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      Mechanistic Insights on GAPDH, Hsp90, and NO-Assisted Heme delivery to sGC in a Defined Reconstitution System

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      cGMP: Generators, Effectors and Therapeutic Implications Conference
      cGMP: Generators, Effectors and Therapeutic Implications
      June 28-30, 2024
      nitric oxide, heme, soluble guanylyl cyclase, cGMP, heterodimer, hsp90
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            Abstract

            To function in cGMP generation the sGCb subunit must incorporate heme and form a heterodimer with the partner a-subunit. Cellular studies have shown that GAPDH associates with and supplies heme to the apo-sGCb subunit, which is in complex with the cell chaperone Hsp90. The Hsp90 then drives heme insertion into the apo-sGCb subunit in an ATP-driven process, which in turn enables sGCb to interact with the a-subunit and form a functional sGC heterodimer. NO at physiologic levels enhances cellular heme delivery into apo-sGCb and the consequent formation of the heterodimer, by a process that also depends on GAPDH and Hsp90.To gain mechanistic understanding, we developed an in vitro reconstitution system that utilizes purified proteins and allows us to follow in real time heme transfer from a pre-formed GAPDH-heme complex into apo-GCb by monitoring fluorescence changes in their respective FlAsH tags. Results from the defined system show that the heme transfer mechanism greatly depends on direct protein-protein interactions of apo-sGCb with GAPDH & with Hsp90 and requires ATP hydrolysis by Hsp90. We also found that added NO drives a faster and greater heme transfer into the apo-sGCb subunit, and its ability to do so depends on NO binding to the heme in the GAPDH. Our findings clarify the processes by which sGC matures inside cells and provide a platform for molecular-level investigations.

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            Author and article information

            Conference
            cGMP: Generators, Effectors and Therapeutic Implications Conference
            29 April 2024
            Affiliations
            [1 ] Dept. Inflammation & Immunity, Lerner Research Institute, Cleveland Clinic, Cleveland Ohio USA ( https://ror.org/03xjacd83)
            Author notes
            Author information
            https://orcid.org/0000-0002-0960-6848
            Article
            10.14293/CGMP.24000001.v1
            9bb1da6c-bf1c-40c8-8c7f-f27b52b6cdd3

            This work has been published open access under Creative Commons Attribution License CC BY 4.0 , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Conditions, terms of use and publishing policy can be found at www.scienceopen.com .

            cGMP: Generators, Effectors and Therapeutic Implications
            11
            Lübeck
            June 28-30, 2024
            History
            : 29 April 2024
            Categories

            The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.
            Chemistry
            nitric oxide,heme,soluble guanylyl cyclase,cGMP,heterodimer,hsp90

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