Functional Role And Mechanism Of ORP4L In PIP₂ Extraction And Presentation For PLC?3 Catalysis In Leukemia Stem Cells

Acute myeloid leukemia?AML?is a cancer of bone marrow blood cells that is characterized by the rapid growth of abnormal cells that accumulate in the bone marrow and blood,interfering with normal blood cells.Symptoms can include feeling tired,shortness of breath,being prone to bruises and bleeding,and an increased risk of infection,a common form of leukemia that occurs most frequently in adults.Leukemia stem cells?LSCs?are a rare subpopulation of abnormal hematopoietic stem cells?HSCs?that propagates leukemia and are responsible for the high frequency of relapse in therapies.ORP4L is a member of the Oxysterol Binding Protein-Related Protein Family?ORPs?,which is selectively expressed in LSCs but not in normal HSCs,and is essential for the survival of LSCs.Previous studies have shown that phospholipase C-?3?PLC?3?translocation from the nucleus to the plasma membrane in Jurkat T-cells,and further regulates pyruvate dehydrogenase activity on mitochondria and leads leukemia cells to use the oxidative phosphorylation pathway for ATP generation by forming ORP4L/G?q/11/PLC?3 complex to regulate phosphatidylinositol 4,5-bisphosphate?PIP₂?metabolism and downstream Ca²⁺release.But how PIP₂is extracted from the plasma membrane and presented to PLC?3protein remains unclear.This study provides a new perspective for ORP4L in the metabolic balance of PIP₂.In this experiment,KG1-?cells were selected as the research subjects of LSCs,and it was verified that KG1-?cells expressed ORP4L as high as LSCs.First,we knocked down ORP4L in KG1-?cells to investigate whether ORP4L has an effect on the phospholipid content regulation of the plasma membrane?PM?.UPLC-HRMS analysis of the PM revealed that knocking down ORP4L increased the content of phosphatidylinositol?PIs?of PM,suggesting that ORP4L plays a role in the metabolism of PM PIs.Next,we overexpressed or knocked down ORP4L to visualize the dynamic process of PIP₂ clearance on the plasma membrane under SDF-1?stimulation by laser confocal microscopy.It was showed that PM PIP₂ level declined rapidly when overexpressing ORP4L while ORP4L knockdown reduced the clearance of PIP₂.The experiment further explored the PM PIP₂ metabolism mechanism by using Molecular Dynamics to simulate apo-ORP4L protein and ORP4/PIP₂ complex.The pocket entrance of apo-ORP4L expanded during 30–60 ns and then restored a closed conformation.However,as for the ORP4L/PIP₂ complex,the pocket entrance expanded over time.Key amino acid residues at the binding site were found by molecular docking.Then the SRP assay was used to detect the binding capacity of ORP4L,ORP4L domain and PLC?3 domain to PIP₂.It was found that the binding of PIP₂ and ORP4L-ORD was prior to PLC?3,while ORP4L-PH cannot bind to PIP₂.Next,the plasma membrane separation technique was used to study the ability of PLC?3 and ORP4L to extract PM PIP₂.The experiment first added PLC?3 alone,the PIP₂ on the plasma membrane was not reduced upon PLC?3 activated or inactivated.The addition of ORP4L protein alone revealed that PM PIP₂ was extracted,proving the ability of ORP4L-ORD to extract PIP₂ on the PM.Finally,a cell-free system was constructed to study the process of PIP₂ presented to the PLC?3 for catalysis.In the absence of the ORP4L protein,there was no reduction in PIP₂ on the PM and 2^ndmessenger.The addition of ORP4L?PLC?3 and ORP4L?PIP₂ also failed to induce IP₃ and DAG generation.Only when the full-length ORP4L protein was added,the PIP₂ can be hydrolyzed on the PM,suggesting the role of ORP4L in the presentation of PIP₂.In summary,ORP4L,as a phospholipid transporter,extracts and presents PM PIP₂ to PLC?3 for catalysis,This study revealed for the first time how the PM PIP₂metabolism is maintained,provided a more detailed mechanism for the production of downstream IP₃ and Ca²⁺-dependent bioenergetics.It also provides a potential target for the treatment of leukemia.