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Phosphoinositide kinases regulate diverse cellular functions and are important targets for therapeutic development for diseases, such as diabetes and cancer. Preparation of the lipid substrate is crucial for the development of a robust and miniaturizable lipid kinase assay. Enzymatic assays for phosphoinositide kinases often use lipid substrates prepared from lyophilized lipid preparations by sonication, which result in variability in the liposome size from preparation to preparation. Herein, we report a homogeneous 1536-well luciferase-coupled bioluminescence assay for PI5P4Kα. The substrate preparation is novel and allows the rapid production of a DMSO-containing substrate solution without the need for lengthy liposome preparation protocols, thus enabling the scale-up of this traditionally difficult type of assay. The Z'-factor value was greater than 0.7 for the PI5P4Kα assay, indicating its suitability for high-throughput screening applications. Tyrphostin AG-82 had been identified as an inhibitor of PI5P4Kα by assessing the degree of phospho transfer of γ-(32)P-ATP to PI5P; its inhibitory activity against PI5P4Kα was confirmed in the present miniaturized assay. From a pilot screen of a library of bioactive compounds, another tyrphostin, I-OMe tyrphostin AG-538 (I-OMe-AG-538), was identified as an ATP-competitive inhibitor of PI5P4Kα with an IC(50) of 1 µM, affirming the suitability of the assay for inhibitor discovery campaigns. This homogeneous assay may apply to other lipid kinases and should help in the identification of leads for this class of enzymes by enabling high-throughput screening efforts.

Vps13 family proteins are proposed to function in bulk lipid transfer between membranes, but little is known about their regulation. During sporulation of Saccharomyces cerevisiae , Vps13 localizes to the prospore membrane (PSM) via the Spo71–Spo73 adaptor complex. We previously reported that loss of any of these proteins causes PSM extension and subsequent sporulation defects, yet their precise function remains unclear. Here, we performed a genetic screen and identified genes coding for a fragment of phosphatidylinositol (PI) 4-kinase catalytic subunit and PI 4-kinase noncatalytic subunit as multicopy suppressors of spo73 Δ. Further genetic and cytological analyses revealed that lowering PI4P levels in the PSM rescues the spo73 Δ defects. Furthermore, overexpression of VPS13 and lowering PI4P levels synergistically rescued the defect of a spo71 Δ spo73 Δ double mutant, suggesting that PI4P might regulate Vps13 function. In addition, we show that an N-terminal fragment of Vps13 has affinity for the endoplasmic reticulum (ER), and ER-plasma membrane (PM) tethers localize along the PSM in a manner dependent on Vps13 and the adaptor complex. These observations suggest that Vps13 and the adaptor complex recruit ER-PM tethers to ER-PSM contact sites. Our analysis revealed that involvement of a phosphoinositide, PI4P, in regulation of Vps13, and also suggest that distinct contact site proteins function cooperatively to promote de novo membrane formation.

Normal leaf margin development is important for leaf morphogenesis and contributes to diverse leaf shapes in higher plants. We here show the crucial roles of an atypical type II phosphatidylinositol 4-kinase, PI4Kγ5, in Arabidopsis leaf margin development. PI4Kγ5 presents a dynamics expression pattern along with leaf development and a T-DNA mutant lacking PI4Kγ5, pi4kγ5-1, presents serrated leaves, which is resulted from the accelerated cell division and increased auxin concentration at serration tips. Studies revealed that PI4Kγ5 interacts with and phosphorylates a membrane-bound NAC transcription factor, ANAC078. Previous studies demonstrated that membrane-bound transcription factors regulate gene transcription by undergoing proteolytic process to translocate into nucleus, and ANAC078 undergoes proteolysis by cleaving off the transmembrane region and carboxyl terminal. Western blot analysis indeed showed that ANAC078 deleting of carboxyl terminal is significantly reduced in pi4kγ5-1, indicating that PI4Kγ5 is important for the cleavage of ANAC078. This is consistent with the subcellular localization observation showing that fluorescence by GFP-ANAC078 is detected at plasma membrane but not nucleus in pi4kγ5-1 mutant and that expression of ANAC078 deleting of carboxyl terminal, driven by PI4Kγ5 promoter, could rescue the leaf serration defects of pi4kγ5-1. Further analysis showed that ANAC078 suppresses the auxin synthesis by directly binding and regulating the expression of auxin synthesis-related genes. These results indicate that PI4Kγ5 interacts with ANAC078 to negatively regulate auxin synthesis and hence influences cell proliferation and leaf development, providing informative clues for the regulation of in situ auxin synthesis and cell division, as well as the cleavage and functional mechanism of membrane-bound transcription factors.

Phosphoinositides (PIs) constitute a minor fraction of total cellular lipids in all eukaryotic cells. They fulfill many important functions through interaction with a wide range of cellular proteins. Members of distinct inositol lipid kinase families catalyze the synthesis of these phospholipids from phosphatidylinositol. The hydrolysis of PIs involves phosphatases and isoforms of PI-specific phospholipase C. Although our knowledge of the roles played by plant PIs is clearly limited at present, there is no doubt that they are involved in many physiological processes during plant growth and development. In this review, we concentrate on inositol lipid-metabolizing enzymes from the model plant Arabidopsis for which biochemical characterization data are available, namely the inositol lipid kinases and PI-specific phospholipase Cs. The biochemical properties and structure of characterized and genome-predicted isoforms are presented and compared with those of the animal enzymes to show that the plant enzymes have some features clearly unique to this kingdom.

Subcellular machinery of NLRP3 is essential for inflammasome assembly and activation. However, the stepwise process and mechanistic basis of NLRP3 engagement with organelles remain unclear. Herein, we demonstrated glycogen synthase kinase 3β (GSK3β) as a molecular determinant for the spatiotemporal dynamics of NLRP3 inflammasome activation. Using live cell multispectral time-lapse tracking acquisition, we observed that upon stimuli NLRP3 was transiently associated with mitochondria and subsequently recruited to the Golgi network (TGN) where it was retained for inflammasome assembly. This occurred in relation to the temporal contact of mitochondria to Golgi apparatus. NLRP3 stimuli initiate GSK3β activation with subsequent binding to NLRP3, facilitating NLRP3 recruitment to mitochondria and transition to TGN. GSK3β activation also phosphorylates phosphatidylinositol 4-kinase 2 Α (PI4k2A) in TGN to promote sustained NLRP3 oligomerization. Our study has identified the interplay between GSK3β signaling and the organelles dynamics of NLRP3 required for inflammasome activation and opens new avenues for therapeutic intervention.

Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) is generated through phosphorylation of phosphatidylinositol 4-phosphate (PtdIns(4)P) by Mss4p, the only PtdIns phosphate 5-kinase in yeast cells. PtdIns(4,5)P2 is involved in various kinds of yeast functions. PtdIns(4)P is not only the immediate precursor of PtdIns(4,5)P2, but also an essential signaling molecule in the plasma membrane, Golgi, and endosomal system. To analyze the distribution of PtdIns(4,5)P2 and PtdIns(4)P in the yeast plasma membrane at a nanoscale level, we employed a freeze–fracture electron microscopy (EM) method that physically immobilizes lipid molecules in situ. It has been reported that the plasma membrane of budding yeast can be divided into three distinct areas: furrowed, hexagonal, and undifferentiated flat. Previously, using the freeze–fracture EM method, we determined that PtdIns(4)P is localized in the undifferentiated flat area, avoiding the furrowed and hexagonal areas of the plasma membrane. In the present study, we found that PtdIns(4,5)P2 was localized in the cytoplasmic leaflet of the plasma membrane, and concentrated in the furrowed area. There are three types of PtdIns 4-kinases which are encoded by stt4, pik1, and lsb6. The labeling density of PtdIns(4)P in the plasma membrane significantly decreased in both pik1ts and stt4ts mutants. However, the labeling densities of PtdIns(4,5)P2 in the plasma membrane of both the pik1ts and stt4ts mutants were comparable to that of the wild type yeast. These results suggest that PtdIns(4)P produced by either Pik1p or Stt4p is immediately phosphorylated by Mss4p and converted to PtdIns(4,5)P2 at the plasma membrane.

Infection by positive-strand RNA viruses necessitates membrane expansion and elevated phospholipid biosynthesis, whereby fatty acids stored as triacylglycerols in lipid droplets (LDs) are mobilized to promote metabolic processes and membrane biogenesis. The replication organelles (ROs) of coronavirus associate with modified host endomembrane; however, the molecular mechanisms underlying the expansion and modification of these membranes remain poorly understood. Here, we show that viral protein orf3a collaborates with nsp3, nsp4, nsp6 to facilitate the formation of ROs in SARS-CoV-2. Importantly, orf3a targets LDs to ROs, establishing novel membrane contact sites and induces host cell microlipophagy, which supplies essential lipids for RO biogenesis. Subsequently, Following the formation of ROs, nsp3, with assistance from nsp12, indirectly recruits phosphatidylinositol 4-kinase beta (PI4KB) to ROs, to produce phosphatidylinositol 4-phosphate (PI4P). This action creates a PI4P-enriched microenvironment that enhances SARS-CoV-2 replication. Our findings elucidate the mechanism governing RO generation during SARS-CoV-2 infection and suggest that targeting microlipophagy pharmacologically may represent a promising strategy for the development of anti-coronaviruses therapies.

Background Artemisinin-based combination therapies are the first line of treatment for Plasmodium falciparum infections worldwide, but artemisinin resistance has risen rapidly in Southeast Asia over the past decade. Mutations in the kelch13 gene have been implicated in this resistance. We used longitudinal genomic surveillance to detect signals in kelch13 and other loci that contribute to artemisinin or partner drug resistance. We retrospectively sequenced the genomes of 194 P. falciparum isolates from five sites in Northwest Thailand, over the period of a rapid increase in the emergence of artemisinin resistance (2001–2014). Results We evaluate statistical metrics for temporal change in the frequency of individual SNPs, assuming that SNPs associated with resistance increase in frequency over this period. After Kelch13 -C580Y, the strongest temporal change is seen at a SNP in phosphatidylinositol 4-kinase, which is involved in a pathway recently implicated in artemisinin resistance. Furthermore, other loci exhibit strong temporal signatures which warrant further investigation for involvement in artemisinin resistance evolution. Through genome-wide association analysis we identify a variant in a kelch domain-containing gene on chromosome 10 that may epistatically modulate artemisinin resistance. Conclusions This analysis demonstrates the potential of a longitudinal genomic surveillance approach to detect resistance-associated gene loci to improve our mechanistic understanding of how resistance develops. Evidence for additional genomic regions outside of the kelch13 locus associated with artemisinin-resistant parasites may yield new molecular markers for resistance surveillance, which may be useful in efforts to reduce the emergence or spread of artemisinin resistance in African parasite populations.

Hepatitis C virus (HCV), like other positive-sense RNA viruses, replicates on an altered host membrane compartment that has been called the \"membranous web.\" The mechanisms by which the membranous web are formed from cellular membranes are poorly understood. Several recent RNA interference screens have demonstrated a critical role for the host phosphatidylinositol 4-kinase PI4KA in HCV replication. We have sought to define the function of PI4KA in viral replication. Using a nonreplicative model of membranous web formation, we show that PI4KA silencing leads to aberrant web morphology. Furthermore, we find that PI4KA and its product, phosphatidylinositol 4-phosphate, are enriched on membranous webs and that PI4KA is found in association with NS5A in HCV-infected cells. While the related lipid kinase PI4KB also appears to support HCV replication, it does not interact with NS5A. Silencing of PI4KB does not overtly impair membranous web morphology or phosphatidylinositol 4-phosphate enrichment at webs, suggesting that it acts at a different point in viral replication. Finally, we demonstrate that the aberrant webs induced by PI4KA silencing require the activity of the viral NS3-4A serine protease but not integrity of the host secretory pathway. PI4KA is necessary for the local enrichment of PI 4-phosphate at the HCV membranous web and for the generation of morphologically normal webs. We also show that nonreplicative systems of web formation can be used to order molecular events that drive web assembly.

Background Phosphoinositide lipid kinases (PIKs) generate specific phosphorylated variants of phosatidylinositols (PtdIns) that are critical for second messenger signaling and cellular membrane remodeling. Mammals have 19 PIK isoforms spread across three major families: the PtIns 3-kinases (PI3Ks), PtdIns 4-kinases (PI4Ks), and PtdIns-P (PIP) kinases (PIPKs). Other eukaryotes have fewer yet varying PIK complements. PIKs are also an important, emerging class of drug targets for many therapeutic areas including cancer, inflammatory and metabolic diseases and host-pathogen interactions. Here, we report the genomic occurrences and evolutionary relationships or phylogenomics of all three PIK families across major eukaryotic groups and suggest potential ramifications for drug discovery. Results Our analyses reveal four core eukaryotic PIKs which are type III PIK4A and PIK4B, and at least one homolog each from PI3K (possibly PIK3C3 as the ancestor) and PIP5K families. We also applied evolutionary analyses to PIK disease ontology and drug discovery. Mutated PIK3CA are known to be oncogenic and several inhibitors are in anti-cancer clinical trials. We found conservation of activating mutations of PIK3CA in paralogous isoforms suggesting specific functional constraints on these residues. By mapping published compound inhibition data (IC50s) onto a phylogeny of PI3Ks, type II PI4Ks and distantly related, MTOR, ATM, ATR and PRKDC kinases, we also show that compound polypharmacology corresponds to kinase evolutionary relationships. Finally, we extended the rationale for drugs targeting PIKs of malarial Plasmodium falciparum , and the parasites, Leishmania sp. and Trypanosoma sp. by identifying those PIKs highly divergent from human homologs. Conclusion Our phylogenomic analysis of PIKs provides new insights into the evolution of second messenger signaling. We postulate two waves of PIK diversification, the first in metazoans with a subsequent expansion in cold-blooded vertebrates that was post-emergence of Deutrostomia\\Chordata but prior to the appearance of mammals. Reconstruction of the evolutionary relationships among these lipid kinases also adds to our understanding of their roles in various diseases and assists in their development as potential drug targets.