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Tax, G.

Publications and source records attributed to Tax, G..

5 recordsLinked to original sources

CBL mutations in pediatric solid and CNS tumors are a marker of receptor tyrosine kinase activation and a potential therapeutic target

Mutations in CBL, an E3 ubiquitin ligase that negatively regulates receptor tyrosine kinases (RTKs) and potentiates intracellular signaling, have been extensively characterized in hematological malignancies. However, the impacts are unknown in other cancer types. We have identified established and novel aberrations in CBL in molecularly diverse pediatric CNS and solid tumors. Additionally, we present novel CBL splice mutations, including in the germline of a high-grade glioma patient, and alternative CBL splicing without corresponding genomic alterations. Functionally, we demonstrate that CBL exon 8/9{Delta}, typically associated with acute myeloid leukemia, is RTK activating in neuroblastoma and that novel CBL variants, CBL E366_E373del and CBL C384G, can cooperate with overexpression of EGFR to transform Ba/F3 cells. Collectively, these data represent a novel form of RTK activation in pediatric patients with solid and CNS tumors who might benefit from RTK-targeted therapies. Statement of significanceMolecularly targeted therapies improve childhood cancer outcomes. Therapies targeting genes called receptor tyrosine kinases are particularly effective. We have identified new genetic alterations that may be a marker for receptor tyrosine kinase activation and drug sensitivity, enabling more patients to benefit from targeted treatments.

cancer biology↗

Structure and function of the EDEM:PDI ERAD checkpoint complex

The ERAD-L checkpoint complex de-mannosylates misfolded glycoproteins with lumenal defects, targeting them to retrotranslocation, ubiquitination, and proteasomal degradation. Commitment to ERAD-L requires an Endoplasmic Reticulum-Degradation Enhancing alpha-Mannosidase-like protein (EDEM) and its associated Protein Disulfide Isomerase (PDI). We determined Cryo-EM structures of the Chaetomium thermophilum EDEM:PDI heterodimer, both by itself and in complex with a classic ERAD substrate, the alpha1-antitrypsin Null Hong Kong mutant (A1AT-NHK). The EDEM catalytic domain nestles within the PDI arc. One intermolecular disulfide, between the a' domain of PDI and the first conserved cysteine of the EDEM linker (Cys A), stably links the two proteins. A second intermolecular disulfide, between the second conserved cysteine of the EDEM linker (Cys B) and the PDI a domain, stabilises the compact apo conformation. In the substrate-bound complex, the Cys B intermolecular disulfide is reduced and the catalytic CXXC motif of the PDI a domain oxidised. Release of the Cys B linkage increases the conformational freedom of the EDEM linker, allowing the EDEM C-terminal domains to adopt both proximal and distal conformations relative to the catalytic domain. The structures provide a framework for discovery of EDEM:PDI modulators, with potential applications in virology, rare genetic disease and cancer, and as reagents for glycoprotein quality-control engineering.

biochemistry↗

Engineered paediatric tumours retain patient tumour genotype and phenotype for precision medicine.

Precision medicine for paediatric and adult cancers that includes drug sensitivity profiling, can identify effective therapies for individual patients. However, obtaining adequate biopsy samples for high-throughput (HTP) screening remains challenging, with tumours needing to be expanded in culture or patient-derived xenografts - this is time-consuming and often unsuccessful. Herein, we have developed paediatric patient-derived tumour models using an engineered extracellular matrix (ECM) tissue mimic hydrogel system and HTP 3D bioprinting. Gene expression analysis from neuroblastoma and sarcoma patients identified key components of the ECM in these tumour types. Engineered hydrogels with ECM-mimic peptides were used to create patient-specific tumour organoids, modelling tumour growth conditions. Expanded tumour organoids recapitulated the genetic and phenotypic characteristics of the original tumours and retained tumourgenicity. Screening of these models identified individualised drug sensitivities. Our approach offers a timely and clinically relevant technology platform for precision medicine in paediatric cancers, potentially transforming preclinical testing across cancer types.

cancer biology↗

Rescue of secretion of a rare-disease associated mis-folded mutant glycoprotein in UGGT1 knock-out mammalian cells

Endoplasmic reticulum (ER) retention of mis-folded glycoproteins is mediated by the ER- localised eukaryotic glycoprotein secretion checkpoint, UDP-glucose glycoprotein glucosyl-transferase (UGGT). The enzyme recognises a mis-folded glycoprotein and flags it for ER retention by reglucosylating one of its N-linked glycans. In the background of a congenital mutation in a secreted glycoprotein gene, UGGT-mediated ER retention can cause rare disease even if the mutant glycoprotein retains activity ("responsive mutant"). Here, we investigated the subcellular localisation of the human Trop-2 Q118E variant, which causes gelatinous drop- like corneal dystrophy (GDLD). Compared with the wild type Trop-2, which is correctly localised at the plasma membrane, the Trop-2-Q118E variant is found to be heavily retained in the ER. Using Trop-2-Q118E, we tested UGGT modulation as a rescue-of-secretion therapeutic strategy for congenital rare disease caused by responsive mutations in genes encoding secreted glycoproteins. We investigated secretion of a EYFP-fusion of Trop-2-Q118E by confocal laser scanning microscopy. As a limiting case of UGGT inhibition, mammalian cells harbouring CRISPR/Cas9-mediated inhibition of the UGGT1 and/or UGGT2 gene expressions were used. The membrane localisation of the Trop-2-Q118E-EYFP mutant was successfully rescued in UGGT1-/-and UGGT1/2-/- cells. UGGT1 also efficiently reglucosylated Trop-2-Q118E-EYFP in cellula. The study supports the hypothesis that UGGT1 modulation constitutes a novel therapeutic strategy for the treatment of Trop-2-Q118E associated GDLD, and it encourages the testing of modulators of ER glycoprotein folding Quality Control (ERQC) as broad-spectrum rescue- of-secretion drugs in rare diseases caused by responsive secreted glycoprotein mutants. SynopsisDeletion of the UGGT1 and UGGT1/2 genes in HEK 293T cells rescues secretion of an EYFP-fusion of the human Trop-2-Q118E glycoprotein mutant. The mutant is retained in the secretory pathway in wild type cells and it localises to the cell membrane in UGGT1-/- single and UGGT1/2-/- double knock-out cells. The Trop-2-Q118E glycoprotein disease mutant is efficiently glucosylated by UGGT1 in human cells demonstrating that it is a bona fide cellular UGGT1 substrate. O_FIG O_LINKSMALLFIG WIDTH=120 HEIGHT=200 SRC="FIGDIR/small/542711v1_ufig1.gif" ALT="Figure 1"> View larger version (63K): org.highwire.dtl.DTLVardef@ac29f1org.highwire.dtl.DTLVardef@f5bcbforg.highwire.dtl.DTLVardef@13ab5a1org.highwire.dtl.DTLVardef@16a9406_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

Clamping, bending, and twisting inter-domain motions in the misfold-recognising portion of UDP glucose:glycoprotein glucosyl-transferase.

UDP-glucose:glycoprotein glucosyltransferase (UGGT) is the only known glycoprotein folding quality control checkpoint in the eukaryotic glycoprotein secretory pathway. When the enzyme detects a misfolded glycoprotein in the Endoplasmic Reticulum (ER), it dispatches it for ER retention by re-glucosylating it on one of its N-linked glycans. Recent crystal structures of a fungal UGGT have suggested the enzyme is conformationally mobile. Here, a negative stain electron microscopy reconstruction of UGGT in complex with a monoclonal antibody confirms that the misfold-sensing N-terminal portion of UGGT and its C-terminal catalytic domain are tightly associated. Molecular Dynamics (MD) simulations capture UGGT in so far unobserved conformational states, giving new insights into the molecules flexibility. Principal component analysis of the MD trajectories affords a description of UGGTs overall inter-domain motions, highlighting three types of inter-domain movements: bending, twisting and clamping. These inter-domain motions modify the accessible surface area of the enzymes central saddle, likely enabling the protein to recognize and re-glucosylate substrates of different sizes and shapes, and/or re-glucosylate N-linked glycans situated at variable distances from the site of misfold. We propose to name "Parodi limit" the maximum distance between a site of misfolding on a UGGT glycoprotein substrate and an N-linked glycan that monomeric UGGT can re-glucosylate on the same glycoprotein. MD simulations estimate the Parodi limit to be around 60-70 [A]. Re-glucosylation assays using UGGT deletion mutants suggest that the TRXL2 domain is necessary for activity against urea-misfolded bovine thyroglobulin. Taken together, our findings support a "one-size-fits-all adjustable spanner" substrate recognition model, with a crucial role for the TRXL2 domain in the recruitment of misfolded substrates to the enzymes active site.

biochemistry↗