Signalling role(s) for the unconventional RdgB proteins: are they lipid sensors for phosphatidic acid ?

Lead Research Organisation: University College London
Department Name: Neuroscience Physiology and Pharmacology

Abstract

High blood pressure causes an increase in the size of the heart (cardiac hypertrophy) and is a major risk factor for the development of heart failure. One in five people die from this condition. Angiotensin II is a hormone that stimulates cardiac hypertrophy and it functions by binding to the Angiotensin II type I (ATI) receptor. A complex programme of intracellular signalling is initiated to stimulate hypertrophy and a new protein called ATRAP has been recently identified that protects against the effects of Angiotensin II. ATRAP was discovered because it binds to the ATI receptor but how ATRAP suppresses cardiac hypertrophy is not known. We have made an unexpected connection between ATRAP and a lipid binding protein, RdgB-beta.

We propose to define the connection between the two proteins, ATRAP and RdgB-beta in the context of lipid signalling via enzymes called phospholipases that produce the 'signalling lipid', phosphatidic acid (PA). We will establish how this protein-lipid network operates during Angiotensin II signalling. The activity of phospholipases is stimulated when Angiotensin II binds to the receptor. RdgB-beta is uncharacterised and we have discovered that it has unusual lipid binding properties - it binds PA.

Our concept is that RdgB-beta sequesters the 'PA' signal and therefore restrains the signalling cascade resulting in inhibition of cardiac hypertrophy. We will examine how RdgB-beta binds 'PA' and disposes of it. Because ATRAP binds RdgB-beta we think that a 'bridge' between two membranes is formed. This allows the 'PA' to be removed from the plasma membrane where signalling occurs and sent to the compartment where lipids are re-used for making new lipids. To form the bridge, RdgB-beta has to interact with ATRAP on one membrane and other proteins on the opposite membrane. We will therefore identify these proteins by using RdgB-beta as bait to fish for new proteins.

We will also study the importance of RdgB-beta and ATRAP by increasing or decreasing the protein levels in the cells. This will inform us on how Angiotensin II signalling is affected. If RdgB-beta reinforces the restraint put by ATRAP on Angiotensin II signalling, this will provide strong evidence that the molecular mechanism used by ATRAP is to participate in the removal of the signalling lipid, PA. To further test the model, we will delete the gene for RdgB-beta in a model organism (Drosophila) and examine the phenotype in collaboration with our project partner in Bangalore, India. To determine the importance of PA binding to RdgB-beta, we will make mutant proteins that cannot bind PA. These mutants will be examined for rescue of the fly defect.

The interaction between RdgB-beta and ATRAP together with the binding of PA to RdgB-beta could provide the molecular explanation of how ATRAP is able to suppress the function of Angiotensin II signalling and could therefore offer a novel therapeutic target for intervention in cardiovascular diseases. In the clinic, inhibition of Angiotensin II signalling by ACE inhibitors that prevents the production of Angiotensin II or drugs that prevent binding of Angiotensin II to its receptor are used for treatment for hypertension. Since most drugs have side-effects, drug combination that targets different systems are often used. Therefore the proposed research could well lead to a different molecular target which could provide a more effective treatment. Understanding how the endogenous inhibitor of Angiotensin II signalling, ATRAP, functions, may provide new strategies for drug targeting. Because ATRAP interacts with RdgB-beta, the possibility that targeting RdgB-beta may provide a unique opportunity to generate a new class of drugs that could be based on binding small hydrophobic molecules in the lipid binding pocket of RdgB-beta. The benefit derived from such drugs is huge as high blood pressure is one of the most common diseases that afflict humans.

Technical Summary

Angiotensin-II plays a major role in the progression of cardiac hypertrophy to heart failure. The actions of Angiotensin-II can be suppressed by ATRAP, an integral membrane protein that interacts with the receptor, by an unknown mechanism. We have made an unexpected connection between ATRAP and an uncharacterised lipid binding protein, RdgB-beta. We have also discovered that RdgB proteins are phosphatidylinositol-phosphatidic acid (PA) binding proteins. The interaction between RdgB-beta and ATRAP together with the ability of RdgB-beta to bind PA may provide a molecular explanation of how ATRAP is able to suppress the function of Angiotensin-II signalling and could therefore offer a novel therapeutic target for intervention in cardiovascular diseases.

We propose to define the connection between ATRAP and RdgB-beta in the context of angiotensin-II-stimulated phospholipase C and D. Both phospholipases produce PA. Our working hypothesis is that removal of the signalling lipid PA from the plasma membrane by RdgB-beta provides the molecular mechanism for suppressing angiotensin-II action. Our objectives are:

1. Identify amino acid residues that confer PA binding properties to RdgB-beta.
2. Define the PA species that RdgB-beta binds following phospholipase C and phospholipase D activation.
3. Examine if RdgB proteins are the long sought-after PA transfer proteins required for phosphatidylinositol resynthesis as part of the 'PI cycle'.
4. Examine whether RdgB-beta forms a 'bridge' between membranes for removal of PA from the plasma membrane to turn off PA signalling.
5. Identify binding partners for RdgB-beta and ATRAP required for lipid transfer.
6. Determine whether both splice variants of ATRAP bind RdgB-beta and the importance of the conserved Tyr133 residue in ATRAP function.
7. Determine the physiological function of RdgB-beta in the model organism, Drosophila and use this model system to analyse the importance of PA binding.

Planned Impact

Who will benefit from this research ?
The outcomes of this project will benefit i) other workers investigating angiotensin II cell signalling, ii) investigators studying phosphatidic acid as a cell signalling molecule and as a central metabolite of lipid metabolism, iii) pharmaceutical and biotech companies seeking new therapeutic targets for the treatment of cardiovascular diseases and obesity, iv) clinicians and their patients who might use such therapeutics.


How will they benefit from this research ?
I focus here on groups iii and iv, since these are the beneficiaries whose activities are most likely to directly impact on the nation's health and wealth. Most drugs entering clinical trials from within the private commercial sector are direct outcomes of basic biomedical research. Introduction of new drugs requires the identification of druggable targets and therefore the basic research underpins this activity. The commercial private sectors as well as charities such as British Heart Foundation and Kidney Research UK will be the direct beneficiary of this work - our work has identified a new protein that may play an important role in Angiotensin II signalling. Angiotensin II is a key hormone that plays a major role in the progression of myocardial hypertrophy to heart failure. In the clinic, inhibition of the angiotensin II converting enzyme (ACE) or blockade of the Angiotensin II receptors is used extensively ifor treatment of hypertension but there is scope for refinement of this mode of therapy. The work described here will provide a platform to design and develop novel strategies that could be aimed at developing drugs that could enhance well-being. In addition, RdgB-beta-ATRAP interactions could provide important molecular targets that would contribute to the design of novel drugs for the treatment of cardiovascular diseases.

The underpinning 'blue-skies' research proposed in this application could, in the longer term, translate to treatment of cardiovascular diseases and obesity, thus having the potential to engage the public. It will add to the wider implications of the ever increasing ageing populations where heart-related diseases and obesity have increased, both in the UK and globally. Hence, the wealth implications are significant.

Beneficiaries are essentially long term and can be defined in relation to enhanced knowledge of cell systems in the context of human physiology. The specific steps by which this enhancement will occur are not predictable and depend on the outcome of the research activity devoted to this end. We will, however, regularly review the findings to ensure that any new discoveries that are likely to impact on beneficiaries - for example, those populations with cardiovascular disease or obesity problems - are given due consideration.
 
Description Our major achievement was to demonstrate that RdgB proteins were the 'long sought after' lipid transfer proteins that facilitated the phospholipase C-phosphoinositide cycle. The phospholipase C signalling is central to many cellular functions and is extremely important in neuronal communication. In order to do this study we pioneered methods to monitor specific lipid binding to lipid transfer proteins. This allowed us to contribute our expertise in two other studies. In collaboration with Dr T Levine, we identified a new family of cholesterol transfer proteins that are present in both yeast and mammals. Collaborating with Dr Griac in Slovakia, we showed a lipid transfer protein in yeast that was responsible for resistance to azole anti-fungals commonly used clinically.
To analyse the biological function of these protein in free living organisms, we have developed a zebrafish model using gene editing. We have knocked out a specific isoform (PITPNC1) and the homozygous fish show disturbances in their sleep-awake behavior. PITPNC1 is only expressed in the zebrafish brain and not in any other tissue. The fish are more active - they sleep less during the night. We have identified hyper activation of neuronal activity in the knockout fish. Further analysis in a neuronal cell line show hyper activation of PI 3kinase pathway in cells where PITPNC1 is down regulated using RNAi.
Exploitation Route The transgenic Zebrafish can now be used to identify the neuronal signalling pathways that regulate the sleep-wake cycle.
Sectors Other
 
Description BHF PhD Studentship
Amount £123,214 (GBP)
Funding ID FS/15/73/31672 
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 11/2015 
End 10/2018
 
Title Transgenic Zebrafish using Cas/CRISPR technology 
Description Transgenic zebrafish lacking the lipid transfer protein, RdgBb (PITPNC1). Zebrafish have two homologues of RdgBb, referred to as RdgBb-A and RdgBb-B compared to the single mammalian gene. RdgBb-A was targeted using CRISPR/Cas9 gene editing and homozygous fish have been obtained. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Provided To Others? No  
Impact The expression of the wild type protein is highly enriched in the dorsal telencephalon (part of the brain). The homozygous fish that have been produced elicit a behavioral phenotype when analysed when using a long term sleep-wake assay. The research tool has the potential to make predictions of druggable pathways that either phenocopy or reverse the mutant phenotype. The analysis is still in progress. 
 
Description Identification of a new family of StART domain proteins at membrane contact sites that has a role in Endoplasmic reticulum-plasma membrane sterol transport 
Organisation University College London (UCL)
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution We demonstrated that this new family of lipid transfer proteins were able to bind cholesterol
Collaborator Contribution They identified a family of proteins using Bioinformatics.
Impact Output: Publication in Elife PMID: 26001273
Start Year 2013
 
Description Phosphatidylinositol binding to Yeast lipid binding protein, Pdr16p that provides protection against azole antifungals 
Organisation Academy of Medical Sciences (AMS)
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Charity/Non Profit 
PI Contribution We used protocols established in our laboratory to study the lipid binding properties of mutant proteins of Pdr16p, a protein that is a consideered a factor of clinical azole resistance in fungal pathogens. The most distinct phenotype of yeast cells lacking Pdr16p is their increased susceptibility to azole fungals. Thus Pdr16p, a PI transfer protein, provides protection against azole fungals. Mutants that do not bind phosphatidylinositol is unable to provide this protection and yeast are now susceptible to these antifungals. An important observation made was that phosphatidylinositol binding is critical for Pdr16p function in modulation of sterol metabolism. Mutants deficient in phosphatidylinositol binding were found to bind sterols instead.
Collaborator Contribution Our partners had initiated the study and made the yeast mutants and studies their growth properties after treatment with the azole fungals.
Impact The work was published in Biochim Biophys acta 2014 vol 1841 p1483-1490 PMID: 25066473 Multi disciplinary: Cockcroft Lab: Biochemistry and Griac lab: Genetics
Start Year 2013
 
Description Phospholipase C Signalling during phototransduction in Drosophila 
Organisation National Centre for Biological Science (NCBS)
Country India, Republic of 
Sector Academic/University 
PI Contribution We have identified point mutants in the PITP domain of RdgB and characterised their lipid binding and transfer activities in the RdgB proteins (also known as PITPNM proteins). RdgB mutants in Drosophila show defects in phototransduction. The response to light is defective and is accompanied by retinal degeneration. RdgB proteins contain a PITP domain that is able to bind and transfer phosphatidylinositol. Phosphatidylinositol is synthesised at the endoplasmic reticulum and has to be transported to the rhabdomere where the light sensing machinery resides
Collaborator Contribution Our partners have made transgenic flies with the mutants and analysed retinal degeneration, the electrical response to light and the synthesis of phosphatidylinositol 4,5 bisphosphate.
Impact The work has been published in 2015 in J Cell Science. The work was presented at two biochemical Society meetings and two separate reviews have been written for Biochem Soc Transactions to be published in 2016. Cockcroft was invited as a speaker at the Biochemical Society meeting on Phosphoinositides which took place in Sept 2015 in Cambridge UK and Raghu was selected as a speaker at the Biochemical society meeting on lipid transport proteins which took place in Edinburgh UK in Nov 2015. The collaboration is multi-disciplinary with the Cockcroft Lab doing the Biochemistry and the Raghu lab doing the fly genetics. Cockcroft spent three months at NCBS as part of the collabotation
Start Year 2010