Human sperm phosphatidylinositol 4-phosphate 5-kinase type I gamma (PI4P- 5KIgamma) activity is crucial for the acrosome granule exocytosis.

PELLETAN, LEONARDO E; SUHAIMAN LAILA; ALTAMIRANO, KARINA N; MAYORGA LS; BELMONTE SILVIA A

Congreso; CONGRESO CONJUNTO SAIB-SAMIGE; 2021

SAIB

The acrosome is a cap-shaped granule that overlies the sperm nucleus. The acrosome reaction (AR) is a regulated calcium-dependent exocytosis necessary for fertilization. Our previous publications demonstrate that diacylglycerol (DAG) stimulates the AR, in part, by feeding into a PKC- and PLD1-dependent positive loop that continuously supplies phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidic acid (PA). ARF6 regulates the synthesis of these lipids. Synthesis and turnover of PIP2 involve a network of kinases, phosphatases, and phospholipases that keep the equilibrium of this phosphoinositide. We aimed to identify the molecule responsible for the PIP2 increase after the exocytic stimuli. We hypothesized that PA and ARF6 activate PI4P-5KIgamma and considered its presence in the positive feedback curl. Here, we evaluated the role of this kinase in AR. First, we used pleckstrin homology (PH) domains to disturb PIP2 availability in exocytosis assays in streptolysin O (SLO)-permeabilized sperm. Both, PH-PLCgamma1 (IC50, 5 μg/ml) and PH-PLCgamma4 (IC50,10 μg/ml) domains abrogated DAG and ARF6-induced AR. PIP2 addition rescued exocytosis suggesting a specific effect of these probes. By using biochemical, exocytosis assays, and microscopy techniques we demonstrated that the PI4P-5KI is present, localizes to the acrosome region and is required for calcium, ARF6, and DAG-triggered exocytosis in human sperm. Sequestration of the protein with specific antibodies introduced into permeabilized sperm impairs exocytosis. To analyze its function deeply during exocytosis, we synthesized the recombinant hPIPKIgamma-5 (a long-isoform of PIPKIgamma), measured its activity in vitro, and analyzed its regulation. The enzyme was active and catalyzed PIP2 synthesis. Its activity was regulated by ARF6 and PA in vitro. The recombinant kinase added to SLO-permeabilized sperm did not induce the AR by itself. Still, it reversed the inhibition of PLD and PKC activity on DAG-elicited exocytosis confirming its participation in the loop proposed and its ability to substitute PIP2 function. Measurements of PIP2 synthesis in sperm exocytosis assays corroborate the involvement and contribution of the kinase during exocytosis. To confirm our experimental predictions, we used COPASI (Complex Pathway Simulator) considering the chemical reactions catalyzed by known enzymes in the complex network involved in PIP2 synthesis. The model replicates the steady state of the pathway and most known dynamic phenomena. Model analysis suggests that the greatest contributor to PIP2 production in our biological system is a flux representing the direct transformation of PI into PIP2 through phosphatidylinositol 4-phosphate phosphorylation including the PI4P-5KIgamma. Here, we present direct evidence showing the presence and function of PI4P-5KIgamma in human sperm. Our findings highlight the synthesis pathway of the multitasking lipid, PIP2, during sperm exocytosis.