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• W3194705295 abstract "Fic domain-containing AMP transferases (fic AMPylases) are conserved enzymes that catalyze the covalent transfer of AMP to proteins. This posttranslational modification regulates the function of several proteins, including the ER-resident chaperone Grp78/BiP. Here we introduce a mouse FICD (mFICD) AMPylase knockout mouse model to study fic AMPylase function in vertebrates. We find that mFICD deficiency is well tolerated in unstressed mice. We also show that mFICD-deficient mouse embryonic fibroblasts are depleted of AMPylated proteins. mFICD deletion alters protein synthesis and secretion in splenocytes, including that of IgM, an antibody secreted early during infections, and the proinflammatory cytokine IL-1β, without affecting the unfolded protein response. Finally, we demonstrate that visual nonspatial short-term learning is stronger in old mFICD−/− mice than in wild-type controls while other measures of cognition, memory, and learning are unaffected. Together, our results suggest a role for mFICD in adaptive immunity and neuronal plasticity in vivo. Fic domain-containing AMP transferases (fic AMPylases) are conserved enzymes that catalyze the covalent transfer of AMP to proteins. This posttranslational modification regulates the function of several proteins, including the ER-resident chaperone Grp78/BiP. Here we introduce a mouse FICD (mFICD) AMPylase knockout mouse model to study fic AMPylase function in vertebrates. We find that mFICD deficiency is well tolerated in unstressed mice. We also show that mFICD-deficient mouse embryonic fibroblasts are depleted of AMPylated proteins. mFICD deletion alters protein synthesis and secretion in splenocytes, including that of IgM, an antibody secreted early during infections, and the proinflammatory cytokine IL-1β, without affecting the unfolded protein response. Finally, we demonstrate that visual nonspatial short-term learning is stronger in old mFICD−/− mice than in wild-type controls while other measures of cognition, memory, and learning are unaffected. Together, our results suggest a role for mFICD in adaptive immunity and neuronal plasticity in vivo. The posttranslational regulation of protein function is a fundamental concept in biology. To manage protein activity, dedicated enzymes attach specific chemical modifications to individual proteins, the presence of which affects the behavior and activity of the modified proteins. These modifications, called posttranslational modifications (PTMs), govern essential biological processes. They are implicated in cancer, neurodegeneration, and cardiovascular diseases, among others. The covalent addition of an AMP moiety to the side chain of exposed threonine and serine residues has emerged as a new paradigm to control the activity of the essential ER-resident chaperone BiP. This process, AMPylation, is catalyzed by metazoan AMP transferases (AMPylases) that contain a filamentation induced by c-AMP (fic) domain. Fic domain-containing AMPylases (fic AMPylases) are highly conserved and are present in a single copy in most metazoans, including Caenorhabditis elegans (FIC-1), Drosophila melanogaster (dfic), Mus musculus (mFICD), and Homo sapiens (FICD) (1Woolery A.R. Luong P. Broberg C.A. Orth K. AMPylation: Something old is new again.Front. Microbiol. 2010; 1: 113Crossref PubMed Scopus (51) Google Scholar, 2Itzen A. Blankenfeldt W. Goody R.S. Adenylylation: Renaissance of a forgotten post-translational modification.Trends Biochem. Sci. 2011; 36: 221-228Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 3Truttmann M.C.P.H.L. rAMPing up stress signaling: Protein AMPylation in metazoans.Trends Cell Biol. 2017; 27: 608-620Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Metazoan fic AMPylases are bifunctional: using a single active site, these enzymes catalyze both the transfer of AMP to surface-exposed threonine and serine hydroxyl groups and the removal of AMP groups from modified residues (deAMPylation) (4Casey A.K. Moehlman A.T. Zhang J. Servage K.A. Krämer H. Orth K. Fic-mediated deAMPylation is not dependent on homodimerization and rescues toxic AMPylation in flies.J. Biol. Chem. 2018; 293: 1550Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar, 5Moehlman A.T. Casey A.K. Servage K. Orth K. Krämer H. Adaptation to constant light requires Fic-mediated AMPylation of BiP to protect against reversible photoreceptor degeneration.Elife. 2018; 7e38752Crossref PubMed Scopus (12) Google Scholar, 6Preissler S. Rato C. Perera L.A. Saudek V. Ron D. FICD acts bifunctionally to AMPylate and de-AMPylate the endoplasmic reticulum chaperone BiP.Nat. Struct. Mol. Biol. 2017; 24: 23-29Crossref PubMed Scopus (45) Google Scholar, 7Preissler S. Rohland L. Yan Y. Chen R. Read R.J. Ron D. AMPylation targets the rate-limiting step of BiP's ATPase cycle for its functional inactivation.Elife. 2017; 6e29428Crossref PubMed Scopus (41) Google Scholar, 8Veyron S. Oliva G. Rolando M. Buchrieser C. Peyroche G. Cherfils J. A Ca2+-regulated deAMPylation switch in human and bacterial FIC proteins.Nat. Commun. 2019; 10: 1142Crossref PubMed Scopus (14) Google Scholar). The switch between AMPylation and deAMPylation is proposed to involve enzyme dimerization, the exchange of Mg2+ with Ca2+ ions in the active site, and the subsequent switch from an open to a closed conformation (4Casey A.K. Moehlman A.T. Zhang J. Servage K.A. Krämer H. Orth K. Fic-mediated deAMPylation is not dependent on homodimerization and rescues toxic AMPylation in flies.J. Biol. Chem. 2018; 293: 1550Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar, 5Moehlman A.T. Casey A.K. Servage K. Orth K. Krämer H. Adaptation to constant light requires Fic-mediated AMPylation of BiP to protect against reversible photoreceptor degeneration.Elife. 2018; 7e38752Crossref PubMed Scopus (12) Google Scholar, 6Preissler S. Rato C. Perera L.A. Saudek V. Ron D. FICD acts bifunctionally to AMPylate and de-AMPylate the endoplasmic reticulum chaperone BiP.Nat. Struct. Mol. Biol. 2017; 24: 23-29Crossref PubMed Scopus (45) Google Scholar, 7Preissler S. Rohland L. Yan Y. Chen R. Read R.J. Ron D. AMPylation targets the rate-limiting step of BiP's ATPase cycle for its functional inactivation.Elife. 2017; 6e29428Crossref PubMed Scopus (41) Google Scholar, 8Veyron S. Oliva G. Rolando M. Buchrieser C. Peyroche G. Cherfils J. A Ca2+-regulated deAMPylation switch in human and bacterial FIC proteins.Nat. Commun. 2019; 10: 1142Crossref PubMed Scopus (14) Google Scholar, 9Perera L.A. Rato C. Yan Y. Neidhardt L. McLaughlin S.H. Read R.J. Preissler S. Ron D. An oligomeric state-dependent switch in the ER enzyme FICD regulates AMPylation and deAMPylation of BiP.EMBO J. 2019; 38e102177Crossref PubMed Scopus (17) Google Scholar). The latter is stabilized by interactions between an inhibitory glutamate and a nearby arginine residue, which aligns an inhibitory α-helix such that the catalytic core preferentially binds AMP over ATP, catalyzing deAMPylation (6Preissler S. Rato C. Perera L.A. Saudek V. Ron D. FICD acts bifunctionally to AMPylate and de-AMPylate the endoplasmic reticulum chaperone BiP.Nat. Struct. Mol. Biol. 2017; 24: 23-29Crossref PubMed Scopus (45) Google Scholar). If the interactions between these residues are prevented or resolved, fic AMPylases adopt an open conformation that favors Mg2+ and ATP recruitment to the active site, enabling AMPylation of target proteins (10Engel P. Goepfert A. Stanger F.V. Harms A. Schmidt A. Schirmer T. Dehio C. Adenylylation control by intra- or intermolecular active-site obstruction in Fic proteins.Nature. 2012; 482: 107-110Crossref PubMed Scopus (108) Google Scholar). Thus, replacing the critical inhibitory glutamate residue with a glycine (FICD(E234G)) converts the enzyme to a constitutively active AMPylase (10Engel P. Goepfert A. Stanger F.V. Harms A. Schmidt A. Schirmer T. Dehio C. Adenylylation control by intra- or intermolecular active-site obstruction in Fic proteins.Nature. 2012; 482: 107-110Crossref PubMed Scopus (108) Google Scholar, 11Truttmann M.C. Cruz V.E. Guo X. Engert C. Schwartz T.U. Ploegh H.L. The Caenorhabditis elegans protein FIC-1 is an AMPylase that covalently modifies heat-shock 70 family proteins, translation elongation factors and histones.PLoS Genet. 2016; 12e1006023Crossref PubMed Scopus (21) Google Scholar, 12Ham H. Woolery A.R. Tracy C. Stenesen D. Krämer H. Orth K. Unfolded protein response-regulated dFic reversibly AMPylates BiP during endoplasmic reticulum homeostasis.J. Biol. Chem. 2014; 289: 36059-36069Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). AMPylation of the ER-resident HSP70 protein, BiP, on T518 locks this chaperone in an ATP- and HSP40-bound “primed” conformation, rendering it unable to support the (re)folding of client proteins (7Preissler S. Rohland L. Yan Y. Chen R. Read R.J. Ron D. AMPylation targets the rate-limiting step of BiP's ATPase cycle for its functional inactivation.Elife. 2017; 6e29428Crossref PubMed Scopus (41) Google Scholar, 13Preissler S. Rato C. Chen R. Antrobus R. Ding S. Fearnley I.M. Ron D. AMPylation matches BiP activity to client protein load in the endoplasmic reticulum.Elife. 2015; 4e12621Crossref PubMed Scopus (64) Google Scholar). Upon BiP deAMPylation, ATP is hydrolyzed and the ADP-bound form of BiP is again able to engage with client proteins (6Preissler S. Rato C. Perera L.A. Saudek V. Ron D. FICD acts bifunctionally to AMPylate and de-AMPylate the endoplasmic reticulum chaperone BiP.Nat. Struct. Mol. Biol. 2017; 24: 23-29Crossref PubMed Scopus (45) Google Scholar, 14Perera L.A. Rato C. Yan Y. Neidhardt L. McLaughlin S.H. Read R.J. Preissler S. Ron D. An oligomeric state-dependent switch in FICD regulates AMPylation and deAMPylation of the chaperone BiP.EMBO J. 2019; 38: e102177Crossref PubMed Scopus (0) Google Scholar). The consequences of BiP S365/T366 AMPylation remain controversial and may either inhibit (4Casey A.K. Moehlman A.T. Zhang J. Servage K.A. Krämer H. Orth K. Fic-mediated deAMPylation is not dependent on homodimerization and rescues toxic AMPylation in flies.J. Biol. Chem. 2018; 293: 1550Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar, 12Ham H. Woolery A.R. Tracy C. Stenesen D. Krämer H. Orth K. Unfolded protein response-regulated dFic reversibly AMPylates BiP during endoplasmic reticulum homeostasis.J. Biol. Chem. 2014; 289: 36059-36069Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 15Casey A.K. Orth K. Enzymes involved in AMPylation and deAMPylation.Chem. Rev. 2018; 118: 1199-1215Crossref PubMed Scopus (35) Google Scholar) or enhance (16Sanyal A Zbornik E.A Watson B.G. Christoffer C. Ma J. Kihara D. Mattoo S. Kinetic and structural parameters governing fic-mediated adenylylation/AMPylation of the Hsp70 chaperone, BiP/GRP78.Cell Stress Chaperones. 2021; 26: 639-656Crossref PubMed Scopus (5) Google Scholar, 17Sanyal A. Chen A.J. Nakayasu E.S. Lazar C.S. Zbornik E.A. Worby C.A. Koller A. Mattoo S. A novel link between fic (filamentation induced by cAMP)-mediated adenylylation/AMPylation and the unfolded protein response.J. Biol. Chem. 2015; 290: 8482-8499Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) BiP activity. In addition to BiP, fic AMPylases also modify a wide range of non-ER proteins (18Nitika Porter C.M. Truman A.W. Truttmann M.C. Post-translational modifications of Hsp70 family proteins: Expanding the chaperone code.J. Biol. Chem. 2020; 295: 10689-10708Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 19Truttmann M.C. Pincus D. Ploegh H.L. Chaperone AMPylation modulates aggregation and toxicity of neurodegenerative disease-associated polypeptides.Proc. Natl. Acad. Sci. U S A. 2018; 115: E5008-E5017Crossref PubMed Scopus (16) Google Scholar, 20Truttmann M.C. Zheng X. Hanke L. Damon J.R. Grootveld M. Krakowiak J. Pincus D. Ploegh H.L. Unrestrained AMPylation targets cytosolic chaperones and activates the heat shock response.Proc. Natl. Acad. Sci. U S A. 2017; 114: E152-E160Crossref PubMed Scopus (19) Google Scholar, 21Truttmann M.C. Ploegh H.L. rAMPing up stress signaling: Protein AMPylation in metazoans.Trends Cell Biol. 2017; 27: 608-620Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 22Truttmann M.C. Cruz V.E. Guo X. Engert C. Schwartz T.U. Ploegh H.L. The Caenorhabditis elegans protein FIC-1 is an AMPylase that affects susceptibility to Pseudomonas aeruginosa infections.PLoS Genet. 2016; 12e1006023Crossref PubMed Scopus (21) Google Scholar, 23Lewallen D.M. Sreelatha A. Dharmarajan V. Madoux F. Chase P. Griffin P.R. Orth K. Hodder P. Thompson P.R. Inhibiting AMPylation: A novel screen to identify the first small molecule inhibitors of protein AMPylation.ACS Chem. Biol. 2014; 9: 433-442Crossref PubMed Scopus (17) Google Scholar, 24Kielkowski P. Buchsbaum I.Y. Becker T. Bach K. Cappello S. Sieber S.A. A pronucleotide probe for live-cell imaging of protein AMPylation.Chembiochem. 2020; 21: 1285-1287Crossref PubMed Scopus (7) Google Scholar, 25Kielkowski P. Buchsbaum I.Y. Kirsch V.C. Bach N.C. Drukker M. Cappello S. Sieber S.A. FICD activity and AMPylation remodelling modulate human neurogenesis.Nat. Commun. 2020; 11: 517Crossref PubMed Scopus (24) Google Scholar, 26Broncel M. Serwa R.A. Bunney T.D. Katan M. Tate E.W. Global profiling of HYPE mediated AMPylation through a chemical proteomic approach.Mol. Cell Proteomics. 2016; 15: 715-725Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 27Gulen B. Rosselin M. Fauser J. Albers M.F. Pett C. Krisp C. Pogenberg V. Schluter H. Hedberg C. Itzen A. Identification of targets of AMPylating Fic enzymes by co-substrate-mediated covalent capture.Nat. Chem. 2020; 12: 732-739Crossref PubMed Scopus (11) Google Scholar). Indeed, fic AMPylases are also present in the nuclear envelope and the cytoplasm (11Truttmann M.C. Cruz V.E. Guo X. Engert C. Schwartz T.U. Ploegh H.L. The Caenorhabditis elegans protein FIC-1 is an AMPylase that covalently modifies heat-shock 70 family proteins, translation elongation factors and histones.PLoS Genet. 2016; 12e1006023Crossref PubMed Scopus (21) Google Scholar, 20Truttmann M.C. Zheng X. Hanke L. Damon J.R. Grootveld M. Krakowiak J. Pincus D. Ploegh H.L. Unrestrained AMPylation targets cytosolic chaperones and activates the heat shock response.Proc. Natl. Acad. Sci. U S A. 2017; 114: E152-E160Crossref PubMed Scopus (19) Google Scholar, 28Sengupta R. Poderycki M.J. Mattoo S. CryoAPEX - an electron tomography tool for subcellular localization of membrane proteins.J. Cell Sci. 2019; 132jcs222315Crossref PubMed Scopus (19) Google Scholar). Changes in cellular AMPylation levels affect cellular fitness and organismal survival: Overexpression of constitutively active fic AMPylases is toxic and kills human (17Sanyal A. Chen A.J. Nakayasu E.S. Lazar C.S. Zbornik E.A. Worby C.A. Koller A. Mattoo S. A novel link between fic (filamentation induced by cAMP)-mediated adenylylation/AMPylation and the unfolded protein response.J. Biol. Chem. 2015; 290: 8482-8499Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 29Truttmann M.C. Wu Q. Stiegeler S. Duarte J.N. Ingram J. Ploegh H.L. HypE-specific nanobodies as tools to modulate HypE-mediated target AMPylation.J. Biol. Chem. 2015; 290: 9087-9100Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 30Bunney T.D. Cole A.R. Broncel M. Esposito D. Tate E.W. Katan M. Crystal structure of the human, FIC-domain containing protein HYPE and implications for its functions.Structure. 2014; 22: 1831-1843Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) and yeast cells (20Truttmann M.C. Zheng X. Hanke L. Damon J.R. Grootveld M. Krakowiak J. Pincus D. Ploegh H.L. Unrestrained AMPylation targets cytosolic chaperones and activates the heat shock response.Proc. Natl. Acad. Sci. U S A. 2017; 114: E152-E160Crossref PubMed Scopus (19) Google Scholar), as well as worm (C. elegans) embryos (19Truttmann M.C. Pincus D. Ploegh H.L. Chaperone AMPylation modulates aggregation and toxicity of neurodegenerative disease-associated polypeptides.Proc. Natl. Acad. Sci. U S A. 2018; 115: E5008-E5017Crossref PubMed Scopus (16) Google Scholar) and flies (D. melanogaster) (4Casey A.K. Moehlman A.T. Zhang J. Servage K.A. Krämer H. Orth K. Fic-mediated deAMPylation is not dependent on homodimerization and rescues toxic AMPylation in flies.J. Biol. Chem. 2018; 293: 1550Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar). In contrast, fic AMPylase deficiency is well tolerated in unstressed human cells but impairs the activation of the unfolded protein response (UPR) under stress (17Sanyal A. Chen A.J. Nakayasu E.S. Lazar C.S. Zbornik E.A. Worby C.A. Koller A. Mattoo S. A novel link between fic (filamentation induced by cAMP)-mediated adenylylation/AMPylation and the unfolded protein response.J. Biol. Chem. 2015; 290: 8482-8499Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) and reduces neuronal differentiation (25Kielkowski P. Buchsbaum I.Y. Kirsch V.C. Bach N.C. Drukker M. Cappello S. Sieber S.A. FICD activity and AMPylation remodelling modulate human neurogenesis.Nat. Commun. 2020; 11: 517Crossref PubMed Scopus (24) Google Scholar). Further, Fic-1 deficient worms show enhanced sensitivity to the presence of aggregation-prone poly-glutamine repeat proteins in neurons (19Truttmann M.C. Pincus D. Ploegh H.L. Chaperone AMPylation modulates aggregation and toxicity of neurodegenerative disease-associated polypeptides.Proc. Natl. Acad. Sci. U S A. 2018; 115: E5008-E5017Crossref PubMed Scopus (16) Google Scholar). Perhaps the most significant in vivo fic AMPylase knockout (KO) phenotype is found in dfic-deficient flies, which show significant defects in visual signaling and suffer from light-induced blindness caused by BiP deregulation (5Moehlman A.T. Casey A.K. Servage K. Orth K. Krämer H. Adaptation to constant light requires Fic-mediated AMPylation of BiP to protect against reversible photoreceptor degeneration.Elife. 2018; 7e38752Crossref PubMed Scopus (12) Google Scholar, 31Rahman M. Ham H. Liu X. Sugiura Y. Orth K. Krämer H. Visual neurotransmission in Drosophila requires expression of Fic in glial capitate projections.Nat. Neurosci. 2012; 15: 871-875Crossref PubMed Scopus (55) Google Scholar). Despite the emerging role of fic AMPylases in proteostasis, our understanding of how these enzymes affect mammalian physiology is lacking. Here we describe the generation and characterization of an mFICD-deficient mouse strain. mFICD−/− mice are viable and are not visually impaired. We further show that mFICD deletion alters IgM synthesis and perturbs IL-1β secretion. Finally, we provide evidence that mFICD is involved in regulating nonspatial short-term memory in vivo. Together, our results support a modulatory role for mFICD function in adaptive immunity and neuronal plasticity in vertebrates. To investigate the role of mFICD-mediated protein AMPylation in vivo, we attempted to generate both mFICD-deficient and constitutively active mFICD(E234G)-expressing transgenic mouse strains using CRISPR/Cas9 technology. We used an sgRNA that targets a site adjacent to the coding sequence of the regulatory motif (TVAIEG) (Fig. S1A) and a double-stranded repair template to introduce the E234G substitution. The injection of approximately 80 blastocysts resulted in more than 20 independent animals carrying insertions or deletions in the mFICD gene that often resulted in frame shifts. Notably, not a single animal carrying the constitutively active mFICD(E234G) mutation was recovered. Parallel injections using identical experimental conditions but targeting different genes efficiently produced transgenic knock-in strains (32Maruyama T. Dougan S.K. Truttmann M.C. Bilate A.M. Ingram J.R. Ploegh H.L. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining.Nat. Biotechnol. 2015; 33: 538-542Crossref PubMed Scopus (719) Google Scholar). These results suggest that embryonic expression of constitutively active mFICD(E234G), particularly in the absence of a wild-type mFICD copy, may be lethal. For this study, we backcrossed a mouse strain carrying a deletion in mFICD. This deletion introduced a frame shift resulting in a premature stop codon (Fig. S1, B and C). To characterize the mFICD−/− animals, we assessed 6-month-old female control and mFICD−/− animals using the SHIPRA method. SHIPRA is a rapid, comprehensive screening approach, which provides a qualitative behavioral and functional profile for each animal (33Rafael J.A. Nitta Y. Peters J. Davies K.E. Testing of SHIRPA, a mouse phenotypic assessment protocol, on Dmd(mdx) and Dmd(mdx3cv) dystrophin-deficient mice.Mamm. Genome. 2000; 11: 725-728Crossref PubMed Scopus (68) Google Scholar). We found that mFICD−/− mice performed similarly to control animals for all 14 assessed features (Fig. 1A; feature by feature results in Table S1). We found no significant differences in body weight (Fig. 1B), rotarod performance (Fig. 1C), and life span (Fig. S1D) between control and mFICD−/− animals. Together, these results establish that mFICD deficiency is well tolerated by mice under normal growth conditions. AMPylation in vertebrates is conferred by at least two evolutionarily unrelated enzymes: FICD and the mitochondrial pseudokinase SelO (34Sreelatha A. Yee S.S. Lopez V.A. Park B.C. Kinch L.N. Pilch S. Servage K.A. Zhang J. Jiou J. Karasiewicz-Urbańska M. Łobocka M. Grishin N.V. Orth K. Kucharczyk R. Pawłowski K. et al.Protein AMPylation by an evolutionarily conserved pseudokinase.Cell. 2018; 175: 809-821.e819Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). To define how mFICD deficiency alters the vertebrate AMPylome, we supplemented mFICD−/− and control mouse embryonic fibroblast (MEF) lysates with 8-azido-ATP. Following AMPylation, a click reaction was used to install a PEG-biotin handle on the modified proteins. We then recovered AMPylated proteins with streptavidin-coated beads and identified AMPylated proteins by mass spectrometry (Fig. 2A). Comparing results from treated MEFs, mFICD−/− KO MEFs and a cell-free control, we identified 108 proteins that were AMPylated only in wild-type MEFs (Table S2). Among these proteins were several known FICD targets, including BiP (HSPA5), HSC70 (HSPA8), and translation elongation factor EEF-1A (Fig. 2A and S2A). In contrast, only two proteins (transketolase (TKT); protein disulfide isomerase (P4HB)) were identified in both wild-type and mFICD−/− lysates. Gene ontology analysis showed that AMPylated proteins were significantly enriched in metabolic, protein (re-)folding, and stress response processes (Fig. S2B). Together, these results confirm that FICD is required for the AMPylation-mediated regulation of BiP, HSC70, EEF-1A, and other proteins. Having established that WT and mFICD−/− mice are phenotypically similar in overall physiological and morphological terms, and given mFICD's role in the UPR, an important pathway in B and T cell development (35Brunsing R. Omori S.A. Weber F. Bicknell A. Friend L. Rickert R. Niwa M. B- and T-cell development both involve activity of the unfolded protein response pathway.J. Biol. Chem. 2008; 283: 17954-17961Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), we asked whether their humoral immune system was affected by mFICD deficiency. Using flow cytometry, we examined the distribution of B and T cells in the spleen. We found no difference between WT and mFICD−/− mice in either the distribution of B and T cells (Fig. 3A and S3A) nor in any T cell (Fig. 3B and S3B) or B cell (Fig. 3C and S3C) subsets present in the spleen. B cell development in the bone marrow (Fig. 3D and S3D) and T cell development in the thymus (Fig. 3E and S3E) were normal in mFICD−/− mice. BiP, an essential molecular chaperone, discovered as an immunoglobulin-binding protein and regulated by FICD, is required for antibody assembly and maturation. We thus examined the impact of mFICD deficiency on protein secretion. Splenocytes were isolated from WT and mFICD−/− mice and stimulated with lipopolysaccharide (LPS), heparan sulfate (HS), or thapsigargin to induce cytokine secretion, which was then assayed by ELISA (Fig. 4, A–C). While mFICD deletion significantly reduced LPS-induced IL-6 secretion (Fig. 4A), it had no effect on secretion of TNFα (Fig. 4B). We also observed a significant difference in IL-1β production between WT and mFICD−/− mice (Fig. 4C). This is striking because unlike IL-6 and TNFα, which traffic through the ER, IL-1β folds in the cytoplasm, and is secreted by a nonclassical pathway. Immunoblots showed that intracellular levels of the IgM heavy chain (μ) were elevated in mFICD−/− B cells, while the levels of other proteins that fold and traffic through the ER were unchanged (Fig. S4A). To further explore the consequences of mFICD deletion on protein folding and secretion, we focused on immunoglobulins because of their requirement for BiP activity in the course of folding and assembly (36Hendershot L. Bole D. Kearney J.F. The role of immunoglobulin heavy chain binding protein in immunoglobulin transport.Immunol. Today. 1987; 8: 111-114Abstract Full Text PDF PubMed Scopus (30) Google Scholar). Naïve B cells were purified from the spleens, activated using LPS, and cultured for 3 days to allow differentiation into IgM-secreting plasmablasts. We then followed IgM folding by pulse-chase analysis. Briefly, plasmablasts were labeled with 35S-methionine/cysteine for 15 min and chased in the absence of radioactive label for various times to follow protein maturation. IgM was immunoprecipitated from detergent lysates and media samples and analyzed by SDS-PAGE followed by autoradiography (Fig. 3D). mFICD−/− mice showed increased levels of both the soluble μ heavy chain (μs) and the membrane-bound B cell receptor (μm) in detergent lysates. This was accompanied by increased levels of both μs and μm mRNA (Fig. 4, E and F). Similar amounts of IgM were recovered from the media for both WT and mFICD−/− samples, suggesting that the increased levels of μ in the mFICD−/− cells did not pass ER quality control for secretion. These observations were consistent across the five pulse-chase experiments performed. We did not observe differences in the synthesis or glycosylation of other ER-folding proteins (Fig. S4B). AMPylation of BiP exerts fine control over the level of active BiP present in the ER (7Preissler S. Rohland L. Yan Y. Chen R. Read R.J. Ron D. AMPylation targets the rate-limiting step of BiP's ATPase cycle for its functional inactivation.Elife. 2017; 6e29428Crossref PubMed Scopus (41) Google Scholar, 12Ham H. Woolery A.R. Tracy C. Stenesen D. Krämer H. Orth K. Unfolded protein response-regulated dFic reversibly AMPylates BiP during endoplasmic reticulum homeostasis.J. Biol. Chem. 2014; 289: 36059-36069Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 13Preissler S. Rato C. Chen R. Antrobus R. Ding S. Fearnley I.M. Ron D. AMPylation matches BiP activity to client protein load in the endoplasmic reticulum.Elife. 2015; 4e12621Crossref PubMed Scopus (64) Google Scholar, 17Sanyal A. Chen A.J. Nakayasu E.S. Lazar C.S. Zbornik E.A. Worby C.A. Koller A. Mattoo S. A novel link between fic (filamentation induced by cAMP)-mediated adenylylation/AMPylation and the unfolded protein response.J. Biol. Chem. 2015; 290: 8482-8499Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). To examine how abrogation of such control affects the folding capacity and stress tolerance of cells, we examined the physiological UPR induced during B cell activation. As before, we isolated B cells from total splenocytes and stimulated them with LPS. Immunoblots showed no change in the levels of UPR sensors IRE1α or PERK, both before and after stimulation (Fig. 5A). There was also no change in the activation of either receptor as monitored by XBP1 splicing or eif2α phosphorylation or in the expression of downstream targets BiP and Grp94 (Fig. 5A). We further verified this by examining downstream targets of the UPR by qPCR (Fig. 5B). We observed no changes in the expression levels between WT and mFICD−/− samples for any of the genes that are downstream targets of the IRE1α, PERK, and ATF6α pathways. The physiological UPR induced upon B cell activation is anticipatory of enhanced antibody production and differs from a UPR induced by an accumulation of misfolded proteins. To ascertain whether mFICD−/− cells responded differently to the latter type of UPR, we incubated B cells with several chemical initiators of the UPR. As with the physiological UPR from B cell activation, we found no significant difference in any of the UPR receptors or downstream targets assayed by immunoblot (Fig. 5C). In D. melanogaster, the mFICD ortholog dfic regulates reversible photoreceptor degeneration, which is critical for visual neurotransmission and adaptation to constant light exposure (5Moehlman A.T. Casey A.K. Servage K. Orth K. Krämer H. Adaptation to constant light requires Fic-mediated AMPylation of BiP to protect against reversible photoreceptor degeneration.Elife. 2018; 7e38752Crossref PubMed Scopus (12) Google Scholar, 31Rahman M. Ham H. Liu X. Sugiura Y. Orth K. Krämer H. Visual neurotransmission in Drosophila requires expression of Fic in glial capitate projections.Nat. Neurosci. 2012; 15: 871-875Crossref PubMed Scopus (55) Google Scholar). We thus tested whether mFICD−/− mice show signs of visual impairment. As part of the SHIRPA test (see Table S1), we assessed visual placing and the pupillary light reflex, which was normal in all tested 6-month-old control as well as mFICD−/− animals. To confirm these results, we performed optometry tests, in which mice were presented a rotating grating, a condition that elicits h" @default.
• W3194705295 created "2021-08-30" @default.
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• W3194705295 date "2021-09-01" @default.
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• W3194705295 title "Deletion of mFICD AMPylase alters cytokine secretion and affects visual short-term learning in vivo" @default.
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• W3194705295 cites W1994580609 @default.
• W3194705295 cites W2007217754 @default.
• W3194705295 cites W2008147758 @default.
• W3194705295 cites W2008816445 @default.
• W3194705295 cites W2013666305 @default.
• W3194705295 cites W2015556811 @default.
• W3194705295 cites W2028560207 @default.
• W3194705295 cites W2029900089 @default.
• W3194705295 cites W2034001730 @default.
• W3194705295 cites W2036946921 @default.
• W3194705295 cites W2055820698 @default.
• W3194705295 cites W2059884320 @default.
• W3194705295 cites W2074043944 @default.
• W3194705295 cites W2081361850 @default.
• W3194705295 cites W2088745167 @default.
• W3194705295 cites W2090188358 @default.
• W3194705295 cites W2110714820 @default.
• W3194705295 cites W2112078820 @default.
• W3194705295 cites W2121866927 @default.
• W3194705295 cites W2216665307 @default.
• W3194705295 cites W2263255481 @default.
• W3194705295 cites W2346066129 @default.
• W3194705295 cites W2560463443 @default.
• W3194705295 cites W2564636373 @default.
• W3194705295 cites W2605419678 @default.
• W3194705295 cites W2746583906 @default.
• W3194705295 cites W2753879466 @default.
• W3194705295 cites W2765949138 @default.
• W3194705295 cites W2779886179 @default.
• W3194705295 cites W2800982196 @default.
• W3194705295 cites W2884356742 @default.
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• W3194705295 cites W2913219010 @default.
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• W3194705295 cites W3034078364 @default.
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