CRISPRa Analysis of Phosphoinositide Phosphatases Shows That TMEM55A Is a Positive Regulator of Autophagy.

Transcriptional activation, based on Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) and known as CRISPR activation (CRISPRa), is a specific and safe tool to upregulate endogenous genes. Therefore, CRISPRa is valuable not only for analysis of molecular mechanisms of cellular events, but also for treatment of various diseases. Regulating autophagy has been proposed to enhance effects of some therapies.

Discovery of Potent DAG-Lactone Derivatives as HIV Latency Reversing Agents.

Toward human immunodeficiency virus type-1 (HIV-1) cure, cells latently infected with HIV-1 must be eliminated from people living with HIV-1. We previously developed a protein kinase C (PKC) activator, diacylglycerol (DAG)-lactone derivative , with high HIV-1 latency-reversing activity, based on YSE028 () as a lead compound and found that the activity was correlated with binding affinity for PKC and stability against esterase-mediated hydrolysis. Here, we synthesized new DAG-lactone derivatives not only containing a tertiary ester group or an isoxazole surrogate but also several symmetric alkylidene moieties to improve HIV-1 latency reversing activity.

A high dose KRP203 induces cytoplasmic vacuoles associated with altered phosphoinositide segregation and endosome expansion.

In animal cells, vacuoles are absent, but can be induced by diseases and drugs. While phosphoinositides are critical for membrane trafficking, their role in the formation of these vacuoles remains unclear. The immunosuppressive KRP203/Mocravimod, which antagonizes sphingosine-1-phosphate receptors, has been identified as having novel multimodal activity against phosphoinositide kinases.

SpCas9-HF1 enhances accuracy of cell cycle-dependent genome editing by increasing HDR efficiency, and by reducing off-target effects and indel rates.

In genome editing, it is important to avoid off-target mutations so as to reduce unexpected side effects, especially for therapeutic applications. Recently, several high-fidelity versions of SpCas9 have been developed to reduce off-target mutations. In addition to reducing off-target effects, highly efficient intended target gene correction is also essential to rescue protein functions that have been disrupted by single nucleotide polymorphisms.

Cas9-Geminin and Cdt1-fused anti-CRISPR protein synergistically increase editing accuracy.

Genome editing with CRISPR-Cas9, particularly for therapeutic purposes, should be accomplished via the homology-directed repair (HDR) pathway, which exhibits greater precision than other pathways. However, one of the issues to be solved is that genome editing efficiency with HDR is generally low. A Streptococcus pyogenes Cas9 (SpyCas9) fusion with human Geminin (Cas9-Gem) reportedly increases HDR efficiency slightly.

Phosphorylation of TMEM55B by Erk/MAPK regulates lysosomal positioning.

TMEM55B is first identified as phosphatidylinositol-4,5-P24-phosphatases (PtdIns-4,5-P24-phosphatases) that catalyse dephosphorylation of PtdIns-4,5-P2 to PtdIns-5-P. We demonstrate for the first time that TMEM55B is phosphorylated by Erk/MAPK and that this mechanism participates in regulation of lysosomal clustering. Exposure of RAW264.

PIKfyve accelerates phagosome acidification through activation of TRPML1 while arrests aberrant vacuolation independent of the Ca2+ channel.

PIKfyve phosphorylates PtdIns(3)P to PtdIns(3, 5)P2. One of the best characterized effector downstream of PtdIns(3, 5)P2 is a lysosomal Ca2+ channel, TRPML1. Although it has been reported that TRPML1 is involved in phagosome-lysosome fusion, the relevance of the Ca2+ channel in phagosome acidification has been denied.

Phosphoinositide phosphatase Sac3 regulates the cell surface expression of scavenger receptor A and formation of lipid droplets in macrophages.

The findings of this study suggest that the phosphoinositide phosphatase Sac3 maintains the protein level of scavenger receptor A (SR-A) and regulates foam cell formation. RAW264.7 macrophages were transfected with short hairpin RNAs that target Sac3.

Inhibitory receptor FcγRIIb mediates the effects of IgG on a phagosome acidification and a sequential dephosphorylation system comprising SHIPs and Inpp4a.

The relative abundance of phosphoinositide (PI) species on the phagosome membrane fluctuates over the course of phagocytosis. PtdIns(3,4,5)P and PtdIns(3,4)P rapidly increase in the forming of the phagocytic cup, following which they disappear after sealing of the cup. In the present study, we monitored the clearance of these PI species using the enhanced green fluorescent protein-fused pleckstrin homology domain of Akt, a fluorescence probe that binds both PtdIns(3,4,5)P and PtdIns(3,4)P in Raw 264.

Myeloid cell-specific inositol polyphosphate-4-phosphatase type I knockout mice impair bacteria clearance in a murine peritonitis model.

Phosphatidylinositol 3-kinase (PI3K)/Akt signaling has been implicated in the anti-inflammatory response in a mouse model of endotoxemia and sepsis. The present study focused on the role of inositol polyphosphate-4-phosphatase type I (Inpp4a), which dephosphorylates PtdIns(3,4)P2 to PtdIns(3)P, in bacterial infections. We prepared myeloid cell-specific Inpp4a-conditional knockout mice.

Inositol Polyphosphate-4-Phosphatase Type I Negatively Regulates Phagocytosis via Dephosphorylation of Phagosomal PtdIns(3,4)P2.

Phagocytosis is a highly conserved process whereby phagocytic cells engulf pathogens and apoptotic bodies. The present study focused on the role of inositol polyphosphate-4-phosphatase type I (Inpp4a) in phagocytosis. Raw264.

Involvement of class II phosphoinositide 3-kinase α-isoform in antigen-induced degranulation in RBL-2H3 cells.

In this study, we present findings that suggest that PI3K-C2α, a member of the class II phosphoinositide 3-kinase (PI3K) subfamily, regulates the process of FcεRI-triggered degranulation. RBL-2H3 cells were transfected with shRNA targeting PI3K-C2α. The knockdown impaired the FcεRI-induced release of a lysosome enzyme, β-hexosaminidase, without affecting the intracellular Ca2+ mobilization.

Inpp5e increases the Rab5 association and phosphatidylinositol 3-phosphate accumulation at the phagosome through an interaction with Rab20.

Phosphoinositide 5'-phosphatases have been implicated in the regulation of phagocytosis. However, their precise roles in the phagocytic process are poorly understood. We prepared RAW264.

PIKfyve regulates the endosomal localization of CpG oligodeoxynucleotides to elicit TLR9-dependent cellular responses.

TLR9 is a receptor for oligodeoxynucleotides that contain unmethylated CpG motifs (CpG). Because TLR9 resides in the endoplasmic reticulum during the quiescence state, CpG binding to TLR9 requires membrane trafficking, which includes the maturation of the CpG-containing endosome. In the present study, we examined the role of PIKfyve, a phosphatidylinositol 3-phosphate 5-kinase, in the regulation of TLR9 signaling.

Class-IA phosphoinositide 3-kinase p110β Triggers GPCR-induced superoxide production in p110γ-deficient murine neutrophils.

Studies with knockout mice have indicated that the only isoform of phosphoinositide 3-kinase (PI3K) functioning in the oxidative burst of mouse neutrophils in response to heterotrimeric guanine nucleotide-binding protein-coupled receptor (GPCR) agonists is a class-IB PI3K, p110γ. In the present study, we observed that the cells from p110γ(-/-) mice gain a response to N-formyl-Met-Leu-Phe (fMLP) after priming with cytochalasin E. Even the unprimed cells, which show no response to fMLP, produce a significant amount of superoxide, when an effective agonist of the mouse-type fMLP receptors, Trp-Lys-Tyr-Met-Val-D-Met, is used to stimulate the cells.

Essential roles of PIKfyve and PTEN on phagosomal phosphatidylinositol 3-phosphate dynamics.

PtdIns(3)P (phosphatidylinositol 3-phosphate) is a signaling molecule important for phagosome maturation. The major role of Vps34 in production of phagosomal PtdIns(3)P has been indicated. However, the fate of the newly generated PtdIns(3)P has not been well described.

Phosphoinositide 3-kinaseγ controls the intracellular localization of CpG to limit DNA-PKcs-dependent IL-10 production in macrophages.

Synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG) stimulate innate immune responses. Phosphoinositide 3-kinase (PI3K) has been implicated in CpG-induced immune activation; however, its precise role has not yet been clarified. CpG-induced production of IL-10 was dramatically increased in macrophages deficient in PI3Kγ (p110γ(-/-)).

C5a controls TLR-induced IL-10 and IL-12 production independent of phosphoinositide 3-kinase.

The complement system is a classic central player in innate immunity. Most pathogens activate both complement and the toll-like receptor (TLR) pathway. Therefore, to provide a more comprehensive understanding of innate immunity, it is important to understand the crosstalk between these two systems.

Specific role of phosphoinositide 3-kinase p110alpha in the regulation of phagocytosis and pinocytosis in macrophages.

PI3K (phosphoinositide 3-kinase) alpha has been implicated in phagocytosis and fluid-phase pinocytosis in macrophages. The subtype-specific role of PI3K in these processes is poorly understood. To elucidate this issue, we made Raw 264.

Negative regulation of class IA phosphoinositide 3-kinase by protein kinase Cdelta Limits Fcgamma receptor-mediated phagocytosis in macrophages.

Stimulation of macrophages by various ligands results in the activation of both phosphoinositide 3-kinase (PI3K) and protein kinase C (PKC). Here, we showed that PKCdelta selectively inhibits class IA PI3K. Prior exposure of macrophages to a PKC activator, phorbol 12-myristate 13-acetate (PMA) inhibited the PI3K activation induced by the Fcgamma receptor (FcgammaR) ligation but not that induced by C5a.

Critical roles of the p110 beta subtype of phosphoinositide 3-kinase in lipopolysaccharide-induced Akt activation and negative regulation of nitrite production in RAW 264.7 cells.

It has been suggested that PI3K participates in TLR signaling. However, identifying specific roles for individual PI3K subtypes in signaling has remained elusive. In macrophages from the p110gamma(-/-) mouse, LPS-induced phosphorylation of Akt occurred normally despite the fact that the action of anaphylatoxin C5a was impaired markedly.

IRAK-4-dependent degradation of IRAK-1 is a negative feedback signal for TLR-mediated NF-kappaB activation.

The activation of interleukin 1 receptor-associated kinase (IRAK)-1 is a key event in the transmission of signals from Toll-like receptors (TLRs). The catalytic activity of the protein kinase is not essential for its ability to activate nuclear factor (NF) kappaB, because transfection of a kinase-dead mutant of IRAK-1 (IRAK-1KD) is able to activate NF-kappaB in HEK293T cells. In the present study, we observed that the effect of IRAK-1KD was impaired by simultaneous expression of IRAK-4.