Biology subjects

Czabotar, P. E.

Publications and source records attributed to Czabotar, P. E..

5 recordsLinked to original sources

bioRxiv2026

DNA binding by ATPase-adjacent domains stimulates SMCHD1 ATPase activity

SMCHD1 is an epigenetic regulator in which heterozygous variants are reported in facioscapulohumeral muscular dystrophy (FSHD), as well as Bosma arhinia microphthalmia syndrome (BAMS). While we have previously shown that SMCHD1 is able to interact with nucleic acids via its hinge domain, we have now identified a second DNA-binding site that is located C-terminal to the ATPase domain and formed by two domains: the Bromo-adjacent homology (BAH) and immunoglobulin-like 1 (IGL-1) domains. Here, we report their mode of DNA-interaction and we present the first high-resolution structure of the wild-type human SMCHD1 ATPase using site-directed mutagenesis and structural analysis via cryo-EM. We also reveal that DNA-binding at the BAH-IGL-1 domains stimulates the ATPase activity of full-length SMCHD1 in vitro, and demonstrate in a mouse model that ATP hydrolysis is essential for SMCHD1 function in vivo. Together, these findings establish bidentate DNA binding and DNA-stimulated ATP hydrolysis as central features of SMCHD1 function, providing new mechanistic insight into how SMCHD1 regulates gene silencing. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/704097v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@12b2fd5org.highwire.dtl.DTLVardef@5152c7org.highwire.dtl.DTLVardef@111c487org.highwire.dtl.DTLVardef@17d8d0d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Differential regulation of BAX and BAK apoptotic activity revealed by a novel small molecule

Defective apoptosis mediated by BAK or BAX underlies various human pathologies including autoimmune and degenerative conditions. The mitochondrial channel protein VDAC2 interacts with BAK and BAX through a common interface to either inhibit BAK or to facilitate BAX apoptotic activity. Using a newly developed small molecule (WEHI-3773) that inhibits the interaction between VDAC2 and BAK or BAX, we reveal contrasting effects on BAX and BAK apoptotic activity. WEHI-3773 inhibits apoptosis mediated by BAX by blocking VDAC2-mediated BAX recruitment to mitochondria. Conversely, WEHI-3773 primes BAK for apoptosis by impairing its inhibitory sequestration by VDAC2 on the mitochondrial membrane. In cells expressing both BAX and BAK, repressing their association with VDAC2 promotes apoptosis, because once BAK is activated, it further activates BAX through a feed-forward mechanism. In some leukemias, mutation or loss of BAX is a key driver of resistance to the BH3-mimetic anti-cancer drug venetoclax. Strikingly, promoting BAK-mediated killing by small molecule dissociation of the VDAC2 interaction can overcome this resistance in different leukemia models. These data reveal a hitherto unappreciated level of coordination of BAX and BAK apoptotic activity through their interaction with VDAC2 that may be targeted therapeutically.

cell biology↗

bioRxiv2023

Molecular definition of the BAK:VDAC2 interaction as a target to manipulate apoptosis

BAK and BAX execute intrinsic apoptosis by permeabilising the mitochondrial outer membrane. Their activity is regulated through interactions with pro-survival BCL-2 family proteins and with non-BCL-2 proteins including the mitochondrial porin VDAC2. VDAC2 is important for bringing both BAK and BAX to mitochondria where they execute their apoptotic function. Despite this important function in apoptosis, whilst interactions with pro-survival family members are well characterised and have culminated in the development of drugs that target these interfaces to induce cancer cell apoptosis, the interaction between BAK and VDAC2 remains largely undefined. Deep scanning mutagenesis coupled with cysteine linkage identified key residues in the interaction between BAK and VDAC2. Obstructive labelling of specific residues in the BH3 domain or hydrophobic groove of BAK disrupted this interaction. Conversely, mutating specific residues in a cytosol-exposed region of VDAC2 stabilised the interaction with BAK, and inhibited BAK apoptotic activity. Thus, this VDAC2-BAK interaction site can potentially be targeted to either inhibit BAK-mediated apoptosis in scenarios where excessive apoptosis contributes to disease, or to promote BAK-mediated apoptosis for cancer therapy.

cell biology↗

bioRxiv2021

Membrane permeabilization is mediated by distinct epitopes in mouse and human orthologs of the necroptosis effector, MLKL

Necroptosis is a lytic programmed cell death pathway with origins in innate immunity that is frequently dysregulated in inflammatory diseases. The terminal effector of the pathway, MLKL, is licensed to kill following phosphorylation of its pseudokinase domain by the upstream regulator, RIPK3 kinase. Phosphorylation provokes the unleashing of MLKLs N-terminal four-helix bundle (4HB or HeLo) domain, which binds and permeabilizes the plasma membrane to cause cell death. The precise mechanism by which the 4HB domain permeabilizes membranes, and how the mechanism differs between species, remains unclear. Here, we identify the membrane binding epitope of mouse MLKL using NMR spectroscopy. Using liposome permeabilization and cell death assays, we validate K69 in the 3 helix, W108 in the 4 helix, and R137/Q138 in the first brace helix as crucial residues for necroptotic signaling. This epitope differs from the phospholipid binding site reported for human MLKL, which comprises basic residues primarily located in the 1 and 2 helices. In further contrast to human and plant MLKL orthologs, in which the 3-4 loop forms a helix, this loop is unstructured in mouse MLKL in solution. Together, these findings illustrate the versatility of the 4HB domain fold, whose lytic function can be mediated by distinct epitopes in different orthologs.

cell biology↗

bioRxiv2020

Mechanism and inhibition of SARS-CoV-2 PLpro

Coronaviruses, including SARS-CoV-2, encode multifunctional proteases that are essential for viral replication and evasion of host innate immune mechanisms. The papain-like protease PLpro cleaves the viral polyprotein, and reverses inflammatory ubiquitin and anti-viral ubiquitin-like ISG15 protein modifications1,2. Drugs that target SARS-CoV-2 PLpro (hereafter, SARS2 PLpro) may hence be effective as treatments or prophylaxis for COVID-19, reducing viral load and reinstating innate immune responses3. We here characterise SARS2 PLpro in molecular and biochemical detail. SARS2 PLpro cleaves Lys48-linked polyubiquitin and ISG15 modifications with high activity. Structures of PLpro bound to ubiquitin and ISG15 reveal that the S1 ubiquitin binding site is responsible for high ISG15 activity, while the S2 binding site provides Lys48 chain specificity and cleavage efficiency. We further exploit two strategies to target PLpro. A repurposing approach, screening 3727 unique approved drugs and clinical compounds against SARS2 PLpro, identified no compounds that inhibited PLpro consistently or that could be validated in counterscreens. More promisingly, non-covalent small molecule SARS PLpro inhibitors were able to inhibit SARS2 PLpro with high potency and excellent antiviral activity in SARS-CoV-2 infection models.

biochemistry↗