Biology subjects

Sapre, T.

Publications and source records attributed to Sapre, T..

3 recordsLinked to original sources

bioRxiv2024

diaPASEF-Powered Chemoproteomics Enables Deep Kinome Interaction Profiling

Protein-protein interactions (PPIs) underlie most biological functions. Devastating human conditions like cancers, neurological disorders, and infections, hijack PPI networks to initiate disease, and to drive disease progression. Understanding precisely how diseases remodel PPI networks can, therefore, help clarify disease mechanisms and identify therapeutic targets. Protein kinases control most cellular processes through protein phosphorylation. The 518 human kinases, known as the kinome, are frequently dysregulated in disease and highly druggable with ATP-competitive inhibitors. Kinase activity, localization, and substrate recognition are regulated by dynamic PPI networks composed of scaffolding and adapter proteins, other signaling enzymes like small GTPases and E3 ligases, and phospho-substrates. Accordingly, mapping kinase PPI networks can help determine kinome activation states, and, in turn, cellular activation states; this information can be used for studying kinase-mediated cell signaling, and for prioritizing kinases for drug discovery. Previously, we have developed a high-throughput method for kinome PPI mapping based on mass spectrometry (MS)-based chemoproteomics that we named kinobead competition and correlation analysis (kiCCA). Here, we introduce 2nd generation (gen) kiCCA which utilizes data-independent acquisition (dia) with parallel accumulation serial fragmentation (PASEF) MS and a re-designed CCA algorithm with improved selection criteria and the ability to predict multiple kinase interaction partners of the same proteins. Using neuroblastoma cell line models of the noradrenergic-mesenchymal transition (NMT), we demonstrate that 2nd gen kiCCA (1) identified 6.1-times more kinase PPIs in native cell extracts compared to our 1st gen approach, (2) determined kinase-mediated signaling pathways that underly the neuroblastoma NMT, and (3) accurately predicted pharmacological targets for manipulating NMT states. Our 2nd gen kiCCA method is broadly useful for cell signaling research and kinase drug discovery.

biochemistry↗

bioRxiv2024

Mitochondrial Calcium Signaling Regulates Branched-Chain Amino Acid Catabolism in Fibrolamellar Carcinoma

Metabolic adaptations in response to changes in energy supply and demand are essential for survival. The mitochondrial calcium uniporter plays a key role in coordinating metabolic homeostasis by regulating TCA cycle activation, mitochondrial fatty acid oxidation, and cellular calcium signaling. However, a comprehensive analysis of uniporter-regulated mitochondrial pathways has remained unexplored. Here, we investigate metabolic consequences of uniporter loss- and gain-of-function using uniporter knockout cells and the liver cancer fibrolamellar carcinoma (FLC), which we demonstrate to have elevated mitochondrial calcium levels. Our results reveal that branched-chain amino acid (BCAA) catabolism and the urea cycle are uniporter-regulated metabolic pathways. Reduced uniporter function boosts expression of BCAA catabolism genes, and the urea cycle enzyme ornithine transcarbamylase (OTC). In contrast, high uniporter activity in FLC suppresses their expression. This suppression is mediated by reduced expression of the transcription factor KLF15, a master regulator of liver metabolism. Thus, uniporter responsive calcium signaling plays a central role in FLC-associated metabolic changes, including hyperammonemia. Our study identifies an important role for mitochondrial calcium signaling in metabolic adaptation through transcriptional regulation of metabolism and elucidates its importance for BCAA and ammonia metabolism in FLC.

cell biology↗

bioRxiv2022

Neurodevelopmental disorder associated mutations in TAOK1 reveal its function as a plasma membrane remodeling kinase

Mutations in TAOK1, a serine-threonine kinase encoding gene are strongly associated with both autism spectrum disorder (ASD) and neurodevelopmental delay (NDD). However, molecular function of this evolutionarily conserved kinase and the mechanisms through which TAOK1 mutations lead to neuropathology are unknown. Here, we showed that TAOK1 is highly expressed in neurons within the brain, and has a functional role in remodeling the plasma membrane through direct association with phosphoinositides. We characterized four NDD-associated TAOK1 mutations, and demonstrated that these mutations render TAOK1 catalytically dead. Kinase dead TAOK1 mutants were aberrantly trapped in membrane-bound state, which induced exuberant membrane protrusions. Expression of TAOK1 disease mutants in hippocampal neurons led to abnormal growth of the dendritic arbor. The coiled-coil region C-terminal to the kinase domain are predicted to fold into a triple helix. We showed that this triple helix directly bound phospholipids, and was required for both TAOK1 membrane association and induction of aberrant protrusions. Further, TAOK1 mutants were rescued from their membrane-trapped state by exogenous expression of the isolated kinase domain. Utilizing mass-spectrometry, we identified critical residues in the triple helix phosphorylated by TAOK1 that autoregulated its plasma membrane association. These findings define a previously unknown function of TAOK1 as a unique plasma membrane remodeling kinase, and reveal the underlying mechanisms through which TAOK1 dysfunction leads to neurodevelopmental disorders.

neuroscience↗