The InterPro database (http://www.ebi.ac.uk/interpro/) is a freely available resource that can be used to classify sequences into protein families and to predict the presence of important domains and sites. Central to the InterPro database are predictive models, known as signatures, from a range of different protein family databases that have different biological focuses and use different methodological approaches to classify protein families and domains. InterPro integrates these signatures, capitalizing on the respective strengths of the individual databases, to produce a powerful protein classification resource. Here, we report on the status of InterPro as it enters its 15th year of operation, and give an overview of new developments with the database and its associated Web interfaces and software. In particular, the new domain architecture search tool is described and the process of mapping of Gene Ontology terms to InterPro is outlined. We also discuss the challenges faced by the resource given the explosive growth in sequence data in recent years. InterPro (version 48.0) contains 36 766 member database signatures integrated into 26 238 InterPro entries, an increase of over 3993 entries (5081 signatures), since 2012.

The endoplasmic reticulum (ER)-localized Hsp70 chaperone BiP affects protein folding homeostasis and the response to ER stress. Reversible inactivating covalent modification of BiP is believed to contribute to the balance between chaperones and unfolded ER proteins, but the nature of this modification has so far been hinted at indirectly. We report that deletion of FICD, a gene encoding an ER-localized AMPylating enzyme, abolished detectable modification of endogenous BiP enhancing ER buffering of unfolded protein stress in mammalian cells, whilst deregulated FICD activity had the opposite effect. In vitro, FICD AMPylated BiP to completion on a single residue, Thr518. AMPylation increased, in a strictly FICD-dependent manner, as the flux of proteins entering the ER was attenuated in vivo. In vitro, Thr518 AMPylation enhanced peptide dissociation from BiP 6-fold and abolished stimulation of ATP hydrolysis by J-domain cofactor. These findings expose the molecular basis for covalent inactivation of BiP.DOI: http://dx.doi.org/10.7554/eLife.12621.001

SummaryWhen unfolded proteins accumulate in the endoplasmic reticulum (ER), the unfolded protein response (UPR) increases ER-protein-folding capacity to restore protein-folding homeostasis. Unfolded proteins activate UPR signaling across the ER membrane to the nucleus by promoting oligomerization of IRE1, a conserved transmembrane ER stress receptor. However, the coupling of ER stress to IRE1 oligomerization and activation has remained obscure. Here, we report that the ER luminal co-chaperone ERdj4/DNAJB9 is a selective IRE1 repressor that promotes a complex between the luminal Hsp70 BiP and the luminal stress-sensing domain of IRE1α (IRE1LD). In vitro, ERdj4 is required for complex formation between BiP and IRE1LD. ERdj4 associates with IRE1LD and recruits BiP through the stimulation of ATP hydrolysis, forcibly disrupting IRE1 dimers. Unfolded proteins compete for BiP and restore IRE1LD to its default, dimeric, and active state. These observations establish BiP and its J domain co-chaperones as key regulators of the UPR.

Protein folding homeostasis in the endoplasmic reticulum (ER) is defended by an unfolded protein response (UPR) 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 (HYPE), 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.