Biology subjects

Andrews, D. W.

Publications and source records attributed to Andrews, D. W..

5 recordsLinked to original sources

bioRxiv2025

A high-resolution dimeric structure reveals a critical role of alpha-helix 9 in apoptotic Bax pore assembly in the mitochondrial membrane

Bax functions as a proapoptotic protein perforating the mitochondrial membrane to release mitochondrial proteins and DNA that kill the cell. Structures of full-length Bax monomer in solution and core domain dimers in solution and bound to lipid/detergent bicelles reveal how Bax proteins must be activated, unfold, refold and dimerize prior to forming oligomeric pores in the membrane. In particular, amphipathic core dimer forms part of the pore wall between the nonpolar lipid bilayer and the aqueous conduit. However, atomic resolution structures of other parts of the pore were scarce. Here, we elucidate a structure of Bax C-terminal -helix 9 (9) in lysolipid micelles using NMR. According to this high-resolution structure the 9 regions form an amphipathic helical dimer with an extended nonpolar interface and several uncharged polar residues on the surface. Structure-guided mutagenesis and functional assessment demonstrate that the nonpolar interactions are important for Bax dimerization in and perforation of the mitochondrial membrane. Surprisingly the polar residues are also important because they form bifurcated hydrogen bonds between helical turns to stabilize each helix and thereby the dimer. Molecular dynamics simulations of an oligomer constructed with wall-forming core and transmembrane 9 dimers linked by flexible 6-7-8 bridges in a mitochondrial lipid bilayer generate an atomic resolution model for a stable Bax pore capable to release cytochrome C. Thus, we made an important step toward elucidating molecular mechanisms of apoptotic Bax perforation.

biochemistry↗

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

Small Molecule Assembly Modulators with Pan-Cancer Therapeutic Efficacy

Two structurally-unrelated small molecule chemotypes, represented by compounds PAV-617 and PAV-951 with antiviral activity in cell culture against monkeypox virus (MPXV) and human immunodeficiency virus (HIV) respectively, were studied for anti-cancer efficacy. Each exhibited apparent pan-cancer cytotoxicity, reasonable pharmacokinetics, and non-toxicity in mice at active concentrations. Anti-tumor properties of both chemotypes, were validated in mouse xenografts against A549 human lung cancer and, for one of the chemotypes, against HT-29 colorectal cancer. The targets of these compounds are unconventional: each binds to a different transient, energy-dependent multi-protein complex containing the protein TRIM28/KAP1, an allosteric modulator known to regulate mechanisms underlying viral and nonviral disease states including cancer. Treatment with these compounds alters the target multi-protein complexes in a manner consistent with allosteric modulation as their mechanism of action. These compounds appear to remove a block, crucial for cancer survival and progression, on the homeostatic linkage of uncontrolled cellular proliferation to apoptosis. These compounds provide starting points for development of next-generation non-toxic, pan-cancer therapeutics.

cancer biology↗

bioRxiv2021

The PNUTS-PAD domain recruits MYC to the PNUTS:PP1 phosphatase complex via the oncogenic MYC-MB0 region

Despite MYC dysregulation in most human cancers, strategies to target this potent oncogenic driver remains an urgent unmet need. Recent evidence shows the PP1 phosphatase and its regulatory subunit PNUTS control MYC phosphorylation and stability, however the molecular basis remains unclear. Here we demonstrate that MYC interacts directly with PNUTS through the MYC homology Box 0 (MB0), a highly conserved region recently shown to be important for MYC oncogenic activity. MB0 interacts with PNUTS residues 1-148, a functional unit here termed, PNUTS amino-terminal domain (PAD). Using NMR spectroscopy we determined the solution structure of PAD, and characterised its interaction with MYC. Point mutations of residues at the MYC-PNUTS interface significantly weaken their interaction both in vitro and in vivo. These data demonstrate the MB0 binding pocket of the PAD represents an attractive site for pharmacological disruption of the MYC-PNUTS interaction. In BriefSolving the structure of MYC-PNUTS direct interaction reveals how the intrinsically disordered MYC-Box0 (MB0) region anchors into a binding pocket in the N-terminal PAD domain of PNUTS. These data provide insight into the molecular mechanism of how the PNUTS:PP1 phosphatase complex regulates MYC phosphorylation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC="FIGDIR/small/470928v1_ufig1.gif" ALT="Figure 1"> View larger version (86K): org.highwire.dtl.DTLVardef@179b89corg.highwire.dtl.DTLVardef@47338eorg.highwire.dtl.DTLVardef@89b6f4org.highwire.dtl.DTLVardef@14a80bd_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIA region critical for MYC oncogenesis, MYC-Box0 (MB0), directly interacts with PNUTS C_LIO_LIPNUTS amino-terminal domain (PAD) is a structural domain that interacts with MYC MB0 C_LIO_LIMutation of single residues at the interaction interface disrupts MYC-PNUTS binding in cells C_LIO_LIMYC-PNUTS binding releases MYC intramolecular interactions to enable PP1substrate access C_LI

cancer biology↗

bioRxiv2020

Amphipathic Bax core dimer forms part of apoptotic pore wall in the mitochondrial membrane

Bax proteins form pores in the mitochondrial outer membrane to initiate apoptosis. They may embed in the cytosolic leaflet of the lipid bilayer generating tension to induce a lipid pore with radially arranged lipids forming the wall. Alternatively, they may comprise part of the pore wall. However, there is no unambiguous structural evidence for either hypothesis. Using NMR, we determine a high-resolution structure of the Bax core region that forms a dimer with the nonpolar surface covering the lipid bilayer edge and the polar surface exposed to water. The dimer tilts from the bilayer normal, not only maximizing nonpolar interactions with lipid tails but creating polar interactions between charged residues and lipid heads. Structure-guided mutations demonstrate the importance of both protein-lipid interactions in Bax pore assembly and core dimer configuration. Therefore, the Bax core dimer forms part of the proteolipid pore wall to permeabilize mitochondria.

biophysics↗