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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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Related in: MedlinePlus

α sGC domains immunoprecipitation by Flag-tagged PDI-WT and Flag-PDI Ser-x-x-Ser2.A. Representative Western blots of lysates (input) and immunoprecipitates (bound) using PDI-Flag and PDI-Flag mutant. COS-7 cells were transiently co-transfected with Flag-tagged PDI-WT or PDI Ser-x-x-Ser2 (PDI SxxS2) and sGC α domain constructs: αWT: full-length, αΔN: N-terminal truncation, αΔC: C-terminal truncation, α-CD: catalytic domain. Immunoprecipitation of lysates was performed using anti-Flag affinity resin as described in Material and Methods. Blots were probed with anti-sGC α Cayman and anti-sGC α Sigma. Bottom panel showing α constructs with domain boundaries labeled. B. Densitometry analysis of bound vs. input with ImageJ of three to five separate experiments. Black bars: PDI WT; gray bars: PDI SxxS2. Statistical analysis using Student's t-Test: α with PDI WT and α with PDI SxxS2, p = 0.01*; α with PDI WT and αΔC with PDI WT, p = 0.02*; and α-CatDom with PDI WT and α-CatDom with PDI SxxS2 = 0.06§.
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pone.0143523.g004: α sGC domains immunoprecipitation by Flag-tagged PDI-WT and Flag-PDI Ser-x-x-Ser2.A. Representative Western blots of lysates (input) and immunoprecipitates (bound) using PDI-Flag and PDI-Flag mutant. COS-7 cells were transiently co-transfected with Flag-tagged PDI-WT or PDI Ser-x-x-Ser2 (PDI SxxS2) and sGC α domain constructs: αWT: full-length, αΔN: N-terminal truncation, αΔC: C-terminal truncation, α-CD: catalytic domain. Immunoprecipitation of lysates was performed using anti-Flag affinity resin as described in Material and Methods. Blots were probed with anti-sGC α Cayman and anti-sGC α Sigma. Bottom panel showing α constructs with domain boundaries labeled. B. Densitometry analysis of bound vs. input with ImageJ of three to five separate experiments. Black bars: PDI WT; gray bars: PDI SxxS2. Statistical analysis using Student's t-Test: α with PDI WT and α with PDI SxxS2, p = 0.01*; α with PDI WT and αΔC with PDI WT, p = 0.02*; and α-CatDom with PDI WT and α-CatDom with PDI SxxS2 = 0.06§.

Mentions: To determine which domains of the sGC α subunit are involved in interaction with PDI, we made truncated constructs in pCMV5 expression plasmids as follows: the N-terminal deletion (αΔN), C-terminal deletion (αΔC), and catalytic domain alone (αCatDom) [8–10]. These constructs were co-expressed with a Flag-tagged PDI-WT or with a Flag-tagged PDI mutated in the second active site Cys397-x-x-Cys400, which is responsible for mixed-disulfide exchange with sGC (PDI Flag-Ser-x-x-Ser2, both Cys mutated to Ser). The results are shown in Fig 4A. PDI-Flag IP of αΔN and αCatDom were not significantly changed compared to α WT. However, αΔC (no catalytic domain) associated with PDI significantly less than full-length α WT (0.71±0.10 vs. 1.01±0.06: n = 3–4 densitometry analysis, Fig 4B). An interaction of each α domain construct, including α WT, with mutated PDI-Ser-x-x-Ser2 was impaired (Fig 4A). Yet, only the catalytic domain of α (αCatDom, 460-690aa) and α WT were significantly affected in their interaction with PDI-Ser-x-x-Ser2 compared to PDI WT (Fig 4B). The results confirmed that association between sGC and PDI involves PDI second active site interaction with the α catalytic domain (αCatDom, αCD).


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)

α sGC domains immunoprecipitation by Flag-tagged PDI-WT and Flag-PDI Ser-x-x-Ser2.A. Representative Western blots of lysates (input) and immunoprecipitates (bound) using PDI-Flag and PDI-Flag mutant. COS-7 cells were transiently co-transfected with Flag-tagged PDI-WT or PDI Ser-x-x-Ser2 (PDI SxxS2) and sGC α domain constructs: αWT: full-length, αΔN: N-terminal truncation, αΔC: C-terminal truncation, α-CD: catalytic domain. Immunoprecipitation of lysates was performed using anti-Flag affinity resin as described in Material and Methods. Blots were probed with anti-sGC α Cayman and anti-sGC α Sigma. Bottom panel showing α constructs with domain boundaries labeled. B. Densitometry analysis of bound vs. input with ImageJ of three to five separate experiments. Black bars: PDI WT; gray bars: PDI SxxS2. Statistical analysis using Student's t-Test: α with PDI WT and α with PDI SxxS2, p = 0.01*; α with PDI WT and αΔC with PDI WT, p = 0.02*; and α-CatDom with PDI WT and α-CatDom with PDI SxxS2 = 0.06§.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4664405&req=5

pone.0143523.g004: α sGC domains immunoprecipitation by Flag-tagged PDI-WT and Flag-PDI Ser-x-x-Ser2.A. Representative Western blots of lysates (input) and immunoprecipitates (bound) using PDI-Flag and PDI-Flag mutant. COS-7 cells were transiently co-transfected with Flag-tagged PDI-WT or PDI Ser-x-x-Ser2 (PDI SxxS2) and sGC α domain constructs: αWT: full-length, αΔN: N-terminal truncation, αΔC: C-terminal truncation, α-CD: catalytic domain. Immunoprecipitation of lysates was performed using anti-Flag affinity resin as described in Material and Methods. Blots were probed with anti-sGC α Cayman and anti-sGC α Sigma. Bottom panel showing α constructs with domain boundaries labeled. B. Densitometry analysis of bound vs. input with ImageJ of three to five separate experiments. Black bars: PDI WT; gray bars: PDI SxxS2. Statistical analysis using Student's t-Test: α with PDI WT and α with PDI SxxS2, p = 0.01*; α with PDI WT and αΔC with PDI WT, p = 0.02*; and α-CatDom with PDI WT and α-CatDom with PDI SxxS2 = 0.06§.
Mentions: To determine which domains of the sGC α subunit are involved in interaction with PDI, we made truncated constructs in pCMV5 expression plasmids as follows: the N-terminal deletion (αΔN), C-terminal deletion (αΔC), and catalytic domain alone (αCatDom) [8–10]. These constructs were co-expressed with a Flag-tagged PDI-WT or with a Flag-tagged PDI mutated in the second active site Cys397-x-x-Cys400, which is responsible for mixed-disulfide exchange with sGC (PDI Flag-Ser-x-x-Ser2, both Cys mutated to Ser). The results are shown in Fig 4A. PDI-Flag IP of αΔN and αCatDom were not significantly changed compared to α WT. However, αΔC (no catalytic domain) associated with PDI significantly less than full-length α WT (0.71±0.10 vs. 1.01±0.06: n = 3–4 densitometry analysis, Fig 4B). An interaction of each α domain construct, including α WT, with mutated PDI-Ser-x-x-Ser2 was impaired (Fig 4A). Yet, only the catalytic domain of α (αCatDom, 460-690aa) and α WT were significantly affected in their interaction with PDI-Ser-x-x-Ser2 compared to PDI WT (Fig 4B). The results confirmed that association between sGC and PDI involves PDI second active site interaction with the α catalytic domain (αCatDom, αCD).

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