Sergio Lilla

Comparative Large Scale Characterization of Plant versus Mammal Proteins Reveals Similar and Idiosyncratic N-α-Acetylation Features

N-terminal modifications play a major role in the fate of proteins in terms of activity, stability, or subcellular compartmentalization. Such modifications remain poorly described and badly characterized in proteomic studies, and only a few comparison studies among organisms have been made available so far. Recent advances in the field now allow the enrichment and selection of N-terminal peptides in the course of proteome-wide mass spectrometry analyses. These targeted approaches unravel as a result the extent and nature of the protein N-terminal modifications. Here, we aimed at studying such modifications in the model plant Arabidopsis thaliana to compare these results with those obtained from a human sample analyzed in parallel. We applied large scale analysis to compile robust conclusions on both data sets. Our data show strong convergence of the characterized modifications especially for protein N-terminal methionine excision, co-translational N-α-acetylation, or N-myristoylation between animal and plant kingdoms. Because of the convergence of both the substrates and the N-α-acetylation machinery, it was possible to identify the N-acetyltransferases involved in such modifications for a small number of model plants. Finally, a high proportion of nuclear-encoded chloroplast proteins feature post-translational N-α-acetylation of the mature protein after removal of the transit peptide. Unlike animals, plants feature in a dedicated pathway for post-translational acetylation of organelle-targeted proteins. The corresponding machinery is yet to be discovered.

PHD1 regulates p53-mediated colorectal cancer chemoresistance

Overcoming resistance to chemotherapy is a major challenge in colorectal cancer (CRC) treatment, especially since the underlying molecular mechanisms remain unclear. We show that silencing of the prolyl hydroxylase domain protein PHD1, but not PHD2 or PHD3, prevents p53 activation upon chemotherapy in different CRC cell lines, thereby inhibiting DNA repair and favoring cell death. Mechanistically, PHD1 activity reinforces p53 binding to p38α kinase in a hydroxylation‐dependent manner. Following p53–p38α interaction and chemotherapeutic damage, p53 can be phosphorylated at serine 15 and thus activated. Active p53 allows nucleotide excision repair by interacting with the DNA helicase XPB, thereby protecting from chemotherapy‐induced apoptosis. In accord with this observation, PHD1 knockdown greatly sensitizes CRC to 5‐FU in mice. We propose that PHD1 is part of the resistance machinery in CRC, supporting rational drug design of PHD1‐specific inhibitors and their use in combination with chemotherapy.

Cucurbitacin covalent bonding to cysteine thiols: the filamentous-actin severing protein Cofilin1 as an exemplary target

BackgroundCucurbitacins are a class of triterpenoid natural compounds with potent bioactivities that led to their use as traditional remedies, and which continue to attract considerable attention as chemical biology tools and potential therapeutics. One obvious target is the actin-cytoskeleton; treatment with cucurbitacins results in cytoskeletal rearrangements that impact upon motility and cell morphology.FindingsCucurbitacin reacted with protein cysteine thiols as well as dithiothreitol, and we propose that the cucurbitacin mechanism of action is through broad protein thiol modifications that could result in inhibition of numerous protein targets. An example of such a target protein is Cofilin1, whose filamentous actin severing activity is inhibited by cucurbitacin conjugation.ConclusionsThe implications of these results are that cucurbitacins are unlikely to be improved for selectivity by medicinal chemistry and that their use as chemical biology probes to analyse the role of specific signalling pathways should be undertaken with caution.

Tyrosine phosphorylation of WIP releases bound WASP and impairs podosome assembly in macrophages

ABSTRACT Podosomes are integrin-containing adhesion structures commonly found in migrating leukocytes of the monocytic lineage. The actin cytoskeletal organisation of podosomes is based on a WASP- and Arp2/3-mediated mechanism. WASP also associates with a second protein, WIP (also known as WIPF1), and they co-localise in podosome cores. Here, we report for the first time that WIP can be phosphorylated on tyrosine residues and that tyrosine phosphorylation of WIP is a trigger for release of WASP from the WIP–WASP complex. Using a knockdown approach together with expression of WIP phosphomimics, we show that in the absence of WIP–WASP binding, cellular WASP is rapidly degraded, leading to disruption of podosomes and a failure of cells to degrade an underlying matrix. In the absence of tyrosine phosphorylation, the WIP–WASP complex remains intact and podosome lifetimes are extended. A screen of candidate kinases and inhibitor-based assays identified Brutons tyrosine kinase (Btk) as a regulator of WIP tyrosine phosphorylation. We conclude that tyrosine phosphorylation of WIP is a crucial regulator of WASP stability and function as an actin-nucleation-promoting factor.

Secreted CLIC3 drives cancer progression through its glutathione-dependent oxidoreductase activity.

The secretome of cancer and stromal cells generates a microenvironment that contributes to tumour cell invasion and angiogenesis. Here we compare the secretome of human mammary normal and cancer-associated fibroblasts (CAFs). We discover that the chloride intracellular channel protein 3 (CLIC3) is an abundant component of the CAF secretome. Secreted CLIC3 promotes invasive behaviour of endothelial cells to drive angiogenesis and increases invasiveness of cancer cells both in vivo and in 3D cell culture models, and this requires active transglutaminase-2 (TGM2). CLIC3 acts as a glutathione-dependent oxidoreductase that reduces TGM2 and regulates TGM2 binding to its cofactors. Finally, CLIC3 is also secreted by cancer cells, is abundant in the stromal and tumour compartments of aggressive ovarian cancers and its levels correlate with poor clinical outcome. This work reveals a previously undescribed invasive mechanism whereby the secretion of a glutathione-dependent oxidoreductase drives angiogenesis and cancer progression by promoting TGM2-dependent invasion.

Proteome-wide analysis of cysteine oxidation reveals metabolic sensitivity to redox stress

Reactive oxygen species (ROS) are increasingly recognised as important signalling molecules through oxidation of protein cysteine residues. Comprehensive identification of redox-regulated proteins and pathways is crucial to understand ROS-mediated events. Here, we present stable isotope cysteine labelling with iodoacetamide (SICyLIA), a mass spectrometry-based workflow to assess proteome-scale cysteine oxidation. SICyLIA does not require enrichment steps and achieves unbiased proteome-wide sensitivity. Applying SICyLIA to diverse cellular models and primary tissues provides detailed insights into thiol oxidation proteomes. Our results demonstrate that acute and chronic oxidative stress causes oxidation of distinct metabolic proteins, indicating that cysteine oxidation plays a key role in the metabolic adaptation to redox stress. Analysis of mouse kidneys identifies oxidation of proteins circulating in biofluids, through which cellular redox stress can affect whole-body physiology. Obtaining accurate peptide oxidation profiles from complex organs using SICyLIA holds promise for future analysis of patient-derived samples to study human pathologies.Reactive oxygen species can modify cysteine residues on proteins to control cellular signalling. Here, the authors develop a proteomics approach to assess cysteine oxidation dynamics in cells and primary tissues, monitoring oxidative stress-induced metabolic adaptations and oxidation states of biofluid proteins.

The inositol-3-phosphate synthase biosynthetic enzyme has distinct catalytic and metabolic roles

ABSTRACT Inositol levels, maintained by the biosynthetic enzyme inositol-3-phosphate synthase (Ino1), are altered in a range of disorders, including bipolar disorder and Alzheimers disease. To date, most inositol studies have focused on the molecular and cellular effects of inositol depletion without considering Ino1 levels. Here we employ a simple eukaryote, Dictyostelium discoideum, to demonstrate distinct effects of loss of Ino1 and inositol depletion. We show that loss of Ino1 results in an inositol auxotrophy that can be rescued only partially by exogenous inositol. Removal of inositol supplementation from the ino1− mutant resulted in a rapid 56% reduction in inositol levels, triggering the induction of autophagy, reduced cytokinesis, and substrate adhesion. Inositol depletion also caused a dramatic generalized decrease in phosphoinositide levels that was rescued by inositol supplementation. However, loss of Ino1 triggered broad metabolic changes consistent with the induction of a catabolic state that was not rescued by inositol supplementation. These data suggest a metabolic role for Ino1 that is independent of inositol biosynthesis. To characterize this role, an Ino1 binding partner containing SEL1L1 domains (Q54IX5) and having homology to mammalian macromolecular complex adaptor proteins was identified. Our findings therefore identify a new role for Ino1, independent of inositol biosynthesis, with broad effects on cell metabolism.

Colorectal Tumors Require NUAK1 for Protection from Oxidative Stress

Exploiting oxidative stress has recently emerged as a plausible strategy for treatment of human cancer, and antioxidant defenses are implicated in resistance to chemotherapy and radiotherapy. Targeted suppression of antioxidant defenses could thus broadly improve therapeutic outcomes. Here, we identify the AMPK-related kinase NUAK1 as a key component of the antioxidant stress response pathway and reveal a specific requirement for this role of NUAK1 in colorectal cancer. We show that NUAK1 is activated by oxidative stress and that this activation is required to facilitate nuclear import of the antioxidant master regulator NRF2: Activation of NUAK1 coordinates PP1β inhibition with AKT activation in order to suppress GSK3β-dependent inhibition of NRF2 nuclear import. Deletion of NUAK1 suppresses formation of colorectal tumors, whereas acute depletion of NUAK1 induces regression of preexisting autochthonous tumors. Importantly, elevated expression of NUAK1 in human colorectal cancer is associated with more aggressive disease and reduced overall survival.Significance: This work identifies NUAK1 as a key facilitator of the adaptive antioxidant response that is associated with aggressive disease and worse outcome in human colorectal cancer. Our data suggest that transient NUAK1 inhibition may provide a safe and effective means for treatment of human colorectal cancer via disruption of intrinsic antioxidant defenses. Cancer Discov; 8(5); 632-47. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 517.

The trimeric coiled‐coil HSBP1 protein promotes WASH complex assembly at centrosomes

The Arp2/3 complex generates branched actin networks that exert pushing forces onto different cellular membranes. WASH complexes activate Arp2/3 complexes at the surface of endosomes and thereby fission transport intermediates containing endocytosed receptors, such as α5β1 integrins. How WASH complexes are assembled in the cell is unknown. Here, we identify the small coiled‐coil protein HSBP1 as a factor that specifically promotes the assembly of a ternary complex composed of CCDC53, WASH, and FAM21 by dissociating the CCDC53 homotrimeric precursor. HSBP1 operates at the centrosome, which concentrates the building blocks. HSBP1 depletion in human cancer cell lines and in Dictyostelium amoebae phenocopies WASH depletion, suggesting a critical role of the ternary WASH complex for WASH functions. HSBP1 is required for the development of focal adhesions and of cell polarity. These defects impair the migration and invasion of tumor cells. Overexpression of HSBP1 in breast tumors is associated with increased levels of WASH complexes and with poor prognosis for patients.

Fam49/CYRI interacts with Rac1 and locally suppresses protrusions

Actin-based protrusions are reinforced through positive feedback, but it is unclear what restricts their size, or limits positive signals when they retract or split. We identify an evolutionarily conserved regulator of actin-based protrusion: CYRI (CYFIP-related Rac interactor) also known as Fam49 (family of unknown function 49). CYRI binds activated Rac1 via a domain of unknown function (DUF1394) shared with CYFIP, defining DUF1394 as a Rac1-binding module. CYRI-depleted cells have broad lamellipodia enriched in Scar/WAVE, but reduced protrusion–retraction dynamics. Pseudopods induced by optogenetic Rac1 activation in CYRI-depleted cells are larger and longer lived. Conversely, CYRI overexpression suppresses recruitment of active Scar/WAVE to the cell edge, resulting in short-lived, unproductive protrusions. CYRI thus focuses protrusion signals and regulates pseudopod complexity by inhibiting Scar/WAVE-induced actin polymerization. It thus behaves like a ‘local inhibitor’ as predicted in widely accepted mathematical models, but not previously identified in cells. CYRI therefore regulates chemotaxis, cell migration and epithelial polarization by controlling the polarity and plasticity of protrusions.Fort et al. identify CYRI as a conserved negative modulator of Scar–WAVE-induced lamellipodia by interacting directly with active Rac1, thereby conferring pseudopod plasticity and dynamics during motility.