Biology subjects

Ron, D.

Publications and source records attributed to Ron, D..

At least 19 recordsLinked to original sources

bioRxiv2025

A molecular stabiliser of an inhibitory eIF2B-eIF2(αP) complex activates the Integrated Stress Response

Eukaryotic initiation factor 2B (eIF2B), a guanine nucleotide exchange factor (GEF), promotes protein synthesis by charging translation initiation factor 2 (eIF2) with GTP. Stress-induced phosphorylation of eIF2 on its -subunit [eIF2(P)] inhibits this reaction triggering a protective Integrated Stress Response (ISR). A DNA-encoded chemical library (DEL) screen for modulators of eIF2B, led to the identification of a chemical series that inactivates eIF2B, stimulating the ISR. Cryo-EM of compound-bound eIF2B revealed a conformational switch to the inactive state engaged by eIF2(P). In cells, compound activity was sensitive to eIF2s phosphorylation state and to a competing eIF2B ligand (ISRIB) that activates the GEF allosterically. These findings mark the discovery of a first-in-class drug-like allosteric inhibitor of eIF2B, an ISR activator (ISRAC), paving the way to explore the therapeutic potential of eIF2B-directed ISR activation.

molecular biology↗

bioRxiv2025

Metabolite import via SLC33A1 enables ATF6 activation by endoplasmic reticulum stress

The transcription factor ATF6 has a central role in adapting mammalian cells to endoplasmic reticulum (ER) stress via the Unfolded Protein Response (UPR). This has driven efforts to identify modulators of ATF6 signalling. Here, an unbiased genome-wide CRISPR-Cas9 screen performed in Chinese Hamster Ovary (CHO) cells revealed that proteolytic processing of the ATF6 precursor to its active form was impaired in CHO cells lacking the ER-resident solute carrier SLC33A1, a transporter involved in acetyl-CoA import, sialylation and N{varepsilon}-lysine protein acetylation. Cells lacking SLC33A1 constitutively trafficked the ATF6 precursor to the Golgi, but exhibit impaired subsequent Golgi processing, correlating with altered ATF6 Golgi glycosylation. SLC33A1 deficiency also deregulated activation of the IRE1 branch of the UPR, pointing to a selective loss of ATF6-mediated negative feedback in the UPR. Notably, Slc33a1-deleted cells accumulated higher levels of unmodified sialylated N-glycans, precursors to acetylated glycans, likely reflecting impaired glycan processing. By contrast, deletion of ER-localised acetyltransferases NAT8 and NAT8B, which catalyse protein N{varepsilon}-lysine acetylation in the secretory pathway, did not replicate the ATF6 processing defects observed in Slc33a1-deficient cells. Together, our findings highlight a role for SLC33A1-mediated metabolite transport in the post-ER maturation of ATF6 and point direct links between small-molecule metabolism and branch-specific signalling in the UPR.

cell biology↗

bioRxiv2025

A structural basis for chaperone repression of stress signalling from the endoplasmic reticulum

The endoplasmic reticulum (ER) unfolded protein response (UPR) is tuned by the balance between unfolded proteins and chaperones. While reserve chaperones are known to suppress the UPR transducers via their stress-sensing luminal domains, the underlying structural mechanisms remain unclear. Cellular and biophysical analyses established that the ER chaperone AGR2 forms a repressive complex with the luminal domain of the UPR transducer IRE1{beta}. Structural prediction, X-ray crystallography and NMR spectroscopy identify critical interactions between an AGR2 monomer and a regulatory loop in IRE1{beta}s luminal domain. However, in the repressive complex it is an AGR2 dimer that binds IRE1{beta}. Cryo-EM reconstruction reveals a mechanism of unanticipated simplicity: one AGR2 protomer engages the regulatory loop, while the second asymmetrically binds IRE1{beta}s luminal domains C-terminus, blocking IRE1{beta}-activating dimerization. Molecular dynamic simulations indicate that the second, disruptive AGR2 protomer exploits rare fluctuations in the IRE1{beta} dimer that expose its binding site. Thus, AGR2 actively disrupts IRE1{beta} dimers to suppress the UPR, while chaperone clients compete for AGR2 to trigger UPR signalling

biochemistry↗

bioRxiv2025

BDNF in Ventrolateral Orbitofrontal Cortex to Dorsolateral Striatum Circuit Moderates Alcohol Consumption and Gates Alcohol Habit

BDNF plays a crucial role in shaping the structure and function of neurons. BDNF signaling in the dorsolateral striatum (DLS) is part of an endogenous pathway that protects against the development of alcohol use disorder (AUD). Dysregulation of BDNF levels in the cortex or dysfunction of BDNF/TrkB signaling in the DLS results in the escalation of alcohol drinking and compulsive alcohol use. The major source of BDNF in the striatum is the prefrontal cortex. We identified a small ensemble of BDNF-positive neurons in the ventrolateral orbitofrontal cortex (vlOFC), a region involved in AUD, that extend axonal projections to the DLS. We speculated that BDNF in vlOFC-to-DLS circuit may play a role in limiting alcohol drinking and that heavy alcohol use disrupts this protective pathway. We found that BDNF expression is reduced in the vlOFC of male but not female mice after long-term cycles of binge alcohol drinking and withdrawal. We discovered that overexpression of BDNF in vlOFC-to-DLS but not in vlOFC-to-dorsomedial striatum (DMS) or M2 motor cortex-to-DLS circuit reduces alcohol but not sucrose intake and preference. The DLS plays a major role in habitual behaviors. We hypothesized that BDNF in vlOFC-to-DLS circuitry controls alcohol intake by gating habitual alcohol seeking. We found that BDNF over-expression in vlOFC-to-DLS circuit and systemic administration of BDNF receptor TrkB agonist, LM22A-4, biases habitually trained mice towards goal-directed alcohol seeking. Together, our data suggest that BDNF in a small ensemble of vlOFC-to-DLS neurons gates alcohol intake by attenuating habitual alcohol seeking.

neuroscience↗

bioRxiv2024

Prosapip1 in the dorsal hippocampus mediates synaptic protein composition, long-term potentiation, and spatial memory

Prosapip1 is a brain-specific protein localized to the postsynaptic density, where it promotes dendritic spine maturation in primary hippocampal neurons. However, nothing is known about the role of Prosapip1 in vivo. To examine this, we utilized the Cre-loxP system to develop a Prosapip1 neuronal knockout mouse. We found that Prosapip1 controls the synaptic localization of its binding partner SPAR, along with PSD-95 and the GluN2B subunit of the NMDA receptor (NMDAR) in the dorsal hippocampus (dHP). We next sought to identify the potential contribution of Prosapip1 to the activity and function of the NMDAR and found that Prosapip1 plays an important role in NMDAR-mediated transmission and long-term potentiation (LTP) in the CA1 region of the dHP. As LTP is the cellular hallmark of learning and memory, we examined the consequences of neuronal knockout of Prosapip1 on dHP-dependent memory. We found that global or dHP-specific neuronal knockout of Prosapip1 caused a deficit in learning and memory whereas developmental, locomotor, and anxiety phenotypes were normal. Taken together, Prosapip1 in the dHP promotes the proper localization of synaptic proteins which, in turn, facilitates LTP driving recognition, social, and spatial learning and memory.

neuroscience↗

bioRxiv2024

A genome wide CRISPR/Cas9 screen identifies calreticulin as a selective repressor of ATF6

Activating transcription factor 6 (ATF6) is one of three endoplasmic reticulum (ER) transmembrane stress sensors that mediate the unfolded protein response (UPR). Despite its crucial role in long-term ER stress adaptation, regulation of ATF6 alpha () signalling remains poorly understood, possibly because its activation involves ER-to-Golgi and nuclear trafficking. Here, we generated an ATF6/IRE1 dual UPR reporter CHO-K1 cell line and performed an unbiased genome-wide CRISPR/Cas9 mutagenesis screen to systematically profile genetic factors that specifically contribute to ATF6 signalling in the presence and absence of ER stress. The screen identified both anticipated and new candidate genes that regulate ATF6 activation. Among these, calreticulin (CRT), a key ER luminal chaperone, selectively repressed ATF6 signalling: Cells lacking CRT constitutively activated a BiP::sfGFP ATF6-dependent reporter, had higher BiP levels and an increased rate of trafficking and processing of ATF6. Purified CRT interacted with the luminal domain of ATF6 in vitro and the two proteins co-immunoprecipitated from cell lysates. CRT depletion exposed a negative feedback loop implicating ATF6 in repressing IRE1 activity basally and overexpression of CRT reversed this repression. Our findings indicate that CRT, beyond its known role as a chaperone, also serves as an ER repressor of ATF6 to selectively regulate one arm of the UPR.

cell biology↗

bioRxiv2023

Substrate recruitment via eIF2γ enhances catalytic efficiency of a holophosphatase that terminates the Integrated Stress Response

Dephosphorylation of pSer51 of the subunit of translation initiation factor 2 (eIF2P) terminates signalling in the integrated stress response (ISR). A trimeric mammalian holophosphatase comprised of a PP1 catalytic subunit, the conserved C-terminally located [~]70 amino acid core of a substrate-specific regulatory subunit (PPP1R15A/GADD34 or PPP1R15B/CReP) and G-actin (an essential co-factor) efficiently dephosphorylate eIF2P in vitro. Unlike their viral or invertebrate counterparts, with whom they share the conserved 70 residue core, the mammalian PPP1R15s are large proteins of more than 600 residues. Genetic and cellular observations point to a functional role for regions outside the conserved core of mammalian PPP1R15A in dephosphorylating its natural substrate, the eIF2 trimer. We have combined deep learning technology, all-atom molecular dynamics simulations, X-Ray crystallography and biochemistry to uncover binding of the {gamma} subunit of eIF2 to a short helical peptide repeated four times in the functionally-important N-terminus of human PPP1R15A that extends past its conserved core. Binding entails insertion of Phe and Trp residues that project from one face of an -helix formed by the conserved repeats of PPP1R15A into a hydrophobic groove exposed on the surface of eIF2{gamma} in the eIF2 trimer. Replacing these conserved Phe and Trp residues with Ala compromises PPP1R15A function in cells and in vitro. These findings suggest mechanisms by which contacts between a distant subunit of eIF2 and elements of PPP1R15A distant to the holophosphatase active site contribute to dephosphorylation of eIF2P by the core PPP1R15 holophosphatase and to efficient termination of the ISR in mammals.

biochemistry↗

bioRxiv2023

Paradoxical mTORC1-Dependent microRNA-mediated Translation Repression in the Nucleus Accumbens of Mice Consuming Alcohol Attenuates Glycolysis

mTORC1 promotes protein translation, learning and memory, and neuroadaptations that underlie alcohol use and abuse. We report that activation of mTORC1 in the nucleus accumbens (NAc) of mice consuming alcohol promotes the translation of microRNA (miR) machinery components and the upregulation of microRNAs (miRs) expression including miR-34a-5p. In parallel, we detected a paradoxical mTORC1-dependent repression of translation of transcripts including Aldolase A, an essential glycolytic enzyme. We found that miR-34a-5p in the NAc targets Aldolase A for translation repression and promotes alcohol intake. Our data further suggest that glycolysis is inhibited in the NAc manifesting in an mTORC1-dependent attenuation of L-lactate, the end product of glycolysis. Finally, we show that systemic administration of L-lactate attenuates mouse excessive alcohol intake. Our data suggest that alcohol promotes paradoxical actions of mTORC1 on translation and glycolysis which in turn drive excessive alcohol use.

neuroscience↗

bioRxiv2023

Sexual Differences In The Interaction Between Alcohol And mTORC1

The kinase mechanistic target of rapamycin complex 1 (mTORC1) plays an essential role in learning and memory by promoting mRNA to protein translation of a subset of synaptic proteins at dendrites. We generated a large body of data in male rodents indicating that mTORC1 is critically involved in mechanisms that promote numerous adverse behaviors associated with alcohol use disorder (AUD) including heavy alcohol use. For example, we found that mTORC1 is activated in the nucleus accumbens (NAc) and orbitofrontal cortex (OFC) of male mice and rats that were subjected to 7 weeks of intermittent access to 20% alcohol two-bottle choice (IA20%2BC). We further showed that systemic or intra-NAc administration of the selective mTORC1 inhibitor, rapamycin, decreases alcohol seeking and drinking, whereas intra-OFC administration of rapamycin reduces alcohol seeking and habit in male rats. This study aimed to assess mTORC1 activation in these corticostriatal regions of female mice and to determine whether the selective mTORC1 inhibitor, rapamycin, can be used to reduce heavy alcohol use in female mice. We found that mTORC1 is not activated by 7 weeks of intermittent 20% alcohol binge drinking and withdrawal in the NAc and OFC. Like in males, mTORC1 signaling was not activated by chronic alcohol intake and withdrawal in the medial prefrontal cortex (mPFC) of female mice. Interestingly, Pearson correlation comparisons revealed that the basal level of mTORC1 activation between the two prefrontal regions, OFC and mPFC were correlated and that the drinking profile predicts the level of mTORC1 activation in the mPFC after 4-hour binge drinking. Finally, we report that administration of rapamycin does not attenuate heavy alcohol drinking in female animals. Together, our results suggest a sex-dependent contribution of mTORC1 to the neuroadaptation that drives alcohol use and abuse.

neuroscience↗

bioRxiv2023

The small G-protein Rac1 in the dorsomedial striatum promotes alcohol-dependent structural plasticity and goal-directed learning in mice

The small G-protein Rac1 promotes the formation of filamentous actin (F-Actin). Actin is a major component of dendritic spines, and we previously found that alcohol alters actin composition and dendritic spine structure in the nucleus accumbens (NAc) and the dorsomedial striatum (DMS). To examine if Rac1 contributes to these alcohol-mediated adaptations, we measured the level of GTP-bound active Rac1 in the striatum of mice following 7 weeks of intermittent access to 20% alcohol. We found that chronic alcohol intake activates Rac1 in the DMS of male mice. In contrast, Rac1 is not activated by alcohol in the NAc and DLS of male mice, or in the DMS of female mice. Similarly, closely related small G-proteins are not activated by alcohol in the DMS, and Rac1 activity is not increased in the DMS by moderate alcohol or natural reward. To determine the consequences of alcohol-dependent Rac1 activation in the DMS of male mice, we inhibited endogenous Rac1 by infecting the DMS of mice with an AAV expressing a dominant negative form of the small G-protein (Rac1-DN). We found that overexpression of AAV-Rac1-DN in the DMS inhibits alcohol-mediated Rac1 signaling and attenuates alcohol-mediated F-actin polymerization, which corresponded with a decrease in dendritic arborization and spine maturation. Finally, we provide evidence to suggest that Rac1 in the DMS plays a role in alcohol-associated goal-directed learning. Together, our data suggest that Rac1 in the DMS plays an important role in alcohol-dependent structural plasticity and aberrant learning. Significance StatementAddiction, including alcohol use disorder, is characterized by molecular and cellular adaptations that promote maladaptive behaviors. We found that Rac1 was activated by alcohol in the dorsomedial striatum (DMS) of male mice. We show that alcohol-mediated Rac1 signaling is responsible for alterations in actin dynamics and neuronal morphology. We also present data to suggest that Rac1 is important for alcohol-associated learning processes. These results suggest that Rac1 in the DMS is an important contributor to adaptations that promote alcohol use disorder.

neuroscience↗

bioRxiv2022

Male mice carrying the BDNF Val68Met polymorphism exhibit alcohol preferences versus social interaction and acute tolerance through malfunction of BDNF in the ventral hippocampus

The brain-derived neurotrophic factor (BDNF) Valine 66 to Methionine human polymorphism results in impaired activity-dependent BDNF release and has been linked to psychiatric disorders including depression and anxiety. We previously showed that male knock-in mice carrying the mouse Methionine homolog (Met68BDNF) exhibit excessive and compulsive alcohol drinking behaviors as compared to the wild-type Val68BDNF mice. Here, we set out to determine the potential mechanism for the heightened and compulsive alcohol drinking phenotypes detected in Met68BDNF mice. We found that male, but not female Met68BDNF mice exhibit social anxiety-like behaviors. We further show that male Met68BDNF mice exhibit a preference for alcohol over social interaction. In contrast, alcohol place preference without an alternative social reward, is similar in male Met68BDNF and Val68BDNF mice. Since the Met68BDNF mice show social anxiety phenotypes, we tested whether alcohol reliefs anxiety similarly in Met68BDNF and Val68BDNF mice and found that male, but not female Met68BDNF mice are insensitive to the acute anxiolytic action of alcohol. Finally, we show that this acute tolerance to alcohol-dependent anxiolysis can be restored by overexpressing wild-type Val68BDNF in the ventral hippocampus (vHC) of Met68BDNF mice. Together, our results suggest that excessive alcohol drinking in the Met68BDNF may be attributed, in part, to heighted social anxiety and a lack of alcohol-dependent anxiolysis, a phenotype that is associated with malfunction of BDNF signaling in the vHC of male Met68BDNF mice.

neuroscience↗

bioRxiv2022

Leucine-Rich Repeat Kinase 2 limits dopamine D1 receptor signaling in the striatum and biases against heavy persistent alcohol drinking

The transition from hedonic alcohol drinking to problematic drinking is a hallmark of alcohol use disorder that occurs only in a subset of drinkers. This transition is known to require long-lasting changes in the synaptic drive and the activity of striatal neurons expressing dopamine D1 receptor (D1R). The molecular mechanisms that generate vulnerability in some individuals to undergo the transition are less understood. Here, we report that the Parkinsons-related protein leucine-rich repeat kinase 2 (LRRK2) modulates striatal D1R function to affect the behavioral response to alcohol and the likelihood that mice transition to heavy, persistent alcohol drinking. Deletion of the Lrrk2 gene specifically from D1R-expressing neurons potentiates D1R signaling at the cellular and synaptic level, enhancing alcohol-related behaviors and drinking. Mice with cell-specific deletion of Lrrk2 are more prone to heavy alcohol drinking and consumption is insensitive to punishment. These findings identify a novel role for LRRK2 function in the striatum in promoting resilience against heavy and persistent alcohol drinking.

neuroscience↗

bioRxiv2021

DNA dynamics dictates p53 functional outcome

The tumor suppressor protein p53 is situated in the midst of a complex network that is activated in response to cellular stress. An unresolved question is how p53 activates its myriad target genes in response to the severity of the stress signal and consequently coordinates the functional outcome in a timely manner. We have previously shown that DNA torsional flexibility distinguishes among p53 response elements (REs). Here we calculated the flexibility of over 200 p53 REs. By connecting functional pathways of p53-dependent genes to the calculated flexibility of their REs, we show that genes belonging to pathways activated rapidly upon stress (e.g., cell-cycle arrest, energy metabolism and innate immunity) contain REs that are significantly more flexible relative to REs of genes involved in pathways that need to be more strictly regulated or are activated later in the response to stress (e.g., intrinsic apoptosis and p53 negative regulation). The global structural properties of several p53 REs belonging to the different pathways were experimentally validated. Additionally, reporter gene expression driven by flexible p53 REs occurred at lower p53 levels and with faster rates than expression from rigid REs. Moreover, analysis of published endogenous mRNA levels of p53 target genes as a function of the flexibility of their REs support our hypothesis. Overall, we demonstrate that DNA flexibility of p53 REs contributes significantly to the timely expression of p53 target genes and thereby plays an important role in cell-faith decisions in the p53 circuity.

biochemistry↗

bioRxiv2021

BRAIN-DERIVED NEUROTROPHIC FACTOR IN AN ORBITOFRONTAL CORTICAL-DORSOLATERAL STRIATAL CIRCUIT GATES ALCOHOL CONSUMPTION

Brain-derived neurotrophic factor (BDNF) signaling in the dorsolateral striatum (DLS) gates alcohol self-administration in rodents. The major source of BDNF in the striatum is the cortex, and we recently found that BDNF-expressing neurons in the ventrolateral orbitofrontal cortex (vlOFC) extend axonal projections to the DLS. We therefore hypothesized that BDNF in the vlOFC to DLS circuit moderates alcohol intake. We show that overexpression of BDNF in the vlOFC, which activates BDNF signaling in the DLS, is sufficient to attenuate voluntary consumption and seeking of 20% alcohol in the home cage using a two-bottle choice paradigm. Overexpressing BDNF in the vlOFC had no effect on the consumption of a sweetened saccharin solution. In addition, BDNF overexpression in the neighboring motor cortex did not alter alcohol intake. Finally, pathway-specific overexpression of BDNF in DLS-projecting vlOFC neurons significantly reduced alcohol intake and preference. Overall, BDNF in the vlOFC, and specifically in a vlOFC-DLS pathway, keeps alcohol drinking in moderation.

neuroscience↗

bioRxiv2021

IDENTIFICATION OF A NOVEL BDNF-SPECIFIC CORTICOSTRIATAL CIRCUITRY

BDNF is released from axon terminals originating in the cerebral cortex onto striatal neurons. Here, we characterized BDNF neurons in the corticostriatal circuitry. First, we utilized BDNF-Cre and Ribotag transgenic mouse lines to label BDNF-positive neurons in the cortex and detected BDNF expression in all the subregions of the prefrontal cortex (PFC). Next, we used a retrograde viral tracing strategy, in combination with BDNF-Cre knockin mice, to map the cortical outputs of BDNF neurons in the dorsomedial and dorsolateral striatum (DMS and DLS). We found that BDNF-expressing neurons located in the medial prefrontal cortex (mPFC) project mainly to the DMS, and those located in the primary and secondary motor cortices (M1 and M2) and agranular insular cortex (AI) project mainly to the DLS. In contrast, BDNF-expressing orbitofrontal cortical (OFC) neurons differentially target the dorsal striatum (DS) depending on their mediolateral and rostrocaudal location. Specifically, the DMS is mainly innervated by the medial and ventral part of the orbitofrontal cortex (MO and VO) whereas the DLS receives projections specifically from the lateral part of the OFC (LO). Together, our study uncovers previously unknown BDNF corticostriatal circuitries. These findings could have important implications for the role of BDNF signaling in corticostriatal pathways. Significance StatementBDNF is released in axons upon neuronal depolarization. Surprisingly, careful mapping of BDNF projecting neurons in the central nervous system (CNS) has not been conducted. Using retrograde viral strategies in combination with transgenic mice, we mapped out corticostriatal BDNF circuits. We found that, mPFC BDNF neurons project mainly to the DMS whereas the motor cortex and AI project to the DLS. BDNF neurons in the OFC are anatomically segregated. Whereas the DMS receives BDNF-positive projections from the VO, the DLS mainly receives BDNF-positive projections from the LO. Our findings could be important to the study of BDNF in corticostriatal circuitries.

neuroscience↗

bioRxiv2021

Higher order phosphatase-substrate contacts terminate the Integrated Stress Response

Many regulatory PPP1R subunits join few catalytic PP1c subunits to mediate phosphoserine and phosphothreonine dephosphorylation in metazoans. Regulatory subunits are known to engage PP1cs surface, locally affecting flexible phosphopeptides access to the active site. However, catalytic efficiency of holophosphatases towards their natively-folded phosphoprotein substrates is largely unexplained. Here we present a Cryo-EM structure of the tripartite PP1c/PPP1R15A/G-actin holophosphatase that terminates signalling in the Integrated Stress Response (ISR) in pre-dephosphorylation complex with its substrate, translation initiation factor 2 (eIF2). G-actins role in eIF2 dephosphorylation is supported crystallographically by the structure of the binary PPP1R15A-G-actin complex, and by biochemical and genetic confirmation of the essential role of PPP1R15A-G-actin contacts to eIF2P dephosphorylation. In the pre-dephosphorylation CryoEM complex, G-actin aligns the catalytic and regulatory subunits, creating a composite surface that engages eIF2s N-terminal domain to position the distant phosphoserine-51 at the active site. eIF2 residues specifying affinity for the holophosphatase are confirmed here to make critical contacts with the eIF2 kinase PERK. Thus, a convergent process of higher-order substrate recognition specifies functionally-antagonistic phosphorylation and dephosphorylation in the ISR.

biochemistry↗

bioRxiv2021

Structures of a deAMPylation complex rationalise the switch between antagonistic catalytic activities of FICD

The endoplasmic reticulum (ER) Hsp70 chaperone BiP is regulated by AMPylation, a reversible inactivating post-translational modification. Both BiP AMPylation and deAMPylation are catalysed by a single ER-localised enzyme, FICD. Here we present long-sought crystallographic and solution structures of a deAMPylation Michaelis complex formed between mammalian AMPylated BiP and FICD. The latter, via its tetratricopeptide repeat domain, binds a surface that is specific to ATP-state Hsp70 chaperones, explaining the exquisite selectivity of FICD for BiPs ATP-bound conformation both when AMPylating and deAMPylating Thr518. The eukaryotic deAMPylation mechanism thus revealed, rationalises the role of the conserved Fic domain Glu234 as a gatekeeper residue that both inhibits AMPylation and facilitates hydrolytic deAMPylation catalysed by dimeric FICD. These findings point to a monomerisation-induced increase in Glu234 flexibility as the basis of an oligomeric state-dependent switch between FICDs antagonistic activities, despite a similar mode of engagement of its two substrates -- unmodified and AMPylated BiP.

biochemistry↗

bioRxiv2020

Calcium depletion challenges endoplasmic reticulum proteostasis by destabilising BiP-substrate complexes

The metazoan endoplasmic reticulum (ER) serves both as a hub for maturation of secreted proteins and as an intracellular calcium storage compartment, facilitating calcium release-dependent cellular processes. ER calcium depletion robustly activates the unfolded protein response (UPR). However, it is unclear how fluctuations in ER calcium impact organellar proteostasis. Here we report that calcium selectively affects the dynamics of the abundant metazoan ER Hsp70 chaperone BiP, by enhancing its affinity for ADP. In the calcium replete ER, ADP rebinding to post-ATP hydrolysis BiP-substrate complexes competes with ATP binding during both spontaneous and co-chaperone-assisted nucleotide exchange, favouring substrate retention. Conversely, in the calcium depleted ER, relative acceleration of ADP-to-ATP exchange favours substrate release. These findings explain the rapid dissociation of certain substrates from BiP observed in the calcium-depleted ER and suggest a mechanism for tuning ER quality control and coupling UPR activity to signals that mobilise ER calcium in secretory cells.

biochemistry↗