Biology subjects

Harding, H. P.

Publications and source records attributed to Harding, H. P..

9 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↗

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↗

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↗

bioRxiv2020

ISRIB blunts the integrated stress response by allosterically antagonising the inhibitory effect of phosphorylated eIF2 on eIF2B

The small molecule ISRIB antagonises the activation of the integrated stress response (ISR) by phosphorylated translation initiation factor 2, eIF2(P). ISRIB and eIF2(P) bind distinct sites in their common target, eIF2B, a guanine nucleotide exchange factor (GEF) for eIF2. We have found that ISRIB-mediated acceleration of eIF2B activity in vitro is observed preferentially in the presence of eIF2(P) and is attenuated by mutations that desensitise eIF2B to the inhibitory effects of eIF2(P). ISRIBs efficacy as an ISR inhibitor in cells also depends on presence of eIF2(P). Cryo-EM showed that engagement of both eIF2B regulatory sites by two eIF2(P) molecules remodels both the ISRIB-binding pocket and the pockets that would engage eIF2 during active nucleotide exchange, thereby discouraging both binding events. In vitro, eIF2(P) and ISRIB reciprocally opposed each others binding to eIF2B. These findings point to antagonistic allostery in ISRIB action on eIF2B, culminating in inhibition of the ISR.

biochemistry↗

bioRxiv2020

Cargo receptor-assisted endoplasmic reticulum export of pathogenic α1-antitrypsin polymers

Circulating polymers of alpha1-antitrypsin (1AT) are chemo-attractant for neutrophils and contribute to inflammation in pulmonary, vascular and adipose tissues. Cellular factors affecting the intracellular itinerary of mutant polymerogenic 1AT remain obscure. Here, we report on an unbiased genome-wide CRISPR/Cas9 screen for regulators of trafficking of the polymerogenic 1ATH334D variant. Single guide RNAs targeting genes whose inactivation enhanced accumulation of polymeric 1AT were enriched by iterative construction of CRISPR libraries based on genomic DNA from fixed cells selected for high polymer content by fluorescence-activated cell sorting. This approach bypassed the limitation to conventional enrichment schemes imposed by cell fixation and identified 121 genes involved in polymer retention at false discovery rate < 0.1. From that set of genes, the pathway cargo loading into COPII-coated vesicles was overrepresented with 16 significant genes, including two transmembrane cargo receptors, LMAN1 (ERGIG-53) and SURF4. LMAN1 and SURF4-disrupted cells displayed a secretion defect extended beyond 1AT monomers to polymers, whose low-level secretion was especially dependent on SURF4 and correlated with SURF4-1ATH334D physical interaction and with enhanced co-localisation of polymeric 1ATH334D with the endoplasmic reticulum (ER). These findings suggest that ER cargo receptors co-ordinate intracellular progression of 1AT out of the ER and modulate the accumulation of polymeric 1AT not only by controlling the concentration of precursor monomers but also through a previously-unrecognised role in secretion of the polymers themselves.

cell biology↗

bioRxiv2019

GDF15 and the beneficial actions of metformin in pre-diabetes

Metformin, the worlds most prescribed anti-diabetic drug, is also effective in preventing Type 2 diabetes in people at high risk, by lowering body weight, fat mass and circulating insulin levels through mechanisms that are incompletely understood. Recent observational studies reporting the association of metformin use and circulating levels of GDF15 led us to hypothesize that GDF15, which signals through a specific receptor complex in the hindbrain to reduce body weight, might mediate these effects. We measured GDF15 in people without diabetes from a randomized placebo-controlled trial of metformin. Over 18 months, participants allocated metformin lost significant weight and levels of GDF15 were persistently elevated compared to placebo. The change in plasma GDF15 in this study correlated positively with weight loss. In wild-type mice, oral metformin increased circulating GDF15 with GDF15 expression increasing predominantly in the distal intestine and the kidney. Metformin prevented weight gain in response to high fat diet in wild-type mice but not in mice lacking GDF15 or its receptor GFRAL. In obese, high fat-fed mice, the effects of metformin to reduce body weight were reversed by a GFRAL antagonist antibody. Metformin had effects on both energy intake and energy expenditure that required GDF15. The insulin sensitising effects of metformin determined by insulin tolerance were abolished in mice lacking GDF15. Metformin significantly reduced fasting glucose and insulin levels in wild type but not in mice lacking GDF15. In summary, metformin increases the circulating levels of GDF15, which appears to be necessary for many of its actions as a metabolic chemopreventive agent.

physiology↗

bioRxiv2017

Binding of the integrated stress response inhibitor, ISRIB, reveals a regulatory site in the nucleotide exchange factor, eIF2B

The Integrated Stress Response (ISR) is a conserved eukaryotic translational and transcriptional program implicated in mammalian metabolism, memory and immunity. The ISR is mediated by stress-induced phosphorylation of translation initiation factor 2 (eIF2) that attenuates the guanine nucleotide exchange factor eIF2B. A chemical inhibitor of the ISR, ISRIB, a bis-O-arylglycolamide, reverses the attenuation of eIF2B by phosphorylated eIF2, protecting mice from neurodegeneration and traumatic brain injury. We report on a cryo-electron microscopy-based structure of ISRIB-bound human eIF2B revealing an ISRIB-binding pocket at the interface between the {beta} and {delta} regulatory subunits. CRISPR/Cas9 mutagenesis of residues lining this pocket altered the hierarchical cellular response to ISRIB congeners in vivo and ISRIB-binding in vitro, thus providing chemogenetic support for the functional relevance of ISRIB binding at a distance from known eIF2-eIF2B interaction sites. Our findings point to a hitherto unexpected allosteric site in the eIF2B decamer exploited by ISRIB to regulate translation.

molecular biology↗