54 results on '"Shehab Ismail"'
Search Results
2. Bacterial delivery of the anti-tumor azurin-like protein Laz to glioblastoma cells
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Manar Mansour, Shehab Ismail, and Khaled Abou-Aisha
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Hypoxia ,Salmonella VNP20009 ,Azurin-like protein ,Glioblastoma ,Biotechnology ,TP248.13-248.65 ,Microbiology ,QR1-502 - Abstract
Abstract Salmonella typhimurium VNP-20009 (VNP) is a non-pathogenic attenuated strain, which, as a facultative anaerobe, preferentially accumulates in hypoxic regions of solid tumors. Here, VNP was utilized as a delivery vehicle of the anti-tumor protein Lipidated azurin, Laz, which is produced by the meningitis-causing bacterium Neisseria meningitides. In brain cancer cells, Laz has been demonstrated to induce apoptosis through an interaction with the tumor suppressor protein p53. In this study, the laz gene, including its signal sequence, was cloned downstream of a hypoxia inducible promoter (HIP-1), before being electroporated into VNP. Successful ectopic expression and export of the Laz protein by VNP under hypoxic conditions were confirmed by Western blot analysis of the cell-free culture medium. Effective expression of Laz by VNP was investigated in two glioblastoma cell lines: LN-229 and U-373, with the latter line carrying a mutated version of p53; as well as in the breast cancer line MCF-7. Cytotoxicity of the VNP-Laz was assessed by determining the fluorescence of the apoptotic marker caspases 3/7. Compared to the purified Laz, VNP-Laz, significantly induced apoptosis in MCF-7, LN-229 and, to a much lower extent in U-373 cells, suggesting a p53-linked mechanism. Our results might represent a new approach of targeted gene delivery and suggest a potential application in brain tumor therapy.
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- 2020
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3. ARL3 activation requires the co-GEF BART and effector-mediated turnover
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Yasmin ElMaghloob, Begoña Sot, Michael J McIlwraith, Esther Garcia, Tamas Yelland, and Shehab Ismail
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primary cilia ,small GTPases ,arf like proteins ,GEFs ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
The ADP-ribosylation factor-like 3 (ARL3) is a ciliopathy G-protein which regulates the ciliary trafficking of several lipid-modified proteins. ARL3 is activated by its guanine exchange factor (GEF) ARL13B via an unresolved mechanism. BART is described as an ARL3 effector which has also been implicated in ciliopathies, although the role of its ARL3 interaction is unknown. Here, we show that, at physiological GTP:GDP levels, human ARL3GDP is weakly activated by ARL13B. However, BART interacts with nucleotide-free ARL3 and, in concert with ARL13B, efficiently activates ARL3. In addition, BART binds ARL3GTP and inhibits GTP dissociation, thereby stabilising the active G-protein; the binding of ARL3 effectors then releases BART. Finally, using live cell imaging, we show that BART accesses the primary cilium and colocalises with ARL13B. We propose a model wherein BART functions as a bona fide co-GEF for ARL3 and maintains the active ARL3GTP, until it is recycled by ARL3 effectors.
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- 2021
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4. Secreted CLIC3 drives cancer progression through its glutathione-dependent oxidoreductase activity
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Juan R. Hernandez-Fernaud, Elena Ruengeler, Andrea Casazza, Lisa J. Neilson, Ellie Pulleine, Alice Santi, Shehab Ismail, Sergio Lilla, Sandeep Dhayade, Iain R. MacPherson, Iain McNeish, Darren Ennis, Hala Ali, Fernanda G. Kugeratski, Heba Al Khamici, Maartje van den Biggelaar, Peter V.E. van den Berghe, Catherine Cloix, Laura McDonald, David Millan, Aoisha Hoyle, Anna Kuchnio, Peter Carmeliet, Stella M. Valenzuela, Karen Blyth, Huabing Yin, Massimiliano Mazzone, Jim C. Norman, and Sara Zanivan
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Science - Abstract
The secretome from cancer and stromal cells contributes to the creation of a microenvironment, which in turn contributes to invasion and angiogenesis. Here, the authors compare the secretomes of immortalized normal fibroblasts and cancer-derived fibroblast and identify CLIC3 as a driver of cancer progression.
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- 2017
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5. PDE6δ-mediated sorting of INPP5E into the cilium is determined by cargo-carrier affinity
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Eyad Kalawy Fansa, Stefanie Kristine Kösling, Eldar Zent, Alfred Wittinghofer, and Shehab Ismail
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Science - Abstract
PDE6δ regulates the sorting of prenylated cargo proteins. Here Fansa et al. propose that the affinity of the interaction between PDE6δ and its cargo protein determines whether they are released by cytoplasmic or cilia-specific release factors ultimately determining their subcellular localization.
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- 2016
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6. Identification of pyrazolopyridazinones as PDEδ inhibitors
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Björn Papke, Sandip Murarka, Holger A Vogel, Pablo Martín-Gago, Marija Kovacevic, Dina C Truxius, Eyad K Fansa, Shehab Ismail, Gunther Zimmermann, Kaatje Heinelt, Carsten Schultz-Fademrecht, Alaa Al Saabi, Matthias Baumann, Peter Nussbaumer, Alfred Wittinghofer, Herbert Waldmann, and Philippe I.H. Bastiaens
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Science - Abstract
PDEδ is a widely expressed factor that sustains the spatial organization and signalling of Ras family proteins. Here the authors describe the activity of Deltazinone 1, a new highly selective PDEδ inhibitor of KRAS-dependent cancer cell growth with low cytotoxic side effects.
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- 2016
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7. Spatiotemporal Regulation of Signaling: Focus on T Cell Activation and the Immunological Synapse
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Esther Garcia and Shehab Ismail
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signal transduction ,T cells ,immunological synapse ,membrane domains ,diffusion barriers ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
In a signaling network, not only the functions of molecules are important but when (temporal) and where (spatial) those functions are exerted and orchestrated is what defines the signaling output. To temporally and spatially modulate signaling events, cells generate specialized functional domains with variable lifetime and size that concentrate signaling molecules, enhancing their transduction potential. The plasma membrane is a key in this regulation, as it constitutes a primary signaling hub that integrates signals within and across the membrane. Here, we examine some of the mechanisms that cells exhibit to spatiotemporally regulate signal transduction, focusing on the early events of T cell activation from triggering of T cell receptor to formation and maturation of the immunological synapse.
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- 2020
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8. The Engineer as Economist: Sewers and the Making of the Water Consumer in Colonial Cairo, 1890
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Shehab Ismail
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History ,Engineering (miscellaneous) - Published
- 2023
9. The Structural and Biochemical Characterization of UNC119B Cargo Binding and Release Mechanisms
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Youhani H. Samarakoon, Esther Garcia, Shehab Ismail, and Tamas Yelland
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chemistry.chemical_classification ,Biochemistry & Molecular Biology ,Conformational change ,Science & Technology ,COMPLEX ,REFINEMENT ,Chemistry ,PROTEIN ,Peptide ,Peptide binding ,Biochemistry ,TRANSPORT ,CD4 ,Article ,Immunological synapse ,Amino acid ,Cytosol ,ARL2-GTP ,CILIA ,Biophysics ,MEMBRANE ,Release factor ,Life Sciences & Biomedicine ,MUTATION ,Myristoylation - Abstract
Two paralogs of the guanine dissociation inhibitor-like solubilizing factors UNC119, UNC119A and UNC119B, are present in the human genome. UNC119 binds to N-myristoylated proteins and masks the hydrophobic lipid from the hydrophilic cytosol, facilitating trafficking between different membranes. Two classes of UNC119 cargo proteins have been classified: low affinity cargoes, released by the Arf-like proteins ARL2 and ARL3, and high affinity cargoes, which are specifically released by ARL3 and trafficked to either the primary cilium or the immunological synapse. The UNC119 homologues have reported differences in functionality, but the structural and biochemical bases for these differences are unknown. Using myristoylated peptide binding and release assays, we show that peptides sharing the previously identified UNC119A high affinity motif show significant variations of binding affinities to UNC119B of up to 427-fold. Furthermore, we solve the first two crystal structures of UNC119B, one in complex with the high affinity cargo peptide of LCK and a second one in complex with the release factor ARL3. Using these novel structures, we identify a stretch of negatively charged amino acids unique to UNC119B that may undergo a conformational change following binding of a release factor which we propose as an additional release mechanism specific to UNC119B. ispartof: BIOCHEMISTRY vol:60 issue:25 pages:1952-1963 ispartof: location:United States status: published
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- 2021
10. Stabilization of the RAS:PDE6D complex is a novel strategy to inhibit RAS signaling
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Tamas Yelland, Esther Garcia, Charles Parry, Dominika Kowalczyk, Marta Wojnowska, Andrea Gohlke, Matja Zalar, Kenneth Cameron, Gillian Goodwin, Qing Yu, Peng-Cheng Zhu, Yasmin ElMaghloob, Angelo Pugliese, Lewis Archibald, Andrew Jamieson, Yong Xiang Chen, Duncan McArthur, Justin Bower, Shehab Ismail, and University of St Andrews. School of Chemistry
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STRUCTURAL BASIS ,Chemistry, Medicinal ,Proto-Oncogene Proteins p21(ras) ,DOMAIN ,Cell Line, Tumor ,Drug Discovery ,PRENYLATION ,KRAS ,Humans ,TOOL ,QD ,Pharmacology & Pharmacy ,Cyclic Nucleotide Phosphodiesterases, Type 6 ,Science & Technology ,MUTATIONS ,Cell Membrane ,DAS ,QD Chemistry ,AC ,PDE-DELTA ,ARL2-GTP ,Molecular Medicine ,MEMBRANE ,Peptides ,Life Sciences & Biomedicine ,Protein Binding ,Signal Transduction - Abstract
RAS is a major anticancer drug target which requires membrane localization to activate downstream signal transduction. The direct inhibition of RAS has proven to be challenging. Here, we present a novel strategy for targeting RAS by stabilizing its interaction with the prenyl-binding protein PDE6D and disrupting its localization. Using rationally designed RAS point mutations, we were able to stabilize the RAS:PDE6D complex by increasing the affinity of RAS for PDE6D, which resulted in the redirection of RAS to the cytoplasm and the primary cilium and inhibition of oncogenic RAS/ERK signaling. We developed an SPR fragment screening and identified fragments that bind at the KRAS:PDE6D interface, as shown through cocrystal structures. Finally, we show that the stoichiometric ratios of KRAS:PDE6D vary in different cell lines, suggesting that the impact of this strategy might be cell-type-dependent. This study forms the foundation from which a potential anticancer small-molecule RAS:PDE6D complex stabilizer could be developed. ispartof: JOURNAL OF MEDICINAL CHEMISTRY vol:65 issue:3 pages:1898-1914 ispartof: location:United States status: published
- Published
- 2022
11. ARL3, a small GTPase with a functionally conserved role in primary cilia and immune synapses
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Shehab Ismail, Laura Powell, John A. Sayer, and Youhani H. Samarakoon
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0303 health sciences ,Primary (chemistry) ,Immunological Synapses ,ADP-Ribosylation Factors ,Cilium ,Cell Membrane ,Cell Biology ,Mini-Review ,Biology ,Biochemistry ,Immunological synapse ,Cell biology ,03 medical and health sciences ,0302 clinical medicine ,Signalling ,Immune system ,030220 oncology & carcinogenesis ,Animals ,Humans ,Small GTPase ,Cilia ,030304 developmental biology - Abstract
The primary cilium and the immunological synapse are both specialized functional plasma membrane domains that share several similarities. Signalling output of membrane domains is regulated, spatially and temporally, by segregating and focusing lipids and proteins. ARL3, a small GTPase, plays a major role in concentrating lipid-modified proteins in both the immunological synapse and the primary cilia. Here in this review we will introduce the role of ARL3 in health and disease and its role in polarizing signalling at the primary cilia and immunological synapses.
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- 2019
12. PINK1 drives production of mtDNA-containing extracellular vesicles to promote invasiveness
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Nicolas Rabas, Payam A. Gammage, Sarah Palmer, Louise Mitchell, Shehab Ismail, Stephen W.G. Tait, Joel S. Riley, Jim C. Norman, Iain R. Macpherson, Andrea Gohlke, and Leandro Lemgruber Soares
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Endosome ,INVASION ,Cell ,Endosomes ,Mitochondrion ,Biology ,Receptors, Metabotropic Glutamate ,DNA, Mitochondrial ,UBIQUITIN ,rab27 GTP-Binding Proteins ,Article ,Mitochondrial Proteins ,Extracellular Vesicles ,MITOCHONDRIA ,Cell Line, Tumor ,DNA Packaging ,medicine ,Humans ,Neoplasm Invasiveness ,Receptor ,Cancer ,EXOSOMES ,Science & Technology ,Glutaminolysis ,Tetraspanin 30 ,PLATFORM ,Cell Biology ,Mitochondria ,Cell Metabolism ,Cell Death and Autophagy ,Cell biology ,RECEPTORS ,medicine.anatomical_structure ,Toll-Like Receptor 9 ,METASTASIS ,CELLS ,Cancer cell ,Metabotropic glutamate receptor 3 ,Cisplatin ,Life Sciences & Biomedicine ,Protein Kinases ,Intracellular - Abstract
Rabas et al. describe a novel means of intercellular communication in which processes evoked to mitigate cytotoxicity in metabolically stressed cells can promote PINK1-dependent packaging of mitochondrial DNA into exosomes to evoke invasive behavior in other cells., The cystine-glutamate antiporter, xCT, supports a glutathione synthesis program enabling cancer cells to cope with metabolically stressful microenvironments. Up-regulated xCT, in combination with glutaminolysis, leads to increased extracellular glutamate, which promotes invasive behavior by activating metabotropic glutamate receptor 3 (mGluR3). Here we show that activation of mGluR3 in breast cancer cells activates Rab27-dependent release of extracellular vesicles (EVs), which can transfer invasive characteristics to “recipient” tumor cells. These EVs contain mitochondrial DNA (mtDNA), which is packaged via a PINK1-dependent mechanism. We highlight mtDNA as a key EV cargo necessary and sufficient for intercellular transfer of invasive behavior by activating Toll-like receptor 9 in recipient cells, and this involves increased endosomal trafficking of pro-invasive receptors. We propose that an EV-mediated mechanism, through which altered cellular metabolism in one cell influences endosomal trafficking in other cells, is key to generation and dissemination of pro-invasive microenvironments during mammary carcinoma progression.
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- 2021
13. CYRI-A limits invasive migration through macropinosome formation and integrin uptake regulation
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Tamas Yelland, Laura M. Machesky, Shehab Ismail, Savvas Nikolaou, Nikki R. Paul, Loic Fort, and Anh Hoang Le
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rac1 GTP-Binding Protein ,Regulator ,Polymerization ,0302 clinical medicine ,Cell Movement ,Chlorocebus aethiops ,BINDING ,Internalization ,media_common ,Actin nucleation ,0303 health sciences ,biology ,Intracellular Signaling Peptides and Proteins ,BREAST-CANCER CELLS ,Cell migration ,Cell biology ,Protein Transport ,030220 oncology & carcinogenesis ,COS Cells ,Life Sciences & Biomedicine ,Integrin alpha5beta1 ,Signal Transduction ,PLECKSTRIN-HOMOLOGY DOMAINS ,ENDOCYTOSIS ,media_common.quotation_subject ,Integrin ,RAC1 ,Endosomes ,PHAGOCYTOSIS ,MECHANISMS ,Mitochondrial Proteins ,03 medical and health sciences ,Cell Line, Tumor ,Animals ,Humans ,Macropinosome ,Actin ,Cell Proliferation ,rab5 GTP-Binding Proteins ,030304 developmental biology ,Osteoblasts ,Science & Technology ,MACROPINOCYTOSIS ,Cell Biology ,Actins ,Wiskott-Aldrich Syndrome Protein Family ,HEK293 Cells ,Gene Expression Regulation ,I-TASSER ,FAM49B ,biology.protein ,Pinocytosis ,ALPHA-5-BETA-1 INTEGRIN ,Phosphatidylinositol 3-Kinase - Abstract
The Scar/WAVE complex drives actin nucleation during cell migration. Interestingly, the same complex is important in forming membrane ruffles during macropinocytosis, a process mediating nutrient uptake and membrane receptor trafficking. Mammalian CYRI-B is a recently described negative regulator of the Scar/WAVE complex by RAC1 sequestration, but its other paralogue, CYRI-A, has not been characterized. Here, we implicate CYRI-A as a key regulator of macropinosome formation and integrin internalization. We find that CYRI-A is transiently recruited to nascent macropinosomes, dependent on PI3K and RAC1 activity. CYRI-A recruitment precedes RAB5A recruitment but follows sharply after RAC1 and actin signaling, consistent with it being a local inhibitor of actin polymerization. Depletion of both CYRI-A and -B results in enhanced surface expression of the α5β1 integrin via reduced internalization. CYRI depletion enhanced migration, invasion, and anchorage-independent growth in 3D. Thus, CYRI-A is a dynamic regulator of macropinocytosis, functioning together with CYRI-B to regulate integrin trafficking. ispartof: JOURNAL OF CELL BIOLOGY vol:220 issue:9 ispartof: location:United States status: published
- Published
- 2021
14. Efficacy and safety of sofosbuvir plus daclatasvir or ravidasvir in patients with COVID‐19: A randomized controlled trial
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Abbass, Sherif, primary, Kamal, Ehab, additional, Salama, Mohsen, additional, Salman, Tary, additional, Sabry, Alyaa, additional, Abdel‐Razek, Wael, additional, Helmy, Sherine, additional, Abdelgwad, Ahmed, additional, Sakr, Neamt, additional, Elgazzar, Mohamed, additional, Einar, Mohamed, additional, Farouk, Mahmoud, additional, Saif, Mounir, additional, Shehab, Ismail, additional, El‐hosieny, Eman, additional, Mansour, Mai, additional, Mahdi, Doaa, additional, Tharwa, El‐Sayed, additional, Salah, Mostafa, additional, Elrouby, Ola, additional, and Waked, Imam, additional
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- 2021
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15. An ARF GTPase module promoting invasion and metastasis through regulating phosphoinositide metabolism
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Tamas Yelland, Jennifer P. Morton, Eva C Freckmann, Sara Zanivan, Lynn McGarry, Shehab Ismail, Hing Y. Leung, Konstantina Nikolatou, Laura C. A. Galbraith, Karen Blyth, Rachana Patel, David M. Bryant, Álvaro Román-Fernández, Marisa Nacke, Elke Markert, Sergio Lilla, Emma Shanks, Emma Sandilands, and Susan M. Mason
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0301 basic medicine ,Male ,ADP ribosylation factor ,Carcinogenesis ,Science ,Regulator ,General Physics and Astronomy ,Mice, Nude ,Collective cell migration ,Biology ,Phosphatidylinositols ,General Biochemistry, Genetics and Molecular Biology ,Article ,Metastasis ,03 medical and health sciences ,Mice ,0302 clinical medicine ,In vivo ,Cell Line, Tumor ,Neoplasms ,medicine ,Animals ,Guanine Nucleotide Exchange Factors ,Humans ,Neoplasm Metastasis ,Multidisciplinary ,Science & Technology ,Cell growth ,ADP-Ribosylation Factors ,Cancer ,Growth factor signalling ,General Chemistry ,medicine.disease ,In vitro ,Cell biology ,Cell invasion ,Multidisciplinary Sciences ,030104 developmental biology ,Signalling ,ADP-Ribosylation Factor 6 ,030220 oncology & carcinogenesis ,Cell polarity ,Heterografts ,Science & Technology - Other Topics ,Proto-Oncogene Proteins c-akt ,Low Density Lipoprotein Receptor-Related Protein-1 ,Signal Transduction - Abstract
The signalling pathways underpinning cell growth and invasion use overlapping components, yet how mutually exclusive cellular responses occur is unclear. Here, we report development of 3-Dimensional culture analyses to separately quantify growth and invasion. We identify that alternate variants of IQSEC1, an ARF GTPase Exchange Factor, act as switches to promote invasion over growth by controlling phosphoinositide metabolism. All IQSEC1 variants activate ARF5- and ARF6-dependent PIP5-kinase to promote PI(3,4,5)P3-AKT signalling and growth. In contrast, select pro-invasive IQSEC1 variants promote PI(3,4,5)P3 production to form invasion-driving protrusions. Inhibition of IQSEC1 attenuates invasion in vitro and metastasis in vivo. Induction of pro-invasive IQSEC1 variants and elevated IQSEC1 expression occurs in a number of tumour types and is associated with higher-grade metastatic cancer, activation of PI(3,4,5)P3 signalling, and predicts long-term poor outcome across multiple cancers. IQSEC1-regulated phosphoinositide metabolism therefore is a switch to induce invasion over growth in response to the same external signal. Targeting IQSEC1 as the central regulator of this switch may represent a therapeutic vulnerability to stop metastasis., The signalling pathways underpinning cell growth and invasion use overlapping components, yet how mutually exclusive responses occur is unclear. Here, the authors show that alternate isoforms of the ARF GTPase exchange factor IQSEC1 direct phosphoinositide metabolism to control this switch.
- Published
- 2021
16. Author response: ARL3 activation requires the co-GEF BART and effector-mediated turnover
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Shehab Ismail, Esther Garcia, Michael J. McIlwraith, Begoña Sot, Tamas Yelland, and Yasmin ElMaghloob
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Effector ,Chemistry ,Cell biology - Published
- 2021
17. Spatial Distribution of Seismocardiographic Signals
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John D’Angelo, Peshala P. T Gamage, Hansen A. Mansy, Shehab Ismail, Richard H. Sandler, and Khurshidul Azad
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QRS complex ,medicine.anatomical_structure ,Anterior chest ,Ventricle ,business.industry ,medicine ,Intercostal space ,Accelerometer ,business ,Instantaneous phase ,Signal ,Xiphoid process ,Biomedical engineering - Abstract
The measurement of chest surface vibrations resulting from cardiac activity is known as seismocardiography (SCG). These signals, typically measured using accelerometers on the chest surface, appear to be primarily generated by myocardial muscle activity, blood momentum changes, and heart valve movements. SCG signals contain important features associated with cardiac mechanical activity, which can be used for monitoring/predicting cardiac health. However, SCG signals are known to vary with chest surface location, which can confound diagnostic interpretation of SCG longitudinal changes over time. Documenting spatial changes will assist with both the identification of optimal sensor placement locations and the impact of misplacements on SCG interpretation. The purpose of this study is to explore the effect of sensor placement on diagnostically important SCG features and to identify locations on the anterior chest surface with consistently strong signal strength and high signal-to-noise ratio. In the current study, the SCG signals were acquired in 15 healthy male subjects along with ECG and respiratory flow measurements for 5–10 min. Here, SCG signals were acquired using an array of 32 sensors placed on the chest surface between the right and left anterior axillary and between the 2nd and 5th intercostal spaces. In addition, four accelerometers were placed at right and left mid-clavicle, mid-sternum, and xiphoid process. SCG waveforms were segmented into SCG beats (i.e., heart cycles) using the ECG R wave. Prior to extracting features, SCG beats were clustered into two groups using unsupervised machine learning to help reduce the effects of the respiratory SCG variations in order to attain more precise feature extraction. Several SCG features including SCG signal morphological variability, amplitude variation, signal-to-noise ratio, cardiac timing intervals, and other features were calculated, and the spatial variability of these features were analyzed. Results suggested that the magnitude of acceleration and signal-to-noise ratio were consistently higher (50~80%) around a 3 cm wide region lateral to the left lower sternal border ranging from the 3rd to 5th intercostal space (ICS) as well as over the xiphoid process. The SCG signal morphological variability values were approximately 4–5% of the signal peak to peak amplitude in this region. Cardiac timing intervals (pre-ejection period and left ventricle ejection period) were also comparable (within 2–5%) in this region. Spatial variations of other potentially useful SCG features (including maximum instantaneous frequency and S3 amplitude) were small (within 5%) at the left lower sternal border and at the xiphoid process. The optimum sensor locations with maximum signal strength and lowest spatial variation of SCG features were found to be at the left lower sternal border ranging from 3rd to 5th ICS and at the xiphoid process. Documentation of these changes should help future researchers and clinicians optimize SCG sensor placement.
- Published
- 2021
18. CYRI-A regulates macropinocytic cup maturation and mediates integrin uptake, limiting invasive migration
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Tamas Yelland, Loic Fort, Anh Hoang Le, Laura M. Machesky, Savvas Nikolaou, Shehab Ismail, and Nikki R. Paul
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biology ,Chemistry ,Pinocytosis ,Macropinocytic cup ,Integrin ,Regulator ,biology.protein ,RAC1 ,Lamellipodium ,Actin ,Actin nucleation ,Cell biology - Abstract
The Scar/WAVE complex is the major driver of actin nucleation at the plasma membrane, resulting in lamellipodia and membrane ruffles. While lamellipodia aid migration, membrane ruffles can generate macropinosomes - cup-like structures - important for nutrient uptake and regulation of cell surface receptor levels. How macropinosomes are formed and the role of the actin machinery in their formation and resolution is still not well understood. Mammalian CYRI-B is a recently described negative regulator of the Scar/WAVE complex by RAC1 sequestration, but its other paralogue, CYRI-A has not been characterised. Here we implicate CYRI-A as a key regulator of macropinosome maturation and integrin internalisation from the cell surface. We find that CYRI-A is recruited to nascent macropinosomes in a transient but distinct burst, downstream of PIP3-mediated RAC1 activation and the initial burst of actin assembly driving cup formation, but upstream of internalisation and RAB5 recruitment to the macropinosome. Together, our data place CYRI-A as a local suppressor of actin dynamics, enabling the resolution of the macropinocytic cup. The failure of CYRI-depleted cells to resolve their macropinocytic cups results in reduced integrin a5b1 internalisation, leading to enhanced spreading, invasive behaviour and anchorage-independent 3D growth. We thus describe a new role for CYRI-A as a highly dynamic regulator of RAC1 activity at macropinosomes, modulating homeostasis of integrin surface presentation, with important functional consequences.
- Published
- 2020
19. WASP restricts active rac to maintain cells' front-rear polarization
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Peter A. Thomason, Clelia Amato, Laura M. Machesky, Andrew J. Davidson, Shehab Ismail, Robert H. Insall, and Karthic Swaminathan
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0301 basic medicine ,Life Sciences & Biomedicine - Other Topics ,DYNAMICS ,actin cytoskeleton ,Arp2/3 complex ,CDC42 ,ARP2/3 COMPLEX ,0302 clinical medicine ,Cell Movement ,SIGNALS ,Cell polarity ,Dictyostelium ,Pseudopodia ,MEDIATED ENDOCYTOSIS ,CRIB motif ,SMALL-MOLECULE INHIBITOR ,actin polymerization ,uropod ,Cell Polarity ,MEMBRANE TENSION ,Endocytosis ,Cell biology ,CLATHRIN ,General Agricultural and Biological Sciences ,Life Sciences & Biomedicine ,Wiskott-Aldrich Syndrome Protein ,Protein Binding ,Biochemistry & Molecular Biology ,DEPOLYMERIZING PROTEIN ,PROTEIN N-WASP ,macromolecular substances ,Biology ,small GTPases ,Clathrin ,Actin-Related Protein 2-3 Complex ,Article ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,FAMILY PROTEINS ,Animals ,Humans ,Protein Interaction Domains and Motifs ,Amino Acid Sequence ,Actin ,Science & Technology ,fungi ,Cell Biology ,Actin cytoskeleton ,Actins ,030104 developmental biology ,biology.protein ,Proto-Oncogene Proteins c-akt ,030217 neurology & neurosurgery - Abstract
Summary Efficient motility requires polarized cells, with pseudopods at the front and a retracting rear. Polarization is maintained by restricting the pseudopod catalyst, active Rac, to the front. Here, we show that the actin nucleation-promoting factor Wiskott-Aldrich syndrome protein (WASP) contributes to maintenance of front-rear polarity by controlling localization and cellular levels of active Rac. Dictyostelium cells lacking WASP inappropriately activate Rac at the rear, which affects their polarity and speed. WASP’s Cdc42 and Rac interacting binding (“CRIB”) motif has been thought to be essential for its activation. However, we show that the CRIB motif’s biological role is unexpectedly complex. WASP CRIB mutants are no longer able to restrict Rac activity to the front, and cannot generate new pseudopods when SCAR/WAVE is absent. Overall levels of Rac activity also increase when WASP is unable to bind to Rac. However, WASP without a functional CRIB domain localizes normally at clathrin pits during endocytosis, and activates Arp2/3 complex. Similarly, chemical inhibition of Rac does not affect WASP localization or activation at sites of endocytosis. Thus, the interaction between small GTPases and WASP is more complex than previously thought—Rac regulates a subset of WASP functions, but WASP reciprocally restricts active Rac through its CRIB motif., Graphical Abstract, Highlights • WASP exploits its CRIB motif to remove active Rac from the membrane via endocytosis • WASP is recruited to clathrin-coated pits (CCPs) independently of small GTPases • WASP triggers actin polymerization at CCPs independently of small GTPase activation • WASP maintains homeostasis in Dictyostelium by controlling the level of active Rac, Amato et al. have discovered a mechanism that contributes to front-rear polarization in migrating cells. During clathrin-mediated endocytosis, Dictyostelium WASP interacts with active Rac via its CRIB motif. This interaction leads to incorporation of active Rac within endocytic vesicles, which facilitates its removal from inappropriate locations.
- Published
- 2019
20. Maintaining protein composition in cilia
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Shehab Ismail, Yasmin ElMaghloob, and Louise A. Stephen
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0301 basic medicine ,Clinical Biochemistry ,Cell ,Biochemistry ,03 medical and health sciences ,0302 clinical medicine ,Cell-Derived Microparticles ,Organelle ,medicine ,Animals ,Humans ,Cilia ,Receptor ,Molecular Biology ,Chemistry ,Cilium ,Proteins ,Protein composition ,Cell biology ,030104 developmental biology ,medicine.anatomical_structure ,Signalling ,Signalling pathways ,030217 neurology & neurosurgery ,Function (biology) ,Signal Transduction - Abstract
The primary cilium is a sensory organelle that is vital in regulating several signalling pathways. Unlike most organelles cilia are open to the rest of the cell, not enclosed by membranes. The distinct protein composition is crucial to the function of cilia and many signalling proteins and receptors are specifically concentrated within distinct compartments. To maintain this composition, a mechanism is required to deliver proteins to the cilium whilst another must counter the entropic tendency of proteins to distribute throughout the cell. The combination of the two mechanisms should result in the concentration of ciliary proteins to the cilium. In this review we will look at different cellular mechanisms that play a role in maintaining the distinct composition of cilia, including regulation of ciliary access and trafficking of ciliary proteins to, from and within the cilium.
- Published
- 2017
21. Tumor matrix stiffness promotes metastatic cancer cell interaction with the endothelium
- Author
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Colin Nixon, Steven Reid, Vasileios Papalazarou, Laura M. Machesky, Ewan J. McGhee, Lisa J. Neilson, David M. Bryant, Ralf H. Adams, Francesca Patella, Karthic Swaminathan, Sandeep Dhayade, Jens Serneels, Karen Blyth, Álvaro Román-Fernández, Shehab Ismail, Manuel Salmerón-Sánchez, Alice Santi, Massimiliano Mazzone, Sara Zanivan, Yasmin ElMaghloob, Emily J. Kay, Juan Ramon Hernandez-Fernaud, Leo M. Carlin, Dimitris Athineos, Anne Theres Henze, and John B. G. Mackey
- Subjects
0301 basic medicine ,CCN1/CYR61 ,Endothelium ,Cell Communication ,Biology ,Article ,Mass Spectrometry ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,Metastasis ,blood vessels ,Extracellular matrix ,stiffness ,03 medical and health sciences ,proteomics ,cancer metastasis ,medicine ,Humans ,Molecular Biology ,beta Catenin ,Cancer ,General Immunology and Microbiology ,General Neuroscience ,Matricellular protein ,Endothelial Cells ,Articles ,Cadherins ,medicine.disease ,030104 developmental biology ,medicine.anatomical_structure ,Gene Expression Regulation ,Tumor progression ,CYR61 ,Cancer cell ,Immunology ,Cancer research ,Melanocytes ,Cell Adhesion, Polarity & Cytoskeleton ,Cysteine-Rich Protein 61 - Abstract
Tumor progression alters the composition and physical properties of the extracellular matrix. Particularly, increased matrix stiffness has profound effects on tumor growth and metastasis. While endothelial cells are key players in cancer progression, the influence of tumor stiffness on the endothelium and the impact on metastasis is unknown. Through quantitative mass spectrometry, we find that the matricellular protein CCN1/CYR61 is highly regulated by stiffness in endothelial cells. We show that stiffness-induced CCN1 activates β-catenin nuclear translocation and signaling and that this contributes to upregulate N-cadherin levels on the surface of the endothelium, in vitro This facilitates N-cadherin-dependent cancer cell-endothelium interaction. Using intravital imaging, we show that knockout of Ccn1 in endothelial cells inhibits melanoma cancer cell binding to the blood vessels, a critical step in cancer cell transit through the vasculature to metastasize. Targeting stiffness-induced changes in the vasculature, such as CCN1, is therefore a potential yet unappreciated mechanism to impair metastasis. ispartof: EMBO Journal vol:36 issue:16 pages:2373-2389 ispartof: location:England status: published
- Published
- 2017
22. WASP restricts active Rac to maintain cells’ front-rear polarisation
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Laura M. Machesky, Andrew D. Davidson, Shehab Ismail, Clelia Amato, Peter A. Thomason, Karthic Swaminathan, and Robert H. Insall
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biology ,Chemistry ,fungi ,Cell ,Mutant ,Regulator ,Motility ,macromolecular substances ,biology.organism_classification ,Dictyostelium ,Cell biology ,medicine.anatomical_structure ,Cell polarity ,medicine ,Pseudopodia ,Actin - Abstract
SummaryEfficient motility requires polarised cells, with anterior pseudopods and a retracting rear. This polarisation requires that the pseudopod catalyst Rac is restricted to the front. Here we show that the Arp2/3 complex regulator WASP is important for maintaining front–rear polarity, using a mechanism that limits where active Rac localises. Dictyostelium cells lacking WASP inappropriately activate Rac and SCAR/WAVE at their rears, leading to reduced cell speed. WASP facilitates the internalisation of clathrin-coated pits, and its Rac-binding CRIB motif is considered essential for its localisation and activity. However, WASP mutants with deleted CRIB domains, or harbouring a new mutation that prevents Rac binding, localise normally, recruit Arp2/3 complex, and drive actin polymerisation. Similarly, Rac inhibitors do not block WASP localisation or activation. Despite this, WASP CRIB mutants cannot restore polarisation of active Rac. Thus, WASP’s interaction with Rac regulates Rac activity and cell polarity, but is dispensable for activating actin polymerization.
- Published
- 2019
23. Hypoxic cancer-associated fibroblasts increase NCBP2-AS2/HIAR to promote endothelial sprouting through enhanced VEGF signaling
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Shehab Ismail, David M. Bryant, Sergio Lilla, Fernanda G. Kugeratski, Lisa J. Neilson, Elke Markert, Sara Zanivan, Amelie Juin, Samuel J. Atkinson, Owen J. Sansom, Jens Serneels, John R. P. Knight, Massimiliano Mazzone, and Laura M. Machesky
- Subjects
Proteomics ,Vascular Endothelial Growth Factor A ,Proteome ,Angiogenesis ,Cell ,Biochemistry ,ANGIOGENESIS ,Metastasis ,0302 clinical medicine ,Cancer-Associated Fibroblasts ,Hypoxia ,Cells, Cultured ,GENE-EXPRESSION ,0303 health sciences ,Tumor ,Cultured ,Neovascularization, Pathologic ,Chemistry ,TUMOR-GROWTH ,FRACTIONATION ,INDUCTION ,Gene Expression Regulation, Neoplastic ,Vascular endothelial growth factor A ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,medicine.symptom ,Life Sciences & Biomedicine ,Blood vessel ,Signal Transduction ,Breast Neoplasms ,Cell Line, Tumor ,Coculture Techniques ,HEK293 Cells ,Human Umbilical Vein Endothelial Cells ,Humans ,Biochemistry & Molecular Biology ,Cells ,BIOLOGY ,Article ,Cell Line ,03 medical and health sciences ,medicine ,Gene silencing ,Molecular Biology ,Neovascularization ,030304 developmental biology ,Pathologic ,Neoplastic ,Science & Technology ,STROMAL FIBROBLASTS ,Cell Biology ,Hypoxia (medical) ,medicine.disease ,Gene Expression Regulation ,DRIVES ,CELLS ,Cancer research - Abstract
Intratumoral hypoxia causes the formation of dysfunctional blood vessels, which contribute to tumor metastasis and reduce the efficacy of therapeutic treatments. Blood vessels are embedded in the tumor stroma of which cancer-associated fibroblasts (CAFs) constitute a prominent cellular component. We found that hypoxic human mammary CAFs promoted angiogenesis in CAF-endothelial cell cocultures in vitro. Mass spectrometry-based proteomic analysis of the CAF secretome unraveled that hypoxic CAFs contributed to blood vessel abnormalities by altering their secretion of various pro- and anti-angiogenic factors. Hypoxia induced pronounced remodeling of the CAF proteome, including proteins that have not been previously related to this process. Among those, the uncharacterized protein NCBP2-AS2 that we renamed HIAR (hypoxia-induced angiogenesis regulator) was the protein most increased in abundance in hypoxic CAFs. Silencing of HIAR abrogated the pro-angiogenic and pro-migratory function of hypoxic CAFs by decreasing secretion of the pro-angiogenic factor VEGFA and consequently reducing VEGF/VEGFR downstream signaling in the endothelial cells. Our study has identified a regulator of angiogenesis and provides a map of hypoxia-induced molecular alterations in mammary CAFs. ispartof: SCIENCE SIGNALING vol:12 issue:567 ispartof: location:United States status: published
- Published
- 2019
24. ARL3 mutations cause Joubert syndrome by disrupting ciliary protein composition
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Simon A. Ramsbottom, Sumaya Alkanderi, Kathryn White, John A. Sayer, Shehab Ismail, Amal Alhashem, Elisa Molinari, Ranad Shaheen, David H. W. Steel, Colin G. Miles, Shalabh Srivastava, Yasmin ElMaghloob, Noel Edwards, Veronica Sammut, Nour Ewida, Louise A. Stephen, George Cairns, Sarah J. Rice, and Fowzan S. Alkuraya
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0301 basic medicine ,Adult ,Male ,Mutation, Missense ,Biology ,Ciliopathies ,Joubert syndrome ,Retina ,03 medical and health sciences ,ARL3 ,Young Adult ,Mutant protein ,trafficking ,GTP-Binding Proteins ,Report ,Cerebellum ,INPP5E ,Genetics ,medicine ,Missense mutation ,guanine nucleotide exchange factor ,Guanine Nucleotide Exchange Factors ,Humans ,Abnormalities, Multiple ,Exome ,Cilia ,Eye Abnormalities ,Child ,Genetics (clinical) ,Exome sequencing ,ADP-Ribosylation Factors ,Chromosomes, Human, Pair 10 ,Cilium ,ARL13B ,Kidney Diseases, Cystic ,medicine.disease ,Ciliopathy ,Protein Transport ,030104 developmental biology ,Child, Preschool ,Female - Abstract
Joubert syndrome (JBTS) is a genetically heterogeneous autosomal-recessive neurodevelopmental ciliopathy. We investigated further the underlying genetic etiology of Joubert syndrome by studying two unrelated families in whom JBTS was not associated with pathogenic variants in known JBTS-associated genes. Combined autozygosity mapping of both families highlighted a candidate locus on chromosome 10 (chr10: 101569997–109106128, UCSC Genome Browser hg 19), and exome sequencing revealed two missense variants in ARL3 within the candidate locus. The encoded protein, ADP ribosylation factor-like GTPase 3 (ARL3), is a small GTP-binding protein that is involved in directing lipid-modified proteins into the cilium in a GTP-dependent manner. Both missense variants replace the highly conserved Arg149 residue, which we show to be necessary for the interaction with its guanine nucleotide exchange factor ARL13B, such that the mutant protein is associated with reduced INPP5E and NPHP3 localization in cilia. We propose that ARL3 provides a potential hub in the network of proteins implicated in ciliopathies, whereby perturbation of ARL3 leads to the mislocalization of multiple ciliary proteins as a result of abnormal displacement of lipidated protein cargo.
- Published
- 2018
25. Structural Basis of CYRI-B Direct Competition with Scar/WAVE Complex for Rac1
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Shehab Ismail, Laura M. Machesky, Robert H. Insall, Tamas Yelland, Savvas Nikolaou, and Anh Hoang Le
- Subjects
rac1 GTP-Binding Protein ,Biochemistry & Molecular Biology ,Protein family ,CYRIB ,Ratchet ,Biophysics ,Regulator ,RAC1 ,macromolecular substances ,Article ,Mitochondrial Proteins ,03 medical and health sciences ,Mice ,Structural Biology ,Chlorocebus aethiops ,Animals ,Humans ,Cytoskeleton ,Molecular Biology ,Actin ,Conserved Sequence ,030304 developmental biology ,Adaptor Proteins, Signal Transducing ,0303 health sciences ,Science & Technology ,Binding Sites ,Basis (linear algebra) ,MUTATIONS ,Chemistry ,030302 biochemistry & molecular biology ,Intracellular Signaling Peptides and Proteins ,cytoskeleton ,Cell Biology ,invasion ,SCAR/WAVE ,Molecular Docking Simulation ,rho ,Helix ,COS Cells ,Life Sciences & Biomedicine ,actin ,Protein Binding - Abstract
Summary Rac1 is a major regulator of actin dynamics, with GTP-bound Rac1 promoting actin assembly via the Scar/WAVE complex. CYRI competes with Scar/WAVE for interaction with Rac1 in a feedback loop regulating actin dynamics. Here, we reveal the nature of the CYRI-Rac1 interaction, through crystal structures of CYRI-B lacking the N-terminal helix (CYRI-BΔN) and the CYRI-BΔN:Rac1Q61L complex, providing the molecular basis for CYRI-B regulation of the Scar/WAVE complex. We reveal CYRI-B as having two subdomains - an N-terminal Rac1 binding subdomain with a unique Rac1-effector interface and a C-terminal Ratchet subdomain that undergoes conformational changes induced by Rac1 binding. Finally, we show that the CYRI protein family, CYRI-A and CYRI-B can produce an autoinhibited hetero- or homodimers, adding an additional layer of regulation to Rac1 signaling., Graphical Abstract, Highlights • CYRI-B structure reveals homology to CYFIP of the Scar/WAVE complex • CYRI-B shares a conserved interface with CYFIP for Rac1 interaction • CYRI proteins form autoinhibited dimers that compete for Rac1 binding • Rac1-CYFIP model shows potential steric clashes of RAC1 with CYFIP, Yelland et al. determined the structure of CYRI-B alone and in complex with Rac1Q61L. These structures show that the binding interface is conserved with CYFIP of the Scar/WAVE complex providing an explanation for CYRI competition. Finally, CYRI proteins form autoinhibited dimers generating additional complexity to CYRI function.
- Published
- 2021
26. Identification of pyrazolopyridazinones as PDEδ inhibitors
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Philippe I. H. Bastiaens, Carsten Schultz-Fademrecht, Gunther Zimmermann, Peter Nussbaumer, Kaatje Heinelt, Marija Kovacevic, Pablo Martín-Gago, Sandip Murarka, Vogel H, Alaa Al Saabi, Eyad K. Fansa, Herbert Waldmann, Alfred Wittinghofer, Shehab Ismail, Björn Papke, Matthias Baumann, and Dina C. Truxius
- Subjects
0301 basic medicine ,Phosphodiesterase Inhibitors ,Science ,General Physics and Astronomy ,Gene Expression ,Antineoplastic Agents ,Plasma protein binding ,Biology ,Crystallography, X-Ray ,General Biochemistry, Genetics and Molecular Biology ,Article ,Protein Structure, Secondary ,Proto-Oncogene Proteins p21(ras) ,Small Molecule Libraries ,03 medical and health sciences ,Protein structure ,Prenylation ,Cell Line, Tumor ,Humans ,Protein Interaction Domains and Motifs ,Binding site ,Gene knockdown ,Cyclic Nucleotide Phosphodiesterases, Type 6 ,Multidisciplinary ,Binding Sites ,Pancreatic Ducts ,Epithelial Cells ,General Chemistry ,Recombinant Proteins ,3. Good health ,Gene Expression Regulation, Neoplastic ,Molecular Docking Simulation ,030104 developmental biology ,Biochemistry ,Cell culture ,Pyrazines ,Pyrazoles ,Benzimidazoles ,Signal transduction ,Protein Binding ,Signal Transduction - Abstract
The prenyl-binding protein PDEδ is crucial for the plasma membrane localization of prenylated Ras. Recently, we have reported that the small-molecule Deltarasin binds to the prenyl-binding pocket of PDEδ, and impairs Ras enrichment at the plasma membrane, thereby affecting the proliferation of KRas-dependent human pancreatic ductal adenocarcinoma cell lines. Here, using structure-based compound design, we have now identified pyrazolopyridazinones as a novel, unrelated chemotype that binds to the prenyl-binding pocket of PDEδ with high affinity, thereby displacing prenylated Ras proteins in cells. Our results show that the new PDEδ inhibitor, named Deltazinone 1, is highly selective, exhibits less unspecific cytotoxicity than the previously reported Deltarasin and demonstrates a high correlation with the phenotypic effect of PDEδ knockdown in a set of human pancreatic cancer cell lines., PDEδ is a widely expressed factor that sustains the spatial organization and signalling of Ras family proteins. Here the authors describe the activity of Deltazinone 1, a new highly selective PDEδ inhibitor of KRAS-dependent cancer cell growth with low cytotoxic side effects.
- Published
- 2016
27. Fam49/CYRI interacts with Rac1 and locally suppresses protrusions
- Author
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Loic Fort, Matthew Neilson, Peter A. Thomason, Grant S. Mastick, Luke H. Chamberlain, Kirsty J. Martin, David M. Bryant, Petra Tafelmeyer, Jennifer Greaves, Shehab Ismail, José Miguel Batista, Luke Tweedy, Heather J. Spence, Kurt I. Anderson, Sara Zanivan, Nicholas C. O. Tomkinson, Peter Brown, Sergio Lilla, Jamie Whitelaw, Laura M. Machesky, and Robert H. Insall
- Subjects
0301 basic medicine ,rac1 GTP-Binding Protein ,RM ,RAC1 ,macromolecular substances ,Article ,Madin Darby Canine Kidney Cells ,Polymerization ,03 medical and health sciences ,Dogs ,Cell Movement ,Cell Line, Tumor ,Chlorocebus aethiops ,Animals ,Humans ,Pseudopodia ,Actin ,COS cells ,Chemistry ,Chemotaxis ,HEK 293 cells ,Intracellular Signaling Peptides and Proteins ,Cell migration ,Cell Biology ,Actins ,Cell biology ,030104 developmental biology ,HEK293 Cells ,COS Cells ,Lamellipodium ,Protein Binding ,Signal Transduction - Abstract
Actin-based protrusions driving cell migration are reinforced through positive feedback, but it is unclear how the cell restricts the eventual size of protrusions or limits positive signals to allow them to split or retract. We have identified an evolutionarily conserved regulator of the protrusion machinery, which we name CYRI (CYFIP-related Rac interactor). CYRI binds specifically to activated Rac1 via a common motif that is also found in CYFIP, the Domain of Unknown Function DUF1394; we demonstrate that DUF1394 is a new class of Rac1 binding module. CYRI-depleted cells have broad lamellipodia enriched in Scar/WAVE, but exhibit reduced protrusion-retraction dynamics. Pseudopods induced by optogenetic Rac1 activation are larger and longer-lived in the absence of CYRI. Conversely, CYRI overexpression suppresses recruitment of active Scar/WAVE complex to the cell edge, resulting in short-lived, unproductive protrusions. CYRI’s role in cell behaviour is therefore to focus positive protrusion signals and regulate pseudopod complexity and dynamics by inhibiting Scar/WAVE induced actin. As such it behaves like a “local inhibitor” predicted and described in widely accepted mathematical models, but not previously identified in living cells. CYRI is important for biological processes requiring polarity and plasticity of protrusions, including directional migration and polarization of epithelial cysts.
- Published
- 2018
28. Abstract AP18: PRO-INVASIVE TUMOUR-STROMA INTERACTIONS: ROLE OF THE SECRETED OXIDOREDUCTASE CLIC3
- Author
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Lisa J. Neilson, Juan Ramon Hernandez-Fernaud, Huabing Yin, Shehab Ismail, Sergio Lilla, Massimiliano Mazzone, Elena Ruengeler, Jim C. Norman, Stella M. Valenzuela, Alice Santi, Sara Zanivan, Samuel J. Atkinson, Iain A. McNeish, and Karen Blyth
- Subjects
chemistry.chemical_classification ,Cancer Research ,Oncology ,chemistry ,Oxidoreductase ,Tumour stroma ,Cancer research - Abstract
Cancer cells are embedded within a microenvironment populated with cancer associated fibroblasts (CAFs) with whom they establish two-way communications through paracrine factors and physical interactions. Through these interactions, CAFs play pivotal roles in cancer and have emerged as promising therapeutic target. To unravel key contributors of CAF-tumour/stroma cell interactions, we have developed mass spectrometry (MS)-based approaches to map proteins secreted in condition medium and extracellular matrix (ECM). Through an extensive MS-proteomic comparative analysis of CAFs with their normal fibroblasts (NFs) counterpart, we have identified the chloride intracellular channel protein 3 (CLIC3), a protein previously considered an intracellular chloride channel (regulator), as one of the most upregulated proteins in CAFs and deposited in the ECM. Secreted CLIC3 promotes invasive behaviour of endothelial cells to drive blood vessels growth and increases invasiveness of cancer cells, both in vivo and in 3D cell culture models, via activation of the tissue transglutaminase-2 (TGM2). We found that CLIC3 is 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 in primary tumours and omental metastases correlate with poor clinical outcome. Our work has unraveled an unprecedented mechanism of cell invasion to be explored for targeting in ovarian cancer. Citation Format: Juan R Hernandez-Fernaud, Elena Ruengeler, Lisa J Neilson, Shehab Ismail, Iain McNeish, Samuel Atkinson, Sergio Lilla, Alice Santi, Stella M Valenzuela, Karen Blyth, Huabing Yin, Massimiliano Mazzone, Jim C Norman and Sara Zanivan. PRO-INVASIVE TUMOUR-STROMA INTERACTIONS: ROLE OF THE SECRETED OXIDOREDUCTASE CLIC3 [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr AP18.
- Published
- 2019
29. The Ciliary Machinery Is Repurposed for T Cell Immune Synapse Trafficking of LCK
- Author
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Pedro Roda-Navarro, Michael J. McIlwraith, Yasmin ElMaghloob, Tamas Yelland, Shehab Ismail, Louise A. Stephen, and Patricia Castro Sanchez
- Subjects
0301 basic medicine ,Immunological Synapses ,T cell ,Cell ,GDI ,Receptors, Antigen, T-Cell ,Antigen-Presenting Cells ,chemical and pharmacologic phenomena ,Biology ,Lymphocyte Activation ,small GTPases ,Article ,General Biochemistry, Genetics and Molecular Biology ,Immunological synapse ,03 medical and health sciences ,ARL3 ,Jurkat Cells ,Immune system ,UNC119 ,medicine ,Humans ,Phosphorylation ,Molecular Biology ,Myristoylation ,Adaptor Proteins, Signal Transducing ,ADP-Ribosylation Factors ,Cilium ,T-cell receptor ,ARL13B ,cilia ,immunological synapse ,hemic and immune systems ,Cell Biology ,Cell biology ,LCK ,Protein Transport ,030104 developmental biology ,medicine.anatomical_structure ,Lymphocyte Specific Protein Tyrosine Kinase p56(lck) ,protein trafficking ,biological phenomena, cell phenomena, and immunity ,Src kinases ,Developmental Biology ,Signal Transduction - Abstract
Summary Upon engagement of the T cell receptor with an antigen-presenting cell, LCK initiates TCR signaling by phosphorylating its activation motifs. However, the mechanism of LCK activation specifically at the immune synapse is a major question. We show that phosphorylation of the LCK activating Y394, despite modestly increasing its catalytic rate, dramatically focuses LCK localization to the immune synapse. We describe a trafficking mechanism whereby UNC119A extracts membrane-bound LCK by sequestering the hydrophobic myristoyl group, followed by release at the target membrane under the control of the ciliary ARL3/ARL13B. The UNC119A N terminus acts as a “regulatory arm” by binding the LCK kinase domain, an interaction inhibited by LCK Y394 phosphorylation, thus together with the ARL3/ARL13B machinery ensuring immune synapse focusing of active LCK. We propose that the ciliary machinery has been repurposed by T cells to generate and maintain polarized segregation of signals such as activated LCK at the immune synapse., Highlights • LCK immune synapse focusing requires its strong interaction with ciliary UNC119A • ARL3Q71L results in unfocused LCK localization and increased T cell stimulation • The ciliary ARL3-GEF ARL13B localizes to the immune synapse • UNC119A regulatory arm interacts with LCK kinase domain in a phosphoregulated manner, Signaling proteins are focused at the immune synapse, where T cells form an interface with target cells. Stephen, ElMaghloob, and McIlwraith et al. demonstrate that T cells repurpose a mechanism used by the cilia, an organelle that T cells lack, to focus the signaling protein LCK at the immune synapse.
- Published
- 2018
30. CYRI (FAM49) proteins are local inhibitors of Scar/WAVE induced lamellipodia that bind directly to active Rac1
- Author
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Peter A. Thomason, Nicholas C. O. Tomkinson, Shehab Ismail, Jennifer Greaves, Sara Zanivan, José Miguel Batista, Laura M. Machesky, Robert H. Insall, Luke H. Chamberlain, Petra Tafelmeyer, Heather J. Spence, Kirsty J. Martin, Kurt I. Anderson, Peter Brown, Loic Fort, Matthew Neilson, and Sergio Lilla
- Subjects
medicine.anatomical_structure ,Chemistry ,Cell ,Regulator ,medicine ,Cell migration ,RAC1 ,Pseudopodia ,Lamellipodium ,psychological phenomena and processes ,Actin ,Cell biology ,Binding domain - Abstract
Actin-based protrusions driving cell migration are reinforced through positive feedback, but it is unclear how the cell restricts the eventual size of a protrusion or limits positive signals to cause splitting or retraction. We have identified an evolutionarily conserved regulator of the protrusion machinery, which we name CYRI (CYFIP-related Rac interacting) protein. CYRI shows sequence similarity to the Scar/WAVE complex member CYFIP in a Domain of Unknown Function, DUF1394. CYRI binds specifically to activated Rac1 via a common motif shared with CYFIP, establishing DUF1394 as a new Rac1 binding domain. CYRI-depleted cells have broad, Scar/WAVE-enriched lamellipodia and enhanced Rac1 signaling. Conversely, CYRI overexpression suppresses spreading and dramatically sharpens protrusions into unproductive needles. CYRI proteins use dynamic inhibition of Scar/WAVE induced actin to focus positive protrusion signals and regulate pseudopod complexity. CYRI behaves like a “local inhibitor” predicted and described in widely accepted mathematical models, but not previously identified in living cells.
- Published
- 2017
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31. The N‐ and C‐terminal ends of <scp>RPGR</scp> can bind to <scp>PDE</scp> 6δ
- Author
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Shehab Ismail, Alfred Wittinghofer, Eyad K. Fansa, and Nicola O’Reilly
- Subjects
Cyclic Nucleotide Phosphodiesterases, Type 6 ,ADP ribosylation factor ,ADP-Ribosylation Factors ,Chemistry ,Stereochemistry ,Immunoprecipitation ,Biochemistry ,eye diseases ,Dissociation (chemistry) ,Dissociation constant ,GTP-binding protein regulators ,GTP-Binding Proteins ,Correspondence ,Hydrolase ,Genetics ,Animals ,Humans ,Surface plasmon resonance ,Eye Proteins ,Molecular Biology ,Fluorescence anisotropy - Abstract
Lee and Seo propose in their article [1] that RPGR binds to PDE6δ not with the N‐terminal RCC1‐like propeller domain but solely with the C‐terminus. They show, using an immunoprecipitation experiment, that FLAG‐tagged fragments missing the C‐terminal CaaX motif of RPGR fail to co‐immunoprecipitate together with myc‐tagged PDE6δ. We have previously shown that the N‐terminal 400 residues of RPGR form a stable RCC1‐like propeller domain and that this protein forms a complex with PDE6δ. This can be demonstrated by (untagged) pull‐down and gel permeation chromatography experiments. Additionally, the equilibrium dissociation constant was determined to be 500 nM by fluorescence polarization which also agrees with previous results published by Linari et al in which they report the affinity of RPGR (aa 1–392), using surface plasmon resonance, to PDE6δ to be 100 nM [2], [3]. Finally, we have solved the structure of the complex PDE6δ–RPGR (aa 8–368) by X‐ray crystallography and verified the interaction interface by mutational analysis [3]. Since immunoprecipitation experiments reflect dissociation kinetics rates rather than equilibrium dissociation, which are in turn very much dependent on many aspects of the experiments (see discussion below), we feel confident about the results by Watzlich et al [3]. Lee and Seo then show that RPGR interacts with the C‐terminal end of RPGR which contains a CaaX motif. This finding may not be too surprising since we and others have previously shown that PDE6δ is a general prenyl‐binding protein that is required to shuttle lipidated proteins between membranes and that the binding is released by Arl2/3•GTP [4], [5], [6], [7]. Our earlier structural studies revealed molecular details of the binding of farnesylated peptides/proteins to PDE6δ, showing how the farnesyl moiety is inserted into the hydrophobic cavity …
- Published
- 2015
32. Structure Guided Design and Kinetic Analysis of Highly Potent Benzimidazole Inhibitors Targeting the PDEδ Prenyl Binding Site
- Author
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Peter Nussbaumer, Philipp Küchler, Shehab Ismail, Herbert Waldmann, Alfred Wittinghofer, Gemma Triola, Sandip Murarka, Carsten Schultz-Fademrecht, and Gunther Zimmermann
- Subjects
Models, Molecular ,Protein Prenylation ,GTPase ,Structure-Activity Relationship ,Prenylation ,Cell Line, Tumor ,Drug Discovery ,Atorvastatin ,Humans ,Computer Simulation ,Pyrroles ,Binding site ,Cellular localization ,Cyclic Nucleotide Phosphodiesterases, Type 6 ,Binding Sites ,Chemistry ,Drug discovery ,Binding protein ,Esters ,Stereoisomerism ,Small molecule ,Kinetics ,Biochemistry ,Heptanoic Acids ,Drug Design ,ras Proteins ,Thermodynamics ,Molecular Medicine ,Protein prenylation ,Benzimidazoles ,Ethers - Abstract
K-Ras is one of the most frequently mutated signal transducing human oncogenes. Ras signaling activity requires correct cellular localization of the GTPase. The spatial organization of K-Ras is controlled by the prenyl binding protein PDEδ, which enhances Ras diffusion in the cytosol. Inhibition of the Ras-PDEδ interaction by small molecules impairs Ras localization and signaling. Here we describe in detail the identification and structure guided development of Ras-PDEδ inhibitors targeting the farnesyl binding pocket of PDEδ with nanomolar affinity. We report kinetic data that characterize the binding of the most potent small molecule ligands to PDEδ and prove their binding to endogenous PDEδ in cell lysates. The PDEδ inhibitors provide promising starting points for the establishment of new drug discovery programs aimed at cancers harboring oncogenic K-Ras.
- Published
- 2014
33. A PDE6δ-KRas Inhibitor Chemotype with up to Seven H-Bonds and Picomolar Affinity that Prevents Efficient Inhibitor Release by Arl2
- Author
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Shehab Ismail, Pablo Martín-Gago, Malte Metz, Sandip Murarka, Herbert Waldmann, Philippe I. H. Bastiaens, Carsten Schultz-Fademrecht, Matthias Baumann, Christian Klein, Petra Janning, Alfred Wittinghofer, Marc Schürmann, and Eyad K. Fansa
- Subjects
0301 basic medicine ,medicine.disease_cause ,010402 general chemistry ,01 natural sciences ,Catalysis ,Fast release ,chemistry.chemical_compound ,03 medical and health sciences ,0302 clinical medicine ,medicine ,biology ,Chemotype ,Chemistry ,General Chemistry ,General Medicine ,Highly selective ,Combinatorial chemistry ,digestive system diseases ,In vitro ,Cell biology ,0104 chemical sciences ,030104 developmental biology ,030220 oncology & carcinogenesis ,Chaperone (protein) ,biology.protein ,KRAS ,Growth inhibition ,Release factor - Abstract
Small-molecule inhibition of the interaction between the KRas oncoprotein and the chaperone PDE6δ impairs KRas spatial organization and signaling in cells. However, despite potent binding in vitro (KD
- Published
- 2016
34. Small molecule inhibition of the KRAS–PDEδ interaction impairs oncogenic KRAS signalling
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Stephan A. Hahn, Gemma Triola, Philippe I. H. Bastiaens, Anchal Chandra, Maike Hoffmann, Herbert Waldmann, Nachiket Vartak, Shehab Ismail, Gunther Zimmermann, Björn Papke, and Alfred Wittinghofer
- Subjects
Models, Molecular ,MAP Kinase Signaling System ,Molecular Conformation ,Mice, Nude ,Adenocarcinoma ,Oncogene Protein p21(ras) ,Biology ,medicine.disease_cause ,Cell Line ,Mice ,03 medical and health sciences ,Dogs ,0302 clinical medicine ,In vivo ,Cell Line, Tumor ,medicine ,Animals ,Humans ,Binding site ,neoplasms ,Cell Proliferation ,030304 developmental biology ,Cyclic Nucleotide Phosphodiesterases, Type 6 ,0303 health sciences ,Binding Sites ,Multidisciplinary ,Oncogene ,Drug discovery ,Hydrogen Bonding ,Small molecule ,Molecular biology ,digestive system diseases ,Cytoplasm ,Cell culture ,030220 oncology & carcinogenesis ,Cancer research ,Benzimidazoles ,KRAS ,Mitogen-Activated Protein Kinases ,Neoplasm Transplantation ,Carcinoma, Pancreatic Ductal ,Protein Binding ,Signal Transduction - Abstract
The KRAS oncogene product is considered a major target in anticancer drug discovery. However, direct interference with KRAS signalling has not yet led to clinically useful drugs. Correct localization and signalling by farnesylated KRAS is regulated by the prenyl-binding protein PDEδ, which sustains the spatial organization of KRAS by facilitating its diffusion in the cytoplasm. Here we report that interfering with binding of mammalian PDEδ to KRAS by means of small molecules provides a novel opportunity to suppress oncogenic RAS signalling by altering its localization to endomembranes. Biochemical screening and subsequent structure-based hit optimization yielded inhibitors of the KRAS-PDEδ interaction that selectively bind to the prenyl-binding pocket of PDEδ with nanomolar affinity, inhibit oncogenic RAS signalling and suppress in vitro and in vivo proliferation of human pancreatic ductal adenocarcinoma cells that are dependent on oncogenic KRAS. Our findings may inspire novel drug discovery efforts aimed at the development of drugs targeting oncogenic RAS.
- Published
- 2013
35. Development of Pyridazinone Chemotypes Targeting the PDEδ Prenyl Binding Site
- Author
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Sandip Murarka, Carsten Schultz-Fademrecht, Matthias Baumann, Shehab Ismail, Alfred Wittinghofer, Alaa Al Saabi, Eyad K. Fansa, Herbert Waldmann, Peter Nussbaumer, and Pablo Martín-Gago
- Subjects
0301 basic medicine ,010405 organic chemistry ,Chemistry ,Drug discovery ,Binding protein ,Organic Chemistry ,General Chemistry ,GTPase ,01 natural sciences ,Anticancer drug ,Small molecule ,Catalysis ,0104 chemical sciences ,03 medical and health sciences ,030104 developmental biology ,Biochemistry ,Prenylation ,In vivo ,Binding site - Abstract
The K-Ras GTPase is a major target in anticancer drug discovery. However, direct interference with signaling by K-Ras has not led to clinically useful drugs yet. Correct localization and signaling by farnesylated K-Ras is regulated by the prenyl binding protein PDEδ. Interfering with binding of PDEδ to K-Ras by means of small molecules provides a novel opportunity to suppress oncogenic signaling. Here we describe the identification and structure-guided development of novel K-Ras–PDEδ inhibitor chemotypes based on pyrrolopyridazinones and pyrazolopyridazinones that bind to the farnesyl binding pocket of PDEδ with low nanomolar affinity. We delineate the structure–property relationship and in vivo pharmacokinetic (PK) and toxicokinetic (Tox) studies for pyrazolopyridazinone-based K-Ras–PDEδ inhibitors. These findings may inspire novel drug discovery efforts aimed at the development of drugs targeting oncogenic Ras.
- Published
- 2016
36. Shuttling and sorting lipid-modified cargo into the cilia
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Shehab Ismail and Louise A. Stephen
- Subjects
0301 basic medicine ,ADP ribosylation factor ,Membrane lipids ,Cell ,Biology ,Biochemistry ,Models, Biological ,03 medical and health sciences ,Membrane Lipids ,Microtubule ,Organelle ,medicine ,Animals ,Humans ,Cilia ,Adaptor Proteins, Signal Transducing ,Cyclic Nucleotide Phosphodiesterases, Type 6 ,ADP-Ribosylation Factors ,Cilium ,Cell biology ,Transport protein ,Protein Transport ,030104 developmental biology ,medicine.anatomical_structure ,Membrane ,Guanosine Triphosphate - Abstract
Primary cilia are hair-like microtubule-based organelles that can be found on almost all human cell types. Although the cilium is not separated from the cell by membranes, their content is different from that of the cell body and their membrane composition is distinct from that of the plasma membrane. Here, we will introduce a molecular machinery that shuttles and sorts lipid-modified proteins to the cilium, thus contributing in maintaining its distinct composition. The mechanism involves the binding of the GDI-like solubilising factors, uncoordinated (UNC)119a, UNC119b and PDE6D, to the lipid-modified ciliary cargo and the specific release of the cargo in the cilia by the ciliary small G-protein Arl3 in a GTP-dependent manner.
- Published
- 2016
37. Structural basis for Arl3-specific release of myristoylated ciliary cargo from UNC119
- Author
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Ingrid R. Vetter, Yong-Xiang Chen, Shehab Ismail, Mandy Miertzschke, Carolin Koerner, and Alfred Wittinghofer
- Subjects
0303 health sciences ,General Immunology and Microbiology ,ADP ribosylation factor ,General Neuroscience ,030302 biochemistry & molecular biology ,Small G Protein ,Plasma protein binding ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Cell biology ,03 medical and health sciences ,GTP-binding protein regulators ,Protein structure ,Molecular Biology ,Ciliary membrane ,Cellular localization ,030304 developmental biology ,Myristoylation - Abstract
Access to the ciliary membrane for trans-membrane or membrane-associated proteins is a regulated process. Previously, we have shown that the closely homologous small G proteins Arl2 and Arl3 allosterically regulate prenylated cargo release from PDEδ. UNC119/HRG4 is responsible for ciliary delivery of myristoylated cargo. Here, we show that although Arl3 and Arl2 bind UNC119 with similar affinities, only Arl3 allosterically displaces cargo by accelerating its release by three orders of magnitude. Crystal structures of Arl3 and Arl2 in complex with UNC119a reveal the molecular basis of specificity. Contrary to previous structures of GTP-bound Arf subfamily proteins, the N-terminal amphipathic helix of Arl3·GppNHp is not displaced by the interswitch toggle but remains bound on the surface of the protein. Opposite to the mechanism of cargo release on PDEδ, this induces a widening of the myristoyl binding pocket. This leads us to propose that ciliary targeting of myristoylated proteins is not only dependent on nucleotide status but also on the cellular localization of Arl3.
- Published
- 2012
38. The GDI-like solubilizing factor PDEδ sustains the spatial organization and signalling of Ras family proteins
- Author
-
Philippe I. H. Bastiaens, Christian Hedberg, Michael Hanzal-Bayer, Alfred Wittinghofer, Shehab Ismail, David Perera, Ashok R. Venkitaraman, Ferdinandos Skoulidis, Hernãin E. Grecco, Liam D. Cassidy, Venkat Pisupati, and Anchal Chandra
- Subjects
symbols.namesake ,Prenylation ,Cytoplasm ,G protein ,symbols ,Fluorescence recovery after photobleaching ,Cell Biology ,HRAS ,Plasma protein binding ,Signal transduction ,Golgi apparatus ,Biology ,Cell biology - Abstract
We identify a role for the GDI-like solubilizing factor (GSF) PDEδ in modulating signalling through Ras family G proteins by sustaining their dynamic distribution in cellular membranes. We show that the GDI-like pocket of PDEδ binds and solubilizes farnesylated Ras proteins, thereby enhancing their diffusion in the cytoplasm. This mechanism allows more effective trapping of depalmitoylated Ras proteins at the Golgi and polycationic Ras proteins at the plasma membrane to counter the entropic tendency to distribute these proteins over all intracellular membranes. Thus, PDEδ activity augments K/Hras signalling by enriching Ras at the plasma membrane; conversely, PDEδ down-modulation randomizes Ras distributions to all membranes in the cell and suppresses regulated signalling through wild-type Ras and also constitutive oncogenic Ras signalling in cancer cells. Our findings link the activity of PDEδ in determining Ras protein topography to Ras-dependent signalling.
- Published
- 2011
39. Arl2-GTP and Arl3-GTP regulate a GDI-like transport system for farnesylated cargo
- Author
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Gemma Triola, Lothar Gremer, Herbert Waldmann, Philippe I. H. Bastiaens, Alfred Wittinghofer, Shehab Ismail, Anchal Chandra, Alexandra Rusinova, Yong-Xiang Chen, and Martin Bierbaum
- Subjects
Models, Molecular ,GTPase-activating protein ,ADP ribosylation factor ,GTP' ,Protein Conformation ,G protein ,03 medical and health sciences ,Dogs ,0302 clinical medicine ,GTP-binding protein regulators ,GTP-Binding Proteins ,Animals ,Humans ,Molecular Biology ,Cells, Cultured ,030304 developmental biology ,Prenylation ,Cyclic Nucleotide Phosphodiesterases, Type 6 ,0303 health sciences ,Guanine Nucleotide Dissociation Inhibitors ,biology ,ADP-Ribosylation Factors ,Biological Transport ,Cell Biology ,Cell biology ,030220 oncology & carcinogenesis ,biology.protein ,Guanosine Triphosphate ,Rab ,RHEB - Abstract
Lipidated Rho and Rab GTP-binding proteins are transported between membranes in complex with solubilizing factors called 'guanine nucleotide dissociation inhibitors' (GDIs). Unloading from GDIs using GDI displacement factors (GDFs) has been proposed but remains mechanistically elusive. PDEδ is a putative solubilizing factor for several prenylated Ras-subfamily proteins. Here we report the structure of fully modified farnesylated Rheb-GDP in complex with PDEδ. The structure explains the nucleotide-independent binding of Rheb to PDEδ and the relaxed specificity of PDEδ. We demonstrate that the G proteins Arl2 and Arl3 act in a GTP-dependent manner as allosteric release factors for farnesylated cargo. We thus describe a new transport system for farnesylated G proteins involving a GDI-like molecule and an unequivocal GDF. Considering the importance of PDEδ for proper Ras and Rheb signaling, this study is instrumental in developing a new target for anticancer therapy.
- Published
- 2011
40. Effect of the N-Terminal Helix and Nucleotide Loading on the Membrane and Effector Binding of Arl2/3
- Author
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Katrin Weise, Eyad K. Fansa, Simone Möbitz, Roland Winter, Shobhna Kapoor, Alfred Wittinghofer, and Shehab Ismail
- Subjects
Membranes ,Effector ,Protein Conformation ,Allosteric regulation ,Optical Imaging ,Biophysics ,Membranes, Artificial ,Biology ,Microscopy, Atomic Force ,Guanosine Diphosphate ,Fluorescence ,Cell biology ,Kinetics ,Membrane ,GTP-binding protein regulators ,Protein structure ,Membrane Microdomains ,Membrane protein ,GTP-Binding Proteins ,Helix ,Membrane fluidity ,Adaptor Proteins, Signal Transducing - Abstract
The small GTP-binding proteins Arl2 and Arl3, which are close homologs, share a number of interacting partners and act as displacement factors for prenylated and myristoylated cargo. Nevertheless, both proteins have distinct biological functions. Whereas Arl3 is considered a ciliary protein, Arl2 has been reported to be involved in tubulin folding, mitochondrial function, and Ras signaling. How these different roles are attained by the two homolog proteins is not fully understood. Recently, we showed that the N-terminal amphipathic helix of Arl3, but not that of Arl2, regulates the release of myristoylated ciliary proteins from the GDI-like solubilizing factor UNC119a/b. In the biophysical study presented here, both proteins are shown to exhibit a preferential localization and clustering in liquid-disordered domains of phase-separated membranes. However, the membrane interaction behavior differs significantly between both proteins with regard to their nucleotide loading. Whereas Arl3 and other Arf proteins with an N-terminal amphipathic helix require GTP loading for the interaction with membranes, Arl2 binds to membranes in a nucleotide-independent manner. In contrast to Arl2, the N-terminal helix of Arl3 increases the binding affinity to UNC119a. Furthermore, UNC119a impedes membrane binding of Arl3, but not of Arl2. Taken together, these results suggest an interplay among the nucleotide status of Arl3, the location of the N-terminal helix, membrane fluidity and binding, and the release of lipid modified cargos from carriers such as UNC119a. Since a specific Arl3-GEF is postulated to reside inside cilia, the N-terminal helix of Arl3•GTP would be available for allosteric regulation of UNC119a cargo release only inside cilia.
- Published
- 2015
41. Kant’s Politics
- Author
-
Shehab Ismail
- Subjects
Politics ,Philosophy ,Epistemology - Published
- 2006
42. Structural analysis of human liver glyceraldehyde-3-phosphate dehydrogenase
- Author
-
H. W. Park and Shehab Ismail
- Subjects
Models, Molecular ,Trypanosoma ,Protein Conformation ,Protein subunit ,Dehydrogenase ,Nicotinamide adenine dinucleotide ,Crystallography, X-Ray ,Structure-Activity Relationship ,chemistry.chemical_compound ,Protein structure ,stomatognathic system ,Trypanosomiasis ,Structural Biology ,Animals ,Humans ,Enzyme Inhibitors ,Muscle, Skeletal ,Glyceraldehyde 3-phosphate dehydrogenase ,chemistry.chemical_classification ,biology ,Glyceraldehyde-3-Phosphate Dehydrogenases ,Parkinson Disease ,General Medicine ,Molecular biology ,Enzyme ,Liver ,chemistry ,Biochemistry ,Drug Design ,biology.protein ,NAD+ kinase ,Homotetramer - Abstract
The crystal structure of human liver glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been determined. This structure represents the first moderate-resolution (2.5 A) and crystallographically refined (Rfree = 22.9%) human GAPDH structure. The liver GAPDH structure consists of a homotetramer, each subunit of which is bound to a nicotinamide adenine dinucleotide (NAD+) molecule. The GAPDH enzyme has glycolytic and non-glycolytic functions, both of which are of chemotherapeutic interest. The availability of a high-quality human GAPDH structure is a necessity for structure-based drug design. In this study, structural differences between human liver and skeletal muscle GAPDHs are reported in order to understand how these two enzymes might respond to anti-trypanosomatid GAPDH inhibitors.
- Published
- 2005
43. Post-proteomic identification of a novel phage-encoded streptodornase, Sda1, in invasive M1T1 Streptococcus pyogenes
- Author
-
Ramy K. Aziz, Shehab Ismail, Malak Kotb, and Hee-Won Park
- Subjects
biology ,Nucleic acid sequence ,Virulence ,medicine.disease_cause ,biology.organism_classification ,Microbiology ,Fusion protein ,Bacteriophage ,Streptococcus pyogenes ,medicine ,Molecular Biology ,Gene ,Peptide sequence ,Prophage - Abstract
The M1T1 strain remains the most frequently isolated strain from group A streptococcal (GAS) infection cases worldwide. We previously reported that M1T1 differs from the fully sequenced M1 SF370 strain. To better understand the reason for the persistence and increased virulence of M1T1, we analysed its secreted proteome and identified two virulence proteins that are not present in the sequenced M1 SF370 strain: streptococcal pyrogenic exotoxin A (SpeA) and a streptodornase D (SdaD) homologue. In the present study, we determined the nucleotide sequence of the M1T1 streptodornase and found that its deduced amino acid sequence is highly similar to other streptococcal streptodornases, and is most closely related to the SdaD of GAS strain M49. M1T1 Sda shares two highly conserved domains with several DNases and putative DNases in streptococci; however, it possesses a unique C-terminal amino acid sequence. Thus, we named the protein Sda1, and we detected the presence of the sda1 gene in 16 M1T1 clinical isolates. The cloned and expressed Sda1 degrades both streptococcal and mammalian DNA at physiological pH. Amino acid similarity analyses of known GAS deoxyribonucleases suggest that Sda1 may be a chimeric protein created through recombination events. Moreover, a natural mutation that resulted in longer Sda1 and SdaD as compared to other GAS DNases was found to confer increased activity on the protein. Analysis of the sequences flanking sda1 determined that it is carried by a prophage or a prophage-like element inserted in the tRNA-Ser gene of M1T1 GAS. Ongoing studies in our laboratory aim to determine the contribution of Sda1 to the virulence of this globally disseminated M1T1 strain.
- Published
- 2004
44. Thermodynamic, dynamic and solvational properties of PDEδ binding to farnesylated cystein: a model study for uncovering the molecular mechanism of PDEδ interaction with prenylated proteins
- Author
-
Roland Winter, Shehab Ismail, and Saba Suladze
- Subjects
Models, Molecular ,Cyclic Nucleotide Phosphodiesterases, Type 6 ,Binding Sites ,Chemistry ,Stereochemistry ,Model study ,Protein Prenylation ,Surfaces, Coatings and Films ,Accessible surface area ,Prenylation ,Solubility ,Materials Chemistry ,Molecular mechanism ,ras Proteins ,Ras subfamily ,Thermodynamics ,Cysteine ,Physical and Theoretical Chemistry - Abstract
The protein PDEδ is an important solubilizing factor for several prenylated proteins including the Ras subfamily members. The binding occurs mainly through the farnesyl anchor of Ras proteins, which is recognized by a hydrophobic pocket of PDEδ. In this study, we carried out a detailed study of the thermodynamic and solvational properties of PDEδ binding to farnesyl-cystein, which serves as a model for PDEδ association to prenylated proteins. Using various biophysical approaches in conjunction with theoretical considerations, we show here that binding of the largely hydrophobic ligand surprisingly has enthalpy-driven signature, and the entropy change is largely controlled by the fine balance between the hydrational and conformational terms. Moreover, binding of PDEδ to farnesyl-cystein is accompanied by an increase in thermal stability, the release of about 150 water molecules from the interacting species, a decrease in solvent accessible surface area, and a marked decrease of the volume fluctuations and hence dynamics of the protein. Altogether, our results shed more light on the molecular mechanism of PDEδ interaction with prenylated Ras proteins, which is also prerequisite for an optimization of the structure-based molecular design of drugs against Ras related diseases and for understanding the multitude of biological functions of PDEδ.
- Published
- 2014
45. Predicted incorporation of non-native substrates by a polyketide synthase yields bioactive natural product derivatives
- Author
-
Alfred Wittinghofer, Elsa Sanchez-Garcia, Kenny Bravo-Rodriguez, Shehab Ismail, Eyad K. Fansa, Frank Schulz, Susanna Kushnir, A. Ismail-Ali, and Stephan Klopries
- Subjects
Models, Molecular ,Protein Conformation ,Stereochemistry ,Ionophore ,Malonic acid ,Biochemistry ,Substrate Specificity ,chemistry.chemical_compound ,Polyketide synthase ,Escherichia coli ,Monensin ,Molecular Biology ,Biological Products ,Natural product ,biology ,Organic Chemistry ,Computational Biology ,Substrate (chemistry) ,Malonates ,Streptomyces ,chemistry ,Acyltransferase ,Fermentation ,biology.protein ,Molecular Medicine ,Polyketide Synthases ,Biologie ,Acyltransferases ,Macromolecule - Abstract
The polyether ionophore monensin is biosynthesized by a polyketide synthase that delivers a mixture of monensins A and B by the incorporation of ethyl- or methyl-malonyl-CoA at its fifth module. Here we present the first computational model of the fifth acyltransferase domain (AT5mon ) of this polyketide synthase, thus affording an investigation of the basis of the relaxed specificity in AT5mon , insights into the activation for the nucleophilic attack on the substrate, and prediction of the incorporation of synthetic malonic acid building blocks by this enzyme. Our predictions are supported by experimental studies, including the isolation of a predicted derivative of the monensin precursor premonensin. The incorporation of non-native building blocks was found to alter the ratio of premonensins A and B. The bioactivity of the natural product derivatives was investigated and revealed binding to prenyl-binding protein. We thus show the potential of engineered biosynthetic polyketides as a source of ligands for biological macromolecules.
- Published
- 2014
46. The interplay between RPGR, PDEδ and Arl2/3 regulate the ciliary targeting of farnesylated cargo
- Author
-
Shehab Ismail, Ingrid R. Vetter, Katja Gotthardt, Denise Wätzlich, Mandy Miertzschke, Yong-Xiang Chen, and Alfred Wittinghofer
- Subjects
Scaffold protein ,Genetics ,0303 health sciences ,genetic structures ,Cilium ,Scientific Reports ,macromolecular substances ,Retinitis pigmentosa GTPase regulator ,Plasma protein binding ,Biology ,Biochemistry ,Ciliopathies ,eye diseases ,3. Good health ,Conserved sequence ,Cell biology ,Transport protein ,03 medical and health sciences ,0302 clinical medicine ,Protein prenylation ,sense organs ,Molecular Biology ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
Defects in primary cilia result in human diseases known as ciliopathies. The retinitis pigmentosa GTPase regulator (RPGR), mutated in the most severe form of the eye disease, is located at the transition zone of the ciliary organelle. The RPGR-interacting partner PDEδ is involved in trafficking of farnesylated ciliary cargo, but the significance of this interaction is unknown. The crystal structure of the propeller domain of RPGR shows the location of patient mutations and how they perturb the structure. The RPGR·PDEδ complex structure shows PDEδ on a highly conserved surface patch of RPGR. Biochemical experiments and structural considerations show that RPGR can bind with high affinity to cargo-loaded PDEδ and exposes the Arl2/Arl3-binding site on PDEδ. On the basis of these results, we propose a model where RPGR is acting as a scaffold protein recruiting cargo-loaded PDEδ and Arl3 to release lipidated cargo into cilia.
- Published
- 2013
47. Arl3 regulates a transport system for farnesylated cargo
- Author
-
Anchal Chandra, PB Batiaens, AR Rusinnova, Shehab Ismail, Lothar Gremer, Gemma Triola, HW Waldmann, Yong-Xiang Chen, AW Winttinghofer, and Martin Bierbaum
- Subjects
biology ,lcsh:Cytology ,Cilium ,Allosteric regulation ,Cell Biology ,Cell biology ,Prenylation ,Live cell imaging ,Poster Presentation ,biology.protein ,lcsh:QH573-671 ,Receptor ,Developmental biology ,Function (biology) ,RHEB - Abstract
Arl3 is a small G-protein that is found exclusively in ciliated organisms. In addition, knocking out of Arl3 results in a plethora of ciliopathies. Arl3 is known to bind the photoreceptor (specialized cilia) specific PDE delta subunit (PDE6D), which in turn bind to prenylated proteins. The significance of this interaction and the function of Arl3 in cilia are poorly understood. Here in this study, by solving the crystal structure of a fully modified prenylated (farnesylated) Rheb in complex with PDE6D and comparing it to a structure of PDE6D in complex with the Arl3 homologue Arl2, we show that Arl3 is an allosteric regulator of PDE6D. Arl3, in a nucleotide dependent manner, releases the farnesylated cargo bound to PDE6D. We explain the molecular mechanism of this release and we further verify the mechanism in vitro and by live cell imaging. Based on this study we hypothesize that Arl3 regulate the targeting of prenylated cargo in and out the cilia.
- Published
- 2012
48. The structure of an Arf-ArfGAP complex reveals a Ca2+ regulatory mechanism
- Author
-
Ingrid R. Vetter, Alfred Wittinghofer, Shehab Ismail, and Begoña Sot
- Subjects
Models, Molecular ,GTPase-activating protein ,ADP ribosylation factor ,PROTEINS ,Molecular Sequence Data ,Small G Protein ,GTPase ,Biology ,Guanosine triphosphate ,Crystallography, X-Ray ,General Biochemistry, Genetics and Molecular Biology ,chemistry.chemical_compound ,Humans ,Protein Interaction Domains and Motifs ,Amino Acid Sequence ,Cytoskeleton ,Biochemistry, Genetics and Molecular Biology(all) ,ADP-Ribosylation Factors ,GTPase-Activating Proteins ,Transport protein ,Cell biology ,Crosstalk (biology) ,chemistry ,SIGNALING ,ADP-Ribosylation Factor 6 ,CELLBIO ,Calcium ,Guanosine Triphosphate ,Sequence Alignment - Abstract
SummaryArfs are small G proteins that have a key role in vesicle trafficking and cytoskeletal remodeling. ArfGAP proteins stimulate Arf intrinsic GTP hydrolysis by a mechanism that is still unresolved. Using a fusion construct we solved the structure of the ArfGAP ASAP3 in complex with Arf6 in the transition state. This structure clarifies the ArfGAP catalytic mechanism and shows a glutamine(Arf6) and an arginine finger(ASAP3) as the important catalytic residues. Unexpectedly the structure shows a calcium ion, liganded by both proteins in the complex interface, stabilizing the interaction and orienting the catalytic machinery. Calcium stimulates the GAP activity of ASAPs, but not other members of the ArfGAP family. This type of regulation is unique for GAPs and any other calcium-regulated processes and hints at a crosstalk between Ca2+ and Arf signaling.
- Published
- 2009
49. Erratum: The GDI-like solubilizing factor PDEδ sustains the spatial organization and signalling of Ras family proteins
- Author
-
Philippe I. H. Bastiaens, Shehab Ismail, Christian Hedberg, Alfred Wittinghofer, Venkat Pisupati, Liam D. Cassidy, Michael Hanzal-Bayer, David Perera, Ferdinandos Skoulidis, Ashok R. Venkitaraman, Anchal Chandra, and Hernán E. Grecco
- Subjects
Signalling ,Cell Biology ,Biology ,Spatial organization ,Cell biology - Published
- 2012
50. Specificity of Arl2/Arl3 signaling is mediated by a ternary Arl3-effector-GAP complex
- Author
-
Alfred Wittinghofer, Stefan Veltel, Shehab Ismail, and Aleksandra Kravchenko
- Subjects
Subfamily ,GTPase-activating protein ,Protein Conformation ,Molecular Sequence Data ,Biophysics ,Small G Protein ,Biology ,Signal transduction ,Biochemistry ,Guanosine Diphosphate ,chemistry.chemical_compound ,Structural Biology ,GTP-Binding Proteins ,Retinitis pigmentosa ,Genetics ,medicine ,Humans ,Amino Acid Sequence ,Eye Proteins ,Molecular Biology ,Ternary complex ,Adaptor Proteins, Signal Transducing ,Cyclic Nucleotide Phosphodiesterases, Type 6 ,Photoreceptor ,Effector ,ADP-Ribosylation Factors ,GTPase-Activating Proteins ,Intracellular Signaling Peptides and Proteins ,Membrane Proteins ,Arf ,Retinal ,GAP ,Cell Biology ,medicine.disease ,Cell biology ,chemistry ,Function (biology) ,Photoreceptor Cells, Vertebrate ,G proteins - Abstract
Arl2 and Arl3, members of the Arf subfamily of small G proteins, are believed to be involved in ciliary and microtubule-dependent processes. Recently, we could identify RP2, responsible for a variant of X-linked retinitis pigmentosa, as the Arl3-specific GAP. Here, we have characterized Arl2/3 interactions. We show the formation of a ternary complex between Arl3, its cognate GAP RP2 and its retinal effector HRG4. This complex seems to be important for photoreceptor function.Structured summaryMINT-6602303:ARL2 (uniprotkb:P36404) binds (MI:0407) to HRG4 (uniprotkb:Q13432) by pull down (MI:0096)MINT-6602333:ARL3 (uniprotkb:P36405) binds (MI:0407) to CoD (uniprotkb:Q9BTW9) by pull down (MI:0096)MINT-6602347, MINT-6602369:RP2 (uniprotkb:O75695), ARL3 (uniprotkb:P36405) and HRG4 (uniprotkb:Q13432) physically interact (MI:0218) by fluorescence polarization spectroscopy (MI:0053)MINT-6602195:PDE delta (uniprotkb:O43924) and ARL2 (uniprotkb:P36404) bind (MI:0407) by fluorescence polarization spectroscopy (MI:0053)MINT-6602213:BART (uniprotkb:Q8WZ55) and ARL2 (uniprotkb:P36404) bind (MI:0407) by fluorescence polarization spectroscopy (MI:0053)MINT-6602239:ARL3 (uniprotkb:P36405) and BART (uniprotkb:Q8WZ55) bind (MI:0407) by fluorescence polarization spectroscopy (MI:0053)MINT-6602322:ARL2 (uniprotkb:P36404) binds (MI:0407) to CoD (uniprotkb:Q9BTW9) by pull down (MI:0096)MINT-6602258:RP2 (uniprotkb:Q8IWN7) and ARL3 (uniprotkb:P36405) bind (MI:0407) by fluorescence polarization spectroscopy (MI:0053)MINT-6602233:ARL3 (uniprotkb:P36405) and HRG4 (uniprotkb:Q13432) bind (MI:0407) by fluorescence polarization spectroscopy (MI:0053)MINT-6602360:HRG4 (uniprotkb:Q13432), ARL3 (uniprotkb:P36405) and RP2 (uniprotkb:O75695) physically interact (MI:0218) by molecular sieving (MI:0071)MINT-6602297:ARL2 (uniprotkb:P36404) binds (MI:0407) to PDE delta (uniprotkb:O43924) by pull down (MI:0096)MINT-6602227:ARL3 (uniprotkb:P36405) and PDE delta (uniprotkb:O43924) bind (MI:0407) by fluorescence polarization spectroscopy (MI:0053)MINT-6602204:HRG4 (uniprotkb:Q13432) and ARL2 (uniprotkb:P36404) bind (MI:0407) by fluorescence polarization spectroscopy (MI:0053)
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