Mechanisms and functions of CRACR2A-L Rab GTPase in vascular endothelial cells
Lead Research Organisation:
University of Leeds
Department Name: School of Medicine
Abstract
Thrombus formation (blood clotting) is an essential process to maintain vascular integrity and prevent excessive bleeding after vessel damage. However abnormal thrombi formation leads to blood vessel occlusions and development of atherosclerotic plaques with subsequent hypertension, stroke or ischaemia. Von-Willebrand Factor (vWF)-mediated platelet adhesion is a major regulator of thrombus formation, therefore vWF secretion from endothelial cells is highly regulated. Increased plasma levels of vWF have been proposed as a risk factor for cardiovascular disease, especially in high risk groups such as diabetics. vWF is contained in special storage vesicles called Weibel Palade Bodies (WPBs) which rapidly release their constituents upon stimulation. Agonist induced vWF secretion is a Ca2+-dependent process and is regulated by molecular switches called Rab GTPases. The protein that couples the rise in agonist-induced intracellular Ca2+ to Rab GTPase activation has remained elusive despite being the focus of several research groups. However, we recently identified a novel Rab GTPase expressed in endothelial cells that, unlike the majority of the Rab GTPase family, contains Ca2+ binding domains. This research proposes that the Ca2+ binding Rab GTPase, CRACR2A-L, has important functions and could potentially be offered as a target for novel therapeutics for athero-thrombotic diseases.
Technical Summary
Objective 1: To determine the contribution of CRACR2A-L to Weibel Palade body trafficking. The general strategy for this project will be to use high resolution imaging to quantify and measure the distribution of CRACR2A-L and vWF in endothelial cells. Contribution of CRACR2A-L to Weibel Palade body trafficking in response to thrombotic stimuli will be assessed by overexpressing wtCRACR2A-L or GTP/Ca2+-binding mutants or alternatively, cellular levels of protein will be reduced by short interfering RNA. Secretion of vWF as a measure of Weibel Palade body exocytosis will be assayed using ELISA. The effect of CRACR2A-L on other endothelial functions such as apoptosis and proliferation will also be assessed.
Objective 2: To discover the fundamental mechanisms by which CRACR2A-L regulates Weibel Palade bodies. Affinity chromatography followed by LC-MS/MS will be used to identify downstream effector proteins. Potential effector proteins will be identified and ranked according to peptide abundance and pathway analysis. Confirmed effectors will be verified by co-immunoprecipitation and rescue of function experiments using high resolution imaging. To be able to understand the structural mechanisms of CRACR2A-L action, structural models will be created by X-ray crystallography and homology modelling.
Objective 3: To verify the in vivo role of CRACR2A-L using an endothelial specific knockout mouse. First, production of a CRACR2A global 'knockout first' (with conditional potential) mouse will be outsourced. Subsequent breeding with FLP deleters and endothelial specific Cre recombinase mice will generate an endothelial specific CRACR2A-L knockout mouse. The in vivo contribution of global and endothelial CRACR2A-L to Weibel-Palade body exocytosis will be analysed by measuring clotting in response to tail injury. In addition, real time intravital imaging of clot formation will be performed in the femoral artery and in the mouse ear. Angiogenesis will also be assessed.
Objective 2: To discover the fundamental mechanisms by which CRACR2A-L regulates Weibel Palade bodies. Affinity chromatography followed by LC-MS/MS will be used to identify downstream effector proteins. Potential effector proteins will be identified and ranked according to peptide abundance and pathway analysis. Confirmed effectors will be verified by co-immunoprecipitation and rescue of function experiments using high resolution imaging. To be able to understand the structural mechanisms of CRACR2A-L action, structural models will be created by X-ray crystallography and homology modelling.
Objective 3: To verify the in vivo role of CRACR2A-L using an endothelial specific knockout mouse. First, production of a CRACR2A global 'knockout first' (with conditional potential) mouse will be outsourced. Subsequent breeding with FLP deleters and endothelial specific Cre recombinase mice will generate an endothelial specific CRACR2A-L knockout mouse. The in vivo contribution of global and endothelial CRACR2A-L to Weibel-Palade body exocytosis will be analysed by measuring clotting in response to tail injury. In addition, real time intravital imaging of clot formation will be performed in the femoral artery and in the mouse ear. Angiogenesis will also be assessed.
Planned Impact
Primarily the impact flows from the basic understanding of cellular trafficking to translational research in the vasculature where novel approaches could be devised to manipulate the secretion of vWF to improve cardiovascular health.
The research on a fundamental mechanism in Rab GTPase regulation of cellular trafficking will be: publishable in text books to enhance student learning; used to encourage further funding applications from vascular biologists, cellular biologists and researchers of many disease areas; result in the publication of several peer reviewed papers for international dissemination. In addition, spin-off companies could specialise in drug discovery programmes using findings from the structural analysis.
Clinically, there is a potential to enhance the treatment of thrombosis in cardiovascular disease, especially in susceptible populations such as diabetics by manipulating vWF release. This would foster further collaborations and investment with clinicians and translational scientists and could involve the exploitation of intellectual property and patenting.
The research on a fundamental mechanism in Rab GTPase regulation of cellular trafficking will be: publishable in text books to enhance student learning; used to encourage further funding applications from vascular biologists, cellular biologists and researchers of many disease areas; result in the publication of several peer reviewed papers for international dissemination. In addition, spin-off companies could specialise in drug discovery programmes using findings from the structural analysis.
Clinically, there is a potential to enhance the treatment of thrombosis in cardiovascular disease, especially in susceptible populations such as diabetics by manipulating vWF release. This would foster further collaborations and investment with clinicians and translational scientists and could involve the exploitation of intellectual property and patenting.
Publications
Miteva KT
(2019)
Rab46 integrates Ca2+ and histamine signaling to regulate selective cargo release from Weibel-Palade bodies.
in The Journal of cell biology
Pedicini L
(2018)
Homotypic endothelial nanotubes induced by wheat germ agglutinin and thrombin.
in Scientific reports
Pedicini L
(2021)
Affinity-based proteomics reveals novel binding partners for Rab46 in endothelial cells
in Scientific Reports
Pedicini Lucia
(2019)
Functions and mechanisms of Rab46 in endothelial cells
Description | BHF 4yr Non-clinical PhD studentship |
Amount | £150,000 (GBP) |
Funding ID | FS/18/61/34182 |
Organisation | British Heart Foundation (BHF) |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 09/2019 |
End | 09/2023 |
Description | BHF Non-clinical PhD studentship |
Amount | £181,000 (GBP) |
Funding ID | FS\PhD\21\29176 |
Organisation | University of Leeds |
Sector | Academic/University |
Country | United Kingdom |
Start | 04/2022 |
End | 03/2025 |
Description | BHF non clinical PhD studentship |
Amount | £107,808 (GBP) |
Funding ID | FS/17/43/33003 |
Organisation | British Heart Foundation (BHF) |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 09/2017 |
End | 08/2020 |
Description | High Flyer Fellowship award |
Amount | £5,000 (GBP) |
Organisation | University of Leeds |
Sector | Academic/University |
Country | United Kingdom |
Start | 03/2016 |
End | 02/2017 |
Description | Lattice Light Sheet Microscopy for the Biosciences |
Amount | £651,012 (GBP) |
Funding ID | BB/V01904X/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 08/2021 |
End | 05/2022 |
Description | Leeds Anniversary Research Scholarship |
Amount | £51,000 (GBP) |
Organisation | University of Leeds |
Sector | Academic/University |
Country | United Kingdom |
Start | 09/2016 |
End | 09/2019 |
Description | Leeds Institute of Cardiovascular and Metabolic Medicine 4yr funded PhD studentship |
Amount | £70,000 (GBP) |
Organisation | University of Leeds |
Sector | Academic/University |
Country | United Kingdom |
Start | 09/2016 |
End | 09/2020 |
Description | Royal Society Project Grant |
Amount | £18,602 (GBP) |
Funding ID | RGS\R2\202259 |
Organisation | University of Leeds |
Sector | Academic/University |
Country | United Kingdom |
Start | 04/2021 |
End | 03/2022 |
Description | The Biochemical Society bursary |
Amount | £75 (GBP) |
Organisation | Biochemical Society |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 03/2021 |
End | 03/2021 |
Description | The role of Rab46 in immune-mediated inflammatory diseases |
Amount | £192,000 (GBP) |
Funding ID | MR/T004134/1 |
Organisation | Medical Research Council (MRC) |
Sector | Public |
Country | United Kingdom |
Start | 12/2019 |
End | 11/2021 |
Description | University of Leeds BHF 4yr PhD programme |
Amount | £170,000 (GBP) |
Funding ID | FS/4yPhD/F/21/34153 |
Organisation | University of Leeds |
Sector | Academic/University |
Country | United Kingdom |
Start | 09/2021 |
End | 09/2025 |
Title | Affinity-based proteomics reveals novel binding partners for Rab46 in endothelial cells |
Description | Proteomic Analysis of Rab46 GFP pulldowns. Added as supplementary to the publication |
Type Of Material | Database/Collection of data |
Year Produced | 2021 |
Provided To Others? | Yes |
Impact | publication |
Description | Alex Breeze |
Organisation | University of Leeds |
Department | School of Design |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Molecular biology |
Collaborator Contribution | In cell NMR |
Impact | Multidisciplinary from School of Medicine (Mckeown) and Faculty of Biological Sciences Astbury Centre for Structural Molecular Biology (Breeze) |
Start Year | 2017 |
Description | BBSRC NAADP signaling in endothelial cells |
Organisation | University of Oxford |
Department | Oxford Hub |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | BBSRC project grant application to study NAADP Ca2+ signalling in endothelial cells. Lynn Mckeown as PI, provides post doc assigned to this project, cell biology skills, grant applications |
Collaborator Contribution | Prof Antony Galione as collaborator. Provides expertise, vectors, antibodies and training for PI and post doc |
Impact | BBSRC project grant application |
Start Year | 2019 |
Description | Bernadette Moore and Mar Pujades Rodriguez |
Organisation | University of Leeds |
Department | Astbury Centre for Structural Molecular Biology |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Expertise in vascular health and endothelial cell biology Bioimaging techniques Biochemistry techniques |
Collaborator Contribution | Expertise in Non alcoholic Liver Disease (NAFLD) and state of the art omics techniques Data analysis and access to large human patient datasets |
Impact | Multidisciplinary across School of Medicine; Leeds Institute of Cardiovascular and Metabolic Medicine (Mckeown), Leeds Institute of Data Analysis (Rodriguez). School of Food Science (Moore) |
Start Year | 2017 |
Description | Bernadette Moore and Mar Pujades Rodriguez |
Organisation | University of Leeds |
Department | Leeds Institute of Data Analysis |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Expertise in vascular health and endothelial cell biology Bioimaging techniques Biochemistry techniques |
Collaborator Contribution | Expertise in Non alcoholic Liver Disease (NAFLD) and state of the art omics techniques Data analysis and access to large human patient datasets |
Impact | Multidisciplinary across School of Medicine; Leeds Institute of Cardiovascular and Metabolic Medicine (Mckeown), Leeds Institute of Data Analysis (Rodriguez). School of Food Science (Moore) |
Start Year | 2017 |
Description | Calcium signalling and Weibel Palade bodies |
Organisation | University of Texas |
Department | Health Science Center at Houston |
Country | United States |
Sector | Academic/University |
PI Contribution | We are investigating the role of differential calcium signatures on the exocytosis of Weibel Palade bodies. We have shown that histamine confers a different response to thrombin and this is regulated by a EF-hand containing Rab GTPase Rab46. |
Collaborator Contribution | Our partners have made knockout mice and cells of channels that we think are important in regulating intracellular calcium release |
Impact | BBSRC project grant application to be submitted april 2018. |
Start Year | 2017 |
Description | Dan Cutler |
Organisation | University College London |
Department | MRC Laboratory for Molecular Cell Biology |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Shared knowledge re the discovery of a Rab GTPase involved in regulating Weibel Palade Bodies and vWF release and discussions re possible future publications. |
Collaborator Contribution | Discussions re the possible role of Rab46 in WPB trafficking and experimental approach. Input into high-through put imaging and data analysis. Invitations to network with UK tracking researchers. |
Impact | no outputs yet |
Start Year | 2015 |
Description | Izzy Jayasinghe |
Organisation | University of Leeds |
Department | Faculty of Biological Sciences |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | vascular surgery endothelial cell biology |
Collaborator Contribution | State of the art microscope techniques using super-resolution with Ca2+ imaging Novel software to analyse microscopy data Expertise in imaging analysis |
Impact | Multidisciplinary across biological sciences and medicine |
Start Year | 2016 |
Description | Rab46 and mast cells |
Organisation | University of Manchester |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Collaboration on BBSRC grant application |
Collaborator Contribution | provide expertise |
Impact | UKRI future leaders fellowship application BBSRC project grant application |
Start Year | 2018 |
Description | Rab46 and mast cells |
Organisation | University of Texas |
Department | Health Science Center at Houston |
Country | United States |
Sector | Academic/University |
PI Contribution | Collaboration on BBSRC grant application |
Collaborator Contribution | provide expertise |
Impact | UKRI future leaders fellowship application BBSRC project grant application |
Start Year | 2018 |
Description | A visit, tour and demonstrations by the Mckeown lab for BHF pledgers |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | People who have pledged money to the BHF were invited to visit my lab to inform them of how their contributions could be used and to give them a better understanding of the research that we undertake. The day consists of presentations from the BHF and Lynn McKeown, followed by four hands on demonstrations to illustrate the equipment we use and how and why we use it. After a networking lunch we had a Q&A session and informal chats regarding the research. |
Year(s) Of Engagement Activity | 2019 |