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Olivier Kocher - One of the best experts on this subject based on the ideXlab platform.

  • Molecular Analysis of the Prostacyclin Receptor’s Interaction with the PDZ1 Domain of Its Adaptor Protein
    2016
    Co-Authors: Gabriel Birrane, Eamon P. Mulvaney, Therese B Kinsella, Rinku Pal, Olivier Kocher
    Abstract:

    The prostanoid prostacyclin, or prostaglandin I2, plays an essential role in many aspects of cardiovascular disease. The actions of prostacyclin are mainly mediated through its activation of the prostacyclin receptor or, in short, the IP. In recent studies, the cytoplasmic carboxy-terminal domain of the IP was shown to bind several PDZ domains of the multi-PDZ adaptor PDZK1. The interaction between the two proteins was found to enhance cell surface expression of the IP and to be functionally important in promoting prostacyclin-induced endothelial cell migration and angiogenesis. To investigate the interaction of the IP with the first PDZ domain (PDZ1) of PDZK1, we generated a nine residue peptide (KK411IAACSLC417) containing the seven carboxy-terminal amino acids of the IP and measured its binding affinity to a recombinant protein corresponding to PDZ1 by isothermal titration calorimetry. We determined that the IP interacts with PDZ1 with a binding affinity of 8.2 mM. Using the same technique, we also determined that the farnesylated form of carboxy-terminus of the IP does not bind to PDZ1. To understand the molecular basis of these findings, we solved the high resolution crystal structure of PDZ1 bound to a 7-residue peptide derived from the carboxy-terminus of the non-farnesylated form of IP (411IAACSLC417). Analysis of the structure demonstrates a critical role for the three carboxy-terminal amino acids in establishing a strong interaction with PDZ1 and explains the inability of the farnesylated form of IP to interact with the PDZ1 domain of PDZK1 a

  • Loss of PDZK1 Causes Coronary Artery Occlusion and Myocardial Infarction in Paigen Diet-Fed Apolipoprotein E Deficient Mice
    2013
    Co-Authors: Ayce Yesilaltay, Monty Krieger, Rinku Pal, Kathleen Daniels, Olivier Kocher
    Abstract:

    Background: PDZK1 is a four PDZ-domain containing protein that binds to the carboxy terminus of the HDL receptor, scavenger receptor class B type I (SR-BI), and regulates its expression, localization and function in a tissue-specific manner. PDZK1 knockout (KO) mice are characterized by a marked reduction of SR-BI protein expression (,95%) in the liver (lesser or no reduction in other organs) with a concomitant 1.7 fold increase in plasma cholesterol. PDZK1 has been shown to be atheroprotective using the high fat/high cholesterol (‘Western’) diet-fed murine apolipoprotein E (apoE) KO model of atherosclerosis, presumably because of its role in promoting reverse cholesterol transport via SR-BI. Principal Findings: Here, we have examined the effects of PDZK1 deficiency in apoE KO mice fed with the atherogenic ‘Paigen ’ diet for three months. Relative to apoE KO, PDZK1/apoE double KO (dKO) mice showed increased plasma lipids (33 % increase in total cholesterol; 49 % increase in unesterified cholesterol; and 36 % increase in phospholipids) and a 26% increase in aortic root lesions. Compared to apoE KO, dKO mice exhibited substantial occlusive coronary artery disease: 375 % increase in severe occlusions. Myocardial infarctions, not observed in apoE KO mice (although occasional minimal fibrosis was noted), were seen in 7 of 8 dKO mice, resulting in 12 times greater area of fibrosis in dKO cardiac muscle. Conclusions: These results show that Paigen-diet fed PDZK1/apoE dKO mice represent a new animal model useful fo

  • challenges in using cultured primary rodent hepatocytes or cell lines to study hepatic hdl receptor sr bi regulation by its cytoplasmic adaptor PDZK1
    PLOS ONE, 2013
    Co-Authors: Kosuke Tsukamoto, Olivier Kocher, Lorenna D Buck, Walker Inman, Linda G Griffith, Monty Krieger
    Abstract:

    Background: PDZK1 is a four PDZ-domain containing cytoplasmic protein that binds to a variety of membrane proteins via their C-termini and can influence the abundance, localization and/or function of its target proteins. One of these targets in hepatocytes in vivo is the HDL receptor SR-BI. Normal hepatic expression of SR-BI protein requires PDZK1 - ,5% of normal hepatic SR-BI is seen in the livers of PDZK1 knockout mice. Progress has been made in identifying features of PDZK1 required to control hepatic SR-BI in vivo using hepatic expression of wild-type and mutant forms of PDZK1 in wild-type and PDZK1 KO transgenic mice. Such in vivo studies are time consuming and expensive, and cannot readily be used to explore many features of the underlying molecular and cellular mechanisms. Methodology/Principal Findings: Here we have explored the potential to use either primary rodent hepatocytes in culture using 2D collagen gels with newly developed optimized conditions or PDZK1/SR-BI co-transfected cultured cell lines (COS, HEK293) for such studies. SR-BI and PDZK1 protein and mRNA expression levels fell rapidly in primary hepatocyte cultures, indicating this system does not adequately mimic hepatocytes in vivo for analysis of the PDZK1 dependence of SR-BI. Although PDZK1 did alter SR-BI protein expression in the cell lines, its influence was independent of SR-BI’s C-terminus, and thus is not likely to occur via the same mechanism as that which occurs in hepatocytes in vivo. Conclusions/Significance: Caution must be exercised in using primary hepatocytes or cultured cell lines when studying the mechanism underlying the regulation of hepatic SR-BI by PDZK1. It may be possible to use SR-BI and PDZK1 expression as sensitive markers for the in vivo-like state of hepatocytes to further improve primary hepatocyte cell culture conditions.

  • noncanonical role of the pdz4 domain of the adaptor protein PDZK1 in the regulation of the hepatic high density lipoprotein receptor scavenger receptor class b type i sr bi
    Journal of Biological Chemistry, 2013
    Co-Authors: Kosuke Tsukamoto, Monty Krieger, Rinku Pal, Kathleen Daniels, Thomas E Wales, Ren Sheng, Wonhwa Cho, Walter F Stafford, John R Engen, Olivier Kocher
    Abstract:

    The four PDZ (PDZ1 to PDZ4) domain-containing adaptor protein PDZK1 controls the expression, localization, and function of the HDL receptor scavenger receptor class B, type I (SR-BI), in hepatocytes in vivo. This control depends on both the PDZ4 domain and the binding of SR-BI's cytoplasmic C terminus to the canonical peptide-binding sites of either the PDZ1 or PDZ3 domain (no binding to PDZ2 or PDZ4). Using transgenic mice expressing in the liver domain deletion (ΔPDZ2 or ΔPDZ3), domain replacement (PDZ2→1), or target peptide binding-negative (PDZ4(G389P)) mutants of PDZK1, we found that neither PDZ2 nor PDZ3 nor the canonical target peptide binding activity of PDZ4 were necessary for hepatic SR-BI regulatory activity. Immunohistochemical studies established that the localization of PDZK1 on hepatocyte cell surface membranes in vivo is dependent on its PDZ4 domain and the presence of SR-BI. Analytical ultracentrifugation and hydrogen deuterium exchange mass spectrometry suggested that the requirement of PDZ4 for localization and SR-BI regulation is not due to PDZ4-mediated oligomerization or induction of conformational changes in the PDZ123 portion of PDZK1. However, surface plasmon resonance analysis showed that PDZ4, but not the other PDZ domains, can bind vesicles that mimic the plasma membrane. Thus, PDZ4 may potentiate PDZK1's regulation of SR-BI by promoting its lipid-mediated attachment to the cytoplasmic membrane. Our results show that not all of the PDZ domains of a multi-PDZ domain-containing adaptor protein are required for its biological activities and that both canonical target peptide binding and noncanonical (peptide binding-independent) capacities of PDZ domains may be employed by a single such adaptor for optimal in vivo activity. Background: PDZK1 (four PDZ domains) regulates the hepatic HDL receptor SR-BI. Results: PDZK1's PDZ2 and PDZ3 domains are not required, whereas PDZ4 is, possibly because PDZ4 mediates membrane binding. Conclusion: Regulation of SR-BI via PDZK1's PDZ domains is complex. Significance: Combined canonical (target peptide binding) and noncanonical (peptide binding-independent) PDZ domain functions can result in optimal activity of a PDZ domain-containing adaptor protein.

  • noncanonical role of the pdz4 domain of the adaptor protein PDZK1 in the regulation of the hepatic high density lipoprotein receptor scavenger receptor class b type i sr bi
    Journal of Biological Chemistry, 2013
    Co-Authors: Kosuke Tsukamoto, Monty Krieger, Rinku Pal, Kathleen Daniels, Thomas E Wales, Ren Sheng, Wonhwa Cho, Walter F Stafford, John R Engen, Olivier Kocher
    Abstract:

    Abstract The four PDZ (PDZ1-PDZ4) domain-containing-adaptor protein PDZK1 controls the expression, localization and function of the HDL receptor SR-BI in hepatocytes in vivo via a PDZ4-dependent mechanism involving the binding of SR-BI′s cytoplasmic carboxy terminus to the canonical peptide binding sites of the PDZ1 or PDZ3 domains (no binding to PDZ2 or PDZ4). Using transgenic mice expressing in the liver domain deletion (ΔPDZ2 or ΔPDZ3), domain replacement (PDZ2→1) or target peptide binding-negative (PDZ4[G389P]) mutants of PDZK1, we found that neither PDZ2 nor PDZ3, nor the canonical target peptide binding activity of PDZ4 were necessary for hepatic SR-BI regulatory activity. Immunohistochemical studies established that the localization of PDZK1 on hepatocyte cell-surface membranes in vivo is dependent on its PDZ4 domain and the presence of SR-BI. Analytical ultracentrifugation and hydrogen deuterium exchange mass spectrometry suggested that the requirement for PDZ4 for localization and SR-BI regulation is not due to PDZ4-mediated oligomerization or induction of conformational changes in the PDZ123 portion of PDZK1. However, Surface Plasmon Resonance analysis showed that PDZ4, but not the other PDZ domains, can bind vesicles that mimic the plasma membrane. Thus, PDZ4 may potentiate PDZK1′s regulation of SR-BI by promoting its lipid-mediated attachment to the cytoplasmic membrane. Our results show that not all of the PDZ domains of a multi-PDZ domain-containing adaptor protein are required for its biological activities and that both canonical target peptide binding and non-canonical (peptide binding independent) capacities of PDZ domains may be employed by a single such adaptor for optimal in vivo activity.

Monty Krieger - One of the best experts on this subject based on the ideXlab platform.

  • Loss of PDZK1 Causes Coronary Artery Occlusion and Myocardial Infarction in Paigen Diet-Fed Apolipoprotein E Deficient Mice
    2013
    Co-Authors: Ayce Yesilaltay, Monty Krieger, Rinku Pal, Kathleen Daniels, Olivier Kocher
    Abstract:

    Background: PDZK1 is a four PDZ-domain containing protein that binds to the carboxy terminus of the HDL receptor, scavenger receptor class B type I (SR-BI), and regulates its expression, localization and function in a tissue-specific manner. PDZK1 knockout (KO) mice are characterized by a marked reduction of SR-BI protein expression (,95%) in the liver (lesser or no reduction in other organs) with a concomitant 1.7 fold increase in plasma cholesterol. PDZK1 has been shown to be atheroprotective using the high fat/high cholesterol (‘Western’) diet-fed murine apolipoprotein E (apoE) KO model of atherosclerosis, presumably because of its role in promoting reverse cholesterol transport via SR-BI. Principal Findings: Here, we have examined the effects of PDZK1 deficiency in apoE KO mice fed with the atherogenic ‘Paigen ’ diet for three months. Relative to apoE KO, PDZK1/apoE double KO (dKO) mice showed increased plasma lipids (33 % increase in total cholesterol; 49 % increase in unesterified cholesterol; and 36 % increase in phospholipids) and a 26% increase in aortic root lesions. Compared to apoE KO, dKO mice exhibited substantial occlusive coronary artery disease: 375 % increase in severe occlusions. Myocardial infarctions, not observed in apoE KO mice (although occasional minimal fibrosis was noted), were seen in 7 of 8 dKO mice, resulting in 12 times greater area of fibrosis in dKO cardiac muscle. Conclusions: These results show that Paigen-diet fed PDZK1/apoE dKO mice represent a new animal model useful fo

  • challenges in using cultured primary rodent hepatocytes or cell lines to study hepatic hdl receptor sr bi regulation by its cytoplasmic adaptor PDZK1
    PLOS ONE, 2013
    Co-Authors: Kosuke Tsukamoto, Olivier Kocher, Lorenna D Buck, Walker Inman, Linda G Griffith, Monty Krieger
    Abstract:

    Background: PDZK1 is a four PDZ-domain containing cytoplasmic protein that binds to a variety of membrane proteins via their C-termini and can influence the abundance, localization and/or function of its target proteins. One of these targets in hepatocytes in vivo is the HDL receptor SR-BI. Normal hepatic expression of SR-BI protein requires PDZK1 - ,5% of normal hepatic SR-BI is seen in the livers of PDZK1 knockout mice. Progress has been made in identifying features of PDZK1 required to control hepatic SR-BI in vivo using hepatic expression of wild-type and mutant forms of PDZK1 in wild-type and PDZK1 KO transgenic mice. Such in vivo studies are time consuming and expensive, and cannot readily be used to explore many features of the underlying molecular and cellular mechanisms. Methodology/Principal Findings: Here we have explored the potential to use either primary rodent hepatocytes in culture using 2D collagen gels with newly developed optimized conditions or PDZK1/SR-BI co-transfected cultured cell lines (COS, HEK293) for such studies. SR-BI and PDZK1 protein and mRNA expression levels fell rapidly in primary hepatocyte cultures, indicating this system does not adequately mimic hepatocytes in vivo for analysis of the PDZK1 dependence of SR-BI. Although PDZK1 did alter SR-BI protein expression in the cell lines, its influence was independent of SR-BI’s C-terminus, and thus is not likely to occur via the same mechanism as that which occurs in hepatocytes in vivo. Conclusions/Significance: Caution must be exercised in using primary hepatocytes or cultured cell lines when studying the mechanism underlying the regulation of hepatic SR-BI by PDZK1. It may be possible to use SR-BI and PDZK1 expression as sensitive markers for the in vivo-like state of hepatocytes to further improve primary hepatocyte cell culture conditions.

  • noncanonical role of the pdz4 domain of the adaptor protein PDZK1 in the regulation of the hepatic high density lipoprotein receptor scavenger receptor class b type i sr bi
    Journal of Biological Chemistry, 2013
    Co-Authors: Kosuke Tsukamoto, Monty Krieger, Rinku Pal, Kathleen Daniels, Thomas E Wales, Ren Sheng, Wonhwa Cho, Walter F Stafford, John R Engen, Olivier Kocher
    Abstract:

    The four PDZ (PDZ1 to PDZ4) domain-containing adaptor protein PDZK1 controls the expression, localization, and function of the HDL receptor scavenger receptor class B, type I (SR-BI), in hepatocytes in vivo. This control depends on both the PDZ4 domain and the binding of SR-BI's cytoplasmic C terminus to the canonical peptide-binding sites of either the PDZ1 or PDZ3 domain (no binding to PDZ2 or PDZ4). Using transgenic mice expressing in the liver domain deletion (ΔPDZ2 or ΔPDZ3), domain replacement (PDZ2→1), or target peptide binding-negative (PDZ4(G389P)) mutants of PDZK1, we found that neither PDZ2 nor PDZ3 nor the canonical target peptide binding activity of PDZ4 were necessary for hepatic SR-BI regulatory activity. Immunohistochemical studies established that the localization of PDZK1 on hepatocyte cell surface membranes in vivo is dependent on its PDZ4 domain and the presence of SR-BI. Analytical ultracentrifugation and hydrogen deuterium exchange mass spectrometry suggested that the requirement of PDZ4 for localization and SR-BI regulation is not due to PDZ4-mediated oligomerization or induction of conformational changes in the PDZ123 portion of PDZK1. However, surface plasmon resonance analysis showed that PDZ4, but not the other PDZ domains, can bind vesicles that mimic the plasma membrane. Thus, PDZ4 may potentiate PDZK1's regulation of SR-BI by promoting its lipid-mediated attachment to the cytoplasmic membrane. Our results show that not all of the PDZ domains of a multi-PDZ domain-containing adaptor protein are required for its biological activities and that both canonical target peptide binding and noncanonical (peptide binding-independent) capacities of PDZ domains may be employed by a single such adaptor for optimal in vivo activity. Background: PDZK1 (four PDZ domains) regulates the hepatic HDL receptor SR-BI. Results: PDZK1's PDZ2 and PDZ3 domains are not required, whereas PDZ4 is, possibly because PDZ4 mediates membrane binding. Conclusion: Regulation of SR-BI via PDZK1's PDZ domains is complex. Significance: Combined canonical (target peptide binding) and noncanonical (peptide binding-independent) PDZ domain functions can result in optimal activity of a PDZ domain-containing adaptor protein.

  • noncanonical role of the pdz4 domain of the adaptor protein PDZK1 in the regulation of the hepatic high density lipoprotein receptor scavenger receptor class b type i sr bi
    Journal of Biological Chemistry, 2013
    Co-Authors: Kosuke Tsukamoto, Monty Krieger, Rinku Pal, Kathleen Daniels, Thomas E Wales, Ren Sheng, Wonhwa Cho, Walter F Stafford, John R Engen, Olivier Kocher
    Abstract:

    Abstract The four PDZ (PDZ1-PDZ4) domain-containing-adaptor protein PDZK1 controls the expression, localization and function of the HDL receptor SR-BI in hepatocytes in vivo via a PDZ4-dependent mechanism involving the binding of SR-BI′s cytoplasmic carboxy terminus to the canonical peptide binding sites of the PDZ1 or PDZ3 domains (no binding to PDZ2 or PDZ4). Using transgenic mice expressing in the liver domain deletion (ΔPDZ2 or ΔPDZ3), domain replacement (PDZ2→1) or target peptide binding-negative (PDZ4[G389P]) mutants of PDZK1, we found that neither PDZ2 nor PDZ3, nor the canonical target peptide binding activity of PDZ4 were necessary for hepatic SR-BI regulatory activity. Immunohistochemical studies established that the localization of PDZK1 on hepatocyte cell-surface membranes in vivo is dependent on its PDZ4 domain and the presence of SR-BI. Analytical ultracentrifugation and hydrogen deuterium exchange mass spectrometry suggested that the requirement for PDZ4 for localization and SR-BI regulation is not due to PDZ4-mediated oligomerization or induction of conformational changes in the PDZ123 portion of PDZK1. However, Surface Plasmon Resonance analysis showed that PDZ4, but not the other PDZ domains, can bind vesicles that mimic the plasma membrane. Thus, PDZ4 may potentiate PDZK1′s regulation of SR-BI by promoting its lipid-mediated attachment to the cytoplasmic membrane. Our results show that not all of the PDZ domains of a multi-PDZ domain-containing adaptor protein are required for its biological activities and that both canonical target peptide binding and non-canonical (peptide binding independent) capacities of PDZ domains may be employed by a single such adaptor for optimal in vivo activity.

  • Aortic root atherosclerosis in Paigen diet-fed apoE KO and PDZK1/apoE dKO mice.
    2013
    Co-Authors: Ayce Yesilaltay, Monty Krieger, Rinku Pal, Kathleen Daniels, Olivier Kocher
    Abstract:

    Hearts were harvested from Paigen diet-fed apoE KO (A, C–D) and PDZK1/apoE dKO (B, E–F) mice as described in Methods (n = 8 per genotype). Left top panels: A–B: representative cross-sections of Oil red O-stained aortic root lesions. (magnification, ×20). Right top panel: quantification of aortic root atherosclerosis by planimetry. Unpaired Student's t-test was used to determine statistical significance. Bottom panels: immunohistochemistry of aortic root atherosclerotic plaques using CD68 (C and E) or alpha-smooth muscle actin α-SMA) (D and F) antibodies show that macrophages compose the overwhelming cell population of aortic root atherosclerotic plaques and that smooth muscle cells are rare in both apoE KO (C–D) and PDZK1/apoE dKO (E–F) mice. “L” indicates the vascular lumen, arrows indicate representative positive cells (magnification, ×100).

Rinku Pal - One of the best experts on this subject based on the ideXlab platform.

  • Molecular Analysis of the Prostacyclin Receptor’s Interaction with the PDZ1 Domain of Its Adaptor Protein
    2016
    Co-Authors: Gabriel Birrane, Eamon P. Mulvaney, Therese B Kinsella, Rinku Pal, Olivier Kocher
    Abstract:

    The prostanoid prostacyclin, or prostaglandin I2, plays an essential role in many aspects of cardiovascular disease. The actions of prostacyclin are mainly mediated through its activation of the prostacyclin receptor or, in short, the IP. In recent studies, the cytoplasmic carboxy-terminal domain of the IP was shown to bind several PDZ domains of the multi-PDZ adaptor PDZK1. The interaction between the two proteins was found to enhance cell surface expression of the IP and to be functionally important in promoting prostacyclin-induced endothelial cell migration and angiogenesis. To investigate the interaction of the IP with the first PDZ domain (PDZ1) of PDZK1, we generated a nine residue peptide (KK411IAACSLC417) containing the seven carboxy-terminal amino acids of the IP and measured its binding affinity to a recombinant protein corresponding to PDZ1 by isothermal titration calorimetry. We determined that the IP interacts with PDZ1 with a binding affinity of 8.2 mM. Using the same technique, we also determined that the farnesylated form of carboxy-terminus of the IP does not bind to PDZ1. To understand the molecular basis of these findings, we solved the high resolution crystal structure of PDZ1 bound to a 7-residue peptide derived from the carboxy-terminus of the non-farnesylated form of IP (411IAACSLC417). Analysis of the structure demonstrates a critical role for the three carboxy-terminal amino acids in establishing a strong interaction with PDZ1 and explains the inability of the farnesylated form of IP to interact with the PDZ1 domain of PDZK1 a

  • Loss of PDZK1 Causes Coronary Artery Occlusion and Myocardial Infarction in Paigen Diet-Fed Apolipoprotein E Deficient Mice
    2013
    Co-Authors: Ayce Yesilaltay, Monty Krieger, Rinku Pal, Kathleen Daniels, Olivier Kocher
    Abstract:

    Background: PDZK1 is a four PDZ-domain containing protein that binds to the carboxy terminus of the HDL receptor, scavenger receptor class B type I (SR-BI), and regulates its expression, localization and function in a tissue-specific manner. PDZK1 knockout (KO) mice are characterized by a marked reduction of SR-BI protein expression (,95%) in the liver (lesser or no reduction in other organs) with a concomitant 1.7 fold increase in plasma cholesterol. PDZK1 has been shown to be atheroprotective using the high fat/high cholesterol (‘Western’) diet-fed murine apolipoprotein E (apoE) KO model of atherosclerosis, presumably because of its role in promoting reverse cholesterol transport via SR-BI. Principal Findings: Here, we have examined the effects of PDZK1 deficiency in apoE KO mice fed with the atherogenic ‘Paigen ’ diet for three months. Relative to apoE KO, PDZK1/apoE double KO (dKO) mice showed increased plasma lipids (33 % increase in total cholesterol; 49 % increase in unesterified cholesterol; and 36 % increase in phospholipids) and a 26% increase in aortic root lesions. Compared to apoE KO, dKO mice exhibited substantial occlusive coronary artery disease: 375 % increase in severe occlusions. Myocardial infarctions, not observed in apoE KO mice (although occasional minimal fibrosis was noted), were seen in 7 of 8 dKO mice, resulting in 12 times greater area of fibrosis in dKO cardiac muscle. Conclusions: These results show that Paigen-diet fed PDZK1/apoE dKO mice represent a new animal model useful fo

  • noncanonical role of the pdz4 domain of the adaptor protein PDZK1 in the regulation of the hepatic high density lipoprotein receptor scavenger receptor class b type i sr bi
    Journal of Biological Chemistry, 2013
    Co-Authors: Kosuke Tsukamoto, Monty Krieger, Rinku Pal, Kathleen Daniels, Thomas E Wales, Ren Sheng, Wonhwa Cho, Walter F Stafford, John R Engen, Olivier Kocher
    Abstract:

    The four PDZ (PDZ1 to PDZ4) domain-containing adaptor protein PDZK1 controls the expression, localization, and function of the HDL receptor scavenger receptor class B, type I (SR-BI), in hepatocytes in vivo. This control depends on both the PDZ4 domain and the binding of SR-BI's cytoplasmic C terminus to the canonical peptide-binding sites of either the PDZ1 or PDZ3 domain (no binding to PDZ2 or PDZ4). Using transgenic mice expressing in the liver domain deletion (ΔPDZ2 or ΔPDZ3), domain replacement (PDZ2→1), or target peptide binding-negative (PDZ4(G389P)) mutants of PDZK1, we found that neither PDZ2 nor PDZ3 nor the canonical target peptide binding activity of PDZ4 were necessary for hepatic SR-BI regulatory activity. Immunohistochemical studies established that the localization of PDZK1 on hepatocyte cell surface membranes in vivo is dependent on its PDZ4 domain and the presence of SR-BI. Analytical ultracentrifugation and hydrogen deuterium exchange mass spectrometry suggested that the requirement of PDZ4 for localization and SR-BI regulation is not due to PDZ4-mediated oligomerization or induction of conformational changes in the PDZ123 portion of PDZK1. However, surface plasmon resonance analysis showed that PDZ4, but not the other PDZ domains, can bind vesicles that mimic the plasma membrane. Thus, PDZ4 may potentiate PDZK1's regulation of SR-BI by promoting its lipid-mediated attachment to the cytoplasmic membrane. Our results show that not all of the PDZ domains of a multi-PDZ domain-containing adaptor protein are required for its biological activities and that both canonical target peptide binding and noncanonical (peptide binding-independent) capacities of PDZ domains may be employed by a single such adaptor for optimal in vivo activity. Background: PDZK1 (four PDZ domains) regulates the hepatic HDL receptor SR-BI. Results: PDZK1's PDZ2 and PDZ3 domains are not required, whereas PDZ4 is, possibly because PDZ4 mediates membrane binding. Conclusion: Regulation of SR-BI via PDZK1's PDZ domains is complex. Significance: Combined canonical (target peptide binding) and noncanonical (peptide binding-independent) PDZ domain functions can result in optimal activity of a PDZ domain-containing adaptor protein.

  • noncanonical role of the pdz4 domain of the adaptor protein PDZK1 in the regulation of the hepatic high density lipoprotein receptor scavenger receptor class b type i sr bi
    Journal of Biological Chemistry, 2013
    Co-Authors: Kosuke Tsukamoto, Monty Krieger, Rinku Pal, Kathleen Daniels, Thomas E Wales, Ren Sheng, Wonhwa Cho, Walter F Stafford, John R Engen, Olivier Kocher
    Abstract:

    Abstract The four PDZ (PDZ1-PDZ4) domain-containing-adaptor protein PDZK1 controls the expression, localization and function of the HDL receptor SR-BI in hepatocytes in vivo via a PDZ4-dependent mechanism involving the binding of SR-BI′s cytoplasmic carboxy terminus to the canonical peptide binding sites of the PDZ1 or PDZ3 domains (no binding to PDZ2 or PDZ4). Using transgenic mice expressing in the liver domain deletion (ΔPDZ2 or ΔPDZ3), domain replacement (PDZ2→1) or target peptide binding-negative (PDZ4[G389P]) mutants of PDZK1, we found that neither PDZ2 nor PDZ3, nor the canonical target peptide binding activity of PDZ4 were necessary for hepatic SR-BI regulatory activity. Immunohistochemical studies established that the localization of PDZK1 on hepatocyte cell-surface membranes in vivo is dependent on its PDZ4 domain and the presence of SR-BI. Analytical ultracentrifugation and hydrogen deuterium exchange mass spectrometry suggested that the requirement for PDZ4 for localization and SR-BI regulation is not due to PDZ4-mediated oligomerization or induction of conformational changes in the PDZ123 portion of PDZK1. However, Surface Plasmon Resonance analysis showed that PDZ4, but not the other PDZ domains, can bind vesicles that mimic the plasma membrane. Thus, PDZ4 may potentiate PDZK1′s regulation of SR-BI by promoting its lipid-mediated attachment to the cytoplasmic membrane. Our results show that not all of the PDZ domains of a multi-PDZ domain-containing adaptor protein are required for its biological activities and that both canonical target peptide binding and non-canonical (peptide binding independent) capacities of PDZ domains may be employed by a single such adaptor for optimal in vivo activity.

  • Steps involved in the isoprenylation and processing of the Prostacyclin Receptor.
    2013
    Co-Authors: Gabriel Birrane, Eamon P. Mulvaney, Therese B Kinsella, Rinku Pal, Olivier Kocher
    Abstract:

    The prostacyclin receptor (IP) contains an evolutionary conserved ‘CaaX motif’ at its cytoplasmic carboxy-terminus, e.g corresponding to C414SLC417 of the mouse IP as shown. During its processing, (i) the IP undergoes isoprenylation through thio-ether attachment of a carbon (C)-15 farnesyl moiety to Cys414 while subsequent (ii) proteolytic cleavage, or aaXing, liberates the terminal 415SLC417 residues and (iii) end-stage carboxy-methylation of the nascent α-carboxy-group on Cys414 generates the fully processed, mature IP in its farnesyl-Cys-carboxymethylated form. Herein, the interaction of peptides based on the mouse IP carboxy-terminus with PDZ domain 1 (PDZ1) or full length PDZK1 was investigated through isothermal titration calorimetry (ITC) where Peptide 1 is a nanopeptide containing the seven carboxy-terminal amino acids (KK411IAACSLC417); Peptide 2 is an octapeptide corresponding to the carboxy-terminus of IP (K407SEAIAAC414) devoid of the 3 terminal amino acids (-aaX/-415SLC417) which are proteolytically cleavage following farnesylation of the IP; Peptide 3 is identical to Peptide 2 (K407SEAIAAC414) except that it was modified by the addition of a C-15 farnesyl group on the carboxy-terminal cysteine (Cys414) and a carboxy-methyl group on the terminal -α-COOH, thereby representing the farnesyl-Cys-carboxymethyl ester form of the C-terminus of the IP.

Ayce Yesilaltay - One of the best experts on this subject based on the ideXlab platform.

  • Loss of PDZK1 Causes Coronary Artery Occlusion and Myocardial Infarction in Paigen Diet-Fed Apolipoprotein E Deficient Mice
    2013
    Co-Authors: Ayce Yesilaltay, Monty Krieger, Rinku Pal, Kathleen Daniels, Olivier Kocher
    Abstract:

    Background: PDZK1 is a four PDZ-domain containing protein that binds to the carboxy terminus of the HDL receptor, scavenger receptor class B type I (SR-BI), and regulates its expression, localization and function in a tissue-specific manner. PDZK1 knockout (KO) mice are characterized by a marked reduction of SR-BI protein expression (,95%) in the liver (lesser or no reduction in other organs) with a concomitant 1.7 fold increase in plasma cholesterol. PDZK1 has been shown to be atheroprotective using the high fat/high cholesterol (‘Western’) diet-fed murine apolipoprotein E (apoE) KO model of atherosclerosis, presumably because of its role in promoting reverse cholesterol transport via SR-BI. Principal Findings: Here, we have examined the effects of PDZK1 deficiency in apoE KO mice fed with the atherogenic ‘Paigen ’ diet for three months. Relative to apoE KO, PDZK1/apoE double KO (dKO) mice showed increased plasma lipids (33 % increase in total cholesterol; 49 % increase in unesterified cholesterol; and 36 % increase in phospholipids) and a 26% increase in aortic root lesions. Compared to apoE KO, dKO mice exhibited substantial occlusive coronary artery disease: 375 % increase in severe occlusions. Myocardial infarctions, not observed in apoE KO mice (although occasional minimal fibrosis was noted), were seen in 7 of 8 dKO mice, resulting in 12 times greater area of fibrosis in dKO cardiac muscle. Conclusions: These results show that Paigen-diet fed PDZK1/apoE dKO mice represent a new animal model useful fo

  • Effects of loss of PDZK1 on coronary atherosclerosis and cardiac fibrosis in apoE KO mice.
    2013
    Co-Authors: Ayce Yesilaltay, Monty Krieger, Rinku Pal, Kathleen Daniels, Olivier Kocher
    Abstract:

    Hearts were harvested from Paigen diet-fed mice as described in Methods (n = 8 per genotype). Left panels: A–B: representative cross-sections of Oil red O-stained (A–B) or trichrome-stained (C) myocardial coronary arterioles, showing unremarkable arterioles in apoE KO (A) and totally occluded arterioles in PDZK1/apoE dKO (B–C) mice (magnification, ×100). The Oil red O stain shows that the coronary arteriole is occluded almost exclusively by lipid-rich lesions (B), while the trichrome stain shows that the arteriole is surrounded by fibrosis in an area of myocardial infarction in a PDZK1/apoE dKO mouse (C). D–E: trichrome stained sections of hearts showing areas of infarction/fibrosis stained blue in PDZK1/apoE dKO (E), while they are absent in apoE KO (D) mice (magnification, ×10). Right top panel: quantification of coronary artery occlusions in apoE KO and PDZK1/apoE dKO mice. Statistically significant differences by ANOVA Tukey posthoc test comparing the two genotypes within a given group are indicated as: P

  • Aortic root atherosclerosis in Paigen diet-fed apoE KO and PDZK1/apoE dKO mice.
    2013
    Co-Authors: Ayce Yesilaltay, Monty Krieger, Rinku Pal, Kathleen Daniels, Olivier Kocher
    Abstract:

    Hearts were harvested from Paigen diet-fed apoE KO (A, C–D) and PDZK1/apoE dKO (B, E–F) mice as described in Methods (n = 8 per genotype). Left top panels: A–B: representative cross-sections of Oil red O-stained aortic root lesions. (magnification, ×20). Right top panel: quantification of aortic root atherosclerosis by planimetry. Unpaired Student's t-test was used to determine statistical significance. Bottom panels: immunohistochemistry of aortic root atherosclerotic plaques using CD68 (C and E) or alpha-smooth muscle actin α-SMA) (D and F) antibodies show that macrophages compose the overwhelming cell population of aortic root atherosclerotic plaques and that smooth muscle cells are rare in both apoE KO (C–D) and PDZK1/apoE dKO (E–F) mice. “L” indicates the vascular lumen, arrows indicate representative positive cells (magnification, ×100).

  • identification of the pdz3 domain of the adaptor protein PDZK1 as a second physiologically functional binding site for the c terminus of the high density lipoprotein receptor scavenger receptor class b type i
    Journal of Biological Chemistry, 2011
    Co-Authors: Olivier Kocher, Gabriel Birrane, Ayce Yesilaltay, Rinku Pal, Sharon Shechter, Kathleen Daniels, Monty Krieger
    Abstract:

    Abstract The normal expression, cell surface localization, and function of the murine high density lipoprotein receptor scavenger receptor class B type I (SR-BI) in hepatocytes in vivo, and thus normal lipoprotein metabolism, depend on its four PDZ domain (PDZ1–PDZ4) containing cytoplasmic adaptor protein PDZK1. Previous studies showed that the C terminus of SR-BI (“target peptide”) binds directly to PDZ1 and influences hepatic SR-BI protein expression. Unexpectedly an inactivating mutation in PDZ1 (Tyr20 → Ala) only partially, rather than completely, suppresses the ability of PDZK1 to control hepatic SR-BI. We used isothermal titration calorimetry to show that PDZ3, but not PDZ2 or PDZ4, can also bind the target peptide (Kd = 37.0 μm), albeit with ∼10-fold lower affinity than PDZ1. This binding is abrogated by a Tyr253 → Ala substitution. Comparison of the 1.5-A resolution crystal structure of PDZ3 with its bound target peptide (505QEAKL509) to that of peptide-bound PDZ1 indicated fewer target peptide stabilizing atomic interactions (hydrogen bonds and hydrophobic interactions) in PDZ3. A double (Tyr20 → Ala (PDZ1) + Tyr253 → Ala (PDZ3)) substitution abrogated all target peptide binding to PDZK1. In vivo hepatic expression of a singly substituted (Tyr253 → Ala (PDZ3)) PDZK1 transgene (Tg) was able to correct all of the SR-BI-related defects in PDZK1 knock-out mice, whereas the doubly substituted [Tyr20 → Ala (PDZ1) + Tyr253 → Ala (PDZ3)]Tg was unable to correct these defects. Thus, we conclude that PDZK1-mediated control of hepatic SR-BI requires direct binding of the SR-BI C terminus to either the PDZ1 or PDZ3 domains, and that binding to both domains simultaneously is not required for PDZK1 control of hepatic SR-BI.

  • in vitro and in vivo analysis of the binding of the c terminus of the hdl receptor scavenger receptor class b type i sr bi to the pdz1 domain of its adaptor protein PDZK1
    Journal of Biological Chemistry, 2010
    Co-Authors: Olivier Kocher, Gabriel Birrane, Ayce Yesilaltay, Rinku Pal, Kathleen Daniels, Kosuke Tsukamoto, Sara A Fenske, John A A Ladias, Monty Krieger
    Abstract:

    The PDZ1 domain of the four PDZ domain-containing protein PDZK1 has been reported to bind the C terminus of the HDL receptor scavenger receptor class B, type I (SR-BI), and to control hepatic SR-BI expression and function. We generated wild-type (WT) and mutant murine PDZ1 domains, the mutants bearing single amino acid substitutions in their carboxylate binding loop (Lys14-Xaa4-Asn19-Tyr-Gly-Phe-Phe-Leu24), and measured their binding affinity for a 7-residue peptide corresponding to the C terminus of SR-BI (503VLQEAKL509). The Y20A and G21Y substitutions abrogated all binding activity. Surprisingly, binding affinities (Kd) of the K14A and F22A mutants were 3.2 and 4.0 μm, respectively, similar to 2.6 μm measured for the WT PDZ1. To understand these findings, we determined the high resolution structure of WT PDZ1 bound to a 5-residue sequence from the C-terminal SR-BI (505QEAKL509) using x-ray crystallography. In addition, we incorporated the K14A and Y20A substitutions into full-length PDZK1 liver-specific transgenes and expressed them in WT and PDZK1 knock-out mice. In WT mice, the transgenes did not alter endogenous hepatic SR-BI protein expression (intracellular distribution or amount) or lipoprotein metabolism (total plasma cholesterol, lipoprotein size distribution). In PDZK1 knock-out mice, as expected, the K14A mutant behaved like wild-type PDZK1 and completely corrected their hepatic SR-BI and plasma lipoprotein abnormalities. Unexpectedly, the 10–20-fold overexpressed Y20A mutant also substantially, but not completely, corrected these abnormalities. The results suggest that there may be an additional site(s) within PDZK1 that bind(s) SR-BI and mediate(s) productive SR-BI-PDZK1 interaction previously attributed exclusively to the canonical binding of the C-terminal SR-BI to PDZ1.

Kathleen Daniels - One of the best experts on this subject based on the ideXlab platform.

  • Loss of PDZK1 Causes Coronary Artery Occlusion and Myocardial Infarction in Paigen Diet-Fed Apolipoprotein E Deficient Mice
    2013
    Co-Authors: Ayce Yesilaltay, Monty Krieger, Rinku Pal, Kathleen Daniels, Olivier Kocher
    Abstract:

    Background: PDZK1 is a four PDZ-domain containing protein that binds to the carboxy terminus of the HDL receptor, scavenger receptor class B type I (SR-BI), and regulates its expression, localization and function in a tissue-specific manner. PDZK1 knockout (KO) mice are characterized by a marked reduction of SR-BI protein expression (,95%) in the liver (lesser or no reduction in other organs) with a concomitant 1.7 fold increase in plasma cholesterol. PDZK1 has been shown to be atheroprotective using the high fat/high cholesterol (‘Western’) diet-fed murine apolipoprotein E (apoE) KO model of atherosclerosis, presumably because of its role in promoting reverse cholesterol transport via SR-BI. Principal Findings: Here, we have examined the effects of PDZK1 deficiency in apoE KO mice fed with the atherogenic ‘Paigen ’ diet for three months. Relative to apoE KO, PDZK1/apoE double KO (dKO) mice showed increased plasma lipids (33 % increase in total cholesterol; 49 % increase in unesterified cholesterol; and 36 % increase in phospholipids) and a 26% increase in aortic root lesions. Compared to apoE KO, dKO mice exhibited substantial occlusive coronary artery disease: 375 % increase in severe occlusions. Myocardial infarctions, not observed in apoE KO mice (although occasional minimal fibrosis was noted), were seen in 7 of 8 dKO mice, resulting in 12 times greater area of fibrosis in dKO cardiac muscle. Conclusions: These results show that Paigen-diet fed PDZK1/apoE dKO mice represent a new animal model useful fo

  • noncanonical role of the pdz4 domain of the adaptor protein PDZK1 in the regulation of the hepatic high density lipoprotein receptor scavenger receptor class b type i sr bi
    Journal of Biological Chemistry, 2013
    Co-Authors: Kosuke Tsukamoto, Monty Krieger, Rinku Pal, Kathleen Daniels, Thomas E Wales, Ren Sheng, Wonhwa Cho, Walter F Stafford, John R Engen, Olivier Kocher
    Abstract:

    The four PDZ (PDZ1 to PDZ4) domain-containing adaptor protein PDZK1 controls the expression, localization, and function of the HDL receptor scavenger receptor class B, type I (SR-BI), in hepatocytes in vivo. This control depends on both the PDZ4 domain and the binding of SR-BI's cytoplasmic C terminus to the canonical peptide-binding sites of either the PDZ1 or PDZ3 domain (no binding to PDZ2 or PDZ4). Using transgenic mice expressing in the liver domain deletion (ΔPDZ2 or ΔPDZ3), domain replacement (PDZ2→1), or target peptide binding-negative (PDZ4(G389P)) mutants of PDZK1, we found that neither PDZ2 nor PDZ3 nor the canonical target peptide binding activity of PDZ4 were necessary for hepatic SR-BI regulatory activity. Immunohistochemical studies established that the localization of PDZK1 on hepatocyte cell surface membranes in vivo is dependent on its PDZ4 domain and the presence of SR-BI. Analytical ultracentrifugation and hydrogen deuterium exchange mass spectrometry suggested that the requirement of PDZ4 for localization and SR-BI regulation is not due to PDZ4-mediated oligomerization or induction of conformational changes in the PDZ123 portion of PDZK1. However, surface plasmon resonance analysis showed that PDZ4, but not the other PDZ domains, can bind vesicles that mimic the plasma membrane. Thus, PDZ4 may potentiate PDZK1's regulation of SR-BI by promoting its lipid-mediated attachment to the cytoplasmic membrane. Our results show that not all of the PDZ domains of a multi-PDZ domain-containing adaptor protein are required for its biological activities and that both canonical target peptide binding and noncanonical (peptide binding-independent) capacities of PDZ domains may be employed by a single such adaptor for optimal in vivo activity. Background: PDZK1 (four PDZ domains) regulates the hepatic HDL receptor SR-BI. Results: PDZK1's PDZ2 and PDZ3 domains are not required, whereas PDZ4 is, possibly because PDZ4 mediates membrane binding. Conclusion: Regulation of SR-BI via PDZK1's PDZ domains is complex. Significance: Combined canonical (target peptide binding) and noncanonical (peptide binding-independent) PDZ domain functions can result in optimal activity of a PDZ domain-containing adaptor protein.

  • noncanonical role of the pdz4 domain of the adaptor protein PDZK1 in the regulation of the hepatic high density lipoprotein receptor scavenger receptor class b type i sr bi
    Journal of Biological Chemistry, 2013
    Co-Authors: Kosuke Tsukamoto, Monty Krieger, Rinku Pal, Kathleen Daniels, Thomas E Wales, Ren Sheng, Wonhwa Cho, Walter F Stafford, John R Engen, Olivier Kocher
    Abstract:

    Abstract The four PDZ (PDZ1-PDZ4) domain-containing-adaptor protein PDZK1 controls the expression, localization and function of the HDL receptor SR-BI in hepatocytes in vivo via a PDZ4-dependent mechanism involving the binding of SR-BI′s cytoplasmic carboxy terminus to the canonical peptide binding sites of the PDZ1 or PDZ3 domains (no binding to PDZ2 or PDZ4). Using transgenic mice expressing in the liver domain deletion (ΔPDZ2 or ΔPDZ3), domain replacement (PDZ2→1) or target peptide binding-negative (PDZ4[G389P]) mutants of PDZK1, we found that neither PDZ2 nor PDZ3, nor the canonical target peptide binding activity of PDZ4 were necessary for hepatic SR-BI regulatory activity. Immunohistochemical studies established that the localization of PDZK1 on hepatocyte cell-surface membranes in vivo is dependent on its PDZ4 domain and the presence of SR-BI. Analytical ultracentrifugation and hydrogen deuterium exchange mass spectrometry suggested that the requirement for PDZ4 for localization and SR-BI regulation is not due to PDZ4-mediated oligomerization or induction of conformational changes in the PDZ123 portion of PDZK1. However, Surface Plasmon Resonance analysis showed that PDZ4, but not the other PDZ domains, can bind vesicles that mimic the plasma membrane. Thus, PDZ4 may potentiate PDZK1′s regulation of SR-BI by promoting its lipid-mediated attachment to the cytoplasmic membrane. Our results show that not all of the PDZ domains of a multi-PDZ domain-containing adaptor protein are required for its biological activities and that both canonical target peptide binding and non-canonical (peptide binding independent) capacities of PDZ domains may be employed by a single such adaptor for optimal in vivo activity.

  • Effects of loss of PDZK1 on coronary atherosclerosis and cardiac fibrosis in apoE KO mice.
    2013
    Co-Authors: Ayce Yesilaltay, Monty Krieger, Rinku Pal, Kathleen Daniels, Olivier Kocher
    Abstract:

    Hearts were harvested from Paigen diet-fed mice as described in Methods (n = 8 per genotype). Left panels: A–B: representative cross-sections of Oil red O-stained (A–B) or trichrome-stained (C) myocardial coronary arterioles, showing unremarkable arterioles in apoE KO (A) and totally occluded arterioles in PDZK1/apoE dKO (B–C) mice (magnification, ×100). The Oil red O stain shows that the coronary arteriole is occluded almost exclusively by lipid-rich lesions (B), while the trichrome stain shows that the arteriole is surrounded by fibrosis in an area of myocardial infarction in a PDZK1/apoE dKO mouse (C). D–E: trichrome stained sections of hearts showing areas of infarction/fibrosis stained blue in PDZK1/apoE dKO (E), while they are absent in apoE KO (D) mice (magnification, ×10). Right top panel: quantification of coronary artery occlusions in apoE KO and PDZK1/apoE dKO mice. Statistically significant differences by ANOVA Tukey posthoc test comparing the two genotypes within a given group are indicated as: P

  • Aortic root atherosclerosis in Paigen diet-fed apoE KO and PDZK1/apoE dKO mice.
    2013
    Co-Authors: Ayce Yesilaltay, Monty Krieger, Rinku Pal, Kathleen Daniels, Olivier Kocher
    Abstract:

    Hearts were harvested from Paigen diet-fed apoE KO (A, C–D) and PDZK1/apoE dKO (B, E–F) mice as described in Methods (n = 8 per genotype). Left top panels: A–B: representative cross-sections of Oil red O-stained aortic root lesions. (magnification, ×20). Right top panel: quantification of aortic root atherosclerosis by planimetry. Unpaired Student's t-test was used to determine statistical significance. Bottom panels: immunohistochemistry of aortic root atherosclerotic plaques using CD68 (C and E) or alpha-smooth muscle actin α-SMA) (D and F) antibodies show that macrophages compose the overwhelming cell population of aortic root atherosclerotic plaques and that smooth muscle cells are rare in both apoE KO (C–D) and PDZK1/apoE dKO (E–F) mice. “L” indicates the vascular lumen, arrows indicate representative positive cells (magnification, ×100).