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Specificity of AMPylation of the human chaperone BiP is mediated by TPR motifs of FICD.

ABSTRACT: To adapt to fluctuating protein folding loads in the endoplasmic reticulum (ER), the Hsp70 chaperone BiP is reversibly modified with adenosine monophosphate (AMP) by the ER-resident Fic-enzyme FICD/HYPE. The structural basis for BiP binding and AMPylation by FICD has remained elusive due to the transient nature of the enzyme-substrate-complex. Here, we use thiol-reactive derivatives of the cosubstrate adenosine triphosphate (ATP) to covalently stabilize the transient FICD:BiP complex and determine its crystal structure. The complex reveals that the TPR-motifs of FICD bind specifically to the conserved hydrophobic linker of BiP and thus mediate specificity for the domain-docked conformation of BiP. Furthermore, we show that both AMPylation and deAMPylation of BiP are not directly regulated by the presence of unfolded proteins. Together, combining chemical biology, crystallography and biochemistry, our study provides structural insights into a key regulatory mechanism that safeguards ER homeostasis.

SUBMITTER: Fauser J

PROVIDER: S-EPMC8065156 | biostudies-literature |

REPOSITORIES: biostudies-literature

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FICD activity and AMPylation remodelling modulate human neurogenesis.

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An oligomeric state-dependent switch in the ER enzyme FICD regulates AMPylation and deAMPylation of BiP.

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Infancy-onset diabetes caused by de-regulated AMPylation of the human endoplasmic reticulum chaperone BiP.

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FICD acts bifunctionally to AMPylate and de-AMPylate the endoplasmic reticulum chaperone BiP.

Project description:Protein folding homeostasis in the endoplasmic reticulum (ER) is defended by an unfolded protein response that matches ER chaperone capacity to the burden of unfolded proteins. As levels of unfolded proteins decline, a metazoan-specific FIC-domain-containing ER-localized enzyme (FICD) rapidly inactivates the major ER chaperone BiP by AMPylating T518. Here we show that the single catalytic domain of FICD can also release the attached AMP, restoring functionality to BiP. Consistent with a role for endogenous FICD in de-AMPylating BiP, FICD-/- hamster cells are hypersensitive to introduction of a constitutively AMPylating, de-AMPylation-defective mutant FICD. These opposing activities hinge on a regulatory residue, E234, whose default state renders FICD a constitutive de-AMPylase in vitro. The location of E234 on a conserved regulatory helix and the mutually antagonistic activities of FICD in vivo, suggest a mechanism whereby fluctuating unfolded protein load actively switches FICD from a de-AMPylase to an AMPylase.

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An oligomeric state-dependent switch in the ER enzyme FICD regulates AMPylation and deAMPylation of BiP

Project description:AMPylation is an inactivating modification that alters the activity of the major endoplasmic reticulum (ER) chaperone BiP to match the burden of unfolded proteins. A single ER-localised Fic protein, FICD (HYPE), catalyses both AMPylation and deAMPylation of BiP. However, the basis for the switch in FICD's activity is unknown. We report on the transition of FICD from a dimeric enzyme, that deAMPylates BiP, to a monomer with potent AMPylation activity. Mutations in the dimer interface, or in residues along an inhibitory pathway linking the dimer interface to the enzyme's active site, favour BiP AMPylation in vitro and in cells. Mechanistically, monomerisation relieves a repressive effect allosterically propagated from the dimer interface to the inhibitory Glu234, thereby permitting AMPylation-competent binding of MgATP. Moreover, a reciprocal signal, propagated from the nucleotide binding site, provides a mechanism for coupling the oligomeric-state and enzymatic activity of FICD to the energy status of the ER.

| S-SCDT-EMBOJ-2019-102177 | biostudies-other

Adaptation to constant light requires Fic-mediated AMPylation of BiP to protect against reversible photoreceptor degeneration.

Project description:In response to environmental, developmental, and pathological stressors, cells engage homeostatic pathways to maintain their function. Among these pathways, the Unfolded Protein Response protects cells from the accumulation of misfolded proteins in the ER. Depending on ER stress levels, the ER-resident Fic protein catalyzes AMPylation or de-AMPylation of BiP, the major ER chaperone and regulator of the Unfolded Protein Response. This work elucidates the importance of the reversible AMPylation of BiP in maintaining the Drosophila visual system in response to stress. After 72 hr of constant light, photoreceptors of fic-null and AMPylation-resistant BiPT366A mutants, but not wild-type flies, display loss of synaptic function, disintegration of rhabdomeres, and excessive activation of ER stress reporters. Strikingly, this phenotype is reversible: photoreceptors regain their structure and function within 72 hr once returned to a standard light:dark cycle. These findings show that Fic-mediated AMPylation of BiP is required for neurons to adapt to transient stress demands.

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J domain co-chaperone specificity defines the role of BiP during protein translocation.

Project description:Hsp70 chaperones can potentially interact with one of several J domain-containing Hsp40 co-chaperones to regulate distinct cellular processes. However, features within Hsp70s that determine Hsp40 specificity are undefined. To investigate this question, we introduced mutations into the ER-lumenal Hsp70, BiP/Kar2p, and found that an R217A substitution in the J domain-interacting surface of BiP compromised the physical and functional interaction with Sec63p, an Hsp40 required for ER translocation. In contrast, interaction with Jem1p, an Hsp40 required for ER-associated degradation, was unaffected. Moreover, yeast expressing R217A BiP exhibited defects in translocation but not in ER-associated degradation. Finally, the genetic interactions of the R217A BiP mutant were found to correlate with those of known translocation mutants. Together, our results indicate that residues within the Hsp70 J domain-interacting surface help confer Hsp40 specificity, in turn influencing distinct chaperone-mediated cellular activities.

| S-EPMC2903355 | biostudies-literature

Diadenosine tetraphosphate as a cosubstrate for AMPylation mediated by FICD

Project description:Diadenosine polyphosphates (ApnAs) are noncanonical nucleotides that are ubiquitous across all forms of life. Due to their strong accumulation upon cellular stress, they are considered alarmones to signal homeostatic imbalance. Nonetheless, their cellular role is poorly understood. In this work, we assessed ApnAs for their usage as cosubstrate for protein AMPylation, a post-translational modification. In humans, AMPylation mediated by the AMPylator FICD with ATP as cosubstrate is a response to ER stress. Here, we demonstrate that Ap4A is proficiently consumed for AMPylation by FICD. By chemical proteomics using a new chemical probe, we identified new potential AMPylation targets. Interestingly, we found that AMPylation targets of FICD may differ depending on the nucleotide cosubstrate. These results may suggest that signaling at elevated Ap4A levels during cellular stress differs from when Ap4A is present at low concentrations allowing response to extracellular cues.

2023-01-27 | PXD036450 | Pride

FICD activity and AMPylation remodelling modulate human neurogenesis

Project description:The AMPylation is novel protein post-translation modification. In consist of attachment of the adenosine monophosphate onto the serine, threonine or tyrosine amino acid residues of the protein. This process is catalysed by bacterial effectors or in human by protein FICD (also called HYPE) which contain conserved fic domain. In our study we have focused on identification of the unknown AMPylated proteins and their function in human cells (HeLa, A549, SH-SY5Y, iPSCs, NPCs and neurons) and cerebral organoids (COs). For this purpose we designed and synthesized the small molecule N6-propargyl phosphoramidate adenosine (pro-N6pA) which was used for in situ labelling of the AMPylation in cells and COs. Subsequnetly the the propargyl tag was employed for preparation of chemical-proteomic samples which were measured by LC-MS/MS. This approach confirmed the known AMPylation of heat shock protein HSPA5 and found diverse group of novel AMPylation targets. Several cathepsins found as AMPylation targets were further characterized by identification of AMP binding site. Based on our chemical proteomic data we found out that neurons and neuronal like cells contain distinct AMPylatino pattern from other studied cell types. This also suggested the importance of the AMPylation in these cell types which were then studied in cerebral organoids by overexpressing the FICD wt and E234G highly active mutant. This led to the accelerated differentiation of progenitors into the neurons. Overall our approach is suitable for identification of the AMPylated proteins in living cells and led to characterization of FICD function.

2019-12-04 | PXD015062 | Pride

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