Biology subjects

Sandow, J. J.

Publications and source records attributed to Sandow, J. J..

4 recordsLinked to original sources

bioRxiv2021

Tankyrase-mediated ADP-ribosylation is a novel regulator of TNF-induced death

Tumor necrosis factor (TNF) is an inflammatory cytokine that, upon binding to its receptor TNFR1, can drive cytokine production, cell survival, or cell death and is a major component of an organisms anti-pathogen repetoire1,2. TNF stimulation leads to the formation of two distinct signalling complexes, a well-defined membrane bound complex (complex 1), and a less well characterised cytosolic death inducing complex (complex 2). Using mass spectrometry, we identified the ADP-ribosyltransferase, tankyrase-1 (TNKS1/TNKS/ARTD5/PARP5a) as a novel native complex 2 component. Following a TNF-induced death stimulus TNKS1 is recruited to complex 2, resulting in complex 2 poly(ADP-ribosyl)ation (PARylation). Tankyrase inhibitors sensitise cells to TNF-induced death, which is correlated with increased complex 2 assembly. Tankyrase-mediated PARylation promotes recruitment of the E3 ligase RNF146 and RNF146 deficiency or proteasome inhibition results in increased levels of complex 2, suggesting that RNF146 causes proteasomal degradation of complex 2. Several viruses express ADP-ribose binding macrodomain proteins, and expression of the SARS-CoV-2 or VEEV macrodomain markedly sensitises cells to TNF-induced death. This suggests that ADP-ribosylation serves as yet another mechanism to detect pathogenic interference of TNF signalling and retaliate with an inflammatory cell death.

cell biology↗

bioRxiv2020

Phosphorylation by Aurora B kinase regulates caspase-2 activity and function

Mitotic catastrophe (MC) is an important oncosuppressive mechanism that serves to eliminate cells that become polyploid or aneuploidy due to aberrant mitosis. Previous studies have demonstrated that the activation and catalytic function of caspase-2 are key steps in MC to trigger apoptosis and/or cell cycle arrest of mitotically defective cells. However, the molecular mechanisms that regulate caspase-2 activation and its function are unclear. Here we identify six new phosphorylation sites in caspase-2 and show that a key mitotic kinase, Aurora B kinase (AURKB), phosphorylates caspase-2 at the highly conserved residue S384. We demonstrate that phosphorylation at S384 blocks caspase-2 catalytic activity and apoptosis function in response to mitotic insults, without affecting caspase-2 dimerisation. Moreover, molecular modelling suggests that phosphorylation at S384 may affect substrate binding by caspase-2. We propose that caspase-2 S384 phosphorylation by AURKB is a key mechanism that controls caspase-2 activation during mitosis.

cell biology↗

bioRxiv2019

Simplified high-throughput methods for deep proteome analysis on the timsTOF Pro

Recent advances in mass spectrometry technology have seen remarkable increases in proteomic sequencing speed, while improvements to dynamic range have remained limited. An exemplar of this is the new timsTOF Pro instrument, which thanks to its trapped ion mobility, pushes effective fragmentation rates beyond 100Hz and provides accurate CCS values as well as impressive sensitivity. Established data dependent methodologies underutilize these advances by relying on long analytical columns and extended LC gradients to achieve comprehensive proteome coverage from biological samples. Here we describe the implementation of methods for short packed emitter columns that fully utilize instrument speed and CCS values by combining rapid generation of deep peptide libraries with enhanced matching of single shot data dependent sample analysis. Impressively, with only a 17 minute separation gradient (50 samples per day), the combination of high performance chromatography and CCS enhanced library based matching resulted in an average of 5,931 protein identifications within individual samples, and 7,244 proteins cumulatively across replicates from HeLa cell tryptic digests. Additionally, an ultra-high throughput setup utilizing 5 min gradients (180 samples per day) yielded an average of 3,666 protein identifications within individual samples and 4,659 proteins cumulatively across replicates. These workflows are simple to implement on available technology and do not require complex software solutions or custom-made consumables to achieve high throughput and deep proteome analysis from biological samples.

biochemistry↗

bioRxiv2018

BAX requires VDAC2 to mediate apoptosis and to limit tumor development

Intrinsic apoptosis is critical for normal physiology including the prevention of tumor formation. BAX and BAK are essential for mediating this process and for the cytotoxic action of many anticancer drugs. BAX and BAK are thought to act in a functionally redundant manner and are considered to be regulated similarly. From an unbiased genome-wide CRISPR/Cas9 screen, we identified VDAC2 (voltage-dependent anion channel 2) as essential for BAX, but not BAK, to function. The genetic deletion of VDAC2 abrogated the association of BAX and BAK with mitochondrial complexes that contain VDAC1, VDAC2 and VDAC3. By disrupting its localization to mitochondria, BAX is rendered completely ineffective. Moreover, we defined an interface unique to VDAC2 that is required to drive BAX activity. Consequently, interfering with this interaction or deleting VDAC2 phenocopied the loss of BAX, including impairing the killing of tumor cells by anti-cancer agents such as the BCL-2 inhibitor venetoclax. Furthermore, the ability of BAX to prevent tumor formation was attenuated in the absence of VDAC2. Taken together, our studies show for the first time that BAX-mediated apoptosis, but not BAK-mediated apoptosis, is critically dependent on VDAC2, hence revealing the differential regulation of BAX and BAK.

cell biology↗