Biology subjects

Suen, K. M.

Publications and source records attributed to Suen, K. M..

9 recordsLinked to original sources

bioRxiv2024

Affinity purification mass spectrometry characterization of the interactome of receptor tyrosine kinase proline rich motifs in cancer

Receptor tyrosine kinase (RTK) overexpression is linked to the development and progression of multiple cancers. RTKs are classically considered to initiate cytoplasmic signalling pathways via ligand-induced tyrosine phosphorylation, however recent evidence points to a second tier of signalling contingent on interactions mediated by the proline-rich motif (PRM) regions of non-activated RTKs. The presence of PRMs on the C-termini of >40% of all RTKs and the abundance of PRM-binding proteins encoded by the human genome suggests that there is likely to be a large number of previously unexplored interactions which add to the RTK intracellular interactome. Here, we explore the RTK PRM interactome and its potential significance using affinity purification mass spectrometry and in silico enrichment analyses. Peptides comprising PRM-containing C-terminal tail regions of EGFR, FGFR2 and HER2 were used as bait to affinity purify bound proteins from different cancer cell line lysates. 490 unique interactors were identified, amongst which proteins with metabolic, homeostatic and migratory functions were overrepresented. This suggests that PRMs from RTKs may sustain a diverse interactome in cancer cells. Since RTK overexpression is common in cancer RTK PRM-derived signalling may be an important, but as yet underexplored, contributor to negative cancer outcomes including resistance to kinase inhibitors.

cell biology↗

bioRxiv2023

Differential labelling of human sub-cellular compartments with fluorescent dye esters and expansion microscopy

Amine-reactive esters of aromatic fluorescent dyes are emerging as imaging probes for nondescript staining of cellular and tissue architectures. We characterised the differential staining patterns of 14 fluorescent dye ester species with varying physical and spectral properties in the broadly studied human cell line - HeLa. When combined with expansion microscopy (ExM), these stains reveal nanoscale features such as the nuclear proteome, membrane-bound compartments and vesicles. Among N-Hydroxysuccinimide (NHS) esters, we observe differential compartment specificity and weighting of labelling density which correlates with the hydrophobicity of the dye ester. We also observe changes in both staining density and compartment specificity for a given dye ester depending on the presence of a second dye ester species and on the timepoint of application in the ExM protocol. Our findings confirm these dye esters as a useful addition to the repertoire of biomedical stains of the cellular proteome, either on their own, or as counterstains to immunofluorescence.

cell biology↗

bioRxiv2022

Expansion microscopy reveals subdomains in C. elegans germ granules

Many membraneless organelles (MLOs) have been shown to form via liquid-liquid phase separation (LLPS). Light and electron microscopy techniques have been indispensable in the identification and characterization of LLPS MLOs. However, for complex MLOs such as the perinuclear germ granule in C. elegans, our understanding of how the organelle as a whole is regulated is hampered by 1) the technical limitations in confocal fluorescence imaging in which only a few granule protein markers can be examined at a time and 2) the inaccessibility of electron microscopy. In this study, we take advantage of the newly developed super-resolution method of expansion microscopy (ExM) and in-situ staining of the whole proteome to examine the C. elegans germ granule, the P granule. We show that in small RNA pathway mutants, the P granule is smaller compared with wild type animals. Furthermore, we investigate the relationship between the P granule and two other germ granules, mutator foci and Z granule, and show that they are located within the same protein-dense regions while occupying distinct subdomains within this ultrastructure. The experimental workflow developed here will serve as an important tool in our understanding of germ granule biology as well as the biological role of LLPS.

cell biology↗

bioRxiv2022

Identification of putative reader proteins of 5-methylcytosine and its derivatives in Caenorhabditis elegans RNA

Methylation of carbon-5 of cytosines (m5C) is a post-transcriptional nucleotide modification of RNA with widespread distribution across organisms. m5C has been shown to participate in mRNA transport and maintain mRNA stability through its recognition by the reader proteins ALYREF and YBX1, respectively. We recently showed that m5C is required for Caenorhabditis elegans development and fertility under heat stress. To contribute to the understanding of how m5C and its oxidative derivatives mediate their functions, we developed RNA baits bearing modified cytosines in diverse structural contexts to pulldown potential readers in C. elegans. Our mass spectrometry analyses reveal unique binding proteins for each of the modifications. We validate our dataset by demonstrating that the nematode ALYREF homologues ALY-1 and ALY-2 preferentially bind m5C in vitro. The dataset presented here serves as an important scientific resource that will support the discovery of new functions of m5C and its derivatives.

biochemistry↗

bioRxiv2020

Regulation of kinase activity by combined action of juxtamembrane and C-terminal regions of receptors

Despite the kinetically-favorable, ATP-rich intracellular environment, the mechanism by which receptor tyrosine kinases (RTKs) repress activation prior to extracellular stimulation is poorly understood. RTKs are activated through a precise sequence of phosphorylation reactions starting with a tyrosine on the activation loop (A-loop) of the intracellular kinase domain (KD). This forms an essential mono-phosphorylated active intermediate state on the path to further phosphorylation of the receptor. We show that this state is subjected to stringent control imposed by the peripheral juxtamembrane (JM) and C-terminal tail (CT) regions. This entails interplay between the intermolecular interaction between JM with KD, which stabilizes the asymmetric active KD dimer, and the opposing intramolecular binding of CT to KD. A further control step is provided by the previously unobserved direct binding between JM and CT. Mutations in JM and CT sites that perturb regulation are found in numerous pathologies, revealing novel sites for potential pharmaceutical intervention.

biochemistry↗

bioRxiv2019

Grb2 binding induces phosphorylation-independent activation of Shp2

The regulation of phosphatase activity is fundamental to the control of intracellular signalling and in particular the tyrosine kinase-mediated mitogen-activated protein kinase (MAPK) pathway. Shp2 is a ubiquitously expressed protein tyrosine phosphatase and its kinase-induced hyperactivity is associated with many cancer types. In non-stimulated cells we find that binding of the adaptor protein, Grb2, in its monomeric state initiates Shp2 activity independent of phosphatase phosphorylation. Grb2 forms a bidentate interaction with both the N-terminal SH2 and the catalytic domains of Shp2, releasing the phosphatase from its auto-inhibited conformation. Grb2 typically exists as a dimer in the cytoplasm. However, its monomeric state prevails under basal conditions when it is expressed at low concentration, or when it is constitutively phosphorylated on a specific tyrosine residue (Y160). Thus, Grb2 can activate Shp2 and downstream signal transduction, in the absence of extracellular growth factor stimulation or kinase-activating mutations, in response to defined cellular conditions. We identify a polypeptide biotool capable of blocking the Grb2-Shp2 interaction. This peptide down-regulates Shp2 activity in vitro and MAPK signalling in a cancer cell line.

cancer biology↗

bioRxiv2019

Receptor tyrosine kinases regulate signal transduction through a liquid-liquid phase separated state

Receptor tyrosine kinases (RTKs), the largest class of transmembrane cell surface receptors, initiate signalling pathways which regulate diverse cellular processes. On activation these receptors rapidly recruit multiple downstream effector proteins to moderate affinity tyrosyl phosphate (pY) binding sites. However, the mechanism for expedient downstream effector protein recruitment via random molecular diffusion through the cytoplasm is not fully understood. One way in which the probabilistic outcome associated with random diffusion could be alleviated is through localized accumulation of high effective concentrations of signalling proteins in discrete pools in the cell (1). The inclusion of interacting proteins into liquid-liquid phase-separated (LLPS), membraneless protein droplets maintains functionally relevant proteins at high concentrations in a liquid phase at the required point of action, enhancing equilibrium binding and enzyme activity (2-6). These LLPS states have been associated with a wide range of cellular functions including regulation of signalling through, for example, nephrin (7, 8), the T-cell receptor (9), mTOR (10), and Sos-Ras (11), however, whether LLPS extends to RTK-mediated signal transduction has not been investigated. Here, we show that an RTK, fibroblast growth factor receptor 2 (FGFR2), forms a signalling competent LLPS state with two downstream effectors, a tandem Src homology 2 (SH2) domain-containing protein tyrosine phosphatase 2 (Shp2), and 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma 1 (Plc{gamma}1). We show that these proteins assemble into a ternary complex which exploits LLPS condensation to simultaneously modulate kinase, phosphatase and phospholipase activities. Therefore, LLPS formation ensures that the requirement for prolonged, high-fidelity signalling is achieved. Additional RTKs also form LLPS with their downstream effectors, suggesting that formation of biological condensates is a key organising principle of RTK-mediated signalling, with broad implications for further mechanistic studies as well as therapeutic intervention.

cell biology↗

bioRxiv2019

Direct interaction of PIWI and DEPS-1 is essential for piRNA function and condensate ultrastructure in Caenorhabditis elegans

Membraneless organelles are platforms for many aspects of RNA biology including small non-coding RNA (ncRNA) mediated gene silencing. How small ncRNAs utilise phase separated environments for their function is unclear. To address this question, we investigated how the PIWI-interacting RNA (piRNA) pathway engages with the membraneless organelle P granule in Caenorhabditis elegans. Proteomic analysis of the PIWI protein PRG-1 revealed an interaction with the constitutive P granule protein DEPS-1. Furthermore we identified a novel motif on DEPS-1, PBS, which interacts directly with the Piwi domain of PRG-1. This protein complex forms intertwining ultrastructures to build elongated condensates in vivo. These sub-organelle ultrastructures depend on the Piwi-interacting motif of DEPS-1 and mediate piRNA function. Additionally, we identify a novel interactor of DEPS-1, EDG-1, which is required for DEPS-1 condensates to form correctly. We show that DEPS-1 is not required for piRNA biogenesis but piRNA function: deps-1 mutants fail to produce the secondary endo-siRNAs required for the silencing of piRNA targets. Our study reveals how specific protein-protein interactions drive the spatial organisation and function of small RNA pathways within membraneless organelles.

cell biology↗

bioRxiv2016

The Aquarius/EMB-4 helicase licenses co-transcriptional gene silencing

Small RNAs (sRNAs) play an ancient role in genome defence against transposable elements. In animals, plants and fungi small RNAs guide Argonaute proteins to nascent RNA transcripts to induce co-transcriptional gene silencing. In animals the link between small RNA pathways and the transcriptional machinery remains unclear. Here we show that the Caenorhabditis elegans germline Argonaute HRDE-1 physically interacts with the conserved RNA helicase Aquarius/EMB-4. We demonstrate that the Aquarius/EMB-4 helicase activity is required to initiate small RNA-induced co-transcriptional gene silencing. HRDE-1 and Aquarius/EMB-4 are required to silence the transcription of overlapping sets of transposable elements. Surprisingly, removal of introns from a small RNA pathway target abolishes the requirement for Aquarius/EMB-4, but not HRDE-1, for gene silencing. We conclude that the Aquarius/EMB-4 helicase activity allows HRDE-1/sRNA complexes to efficiently engage nascent RNA transcripts - in competition with the general RNA processing machinery. We postulate that Aquarius/EMB-4 facilitates the surveillance of the nascent transcriptome to detect and silence transposable elements through small RNA pathways.

molecular biology↗