Knol, J. C. | bioRxiv Science

bioRxiv2024

NUAK1 regulates contraction in vascular smooth muscle cells derived from abdominal aortic aneurysm patients

AimAbdominal aortic aneurysms (AAA) are defined as a dilatation of the aortic wall. Impaired contractile ability of vascular smooth muscle cells (vSMC) is a hallmark of AAA. This study investigates the underlying mechanism of altered in vitro contractility of vSMC derived from AAA patients (AAA-SMC) compared to control vSMC (C-SMC). MethodsContractility of AAA-SMC (n=24) and C-SMC (n=8) was measured using Electric Cell- substrate Impedance Sensing. Large variability in AAA-SMC contraction was observed compared to C-SMC contraction, and AAA-SMC were therefore subdivided into low, intermediate (i.e. comparable to C-SMC contraction) and high contracting. To identify novel proteins involved in altered AAA-SMC contraction, a phosphoproteomic analysis was performed. ResultsThe proteomics data showed that Thrombospondin-1, PDZ and LIM domain protein 4 and ATPase plasma membrane Ca2+ transporting 1 expression correlated with vSMC contraction, but knockdown (KD) of these targets did not affect contraction. Next, the phosphoproteomics data identified NUAK family kinase 1 (NUAK1) as a potential regulator of contraction, since its kinase activity correlated with AAA-SMC contraction. NUAK1 regulates Myosin phosphatase targeting subunit 1 (MYPT1) activity by phosphorylation, as confirmed by the correlation between NUAK1 activity and phosphorylation levels of Ser445 and Ser910 on MYPT1. NUAK1 protein and RNA expression correlated with AAA-SMC contraction. Moreover, NUAK1 KD decreased contraction in AAA-SMC, combined with reduced phosphorylation levels of Myosin light chain (pMLC) and Vinculin gene expression, and increased F-actin cytoskeletal filament levels. ConclusionsNUAK1 regulates AAA-SMC contraction. Low NUAK1 expression decreased pMLC, potentially by higher MYPT1 activity, and subsequently reduced contraction. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/629332v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@41c0f0org.highwire.dtl.DTLVardef@e1dd1borg.highwire.dtl.DTLVardef@7b36d7org.highwire.dtl.DTLVardef@14d65b5_HPS_FORMAT_FIGEXP M_FIG C_FIG Elements were modified from Servier Medical Art, licensed under a Creative Common Attribution 3.0 Generic License. https://smart.servier.com/; https://creativecommons.org/licenses/by/3.0/ HighlightsO_LIQuantification of in vitro vascular smooth muscle cells (vSMC) contractile capacity revealed large variation in contraction of vSMC derived from human AAA tissue (AAA- SMC) compared to vSMC derived from healthy aortic biopsies (C-SMC). C_LIO_LIPhosphoproteomic analysis identified NUAK family kinase 1 (NUAK1) as a novel regulator of contraction in AAA-SMC, since its kinase activity and expression levels correlated with AAA-SMC contraction. C_LIO_LINUAK1 regulates contraction in AAA-SMC by regulating Myosin phosphatase targeting subunit 1 (MYPT1) activity through phosphorylation, which subsequently affects phosphorylation levels of Myosin light chain (pMLC), and by affecting Vinculin expression and F-actin cytoskeletal filament levels. C_LIO_LIGaining more insight into NUAK1 function and mechanisms leading to aortic wall weakening can ultimately contribute to better AAA disease prediction and treatment. C_LI

cell biology↗

bioRxiv2023

Large pan-cancer cell screen coupled to (phospho-) proteomics underscores high-dose vitamin C as a potent anti-cancer agent

Increasing preclinical and clinical evidence has positioned high-dose vitamin C as a promising anti-cancer treatment that merits more clinical attention. Multiple cytotoxicity mechanisms have been described, including pro-oxidant effects. To contribute to the preclinical understanding of the broad pan-cancer effects of high-dose vitamin C in a global manner, we determined the IC50 of a large panel of cancer cell lines (n=51) representing 7 solid tumour types and generated proteome data. The majority of cell lines were highly sensitive (IC50 range 0.036-10mM, mean 1.7 {+/-} 0.4 mM), well below a clinically achievable dose. The proteome data (>5000 proteins per sample), showed that high sensitivity is associated with proliferation, as indicated by functional enrichment of cell cycle, RNA splicing and chromatin organization, while lower sensitivity is linked to extracellular vesicles, glycolysis, fatty acid metabolism and mitochondria. Moreover, (phospho-)proteome analysis of on-treatment vitamin C effects on four pancreatic ductal adenocarcinoma (PDAC) cells dosed at a range of IC50 values (Hs766 T, 2 mM; Capan-2, 0.6 mM; PANC-1, 0.14 mM and Suit-2, 0.1 mM) revealed, next to cell line specific effects, down-modulation of AKT-MTOR signalling and immune suppressive signalling, while IFN- response was enhanced upon vitamin C. Altogether, our comprehensive pharmacological and (phospho-)proteome analysis is the first to assess cancer vulnerabilities and effects of vitamin C on a large cancer cell line panel and underscores the potential of high-dose vitamin C as an anti-cancer agent.

cancer biology↗

bioRxiv2018

INKA, an integrative data analysis pipeline for phosphoproteomic inference of active phosphokinases

Identifying (hyper)active kinases in cancer patient tumors is crucial to enable individualized treatment with specific inhibitors. Conceptually, kinase activity can be gleaned from global protein phosphorylation profiles obtained with mass spectrometry-based phosphoproteomics. A major challenge is to relate such profiles to specific kinases to identify (hyper)active kinases that may fuel growth/progression of individual tumors. Approaches have hitherto focused on phosphorylation of either kinases or their substrates. Here, we combine kinase-centric and substrate-centric information in an Integrative Inferred Kinase Activity (INKA) analysis. INKA utilizes label-free quantification of phosphopeptides derived from kinases, kinase activation loops, kinase substrates deduced from prior experimental knowledge, and kinase substrates predicted from sequence motifs, yielding a single score. This multipronged, stringent analysis enables ranking of kinase activity and visualization of kinase-substrate relation networks in a biological sample. As a proof of concept, INKA scoring of phosphoproteomic data for different oncogene-driven cancer cell lines inferred top activity of implicated driver kinases, and relevant quantitative changes upon perturbation. These analyses show the ability of INKA scoring to identify (hyper)active kinases, with potential clinical significance.

systems biology↗