Periodic Reporting for period 1 - LivFlip (Structural and cellular investigation of the regulation of ATP8B1/CDC50A, a human flippase important for the hepatic function)
Reporting period: 2021-03-01 to 2023-02-28
The inherited intrahepatic hepatic cholestasis is a rare disease characterized by impaired bile flow and a highly toxic accumulation of bile salts in the liver which normally helps digestion and absorption of nutrients in the small intestine. Familial intrahepatic disorders have been linked to five genes implicated in bile salt homeostasis or hepatocyte membrane integrity. Among them, the ATP8B1 gene encodes for a lipid transporter (lipid flippase). However, little is known about the function of ATP8B1 at the canalicular membrane and this is mainly due to the lack of consensus regarding its transport substrate and how its activity is regulated. Thanks to the Marie Curie program, the supported project provided structural and functional information on ATP8B1 autoinhibition, lipid specificity and transport mechanisms.
WP1:
The intention of WP1 was to obtain high-resolution structures of the human ATP8B1-CDC50A complex in different conformations. For this, we optimized the purification procedure of the ATP8B1-CDC50A complex. From the initial protocol we included new steps allowing delipidation of the sample to further characterize the lipid specificity of the flippase. Thanks to this work, we could obtain a sample for cryo-EM single particle analysis. Nine different cryo-EM structures could be solved (1 published, 8 yet unpublished)(Figure 1A), covering the entire catalytic cycle of the lipid flippase. The structures highlight ATP8B1 autoinhibition mechanism, the mechanism of activation based on phosphoinositide binding and the lipid specificity of ATP8B1. Additionnaly, thanks to my secondmenet in Paris-Saclay we could reinforce the intepretation of the structural information by using fluorescence approaches. From these data we could also better understand the structural consequences of mutations found in patients suffering from intrahepatic cholestasis (Figure 1B).
WP2:
In the WP2, we initially intended to identified regulators of ATP8B1. The mouse homologue of ATP8B1 was reported to be phosphorylated on the regulatory part of the C-terminus of ATP8B1 involved in its autoinhibition mechanism. To test the effect of such post-translational modification, we use synthetic peptides carrying the phosphorylation and compare it to the wild type peptide for their ability to inhibit the activity of ATP8B1. We found that the phosphorylation entailed the IC50 of the peptide by approximately 60-fold. To better understand the regulation of ATP8B1 we decided to identify the kinase(s) responsible for ATP8B1 C-terminal tail phosphorylation.
For this we purified the N- and C-terminal tails of ATP8B1, and a kinase screen was conducted by the company Reaction biology. More that 250 different Ser/Thr/Tyr kinases were tested for their ability to phosphorylate the N- an C-terminal tails of ATP8B1. From this screen, the PKC subgroup showed the highest activity. To go further into that direction, we implemented two new techniques in Poul Nissen group for the detection of phosphoprotein (Phostag gels and phosphorylation assay using radioactive ATP). Thanks to our collaboration with the group of Professor Jan J. Enghild, we could further identified the residues phosphorylated by PKC by mass spectrometry. The WP2 was mainly performed by Michelle Laursen as Master students under my supervision and is still part of her Ph.D. project.
WP3:
In the WP3 we intended to characterize the regulation of ATP8B1 in cellulo (Figure 1C). For this we have generated tools to investigate ATP8B1-CDC50A phosphorylation state, localization, and lipid transport function in hepatocyte cell line (HepG2 cells). To isolate ATP8B1 from these cells, we established new tools in the Nissen group with the use of anti-GFP nanobodies for affinity purification of GFP fused proteins. We could kindly obtain HepG2 cell line expressing ATP8B1 fused to GFP from Dr. Coen Paulusma (Amsterdam) and we are currently trying to isolate ATP8B1 to analyze its post-translational modification by mass spectrometry. Additionally, we have developed different baculoviruses to express different constructs of ATP8B1 with its subunit CDC50A for lipid uptake assays.
WP4:
With this project and thanks to the support of the Marie Curie program I receive deep training in electron microscopy and structural biology thanks the training sessions organized by the cryoEM facility at Aarhus University. Additionally, I was introduced to sample preparation for mass spectrometry through our collaboration with Prof Jan J. Enghild. During my secondment in France, I could get trained on fluorescence technics with a high-end fluorimeter. In addition, thanks to the support of my supervisor, Prof Poul Nissen. I could quickly supervise students During the fellowship, I could supervise a student on the LivFlip project. Michelle Laursen could obtain a master degree thanks to her work on the WP2. Michelle Laursen continued on the same project as Research assistant before obtaining a fellowship to start her Ph.D program with me as a daily supervisor.
WP5:
During the project, and despite the COVID-19 pandemic, I was able to communicate our results throughout the duration of the fellowship. First, I could present our result at the online meeting “building bridges” of the Nordic EMBL Partnership for Molecular Medicine in December 2021. After this, and concomitantly to our publication of a preprint on bioRxiv I was invited by the P-type ATPase community to give a webinar. I could also present our results through a talk and a poster at the Gordon Research Seminar and Gordon Research Conference “Ligand Recognition and Molecular Gating” respectively. In 2022, I was selected to present my results at the international conference on “P-type ATPase in health and disease” in Banff, Canada. Finally, during my secondment in France, I was also selected to orally present my result at the Annual membrane proteins French working group meeting. In terms of publication, the project already led to one research article, firstly released as a preprint on the bioRxiv server, and then published in Elife after peer-reviewing. I was also involved in the writing of an open-access review on phosphatidylserine homeostasis. In addition, we are preparing a second research article which will be submitted Q2 of 2023. All publications were also communicated via social networks.
The intention of WP1 was to obtain high-resolution structures of the human ATP8B1-CDC50A complex in different conformations. For this, we optimized the purification procedure of the ATP8B1-CDC50A complex. From the initial protocol we included new steps allowing delipidation of the sample to further characterize the lipid specificity of the flippase. Thanks to this work, we could obtain a sample for cryo-EM single particle analysis. Nine different cryo-EM structures could be solved (1 published, 8 yet unpublished)(Figure 1A), covering the entire catalytic cycle of the lipid flippase. The structures highlight ATP8B1 autoinhibition mechanism, the mechanism of activation based on phosphoinositide binding and the lipid specificity of ATP8B1. Additionnaly, thanks to my secondmenet in Paris-Saclay we could reinforce the intepretation of the structural information by using fluorescence approaches. From these data we could also better understand the structural consequences of mutations found in patients suffering from intrahepatic cholestasis (Figure 1B).
WP2:
In the WP2, we initially intended to identified regulators of ATP8B1. The mouse homologue of ATP8B1 was reported to be phosphorylated on the regulatory part of the C-terminus of ATP8B1 involved in its autoinhibition mechanism. To test the effect of such post-translational modification, we use synthetic peptides carrying the phosphorylation and compare it to the wild type peptide for their ability to inhibit the activity of ATP8B1. We found that the phosphorylation entailed the IC50 of the peptide by approximately 60-fold. To better understand the regulation of ATP8B1 we decided to identify the kinase(s) responsible for ATP8B1 C-terminal tail phosphorylation.
For this we purified the N- and C-terminal tails of ATP8B1, and a kinase screen was conducted by the company Reaction biology. More that 250 different Ser/Thr/Tyr kinases were tested for their ability to phosphorylate the N- an C-terminal tails of ATP8B1. From this screen, the PKC subgroup showed the highest activity. To go further into that direction, we implemented two new techniques in Poul Nissen group for the detection of phosphoprotein (Phostag gels and phosphorylation assay using radioactive ATP). Thanks to our collaboration with the group of Professor Jan J. Enghild, we could further identified the residues phosphorylated by PKC by mass spectrometry. The WP2 was mainly performed by Michelle Laursen as Master students under my supervision and is still part of her Ph.D. project.
WP3:
In the WP3 we intended to characterize the regulation of ATP8B1 in cellulo (Figure 1C). For this we have generated tools to investigate ATP8B1-CDC50A phosphorylation state, localization, and lipid transport function in hepatocyte cell line (HepG2 cells). To isolate ATP8B1 from these cells, we established new tools in the Nissen group with the use of anti-GFP nanobodies for affinity purification of GFP fused proteins. We could kindly obtain HepG2 cell line expressing ATP8B1 fused to GFP from Dr. Coen Paulusma (Amsterdam) and we are currently trying to isolate ATP8B1 to analyze its post-translational modification by mass spectrometry. Additionally, we have developed different baculoviruses to express different constructs of ATP8B1 with its subunit CDC50A for lipid uptake assays.
WP4:
With this project and thanks to the support of the Marie Curie program I receive deep training in electron microscopy and structural biology thanks the training sessions organized by the cryoEM facility at Aarhus University. Additionally, I was introduced to sample preparation for mass spectrometry through our collaboration with Prof Jan J. Enghild. During my secondment in France, I could get trained on fluorescence technics with a high-end fluorimeter. In addition, thanks to the support of my supervisor, Prof Poul Nissen. I could quickly supervise students During the fellowship, I could supervise a student on the LivFlip project. Michelle Laursen could obtain a master degree thanks to her work on the WP2. Michelle Laursen continued on the same project as Research assistant before obtaining a fellowship to start her Ph.D program with me as a daily supervisor.
WP5:
During the project, and despite the COVID-19 pandemic, I was able to communicate our results throughout the duration of the fellowship. First, I could present our result at the online meeting “building bridges” of the Nordic EMBL Partnership for Molecular Medicine in December 2021. After this, and concomitantly to our publication of a preprint on bioRxiv I was invited by the P-type ATPase community to give a webinar. I could also present our results through a talk and a poster at the Gordon Research Seminar and Gordon Research Conference “Ligand Recognition and Molecular Gating” respectively. In 2022, I was selected to present my results at the international conference on “P-type ATPase in health and disease” in Banff, Canada. Finally, during my secondment in France, I was also selected to orally present my result at the Annual membrane proteins French working group meeting. In terms of publication, the project already led to one research article, firstly released as a preprint on the bioRxiv server, and then published in Elife after peer-reviewing. I was also involved in the writing of an open-access review on phosphatidylserine homeostasis. In addition, we are preparing a second research article which will be submitted Q2 of 2023. All publications were also communicated via social networks.
The work done during the project has provided fundamental details on ATP8B1-CDC50A . The structural data obtained as well as the biochemical characterization of the complex help us to better understand the mechanism of transport of all P4-ATPases. Additionally, the project also revealed the strong regulations mechanisms of the complex and provided the first clue of the regulators of the complex activity (kinases and phosphoinostides). In the near future, we hope that the identification of these regulatory mechanisms of ATP8B1, which shares similarities with of human P4-ATPase will be instrumental for specific design of molecules that would reinforce or stimulate the release of the autoinhibitory mechanism. Additionally, our work also provided evidence of the consequence of disease-associated ATP8B1 mutations found in PFIC1 patients and we hope that this will pave the way to a more directed personalized medicine.