Depending on their phosphorylation status, derivatives of phosphatidylinositol play important roles in vesicle identity, recognition and intracellular trafficking processes. In eukaryotic cells, phosphatidylinositol-4 phosphate pools generated by specific kinases are key determinants of the conventional secretion pathways. Earlier work in yeast has classified phosphatidylinositol-4 kinases in two types, Stt4p and Pik1p belonging to type III and Lsb6p to type II, with distinct cellular localizations and functions. Eurotiomycetes appear to lack Pik1p homologues. In Aspergillus nidulans, unlike homologues in other fungi, AnLsb6 is associated to late Golgi membranes and when heterologously overexpressed, it compensates for the thermosensitive phenotype in a Saccharomyces cerevisiae pik1 mutant, whereas its depletion leads to disorganization of Golgi-associated PHOSBP-labelled membranes, that tend to aggregate dependent on functional Rab5 GTPases. Evidence provided herein, indicates that the single type II phosphatidylinositol-4 kinase AnLsb6 is the main contributor for decorating secretory vesicles with relevant phosphatidylinositol-phosphate species, which navigate essential cargoes following the route of apical polarization via endocytic recycling.

Structure-activity relationship studies of 2,8-disubstituted-1,5-naphthyridines, previously reported as potent inhibitors of Plasmodium falciparum (Pf) phosphatidylinositol-4-kinase β (PI4K), identified 1,5-naphthyridines with basic groups at 8-position, which retained Plasmodium PI4K inhibitory activity but switched primary mode of action to the host hemoglobin degradation pathway through inhibition of hemozoin formation. These compounds showed minimal off-target inhibitory activity against the human phosphoinositide kinases and MINK1 and MAP4K kinases, which were associated with the teratogenicity and testicular toxicity observed in rats for the PfPI4K inhibitor clinical candidate MMV390048. A representative compound from the series retained activity against field isolates and lab-raised drug-resistant strains of Pf. It was efficacious in the humanized NSG mouse malaria infection model at a single oral dose of 32 mg/kg. This compound was nonteratogenic in the zebrafish embryo model of teratogenicity and has a low predicted human dose, indicating that this series has the potential to deliver a preclinical candidate for malaria.

Phosphatidylinositol 4-kinase beta (PI4KB) is a member of the PI4K family, which is mainly enriched and functions in the Golgi apparatus. The kinase domain of PI4KB catalyzes the phosphorylation of phosphatidylinositol to form phosphatidylinositol 4-phosphate, a process that regulates various sub-cellular events, such as non-vesicular cholesterol and ceramide transport, protein glycosylation, and vesicle transport, as well as cytoplasmic division. In this study, a strain of PI4KB knockout mouse, immunofluorescence, reverse transcription polymerase chain reaction and microinjection were used to characterize the cytological location and biological function of PI4KB in the mouse embryos. we found that knocking down Pi4kb in mouse embryos resulted in embryonic lethality at around embryonic day (E) 7.5. Additionally, we observed dramatic fluctuations in PI4KB expression during the development of preimplantation embryos, with high expression in the 4-cell and morula stages. PI4KB colocalized with the Golgi marker protein TGN46 in the perinuclear and cytoplasmic regions in early blastomeres. Postimplantation, PI4KB was highly expressed in the epiblast of E7.5 embryos. Treatment of embryos with PI4KB inhibitors was found to inhibit the development of the morula into a blastocyst and the normal progression of cytoplasmic division during the formation of a 4-cell embryo. These findings suggest that PI4KB plays an important role in mouse embryogenesis by regulating various intracellular vital functions of embryonic cells.

Enterovirus 71 (EV71) is one of the major pathogens causing hand, foot, and mouth disease in children under 5 years old, which can result in severe neurological complications and even death. Due to limited treatments for EV71 infection, the identification of novel host factors and elucidation of mechanisms involved will help to counter this viral infection. N-terminal acetyltransferase 6 (NAT6) was identified as an essential host factor for EV71 infection with genome-wide CRISPR/Cas9 screening. NAT6 facilitates EV71 viral replication depending on its acetyltransferase activity but has little effect on viral release. In addition, NAT6 is also required for Echovirus 7 and coxsackievirus B5 infection, suggesting it might be a pan-enterovirus host factor. We further demonstrated that NAT6 is required for Golgi integrity and viral replication organelle (RO) biogenesis. NAT6 knockout significantly inhibited phosphatidylinositol 4-kinase IIIβ (PI4KB) expression and PI4P production, both of which are key host factors for enterovirus infection and RO biogenesis. Further mechanism studies confirmed that NAT6 formed a complex with its substrate actin and one of the PI4KB recruiters-acyl-coenzyme A binding domain containing 3 (ACBD3). Through modulating actin dynamics, NAT6 maintained the integrity of the Golgi and the stability of ACBD3, thereby enhancing EV71 infection. Collectively, these results uncovered a novel mechanism of N-acetyltransferase supporting EV71 infection.IMPORTANCEEnterovirus 71 (EV71) is an important pathogen for children under the age of five, and currently, no effective treatment is available. Elucidating the mechanism of novel host factors supporting viral infection will reveal potential antiviral targets and aid antiviral development. Here, we demonstrated that a novel N-acetyltransferase, NAT6, is an essential host factor for EV71 replication. NAT6 could promote viral replication organelle (RO) formation to enhance viral replication. The formation of enterovirus ROs requires numerous host factors, including acyl-coenzyme A binding domain containing 3 (ACBD3) and phosphatidylinositol 4-kinase IIIβ (PI4KB). NAT6 could stabilize the PI4KB recruiter, ACBD3, by inhibiting the autophagy degradation pathway. This study provides a fresh insight into the relationship between N-acetyltransferase and viral infection.

Phosphatidylinositol 4-kinases (PI4Ks) could phosphorylate phosphatidylinositol (PI) to produce phosphatidylinositol 4-phosphate (PI4P) and maintain its metabolic balance and location. PI4P, the most abundant monophosphate inositol in eukaryotic cells, is a precursor of higher phosphoinositols and an essential substrate for the PLC/PKC and PI3K/Akt signaling pathways. PI4Ks regulate vesicle transport, signal transduction, cytokinesis, and cell unity, and are involved in various physiological and pathological processes, including infection and growth of parasites such as Plasmodium and Cryptosporidium, replication and survival of RNA viruses, and the development of tumors and nervous system diseases. The development of novel drugs targeting PI4Ks and PI4P has been the focus of the research and clinical application of drugs, especially in recent years. In particular, PI4K inhibitors have made great progress in the treatment of malaria and cryptosporidiosis. We describe the biological characteristics of PI4Ks; summarize the physiological functions and effector proteins of PI4P; and analyze the structural basis of selective PI4K inhibitors for the treatment of human diseases in this review. Herein, this review mainly summarizes the developments in the structure and enzyme activity of PI4K inhibitors.

Apolipoproteins L1 and L3 (APOLs) are associated at the Golgi with the membrane fission factors phosphatidylinositol 4-kinase-IIIB (PI4KB) and non-muscular myosin 2A. Either APOL1 C-terminal truncation (APOL1Δ) or APOL3 deletion (APOL3-KO [knockout]) reduces PI4KB activity and triggers actomyosin reorganization. We report that APOL3, but not APOL1, controls PI4KB activity through interaction with PI4KB and neuronal calcium sensor-1 or calneuron-1. Both APOLs are present in Golgi-derived autophagy-related protein 9A vesicles, which are involved in PI4KB trafficking. Like APOL3-KO, APOL1Δ induces PI4KB dissociation from APOL3, linked to reduction of mitophagy flux and production of mitochondrial reactive oxygen species. APOL1 and APOL3, respectively, can interact with the mitophagy receptor prohibitin-2 and the mitophagosome membrane fusion factor vesicle-associated membrane protein-8 (VAMP8). While APOL1 conditions PI4KB and APOL3 involvement in mitochondrion fission and mitophagy, APOL3-VAMP8 interaction promotes fusion between mitophagosomal and endolysosomal membranes. We propose that APOL3 controls mitochondrial membrane dynamics through interactions with the fission factor PI4KB and the fusion factor VAMP8.

BACKGROUND: Tyrosine kinase and phosphoinositide kinase pathways play important roles in asthma formation. As a dual tyrosine and phosphoinositide kinase inhibitor, PP121 has shown anticancer efficacy in multiple tumors. However, the study of PP121 in pulmonary diseases is still limited. Herein, we investigated the therapeutic activities of PP121 in asthma treatment.
METHODS: Tension measurements and patch clamp recordings were made to investigate the anticontractile characteristics of PP121 in vitro. Then, an asthma mouse model was established to further explore the therapeutic characteristics of PP121 via measurement of respiratory system resistance, histological analysis and western blotting.
RESULTS: We discovered that PP121 could relax precontracted mouse tracheal rings (mTRs) by blocking certain ion channels, including L-type voltage-dependent Ca2+ channels (L-VDCCs), nonselective cation channels (NSCCs), transient receptor potential channels (TRPCs), Na+/Ca2+ exchangers (NCXs) and K+ channels, and accelerating calcium mobilization. Furthermore, PP121 relieved asthmatic pathological features, including airway hyperresponsiveness, systematic inflammation and mucus secretion, via downregulation of inflammatory factors, mucins and the mitogen-activated protein kinase (MAPK)/Akt signaling pathway in asthmatic mice.
CONCLUSIONS: In summary, PP121 exerts dual anti-contractile and anti-inflammatory effects in asthma treatment, which suggests that PP121 might be a promising therapeutic compound and shed new light on asthma therapy.

B-cell lymphomas (BCLs) are the most common disease entity among hematological malignancies and have various genetically and molecularly distinct subtypes. In this study, we revealed that the blockade of phosphoinositide-dependent kinase-1 (PDPK1), the master kinase of AGC kinases, induces a growth inhibition via cell cycle arrest and the induction of apoptosis in all eight BCL-derived cell lines examined, including those from activated B-cell-like diffuse large B-cell lymphoma (DLBCL), double expressor DLBCL, Burkitt lymphoma, and follicular lymphoma. We also demonstrated that, in these cell lines, RSK2, AKT, and S6K, but not PLK1, SGK, or PKC, are the major downstream therapeutic target molecules of PDPK1 and that RSK2 plays a central role and AKT and S6K play subsidiary functional roles as the downstream effectors of PDPK1 in cell survival and proliferation. Following these results, we confirmed the antilymphoma efficacy of TAS0612, a triple inhibitor for total RSK, including RSK2, AKT, and S6K, not only in these cell lines, regardless of disease subtypes, but also in all 25 patient-derived B lymphoma cells of various disease subtypes. At the molecular level, TAS0612 caused significant downregulation of MYC and mTOR target genes while inducing the tumor suppressor TP53INP1 protein in these cell lines. These results prove that the simultaneous blockade of RSK2, AKT, and S6K, which are the pivotal downstream substrates of PDPK1, is a novel therapeutic target for the various disease subtypes of BCLs and line up TAS0612 as an attractive candidate agent for BCLs for future clinical development.

The Tubby domain, named after the TUBBY protein in mice, binds to phosphatidylinositol 4,5-bisphosphate. Arabidopsis has 11 Tubby domain-containing proteins referred to as Tubby-Like Proteins (TLPs). Of the 11 TLPs, 10 possess the N-terminal F-box domain, which can interact with SKP-like proteins and form SKP1-Cullin-F-box E3 ligase complexes. Although mice TUBBY has been extensively studied, plant TLPs\' functions are scarcely detailed. In this study, we show that the Arabidopsis Tubby-like protein 6 (TLP6) and its redundant homologs, TLP1, TLP2, TLP5, and TLP10, positively regulate Arabidopsis immune responses. Furthermore, in an immunoprecipitation mass spectrometry analysis to search for ubiquitination substrates of the TLPs, we identified two redundant phosphoinositide biosynthesis enzymes, phosphatidylinositol 4-kinase β proteins (PI4Kβs), PI4Kβ1 and PI4Kβ2, as TLP interactors. Importantly, TLP6 overexpression lines fully phenocopy the phenotypes of the pi4kβ1,2 mutant, while TLP6 overexpression also leads to increased PI4Kβ2 ubiquitination and reduction in its protein level in a proteasome-dependent manner. Most significantly, TLP6 overexpression does not further enhance the autoimmunity of the pi4kβ1,2 double mutant, supporting the hypothesis that TLP6 targets the PI4Kβs for ubiquitination and degradation. Thus, our study reveals a novel mechanism where TLPs promote plant immune responses by modulating the PI4Kβs protein levels.

Chicoric acid (CA), a functional food ingredient, is a caffeic acid derivative that is mainly found in lettuce, pulsatilla, and other natural plants. However, the anti-inflammatory effects of CA in acute lung injury (ALI) remain poorly understood. This study was conducted to investigate potential drug usage of CA for ALI and the underlying molecular mechanisms of inflammation. C57BL/6 mice were given injections of liposaccharide (LPS) to establish the in vivo model. Meanwhile, BMDM cells were stimulated with LPS+ATP to build the in vitro model. CA significantly alleviated inflammation and oxidative stress in both the in vivo and in vitro models of ALI through the inhibition of NLR family pyrin domain-containing 3 (NLRP3)-mediated pyroptosis. In addition, CA attenuated mitochondrial damage to suppress NLRP3-mediated pyroptosis in the in vivo and in vitro models of ALI by suppressing the production of reactive oxygen species (ROS) via inhibiting the Akt/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. CA inhibited the interaction between Akt at T308 and phosphoinositide-dependent kinase-1 (PDPK1) at S549, thus promoting the phosphorylation of the Akt protein. Furthermore, CA directly targeted the PDPK1 protein and accelerated PDPK1 ubiquitination, indicating that 91-GLY, 111-LYS, 126-TYR, 162-ALA, 205-ASP, and 223-ASP might be responsible for the interaction between PDPK1 and CA. In conclusion, CA from Lettuce alleviated NLRP3-mediated pyroptosis in the ALI model through ROS-induced mitochondrial damage by activating Akt/Nrf2 pathway via PDPK1 ubiquitination. The present study suggests that CA might be a potential therapeutic drug to treat or prevent ALI in pneumonia or COVID-19.