A screening-compatible live cell fluorescence resonance energy transfer-based assay for modulation of Rho GTPase activity

Rho family GTPases are central regulators of cytoskeletal dynamics controlled by guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs). This protocol presents a workflow for a robust high-throughput compatible biosensor assay to analyze changes in Rho GTPase activity by these proteins in the native cellular environment. The procedure can be used for semi-quantitative comparison of GEF/GAP function and extended for analysis of additional modulators. The experimental design is applicable also to other monomolecular ratiometric FRET sensors. For complete details on the use and execution of this protocol, please refer to Müller et al. (2020)

Interaction modulation through arrays of clustered methyl-arginine protein modifications

Systematic analysis of human arginine methylation identifies two distinct signaling modes;either isolated modifications akin to canonical post-translational modification regulation, or clustered arrays within disordered protein sequence. Hundreds of proteins contain these methyl-arginine arrays and are more prone to accumulate mutations and more tightly expression-regulated than dispersed methylation targets. Arginines within an array in the highly methylated RNA-binding protein synaptotagmin binding cytoplasmic RNA interacting protein (SYNCRIP) were experimentally shown to function in concert, providing a tunable protein interaction interface. Quantitative immunoprecipitation assays defined two distinct cumulative binding mechanisms operating across 18 proximal arginine-glycine (RG) motifs in SYNCRIP. Functional binding to the methyltransferase PRMT1 was promoted by continual arginine stretches, whereas interaction with the methyl-binding protein SMN1 was arginine content-dependent irrespective of linear position within the unstructured region. This study highlights how highly repetitive modifiable amino acid arrays in low structural complexity regions can provide regulatory platforms, with SYNCRIP as an extreme example how arginine methylation leverages these disordered sequences to mediate cellular interactions

Bimodal antagonism of PKA signalling by ARHGAP36

Protein kinase A is a key mediator of cAMP signalling downstream of G-protein-coupled receptors, a signalling pathway conserved in all eukaryotes. cAMP binding to the regulatory subunits (PKAR) relieves their inhibition of the catalytic subunits (PKAC). Here we report that ARHGAP36 combines two distinct inhibitory mechanisms to antagonise PKA signalling. First, it blocks PKAC activity via a pseudosubstrate motif, akin to the mechanism employed by the protein kinase inhibitor proteins. Second, it targets PKAC for rapid ubiquitin-mediated lysosomal degradation, a pathway usually reserved for transmembrane receptors. ARHGAP36 thus dampens the sensitivity of cells to cAMP. We show that PKA inhibition by ARHGAP36 promotes derepression of the Hedgehog signalling pathway, thereby providing a simple rationale for the upregulation of ARHGAP36 in medulloblastoma. Our work reveals a new layer of PKA regulation that may play an important role in development and disease

A de-/reacylation cycle controls the localisation and compartmentalised activity of palmitoylated ras

Am Beispiel der Ras GTPasen wurde ein neuartiger Mechanismus charakterisiert, der die spezifische subzelluläre Lokalisation palmitoylierter Proteine an der Plasmamembran (PM) und dem Golgi-Apparat ermöglicht. Diese wird durch einen konstitutiven Zyklus von De- und Reazylierungsreaktionen bestimmt, der den raschen Austausch der Proteine zwischen beiden Membrankompartimenten steuert. Nach Depalmitoylierung verteilt sich farnesyliertes Ras lose über alle zellulären Membranen. Repalmitoylierung am Golgi sorgt für eine stabile Membranverankerung der Proteine, die dann über den sekretorischen Weg zurück zur PM gelangen. Die Stabilität der Palmitoylierung reguliert sowohl die Geschwindigkeit des PM/Golgi Austauschs als auch die PM/Golgi Gleichgewichtsverteilung der Proteine. Der Zyklus sorgt außerdem für die Aktivierung von Ras am Golgi-Apparat durch Transport von Ras-GTP von der PM. Verschiedene De- und Reazylierungskinetiken erlauben hierbei Ras Isoform-spezifische Aktivierungsmuster.A novel mechanism was characterised that accounts for the specific subcellular localisation of membrane associated palmitoylated proteins. As shown for Ras GTPases, their localisation to the plasma membrane (PM) and the Golgi apparatus is maintained by a constitutive de-/reacylation cycle that drives their rapid exchange between these membrane compartments. Depalmitoylation redistributes farnesylated Ras in all membranes followed by repalmitoylation and trapping at the Golgi apparatus from where it is redirected to the PM via the secretory pathway. The stability of palmitate attachment thereby dictates both the speed of retrograde PM to Golgi trafficking and the steady-state distribution of palmitoylated proteins between these two compartments. The de-/reacylation cycle also initiates Ras activity at the Golgi by transport of PM Ras-GTP, thereby generating isoform-specific activation responses due to their different de-/repalmitoylation kinetics

Imaging Activation of Two Ras Isoforms Simultaneously in a Single Cell

Fluorescence resonance energy transfer (FRET) microscopy approaches have been used to study protein interactions in living cells. Up to now, due to the spectral requirements for FRET detection, this has been limited to the measurement of single protein interactions. Here we present a novel time-resolved fluorescence imaging method for simultaneously monitoring the activation state of two proteins in a single cell. A Ras sensor, consisting of fluorescently labelled Ras and a fluorescently labelled Ras binding domain (RBD) of Raf, which reads out Ras activation by its interaction with RBD as a FRET signal, has been adapted for this purpose. By using yellow (YFP) and cyan (CFP) versions of the green fluorescent protein from Aquorea victoria as donors and a tandem construct of Heteractis crispa Red (tHcRed) as acceptor for both donors, two independent FRET signals can be measured at the same time. Measuring the YFP and CFP donor lifetimes by fluorescence-lifetime imaging microscopy (FLIM) allows us to distinguish the two different FRET signals in a single cell. Using this approach, we show that different Ras isoforms and mutants that localize to the plasma membrane, to the Golgi or to both compartments display distinct activation profiles upon growth-factor stimulation; this indicates that there is a differential regulation in cellular compartments. The method presented here is especially useful when studying spatiotemporal aspects of protein regulation as part of larger cellular signalling networks

A new role of the Rac-GAP ß2-chimaerin in cell adhesion reveals opposite functions in breast cancer initiation and tumor progression

β2-chimaerin is a Rac1-specific negative regulator and a candidate tumor suppressor in breast cancer but its precise function in mammary tumorigenesis in vivo is unknown. Here, we study for the first time the role of β2-chimaerin in breast cancer using a mouse model and describe an unforeseen role for this protein in epithelial cell-cell adhesion. We demonstrate that expression of β2-chimaerin in breast cancer epithelial cells reduces E-cadherin protein levels, thus loosening cell-cell contacts. In vivo, genetic ablation of β2-chimaerin in the MMTV-Neu/ErbB2 mice accelerates tumor onset, but delays tumor progression. Finally, analysis of clinical databases revealed an inverse correlation between β2-chimaerin and E-cadherin gene expressions in Her2+ breast tumors. Furthermore, breast cancer patients with low β2-chimaerin expression have reduced relapse free survival but develop metastasis at similar times. Overall, our data redefine the role of β2-chimaerin as tumor suppressor and provide the first in vivo evidence of a dual function in breast cancer, suppressing tumor initiation but favoring tumor progression.This work was initially supported by a grant from the Spanish Ministry of Economy and Competitiveness to MJC (BFU2009-08051), but could be finished only thanks to the support from the Castilla-León Autonomous Government to MJC (grants BIO103/VA44/11, BIO/VA22/14, CSI090U14 and BIO/VA34/15).Peer Reviewe

The use of multicriteria decision analysis to support decision making in healthcare: an updated systematic literature review

Objectives Multicriteria decision analysis (MCDA) is increasingly used for decision making in healthcare. However, its application in different decision-making contexts is still unclear. This study aimed to provide a comprehensive review of MCDA studies performed to inform decisions in healthcare and to summarize its application in different decision contexts. Methods We updated a systematic review conducted in 2013 by searching Embase, MEDLINE, and Google Scholar for MCDA studies in healthcare, published in English between August 2013 and November 2020. We also expanded the search by reviewing grey literature found via Trip Medical Database and Google, published between January 1990 and November 2020. A comprehensive template was developed to extract information about the decision context, criteria, methods, stakeholders involved, and sensitivity analyses conducted. Results From the 4295 identified studies, 473 studies were eligible for full-text review after assessing titles and abstracts. Of those, 228 studies met the inclusion criteria and underwent data extraction. The use of MCDA continues to grow in healthcare literature, with most of the studies (49%) informing priority-setting decisions. Safety, cost, and quality of care delivery are the most frequently used criteria, although there are considerable differences across decision contexts. Almost half of the MCDA studies used the linear additive model whereas scales and the analytical hierarchy process were the most used techniques for scoring and weighting, respectively. Not all studies report on each one of the MCDA steps, consider axiomatic properties, or justify the methods used. Conclusions A guide on how to conduct and report MCDA that acknowledges the particularities of the different decision contexts and methods needs to be developed