Evidences of the PI5P Increasing the Expression of HBG and Gamma Globin Concentrations

Efforts have been made to identify modulators of increasing fetal hemoglobin (HbF - α2ɣ2) that may act as promising targets for hemoglobinopathies, such as in sickle cell disease and beta thalassemia. The inositide PI5P is a second messenger from the inositol pathway that comprehends in the main phosphorylation target of the enzyme phosphatidylinositol-4-phosphate-5-kinase-II-alpha (PIPKIIα), found in high concentrations in the erythrocyte. Some evidences have postulated a relation between this enzyme and the expression of the hemoglobin (Hb) genes (1,2). More recently, PI5P was found to stabilize UHRF1 protein (3), which has been reported as a possible repressor of HBG (from ɣ globin gene), in the switch from ɣ to β in the transition HbF- HbA (4). To evaluate the effects of the inositide PI5P (substrate of PIPKIIα) in globins, a preliminary time course experiment was performed. KU812 cells (ATCC CRL-2099), which express all three globin genes (HBA, HBB and HBG), were treated with 1μM of PI5P (Thermo Fischer Scientific, USA) in triplicate (4, 8 and 12 hours of treatment) - time 0h used as control. Gene expression and protein concentrations were determined by qPCR - SYBR Green Master Mix amplification and detection kit (Thermo Fischer Scientific, USA, in equipment StepOnePlus Real Time PCR System, Applied Biosystems - USA) using 2DDCT method with the ACTB and GAPDH genes as internal controls - and immunoblotting. Primers were design according to Zacariotto et al (2). No additional phosphate was imputed into the cell culture and the ATP consumption was monitored by ATP Determination Kit (Thermo Fisher Scientific, EUA). PI5P treatment resulted in significant increasement of HBG RNA transcripts (p= 0.001; 8 hours / treatment) and decrease of PIP4K2A (from PIPKIIα enzyme) (p=0.02; 12 hours / treatment), with no other significant changes into globin genes (HBA and HBB). At the protein level, there was a prominent increase in ɣ globin concentrations according to time course. No other consistent change in the concentrations of PIPKIIα or globin chains were observed. Due to protein similarities between the subfamilies of phosphatidylinositol-phosphate kinases (PIPKins) (5), the gene expression of these enzymes was also monitored by qPCR. There was a significant reduction of PIP5K1A (from PIPKIα enzyme) (p=0.01 - 4h; p=0.01 - 8h, and p=0.009 - 12h) as well as PIP4K2B (from PIPKIIβ enzyme) (p=0.03 - 4 hours/ treatment). No significant changes were observed in the other PIPKins (PIPKIβ, PIPKIɣ, PIPKIIɣ and PIPKIII). Considering that a significant consumption of intracellular ATP was observed in time course (p=0.006) it is possible to infer that the high concentrations of PI5P have shifted the inositol pathway resulting in the downregulation of its own PIPKin genes and culminating into the upregulation of HBG, reflected into the protein level. The mechanisms involving the inositide as second messenger, the role of PIPKins or other genes into the modulation of hemoglobin genes, particularly in HBG, should be further investigated. However, despite preliminary, these results reinforce the involvement of the PIPKins and its molecular targets (mainly PIPKIα and PI5P) in globin gene regulation and could represent a promising target for future therapeutic target for hemoglobinopathies. Financial Support: Fapesp, CNPq, CAPES, Faepex-Unicamp. 1.Wenning MR, Mello MP, Andrade TG, et al. PIP4KIIA and beta-globin: transcripts differentially expressed in reticulocytes and associated with high levels of Hb H in two siblings with Hb H disease. Eur J Haematol. 2009; 83: 490-3. 2.Zaccariotto TR, Lanaro C, Albuquerque DM, Santos MN, Bezerra MA, Cunha FG, et al. Expression profiles of phosphatidylinositol phosphate kinase genes during normal human in vitro erythropoiesis. Genet Mol Res. 2012; 11: 3861-8. 3.Gelato KA, Tauber M, Ong MS, Winter S, et al. Accessibility of different histone H3-binding domains of UHRF1 is allosterically regulated by phosphatidylinositol 5-phosphate. Mol Cell. 2014;54(6):905-19. 4.Ruopeng Feng, Phillip A Doerfler, Yu Yao XT, et al. The DNA Methylation Maintenance Protein UHRF1 Regulates Fetal Globin Expression Independent of H BG Promoter DNA Methylation. Blood. 2018;132:410. 5.Heck JN, Mellman DL, Ling K, et al. A conspicuous connection: structure defines function for the phosphatidylinositol-phosphate kinase family. Crit Rev Biochem Mol Biol. 2007; 42: 15-39. Disclosures Costa: Novartis: Consultancy.

Staphylococcus aureus Lipase 3 (SAL3) is a surface-associated lipase that hydrolyzes short chain fatty acids

Bacterial lipases play important roles during infection. The Staphylococcus aureus genome contains several genes that encode well-characterized lipases and several genes predicted to encode lipases or esterases for which the function has not yet been established. In this study, we sought to define the function of an uncharacterized S. aureus protein, and we propose the annotation S. aureus lipase 3 (SAL3) (SAUSA300_0641). We confirmed that SAL3 is a lipase and that it is surface associated and secreted through an unknown mechanism. We determined that SAL3 specifically hydrolyzes short chain (4-carbon and fewer) fatty acids and specifically binds negatively charged lipids including phosphatidic acid, phosphatidylinositol phosphate, and phosphatidylglycerol, which is the most abundant lipid in the staphylococcal cell membrane. Mutating the catalytic triad S66-A, D167-A, S168-A, and H301-A in the recombinant protein abolished lipase activity without altering binding to host lipid substrates. Taken together we report the discovery of a novel lipase from S. aureus specific to short chain fatty acids with yet to be determined roles in host pathogen interactions.

Inositol lipid synthesis is widespread in host-associated Bacteroidetes

Ubiquitous in eukaryotes, inositol lipids have finely tuned roles in cellular signaling and membrane homeostasis. In Bacteria, however, inositol lipid production is rare. Recently, the prominent human gut bacterium Bacteroides thetaiotaomicron (BT) was reported to produce inositol lipids, including inositol sphingolipids, but the pathways remain ambiguous and their prevalence unclear. Here, we investigated the gene cluster responsible for inositol lipid synthesis in BT using a novel strain with inducible control of sphingolipid synthesis. We characterized the biosynthetic pathway from myo-inositol-phosphate (MIP) synthesis to phosphoinositol-dihydroceramide, including structural and kinetic studies of the enzyme MIP synthase (MIPS). We determined the crystal structure of recombinant BT MIPS with bound NAD cofactor at 2.0 Å resolution, and identified the first reported phosphatase for the conversion of bacterially-derived phosphatidylinositol phosphate (PIP) to phosphatidylinositol (PI). Transcriptomic analysis indicated inositol production is nonessential but its loss alters BT capsule expression. Bioinformatic and lipidomic comparisons of Bacteroidetes species revealed a novel second putative pathway for bacterial PI synthesis without a PIP intermediate. Our results indicate that inositol sphingolipid production, via one of the two pathways, is widespread in host-associated Bacteroidetes, and may be implicated in host interactions both indirectly via the capsule and directly through inositol lipid provisioning.

Phosphatidylinositol phosphate binding domains exhibit complex dissociation properties at the inner leaflet of plasma membrane sheets

The pleckstrin homology (PH) domain is a lipid targeting motif that binds with high specificity to phosphatidylinositol phosphate (PIP) lipids. Using TIRF microscopy, we followed the dissociation of GFP-tagged PH domains from the plasma membranes of rapidly unroofed cells and found that AKT-PH and PLCδ1-PH dissociation kinetics can be distinguished by their effective koff values determined from fitting fluorescence traces to a single exponential equation. Our measurements for the koff of AKT-PH-GFP and PLCδ1-PH-GFP were significantly different (p < 0.05) at 0.39 ± 0.05 s−1 and 0.56 ± 0.17 s−1, respectively. Furthermore, we identified substantial rebinding events in our measurements of PLCδ1-PH-GFP dissociation kinetics. By applying inositol triphosphate (IP3) to samples during the unroofing process, we measured a much faster koff of 1.54 ± 0.42 s−1 for PLCδ1-PH-GFP, indicating that rebinding events are significantly depressed through competitive action by IP3 for the same PH domain binding site as phosphatidylinositol (4,5)-bisphosphate (PIP2). We discuss the complex character of our PLCδ1-PH-GFP fluorescence decays in the context of membrane receptor and ligand theory to address the question of how free PIP2 levels modulate the interaction between membrane associated proteins and the plasma membrane.

The Role of Phosphatidylinositol Phosphate Kinases during Viral Infection

Phosphoinositides account for only a small proportion of cellular phospholipids, but have long been known to play an important role in diverse cellular processes, such as cell signaling, the establishment of organelle identity, and the regulation of cytoskeleton and membrane dynamics. As expected, given their pleiotropic regulatory functions, they have key functions in viral replication. The spatial restriction and steady-state levels of each phosphoinositide depend primarily on the concerted action of specific phosphoinositide kinases and phosphatases. This review focuses on a number of remarkable examples of viral strategies involving phosphoinositide kinases to ensure effective viral replication.

Functional genomic screens identify human host factors for SARS-CoV-2 and common cold coronaviruses

The Coronaviridae are a family of viruses that causes disease in humans ranging from mild respiratory infection to potentially lethal acute respiratory distress syndrome. Finding host factors that are common to multiple coronaviruses could facilitate the development of therapies to combat current and future coronavirus pandemics. Here, we conducted parallel genome-wide CRISPR screens in cells infected by SARS-CoV-2 as well as two seasonally circulating common cold coronaviruses, OC43 and 229E. This approach correctly identified the distinct viral entry factors ACE2 (for SARS-CoV-2), aminopeptidase N (for 229E) and glycosaminoglycans (for OC43). Additionally, we discovered phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis as critical host pathways supporting infection by all three coronaviruses. By contrast, the lysosomal protein TMEM106B appeared unique to SARS-CoV-2 infection. Pharmacological inhibition of phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis reduced replication of all three coronaviruses. These findings offer important insights for the understanding of the coronavirus life cycle as well as the potential development of host-directed therapies.