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Mapping Soluble Guanylyl Cyclase and Protein Disulfide Isomerase Regions of Interaction.

Heckler EJ, Kholodovych V, Jain M, Liu T, Li H, Beuve A - PLoS ONE (2015)

Bottom Line: Together with Flag-immunoprecipitation using sGC domain deletions, wild-type (WT) and mutated PDI, regions of sGC involved in this interaction were identified.Our results indicate that PDI interacts preferentially with the catalytic domain of sGC, thus providing a mechanism for PDI inhibition of sGC.A model in which PDI interacts with either the α or the β catalytic domain is proposed.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Physiology and Neuroscience, New Jersey Medical School, Rutgers University, Newark, NJ, United States of America.

ABSTRACT
Soluble guanylyl cyclase (sGC) is a heterodimeric nitric oxide (NO) receptor that produces cyclic GMP. This signaling mechanism is a key component in the cardiovascular system. NO binds to heme in the β subunit and stimulates the catalytic conversion of GTP to cGMP several hundred fold. Several endogenous factors have been identified that modulate sGC function in vitro and in vivo. In previous work, we determined that protein disulfide isomerase (PDI) interacts with sGC in a redox-dependent manner in vitro and that PDI inhibited NO-stimulated activity in cells. To our knowledge, this was the first report of a physical interaction between sGC and a thiol-redox protein. To characterize this interaction between sGC and PDI, we first identified peptide linkages between sGC and PDI, using a lysine cross-linking reagent and recently developed mass spectrometry analysis. Together with Flag-immunoprecipitation using sGC domain deletions, wild-type (WT) and mutated PDI, regions of sGC involved in this interaction were identified. The observed data were further explored with computational modeling to gain insight into the interaction mechanism between sGC and oxidized PDI. Our results indicate that PDI interacts preferentially with the catalytic domain of sGC, thus providing a mechanism for PDI inhibition of sGC. A model in which PDI interacts with either the α or the β catalytic domain is proposed.

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Flag immunoprecipitation of α and/or β sGC by Flag-tagged PDI-WT.A. Representative Western blots of α, β and Flag-PDI expressed in COS-7 cells (input right panel) and precipitated by PDI-Flag (bound, right panel). COS-7 cells were transiently transfected with Flag-tagged PDI-WT and infected with sGC α and/or β WT adenoviruses 24 hours post-transfection. Immunoprecipitation of lysates was performed using anti-Flag affinity resin as described in Material and Methods. Blots were probed with anti-α and β (top panels) and PDI (bottom panels). B. Densitometry analysis of the blots. Band analysis of bound vs. input with ImageJ of three separate experiments; sGC bound over input: α (1.08 ± 0.13), β (0.74 ± 0.02), and αβ (0.76 ± 0.06).
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pone.0143523.g003: Flag immunoprecipitation of α and/or β sGC by Flag-tagged PDI-WT.A. Representative Western blots of α, β and Flag-PDI expressed in COS-7 cells (input right panel) and precipitated by PDI-Flag (bound, right panel). COS-7 cells were transiently transfected with Flag-tagged PDI-WT and infected with sGC α and/or β WT adenoviruses 24 hours post-transfection. Immunoprecipitation of lysates was performed using anti-Flag affinity resin as described in Material and Methods. Blots were probed with anti-α and β (top panels) and PDI (bottom panels). B. Densitometry analysis of the blots. Band analysis of bound vs. input with ImageJ of three separate experiments; sGC bound over input: α (1.08 ± 0.13), β (0.74 ± 0.02), and αβ (0.76 ± 0.06).

Mentions: The MassMatrix and cross-linking mass spectrometry results showed that the catalytic domains of both subunits could interact with PDI. To determine whether PDI might have a stronger affinity for a particular sGC subunit, i.e. α vs. β, and whether the heterodimer is required for interaction, WT sGC α and β, separately or together were co-expressed with Flag-tagged WT PDI (PDI-WT-Flag) in COS-7 cells, which have no detectable sGC. PDI-Flag immunoprecipitation (Flag-IP, Fig 3) indicated that both α and β, separately or as a heterodimer were pulled down by PDI, yet the signal was stronger with α subunit alone compared to sGC β alone or α+β, as measured by densitometry analysis (1.08 ± 0.13 vs. 0.74 ± 0.02, 0.76 ± 0.06). This result suggests that PDI interaction with sGC does not require the heterodimer conformation and that the α subunit more readily interacts with PDI in an exogenous expression system. Interestingly, the decreased pull-down by PDI of α when co-expressed with β (compared with α subunit expressed alone) while PDI pull-down of β is similar when expressed alone or with α, suggests that there is a competition between PDI binding to sGC subunits and to each other.


Mapping Soluble Guanylyl Cyclase and Protein Disulfide Isomerase Regions of Interaction.

Heckler EJ, Kholodovych V, Jain M, Liu T, Li H, Beuve A - PLoS ONE (2015)

Flag immunoprecipitation of α and/or β sGC by Flag-tagged PDI-WT.A. Representative Western blots of α, β and Flag-PDI expressed in COS-7 cells (input right panel) and precipitated by PDI-Flag (bound, right panel). COS-7 cells were transiently transfected with Flag-tagged PDI-WT and infected with sGC α and/or β WT adenoviruses 24 hours post-transfection. Immunoprecipitation of lysates was performed using anti-Flag affinity resin as described in Material and Methods. Blots were probed with anti-α and β (top panels) and PDI (bottom panels). B. Densitometry analysis of the blots. Band analysis of bound vs. input with ImageJ of three separate experiments; sGC bound over input: α (1.08 ± 0.13), β (0.74 ± 0.02), and αβ (0.76 ± 0.06).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4664405&req=5

pone.0143523.g003: Flag immunoprecipitation of α and/or β sGC by Flag-tagged PDI-WT.A. Representative Western blots of α, β and Flag-PDI expressed in COS-7 cells (input right panel) and precipitated by PDI-Flag (bound, right panel). COS-7 cells were transiently transfected with Flag-tagged PDI-WT and infected with sGC α and/or β WT adenoviruses 24 hours post-transfection. Immunoprecipitation of lysates was performed using anti-Flag affinity resin as described in Material and Methods. Blots were probed with anti-α and β (top panels) and PDI (bottom panels). B. Densitometry analysis of the blots. Band analysis of bound vs. input with ImageJ of three separate experiments; sGC bound over input: α (1.08 ± 0.13), β (0.74 ± 0.02), and αβ (0.76 ± 0.06).
Mentions: The MassMatrix and cross-linking mass spectrometry results showed that the catalytic domains of both subunits could interact with PDI. To determine whether PDI might have a stronger affinity for a particular sGC subunit, i.e. α vs. β, and whether the heterodimer is required for interaction, WT sGC α and β, separately or together were co-expressed with Flag-tagged WT PDI (PDI-WT-Flag) in COS-7 cells, which have no detectable sGC. PDI-Flag immunoprecipitation (Flag-IP, Fig 3) indicated that both α and β, separately or as a heterodimer were pulled down by PDI, yet the signal was stronger with α subunit alone compared to sGC β alone or α+β, as measured by densitometry analysis (1.08 ± 0.13 vs. 0.74 ± 0.02, 0.76 ± 0.06). This result suggests that PDI interaction with sGC does not require the heterodimer conformation and that the α subunit more readily interacts with PDI in an exogenous expression system. Interestingly, the decreased pull-down by PDI of α when co-expressed with β (compared with α subunit expressed alone) while PDI pull-down of β is similar when expressed alone or with α, suggests that there is a competition between PDI binding to sGC subunits and to each other.

Bottom Line: Together with Flag-immunoprecipitation using sGC domain deletions, wild-type (WT) and mutated PDI, regions of sGC involved in this interaction were identified.Our results indicate that PDI interacts preferentially with the catalytic domain of sGC, thus providing a mechanism for PDI inhibition of sGC.A model in which PDI interacts with either the α or the β catalytic domain is proposed.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Physiology and Neuroscience, New Jersey Medical School, Rutgers University, Newark, NJ, United States of America.

ABSTRACT
Soluble guanylyl cyclase (sGC) is a heterodimeric nitric oxide (NO) receptor that produces cyclic GMP. This signaling mechanism is a key component in the cardiovascular system. NO binds to heme in the β subunit and stimulates the catalytic conversion of GTP to cGMP several hundred fold. Several endogenous factors have been identified that modulate sGC function in vitro and in vivo. In previous work, we determined that protein disulfide isomerase (PDI) interacts with sGC in a redox-dependent manner in vitro and that PDI inhibited NO-stimulated activity in cells. To our knowledge, this was the first report of a physical interaction between sGC and a thiol-redox protein. To characterize this interaction between sGC and PDI, we first identified peptide linkages between sGC and PDI, using a lysine cross-linking reagent and recently developed mass spectrometry analysis. Together with Flag-immunoprecipitation using sGC domain deletions, wild-type (WT) and mutated PDI, regions of sGC involved in this interaction were identified. The observed data were further explored with computational modeling to gain insight into the interaction mechanism between sGC and oxidized PDI. Our results indicate that PDI interacts preferentially with the catalytic domain of sGC, thus providing a mechanism for PDI inhibition of sGC. A model in which PDI interacts with either the α or the β catalytic domain is proposed.

Show MeSH
Related in: MedlinePlus