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Biology subjects

George, G.

Publications and source records attributed to George, G..

9 recordsLinked to original sources

Chromosome axis protein SYCP2 recruits HORMAD2 to enable meiotic synapsis quality control in mice

Faithful chromosome segregation during meiosis depends on accurate recombination and synapsis of homologous chromosomes. These processes are monitored in mammals by checkpoint mechanisms involving the meiotic HORMA-domain proteins HORMAD1 and HORMAD2, which bind unsynapsed chromosome axes and promote activation of the DNA damage-response kinase ATR independently of DNA double-strand breaks (DSBs). However, no mechanism for axis recruitment of HORMAD1 or HORMAD2 had been demonstrated, nor had its role in checkpoint function been tested. We establish that a putative HORMAD-interacting region--the closure motif (CM)--within the chromosome-axis component SYCP2 is selectively required for HORMAD2, but not HORMAD1, localization. Deletion of the SYCP2 CM disrupts SYCP2-HORMAD2 complexes and prevents HORMAD2 axis binding without affecting axis assembly or recombination. Consequently, ATR accumulation and signaling on unsynapsed axes are reduced, and the prophase checkpoint malfunctions in a sexually dimorphic manner--causing aberrant elimination of synapsis-proficient spermatocytes and persistence of asynaptic oocytes. The phenotypes of SYCP2-CM-deficient and HORMAD2-null mice are indistinguishable, establishing the requirement for HORMAD2 axis recruitment in synapsis surveillance. We propose that axial recruitment generates a HORMAD2 scaffold that drives clustering-mediated ATR network activation independently of DSBs, thereby linking chromosome-axis architecture to synapsis quality control in mammalian meiosis.

cell biology↗

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↗

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↗

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↗

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↗

iPheGWAS : an intelligent computational framework to integrate and visualise genome-phenome wide association studies

Estimating the genetic correlations by LDSC is computationally demanding and visualising multiple GWAS results along with their genetic relationships is restricted. This study developed iPheGWAS, a novel approach which applied hierarchical clustering to GWAS summary statistics to (i) calculate their genetic relatedness, and (ii) enable three-dimensional visualisation of multiple ordered GWAS plots. Simulation and real-world data analysis demonstrated that when investigating genetic relationships among multiple phenotypes, iPheGWAS can deliver comparable results with LDSC but with 8 times faster computational speed. It can also provide novel findings in studying genetically-correlated comorbidities, such as mental illness and rheumatoid arthritis.

bioinformatics↗

Molecular and functional characterization of buffalo nasal epithelial odorant binding proteins and their structural insights by in-silico and biochemical approach

The olfactory system is capable of detecting and distinguishing thousands of environmental odorants that play a key role in reproduction, social behaviours including pheromones influenced classical events. Membrane secretary odorant binding proteins (OBPs) are soluble lipocalins, localized in the nasal membrane of mammals. They bind and carry odorants within the nasal epithelium to putative olfactory transmembrane receptors (ORs). While the existence of OBPs and their significant functions are very well known in insects and laboratory mammals, there is little information about the species-specific OBPs in buffaloes. In fact, the OBP of nasal epithelium has not yet been exploited to develop a suitable technique to detect estrus which is being reported as a difficult task in buffalo. In the present study, using molecular biology and protein engineering approaches, we have cloned six novel OBP isoforms from buffalo nasal epithelium (bnOBPs). Furthermore, 3D model was developed and molecular-docking, dynamics experiments were performed by In-silico approach. In particular, we found four residues (Phe104, Phe134, Phe69 and Asn118) from OBP1a, which had strong binding affinities towards two sex pheromones, specifically oleic acid and p-cresol. We expressed this protein in Escherichia coli to examine its involvement in the sex pheromone perception from female buffalo urine and validated through fluorescence quenching studies. Interestingly, fluorescence binding experiments also showed similar strong binding affinities of OBP1a to oleic acid and p-cresol. By using structural data, the binding specificity is also verified by site-directed mutagenesis of the four residues followed by in-vitro binding assays. Our results enable to better understand the functions of different nasal epithelium OBPs in buffaloes. They also lead to improved understanding of the interaction between olfactory proteins and odorants to develop highly selective biosensing devices for non-invasive detection of estrus in buffaloes.

bioinformatics↗

A Fyn - specific biosensor reveals localized, pulsatile kinase activity and spatially regulated signaling crosstalk

Cell behavior is controlled through spatio-temporally localized protein activity. Despite unique and often contradictory roles played by Src-family-kinases (SFKs) in regulating cell physiology, activity patterns of individual SFKs have remained elusive. Here, we report a biosensor for specifically visualizing active conformation of SFK-Fyn in live cells. We deployed combinatorial library screening to isolate a binding-protein (F29) targeting activated Fyn. Nuclear-magnetic-resonance (NMR) analysis provides the structural basis of F29 specificity for Fyn over homologous SFKs. Using F29, we engineered a sensitive, minimally-perturbing fluorescence-resonance-energy-transfer (FRET) biosensor (FynSensor) that reveals cellular Fyn activity to be spatially localized, pulsatile and sensitive to adhesion/integrin signaling. Strikingly, growth factor stimulation further enhanced Fyn activity in pre-activated intracellular zones. However, inhibition of focal-adhesion-kinase activity not only attenuates Fyn activity, but abolishes growth-factor modulation. FynSensor imaging uncovers spatially-organized, sensitized signaling clusters, direct crosstalk between integrin and growth-factor-signaling, and clarifies how compartmentalized Src-kinase activity may drive cell fate.

cell biology↗

PheGWAS: A new dimension to visualize GWAS across multiple phenotypes

MotivationPheGWAS was developed to enhance exploration of phenome-wide pleiotropy at the genome-wide level through the efficient generation of a dynamic visualization combining Manhattan plots from GWAS with PheWAS to create a three-dimensional "landscape". Pleiotropy in sub-surface GWAS significance strata can be explored in a sectional view plotted within user defined levels. Further complexity reduction is achieved by confining to a single chromosomal section. Comprehensive genomic and phenomic coordinates can be displayed. ResultsPheGWAS is demonstrated using summary data from Global Lipids Genetics Consortium (GLGC) GWAS across multiple lipid traits. For single and multiple traits PheGWAS highlighted all eight-eight and sixty-nine loci respectively. Further, the genes and SNPs reported in GLGC were identified using additional functions implemented within PheGWAS. Not only is PheGWAS capable of identifying independent signals but also provide insights to local genetic correlation (verified using HESS) and in identifying the potential regions that share causal variants across phenotypes (verified using colocalization tests). Availability and ImplementationThe PheGWAS software and code are freely available at (https://github.com/georgeg0/PheGWAS). Contacta.doney@dundee.ac.uk, g.z.george@dundee.ac.uk

bioinformatics↗