Biology subjects

Barber, L.

Publications and source records attributed to Barber, L..

3 recordsLinked to original sources

bioRxiv2024

BCL-2 and BOK regulate apoptosis by interaction of their C-terminal transmembrane domains

The Bcl-2 family controls apoptosis by direct interactions of pro- and anti-apoptotic proteins. The principle mechanism is binding of the BH3 domain of pro-apoptotic proteins to the hydrophobic groove of anti-apoptotic siblings, which is therapeutically exploited by approved BH3-mimetic anti-cancer drugs. Evidence suggests that also the transmembrane domain (TMD) of Bcl-2 proteins affects Bcl-2 interactions. We developed a highly-specific split luciferase assay, enabling the analysis of TMD interactions of pore-forming apoptosis effectors BAX, BAK, and BOK with anti-apoptotic Bcl-2 proteins in living cells. We confirm homotypic interaction of the BAX-TMD, but also newly identify interaction of the TMD of anti-apoptotic BCL-2 with the TMD of BOK, a so far very peculiar pro-apoptotic Bcl-2 protein. Interaction of BOK-TMD with BCL-2-TMD localizes at the endoplasmic reticulum (ER). Molecular dynamics simulations in an ER membrane model confirm dynamic BOK-TMD and BCL-2-TMD homo- and heterodimers and stable heterotetramers. Inhibition of BOK-induced apoptosis by BCL-2 depends specifically on their TMDs. Thus, TMDs of Bcl-2 proteins are a relevant interaction interface for apoptosis regulation and provide a novel potential drug target.

molecular biology↗

bioRxiv2022

Multifactorial remodeling of the cancer immunopeptidome by interferon gamma

IFN{gamma} alters the immunopeptidome presented on HLA class I (HLA-I), and its activity on cancer cells is known to be important for effective immunotherapy responses. We performed proteomic analyses of untreated and IFN{gamma}-treated colorectal cancer patient-derived organoids (PDOs) and combined this with transcriptomic and HLA-I immunopeptidomics data to dissect mechanisms that lead to remodeling of the immunopeptidome through IFN{gamma}. IFN{gamma}-induced changes in the abundance of source proteins, switching from the constitutive- to the immunoproteasome, and differential upregulation of different HLA alleles explained some, but not all, observed peptide abundance changes. By selecting for peptides which increased or decreased the most in abundance, but originated from proteins with limited abundance changes, we discovered that the amino acid composition of presented peptides also influences whether a peptide is up- or downregulated on HLA-I through IFN{gamma}. The presence of proline within the peptide core was most strongly associated with peptide downregulation. This was validated in an independent dataset. Proline substitution in relevant core positions did not influence the predicted HLA-I binding affinity or stability, indicating that proline effects on peptide processing may be most relevant. Understanding the multiple factors that influence the abundance of peptides presented on HLA-I in the absence or presence of IFN{gamma} is important to identify the best targets for antigen-specific cancer immunotherapies such as vaccines or T-cell receptor engineered therapeutics.

cancer biology↗

bioRxiv2020

A small-molecule inhibitor of the BRCA2-RAD51 interaction modulates RAD51 assembly and potentiates DNA damage-induced cell death

BRCA2 controls RAD51 recombinase during homologous DNA recombination (HDR) through eight evolutionarily-conserved BRC repeats, which individually engage RAD51 via the motif Phe-x-x-Ala. Using structure-guided molecular design, templated on a monomeric thermostable chimera between human RAD51 and archaeal RadA, we identify CAM833, a 529 Da orthosteric inhibitor of RAD51:BRC with a Kd of 366 nM. The quinoline of CAM833 occupies a hotspot, the Phe-binding pocket on RAD51 and the methyl of the substituted -methylbenzyl group occupies the Ala-binding pocket. In cells, CAM833 diminishes formation of damage-induced RAD51 nuclear foci; inhibits RAD51 molecular clustering, suppressing extended RAD51 filament assembly; potentiates cytotoxicity by ionising radiation, augmenting 4N cell-cycle arrest and apoptotic cell death and works with poly-ADP ribose polymerase (PARP)1 inhibitors to suppress growth in BRCA2-wildtype cells. Thus, chemical inhibition of the protein-protein interaction between BRCA2 and RAD51 disrupts HDR and potentiates DNA damage-induced cell death, with implications for cancer therapy.

cancer biology↗