient mice. Contrary to our expectation, Icmt deficiency caused a more severe phenotype than RCE1 deficiency, with virtually all of the knockout embryos (Icmt-/-) dying by mid-gestation. An analysis of chimeric mice produced from Icmt-/- embryonic stem cells showed that the Icmt-/- cells retained the capacity to contribute to some tissues (e.g. skeletal muscle) but not to others (e.g. brain). Lysates from Icmt-/- embryos lacked the ability to methylate either recombinant K-Ras or small molecule substrates (e.g. N-acetyl-S-geranylgeranyl-l-cysteine). In addition, Icmt-/- cells lacked the ability to methylate Rab proteins. Thus, Icmt appears to be the only enzyme participating in the carboxyl methylation of isoprenylated proteins.
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Disruption of the Mouse RCE1 Gene Results in Defective Ras Processing and Mislocalization of Ras within Cells
The Journal of biological chemistry, 1999Co-Authors: Edward Kim, Matthew N. Ashby, James C. Otto, Patrick J. Casey, Brigit R Taylor, Kevin Shannon, Patricia Ambroziak, Stephen G. YoungAbstract:Abstract Little is known about the enzyme(s) required for the endoproteolytic processing of mammalian Ras proteins. We identified a mouse gene (designated RCE1) that shares sequence homology with a yeast gene (RCE1) implicated in the proteolytic processing of Ras2p. To define the role of RCE1in mammalian Ras processing, we generated and analyzedRCE1-deficient mice. RCE1 deficiency was lethal late in embryonic development (after embryonic day 15.5). Multiple lines of evidence revealed that RCE1-deficient embryos and cells lacked the ability to endoproteolytically process Ras proteins. First, Ras proteins from RCE1-deficient cells migrated more slowly on SDS-polyacrylamide gels than Ras proteins from wild-type embryos and fibroblasts. Second, metabolic labeling ofRCE1-deficient cells revealed that the Ras proteins were not carboxymethylated. Finally, membranes fromRCE1-deficient fibroblasts lacked the capacity to proteolytically process farnesylated Ha-Ras, N-Ras, and Ki-Ras or geranylgeranylated Ki-Ras. The processing of two other prenylated proteins, the farnesylated Gγ1 subunit of transducin and geranylgeranylated Rap1B, was also blocked. The absence of endoproteolytic processing and carboxymethylation caused Ras proteins to be mislocalized within cells. These studies indicate thatRCE1 is responsible for the endoproteolytic processing of the Ras proteins in mammals and suggest a broad role for this gene in processing other prenylated CAAX proteins.
