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Inhibition of FicD-mediated AMPylation and deAMPylation by isoprenoid diphosphates.

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Title: Inhibition of FicD-mediated AMPylation and deAMPylation by isoprenoid diphosphates.
Authors: Blevins, Aubrie M.1,2, Peng, Wei1,3, Kinch, Lisa N.1,3, Monshad, Zihan4, Paredes, Andrea G.4, Volz, Christina4, Rutter, Jared4,5, Casey, Amanda K.1, Hicks, Kevin G.6, Orth, Kim1,2,3 kim.orth@utsouthwestern.edu
Source: Proceedings of the National Academy of Sciences of the United States of America. 3/10/2026, Vol. 123 Issue 10, p1-10. 31p.
Subjects: Pyrophosphates, Unfolded protein response, Endoplasmic reticulum stress, Genetic disorders, Adenylation (Biochemistry), Molecular chaperones
Abstract: FicD regulates Unfolded Protein Response (UPR) through reversible AMPylation and deAMPylation of BiP, an HSP70 chaperone and master regulator of the UPR. FicD activity is regulated by endoplasmic reticulum-stress, catalyzing BiP AMPylation under low stress conditions to hold inactive chaperone in reserve. In stressed cells, FicD deAMPylates BiP, acutely increasing its active pool to assist in protein folding. Variants in UPR machinery, including those in the FicD gene, are linked to hereditary diseases. Despite the known role of FicD in UPR, in-vivo regulation of its activity remains elusive, and identifying metabolites that alter FicD activity could prove useful pharmaceutically. We applied an unbiased high-throughput screening platform, known as Mass spectrometry Integrated with equilibrium Dialysis for the discovery of Allostery Systematically (MIDAS), to identify small molecule metabolites that might regulate FicD activity. MIDAS revealed interactions between FicD and two mevalonate pathway intermediates: geranyl-pyrophosphate and farnesyl-pyrophosphate. Biochemical characterization indicates that both potently inhibit FicD-mediated AMPylation and deAMPylation. The crystal structure of FicD bound to farnesyl-pyrophosphate demonstrates a competitive inhibition mechanism, with the pyrophosphate adopting the alpha and beta phosphate positions of adenosine triphosphate (ATP) and the hydrocarbon chain filling the nucleoside pocket. FicD variants previously appeared as biochemically indistinguishable, yet lead to different human pathologies. We demonstrate farnesyl-pyrophosphate inhibits FicDR374H and FicDR374C variants implicated in causing hereditary spastic paraplegia, but not the FicDR371S variant associated with neonatal diabetes. This study furthers our understanding of FicD inhibitors and distinguishes disease causing variants, providing insight into pharmacological targeting of UPR activity. [ABSTRACT FROM AUTHOR]
Copyright of Proceedings of the National Academy of Sciences of the United States of America is the property of National Academy of Sciences and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)
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  Data: Inhibition of FicD-mediated AMPylation and deAMPylation by isoprenoid diphosphates.
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– Name: Abstract
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  Data: FicD regulates Unfolded Protein Response (UPR) through reversible AMPylation and deAMPylation of BiP, an HSP70 chaperone and master regulator of the UPR. FicD activity is regulated by endoplasmic reticulum-stress, catalyzing BiP AMPylation under low stress conditions to hold inactive chaperone in reserve. In stressed cells, FicD deAMPylates BiP, acutely increasing its active pool to assist in protein folding. Variants in UPR machinery, including those in the FicD gene, are linked to hereditary diseases. Despite the known role of FicD in UPR, in-vivo regulation of its activity remains elusive, and identifying metabolites that alter FicD activity could prove useful pharmaceutically. We applied an unbiased high-throughput screening platform, known as Mass spectrometry Integrated with equilibrium Dialysis for the discovery of Allostery Systematically (MIDAS), to identify small molecule metabolites that might regulate FicD activity. MIDAS revealed interactions between FicD and two mevalonate pathway intermediates: geranyl-pyrophosphate and farnesyl-pyrophosphate. Biochemical characterization indicates that both potently inhibit FicD-mediated AMPylation and deAMPylation. The crystal structure of FicD bound to farnesyl-pyrophosphate demonstrates a competitive inhibition mechanism, with the pyrophosphate adopting the alpha and beta phosphate positions of adenosine triphosphate (ATP) and the hydrocarbon chain filling the nucleoside pocket. FicD variants previously appeared as biochemically indistinguishable, yet lead to different human pathologies. We demonstrate farnesyl-pyrophosphate inhibits FicDR374H and FicDR374C variants implicated in causing hereditary spastic paraplegia, but not the FicDR371S variant associated with neonatal diabetes. This study furthers our understanding of FicD inhibitors and distinguishes disease causing variants, providing insight into pharmacological targeting of UPR activity. [ABSTRACT FROM AUTHOR]
– Name: AbstractSuppliedCopyright
  Label:
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  Data: <i>Copyright of Proceedings of the National Academy of Sciences of the United States of America is the property of National Academy of Sciences and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract.</i> (Copyright applies to all Abstracts.)
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        Value: 10.1073/pnas.2533457123
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      – Code: eng
        Text: English
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        PageCount: 31
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    Subjects:
      – SubjectFull: Pyrophosphates
        Type: general
      – SubjectFull: Unfolded protein response
        Type: general
      – SubjectFull: Endoplasmic reticulum stress
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      – SubjectFull: Genetic disorders
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      – SubjectFull: Adenylation (Biochemistry)
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      – SubjectFull: Molecular chaperones
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    Titles:
      – TitleFull: Inhibition of FicD-mediated AMPylation and deAMPylation by isoprenoid diphosphates.
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              Text: 3/10/2026
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