Phosphoinositide Messengers in Cellular Signaling and Trafficking
- Tamás Balla, MD, PhD, Head, Section on Molecular Signal Transduction
- Yeun Ju Kim, PhD, Staff Scientist
- Alejandro Alvarez-Prats, PhD, Postdoctoral Fellow
- Takashi Baba, PhD, Postdoctoral Fellow
- Gergo Gulyas, MD, PhD, Postdoctoral Fellow
- Joshua Pemberton, PhD, Postdoctoral Fellow
- Nivedita Sengupta, PhD, Postdoctoral Fellow
- Marek Korzeniowski, PhD, Special Volunteer
- Ljubisa Vitkovic, PhD, Special Volunteer
- Elizabeth Schott, BS, Postbacccalureate and Pre-Intramural Training Award Fellow
Every biochemical process in a eukaryotic cell relies on a molecular information flow that leads from receptors that inform the cell about its environment all the way to the molecular effectors that determine the appropriate cellular response. Proper information transmission requires a high degree of organization, where the molecular players are organized into different cellular compartments so that the specificity of the cellular response can be properly maintained. Breakdown of this organization is the ultimate cause of all human diseases, even if the affected molecular pathways differ according to the type of disease, such as cancer, diabetes, or neurodegenerative diseases, just to name a few.
Research described in this report has focused on the question of how cells organize their internal membranes to provide a structural framework on which molecular signaling complexes assemble to ensure proper information processing. These cellular processes are often targeted by cellular pathogens, such as viruses, to force the cells to produce the pathogen instead of performing the cell's normal functions. Better understanding of such processes can not only provide new strategies to fight various human diseases but also intercept the life cycle of cellular pathogens, thus offering an alternative to antimicrobial drugs.
Phosphatidylinositol 4,5-bisphosphate controls autophagosome-lysosome fusion.
Inositol phospholipids constitute a class of phospholipids that are present in tiny amounts but that have very important regulatory functions, as they organize protein signaling complexes on specific membrane compartments. They are produced by phosphoinositide kinases that can phosphorylate specifically one of three positions of the inositol ring of phosphatidylinositol (PI). We studied the role of phosphatidylinositol 4-kinase alpha (PI4K2A) in autophagosome-lysosome fusion. Autophagy is an important cellular process that helps clear damaged organelles and also allows cells to recycle useful nutrients from degraded organelles during starvation. In earlier studies we found that PI4K2A interacted with the GABARAP protein, one of a family of proteins that are important for autophagy. Our observation was confirmed in a subsequent study by the Albanesi group, which also showed that PI4K2A was important for the acidification of autophagic vesicles. Therefore, we generated HEK293 cells (human embryonic kidney tissue culture cells) with inactivated PI4K2A using CRISPR/Cas9 gene editing and studied their properties, including those important for autophagy. We found increased number of LC3 (a widely used marker of autophagy)–positive vesicles that failed to acidify in the PI4K2A knockout (KO) cells. We determined the distribution of phosphatidylinositol 4-phosphate (PI4P) along the endosomal network by introducing a novel bioluminescence resonance energy transfer (BRET)–based assay, which permitted the quantification of levels of PI4P within various Rab–positive endosomes (phosphatidylinositol 4,5-bisphosphate controls autophagosome-lysosome fusion by activating cycling of the GTPase Rab7). The analysis showed that the levels of PI4P are highest in Rab7–positive endosomes and are kept very low in Rab4, Rab5, and Rab11 compartments. PI4K2A was responsible for 80% of the PI4P produced in the Rab7 compartment, with the rest produced by PI4K2B. Conversion of PI4P to PI(4,5)P2 caused the inactivation of Rab7 and release of PLEKHM1, an important adapter protein linking autophagic vesicles with lysosomes, from the Rab7 compartment. We also found that PI4P in the Rab7 compartment was converted to PI(4,5)P2 by endogenous PIP5Kgamma in wild-type, but not in PI4K2A KO cells and that PIP5Kgamma knock-down in parental HEK293 reproduced the effects of PI4K2A inactivation by inhibiting autophagic vesicle acidification. These data suggested a sequence of events by which PI(4,5)P2 generation from the PI4P produced by PI4K2A in Rab7–positive endosomes caused Rab7 inactivation, presumably by activating one or more Rab7 GTPase–activating (GAP) protein(s). This chain of events contributes to the cycling of some Rab7 effectors, including PLEKHM1, which is necessary for the fusion of lysosomes with autophagosomes. The importance of these studies is that they reveal a hitherto unknown role of PI4P to PI(4,5)P2 conversion in the control of Rab7 activation state, thus serving as a regulator of trafficking decisions in the endocytic pathway.
Identification of chemical inhibitors of phosphatidylinositol 4-kinase type II alpha
In light of the pivotal role of PI4P in virus replication and the role of type II PI4Ks in endosomal functions, we wanted to identify small-molecule inhibitors for the PI4K2A enzyme. To date, pharmaceutical companies have focused exclusively on inhibitors for type III PI4Ks, such as PI4KA and PI4KB, but no efforts have been devoted by the industry to identifying inhibitors of type II PI4Ks. Part of the reason is that type II PI4Ks represent a different class of lipid kinases, which are resistant to wortmannin and to all known PI3K inhibitors, whereas the type III PI4Ks are sensitive to some PI3K inhibitors, thus providing chemical scaffolds that companies could modify to enhance their selectivity and potency to type III PI4Ks. We developed a PI4K activity assay for a small format suitable for high-throughput screening (HTS) using a bacterially expressed human PI4K2A enzyme produced by Evžen Boura’s group. A high throughput screening was then performed by our collaborators Marc Ferrer and Juan Marugan with about 400,000 compounds from small molecule diversity collections. Sytravon (library of novel small molecules), NPC (NCGC Pharmaceutical Collection of approved and investigational drugs) and MLPCN (NIH’s Molecular Libraries Probe Production Centers Network of thousands of small molecules) collections were screened at top two doses (76 & 15 µM final concentration). Based on the screening results, we identified over 580 compounds with over 50% inhibitory activity. The compounds were re-tested at seven doses in 1:3 serial dilution to confirm their activity. Further, they were counter-screened with ADP-Glo™ reagents (a luminescent ADP detection assay) in the absence of the enzyme to exclude artificially luminescent compounds, and with PI4KB, another structurally unrelated PI4K. The list was further narrowed by eliminating compounds that were known inhibitors of protein kinases or were deemed structurally unsuitable for further development, yielding 14 inhibitors shortlisted for further studies. We then tested the 14 inhibitors in a cellular assay using the BRET method to monitor PI4P levels in Rab7 endosomes that require PI4K2A. Two inhibitors were then found to have an inhibitory effect on PI4K2A in the cell with an IC50 of about 30 µM. The two lead compounds (code named NC03 and NC02) were then modified by NCATS (NC03) and by Radim Nencka, our medicinal chemist collaborator (NC02) to increase the potency and cellular availability. Unfortunately, these significant efforts did not yield any improvement over the lead compounds. Since then, a new screen was performed, and testing is under way to improve the newly identified lead compounds.
Reversible oxidation of the PtdIns(4)P phosphatase Sac1 by H2O2
In a collaborative project with the group of Sue Goo Rhee and Dongmin Kang, the effects of reactive oxygen radicals were examined on the Sac1 phosphatase enzyme. Sac1 dephosphorylates PI4P in the endoplasmic reticulum (ER) and plays crucial roles in the control of non-vesicular lipid transport driven by PI4P gradients between various organelles and the ER. We found that hydrogen peroxide (H2O2) has a profound effect on the cellular level of PI4P, which was most prominent in the Golgi and Rab7–positive endosomes, but also manifested in a PI4P increase in the plasma membrane (PM). Upon H2O2 exposure, Sac1 undergoes reversible inactivation in mammalian cells owing to the oxidation of its catalytic Cys389 residue, which then forms an intramolecular disulfide with Cys392. The Korean group also showed that this oxidation process also takes place during stimulation of cells by EGF and that Duox (dual oxidase) enzymes are responsible for the endogenous H2O2 production under these conditions. The findings revealed an important regulation of the Sac1 phosphatase by reversible oxidation, thereby controlling both the signaling function of PI4P and its effectiveness in driving lipid transport at membrane contact sites.
Additional Funding
- Natural Sciences & Engineering Research Council of Canada (NSERC) Banting Postdoctoral Fellowship supporting Dr. Joshua Pemberton
Publications
- Baba T, Toth DJ, Sengupta N, Kim YJ, Balla T. Phosphatidylinositol 4,5-bisphosphate controls Rab7 and PLEKHM1 membrane cycling during autophagosome-lysosome fusion. EMBO J 2019;38:e100312.
- Balla T, Kim YJ, Alvarez-Prats A, Pemberton J. Lipid Dynamics at Contact Sites Between the Endoplasmic Reticulum and Other Organelles. Annu Rev Cell Dev Biol 2019;35:85-109.
- Balla T, Sengupta N, Kim YJ. Lipid synthesis and transport are coupled to regulate membrane lipid dynamics in the endoplasmic reticulum. Biochim Biophys Acta Mol Cell Biol Lipids 2019;S1388:30075-30077.
- Sengupta N, Jovic M, Barnaeva E, Kim DW, Hu X, Southall N, Dejmek M, Mejdrova I, Nencka R, Baumlova A, Chalupska D, Boura E, Ferrer M, Marugan J, Balla T. A large scale high-throughput screen identifies chemical inhibitors of phosphatidylinositol 4-kinase type II alpha. J Lipid Res 2019;60:683-693.
Collaborators
- Evžen Boura, PhD, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic
- Marc Ferrer, PhD, Division of Pre-Clinical Innovation, NCATS, Bethesda, MD
- Dongmin Kang, PhD, Ewha Womans University, Seoul, South Korea
- Juan Marugan, PhD, Division of Pre-Clinical Innovation, NCATS, Bethesda, MD
- Radim Nencka, PhD, Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague, Czech Republic
- Sue Goo Rhee, PhD, Yonsei Biomedical Research Institute, Yonsei University, Seoul, South Korea
- Mark Stopfer, PhD, Section on Sensory Coding and Neural Ensembles, NICHD, Bethesda, MD
- Péter Várnai, MD, PhD, Semmelweis University, Faculty of Medicine, Budapest, Hungary
Contact
For more information, email ballat@mail.nih.gov or visit http://ballalab.nichd.nih.gov/.
