Key Phosphatase Research Articles

Identification of YigL as a PLP/PNP phosphatase in Escherichia coli.

In various organisms, the coenzyme form of vitamin B6, pyridoxal phosphate (PLP), is synthesized from pyridoxine phosphate (PNP). Control of PNP levels is crucial for metabolic homeostasis because PNP has the potential to inhibit PLP-dependent enzymes and proteins. Although the only known pathway for PNP metabolism in Escherichia coli involves oxidation by PNP oxidase, we detected a strong PNP phosphatase activity in E. coli cell lysate. To identify the unknown PNP phosphatase(s), we performed a multicopy suppressor screening using the E. coli serA pdxH strain, which displays PNP-dependent conditional lethality. The results showed that overexpression of the yigL gene, encoding a putative sugar phosphatase, effectively alleviated the PNP toxicity. Biochemical analysis revealed that YigL has strong phosphatase activity against PNP. A yigL mutant exhibited decreased PNP phosphatase activity, elevated intracellular PNP concentrations, and increased PNP sensitivity, highlighting the important role of YigL in PNP homeostasis. YigL also shows reactivity with PLP. The phosphatase activity of PLP in E. coli cell lysate was significantly reduced by mutation of yigL and nearly abolished by additional mutation of ybhA, which encodes putative PLP phosphatase. These results underscore the important contribution of YigL, in combination with YbhA, as a primary enzyme in the dephosphorylation of both PNP and PLP in E. coli.IMPORTANCEPyridoxine phosphate (PNP) metabolism is critical for both vitamin B6 homeostasis and cellular metabolism. In Escherichia coli, oxidation of PNP was the only known mechanism for controlling PNP levels. This study uncovered a novel phosphatase-mediated mechanism for PNP homeostasis. Multicopy suppressor screening, kinetic analysis of the enzyme, and knockout/overexpression studies identified YigL as a key PNP phosphatase that contributes to PNP homeostasis when facing elevated PNP concentrations in E. coli. This study also revealed a significant contribution of YigL, in combination with YbhA, to PLP metabolism, shedding light on the mechanisms of vitamin B6 regulation in bacteria.

Dynamic regulation of phosphorylation of NMDA receptor GluN2B subunit tyrosine residues mediates ketamine rapid antidepressant effects

The rapid antidepressant effects of ketamine depend on the N-methyl-D-aspartate (NMDA) receptor containing 2B subunit (NR2B), whose function is influenced by its phosphorylated regulation and distribution within and outside synapses. It remains unclear if ketamine’s rapid onset of antidepressant effects relies on the dynamic phosphorylated regulation of NR2B within and outside synapses. Here, we show that ketamine rapidlyalleviated depression-like behaviors and normalized abnormal expression of pTyr1472NR2B and striatal-enriched protein tyrosine phosphatase (STEP) 61 within and outside synapses in the medial prefrontal cortex (mPFC) induced by chronic unpredictable stress (CUS) and conditional knockdown of STEP 61, a key phosphatase of NR2B, within 1 hour after administration Together, our results delineate the rapid initiation of ketamine’s antidepressant effects results from the restoration of NR2B phosphorylation homeostasis within and outside synapses. The dynamic regulation of phosphorylation of NR2B provides a new perspective for developing new antidepressant strategies.

Metabolomic analysis of lipid changes in Bombyx mori infected with Nosema bombycis

The silkworm (Bombyx mori) is a model species of lepidopteran insect. Microsporidium spp. are obligate intracellular eukaryotic parasites. Infection by the microsporidian Nosema bombycis (Nb) results in an outbreak of Pébrine disease in silkworms and causes substantial losses to the sericulture industry. It has been suggested that Nb depends on nutrients from host cells for spore growth. However, little is known about changes in lipid levels after Nb infection. In this study, ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was performed to analyze the effect of Nb infection on lipid metabolism in the midgut of silkworms. A total of 1601 individual lipid molecules were detected in the midgut of silkworms, of which 15 were significantly decreased after Nb challenge. Classification, chain length, and chain saturation analysis revealed that these 15 differential lipids can be classified into different lipid subclasses, of which 13 belong to glycerol phospholipid lipids and two belong to glyceride esters. The results indicated that Nb uses the host lipids to complete its own replication, and the acquisition of host lipid subclasses is selective; not all lipid subclasses are required for microsporidium growth or proliferation. Based on lipid metabolism data, phosphatidylcholine (PC) was found to be an important nutrient for Nb replication. Diet supplementation with lecithin substantially promoted the replication of Nb. Knockdown and overexpression of the key enzyme phosphatidate phosphatase (PAP) and phosphatidylcholine (Bbc) for PC synthesis also confirmed that PC is necessary for Nb replication. Our results showed that most lipids in the host midgut decreased when silkworms were infected with Nb. Reduction of or supplementation with PC may be a strategy to suppress or promote microsporidial replication.

Effects of initial carbon-phosphorus ratio on phosphatase, and phosphorus availability in sludge composting

Phosphorus in wastewater sludge is one of the important nutrients for biological growth and an important non-renewable resource. Most research in the composting field focuses on the C/N ratio, but there are few reports on initial carbon-phosphorus (C/P) ratio regulation. This study investigated the effects of different initial C/P ratios on phosphatase activity, key bacteria, and phosphorus availability in compost. In this study, the activity of phosphatase was measured, and key bacteria secreting phosphatase were identified. The results showed that adjusting the initial C/P ratio could prolong the cycle of action of key bacteria, thus affecting the activity of phosphatase and promoting the production of available phosphorus, but it was also inhibited by the feedback of available phosphorus. This study demonstrated the feasibility of adjusting the initial C/P ratio of sludge composting and provided theoretical support for optimizing the application of sludge composting products with different initial C/P ratios.

Analysis of the effects of curcumin and symbiotic consumption on bones of rats submitted to the use of dexamethasone

AimsGlucocorticoids have adverse side effects that can compromise bone tissue. There is evidence to show that symbiotics and curcumin can prevent bone loss. The aim of this study was to analyze the effects of curcumin and symbiotic to prevent and/or minimize a possible bone impairment in rats submitted to the use of dexamethasone. MethodsFifty Wistar female rats were divided into five groups: control group (CT), dexamethasone control group (D), dexamethasone and symbiotic group (DS), dexamethasone and curcumin group (DC), and dexamethasone and symbiotic/curcumin group (DSC). Dexamethasone was applied three times a week, while the symbiotic and curcumin were administered daily. Alkaline phosphatase and calcium dosages, analysis of structural and material properties, and Raman analysis of femurs were performed. Key findingsAlkaline phosphatase was higher in the DC group. Maximum load and structural stiffness were higher in the CT group. Maximum stress was lower and similar between dexamethasone groups. The CT group had a lower percentage of strain, the D group had greater deformation compared to the DC group and the DS group presented more deformation than the DC group. The D, DS, and DSC groups had a lower elastic modulus compared to the CT group. The 960/1660 ratios of the D, DS, and DSC groups were different from the CT group. The 1070/1660 ratio was higher in the DC group. SignificanceIt was possible to verify that curcumin showed promising effects related to the increase in bone strength and mineralization, mitigating the deleterious effects caused by dexamethasone, when used simultaneously with this drug.

Gypenoside ameliorates insulin resistance and hyperglycemia via the AMPK-mediated signaling pathways in the liver of type 2 diabetes mellitus mice

Gynostemma pentaphyllum, also called “Southern Ginseng” in China, is a traditional Asian folk medicinal plant. Gypenosides (Gps) are the biologically active constituents of G. pentaphyllum, which have been reported with hypoglycemic activity. However, the underlying mechanisms are unclear. The effects of two Gps (Gp-I and Gp-II) on type 2 diabetic mellitus (T2DM) mice, induced by high-fat and high-sugar diet and streptozotocin, were evaluated to explore the mechanism of their hypoglycemic actions. Gps reduced fasting blood glucose and serum lipids, as well as significantly improved T2DM mice glucose tolerance and insulin resistance (IR). After Gps treatment, the severity of liver injury was reduced and liver glycogen content increased. In addition, Gps promoted the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK), and downregulated the key proteins phosphoenolpyruvate carboxy kinase and glucose-6 phosphatase, in the AMPK signaling pathway. Thus, our study suggests that Gps mediate hepatic gluconeogenesis and improve IR via activating AMPK signaling pathway in T2DM mice.

PP2A is activated by cytochrome c upon formation of a diffuse encounter complex with SET/TAF-Iβ

Intrinsic protein flexibility is of overwhelming relevance for intermolecular recognition and adaptability of highly dynamic ensemble of complexes, and the phenomenon is essential for the understanding of numerous biological processes. These conformational ensembles—encounter complexes—lack a unique organization, which prevents the determination of well-defined high resolution structures. This is the case for complexes involving the oncoprotein SET/template-activating factor-Iβ (SET/TAF-Iβ), a histone chaperone whose functions and interactions are significantly affected by its intrinsic structural plasticity. Besides its role in chromatin remodeling, SET/TAF-Iβ is an inhibitor of protein phosphatase 2A (PP2A), which is a key phosphatase counteracting transcription and signaling events controlling the activity of DNA damage response (DDR) mediators. During DDR, SET/TAF-Iβ is sequestered by cytochrome c (Cc) upon migration of the hemeprotein from mitochondria to the cell nucleus. Here, we report that the nuclear SET/TAF-Iβ:Cc polyconformational ensemble is able to activate PP2A. In particular, the N-end folded, globular region of SET/TAF-Iβ (a.k.a. SET/TAF-Iβ ΔC)—which exhibits an unexpected, intrinsically highly dynamic behavior—is sufficient to be recognized by Cc in a diffuse encounter manner. Cc-mediated blocking of PP2A inhibition is deciphered using an integrated structural and computational approach, combining small-angle X-ray scattering, electron paramagnetic resonance, nuclear magnetic resonance, calorimetry and molecular dynamics simulations.

Calcineurin Aα Contributes to IgE-Dependent Mast-Cell Mediator Secretion in Allergic Inflammation

Mast cells (MCs) are key mediators of allergic inflammation through the activation of cross-linked immunoglobulin E (IgE) bound to the high-affinity IgE receptor (FcϵRI) on the cell surface, leading to the release of biologically potent mediators, either from preformed granules or newly synthesized. Pharmacological inhibitors have been developed to target a key signaling protein phosphatase in this pathway, calcineurin, yet there is a lack of genetic and definitive evidence for the various isoforms of calcineurin subunits in FcϵRI-mediated responses. In this study, we hypothesized that deficiency in the calcineurin Aα isoform will result in a decreased allergic immune response by the MCs. In a model of passive cutaneous anaphylaxis, there was a reduction in vascular permeability in MC-deficient mouse tissues reconstituted with calcineurin subunit A (CnAα) gene-knockout (CnAα^−/−) MCs, and in vitro experiments identified a significant reduction in release of preformed mediators from granules. Furthermore, released levels of de novo synthesized cytokines were reduced upon FcϵRI activation of CnAα^−/− MCs in vitro. Characterizing the mechanisms associated with this deficit response, we found a significant impairment of nuclear factor of kappa light polypeptide gene enhancer in B cell phosphorylation and impaired nuclear factor kappa-light-chain-enhancer of activated B-cell inhibitor alpha (NF-κB) activation. Thus, we concluded that CnAα contributes to the release of preformed mediators and newly synthesized mediators from FcϵRI-mediated activation of MCs, and this regulation includes NF-κB signaling.

Old and New Calcineurin Inhibitors in Lupus Nephritis.

Calcineurin inhibitors (CNIs) are drugs that inhibit calcineurin, a key phosphatase that dephosphorylates a transcription factor called the nuclear factor of activated T cells (NFAT), allowing its translocation into the nucleus of quiescent T cells. In the nucleus, NFAT activates interleukin 2, which stimulates the proliferation and differentiation of T-cells. CNIs can also stabilize the actin cytoskeleton of podocytes reducing proteinuria. Thanks to these characteristics, CNIs have been often used in the treatment of autoimmune diseases. However, the therapeutic index of CNIs is narrow, and their interactions with other drugs can increase toxicity or reduce efficacy. In lupus nephritis, cyclosporine and tacrolimus have been used both in induction and maintenance therapies. Observational studies and randomized controlled trials showed that both cyclosporine and tacrolimus can increase efficacy. Tolerance is satisfactory if low doses are used and the patient is carefully monitored. More recently, a new CNI, called voclosporin (VCS), has been approved by the Food and Drug Administration for use in lupus nephritis. VCS offers potential advantages over other CNIs. In two large multiethnic trials, VCS was not associated with adverse renal and metabolic events and obtained positive results despite a novel and rapid corticosteroid tapering regime.

Electroanalysis of Enzyme Activity in Small Biological Samples: Alkaline Phosphatase.

The discovery of sulfite-stabilized anodic current of hydroquinone (HQ) at high pH was used to develop two new methods for measuring the activity of the key biomarker alkaline phosphatase (ALP). Both approaches relied on the monitoring of ALP-triggered release of HQ from a substrate hydroquinone diphosphate (HQDP) into a pH 10.00 solution. One detected the released HQ via the internally calibrated electrochemical continuous enzyme assay (ICECEA) at a glassy carbon (GC) electrode with no sample incubation. The other used sample incubation with HQDP and quantified the released HQ via a coulometric assay at a commercial glucose test strip (GTS). The assay solution was optimized by investigating the ALP/HQDP/HQ system at a GC electrode. The ICECEA revealed high affinity of ALP for HQDP (Kmapp, 87 μM; Vmax, 0.36 μM min-1) and detected ALP down to 0.022 U L-1. At GTS, ALP was detected down to 0.064 U L-1 in a 1 μL sample of human serum after a 20 min incubation at room temperature. The linear range (R2, 0.994) extended at least up to 1.7 U L-1 ALP, which covered more than the clinical range for ALP in serum. The interferences from the sample matrix including those from indigenous glucose were eliminated using a charge difference ΔQ (=Qtotal - Qsamplematrix) as a signal for ALP. Both advances proposed here are direct (no auxiliary enzymes or labels required), accurate (98 ± 3% ALP signal recovery), and precise (relative standard deviation (RSD), <7%). The HQDP-GTS-based assay advances the analysis of ALP activity in microsized real-life samples.

DHCR24 Knockdown Lead to Hyperphosphorylation of Tau at Thr181, Thr231, Ser262, Ser396, and Ser422 Sites by Membrane Lipid-Raft Dependent PP2A Signaling in SH-SY5Y Cells.

Accumulating data suggest that the downregulation of DHCR24 is linked to the pathological risk factors of AD, denoting a potential role of DHCR24 in AD pathogenesis. However, it remains unclear whether the downregulation of DHCR24 affects the abnormal heper-phosphorylation of tau protein, which is involved in tauopathy. In present papers, immunofluorescence and Filipin III fluorescence results showed that DHCR24 knockdown significantly lowered the level of plasma membrane cholesterol and expression level of membrane lipid-raft structural protein caveolin-1; and overexpression of DHCR24 could increase the plasma membrane cholesterol levels and facilitating caveolae structure through increase the expression of caveolin-1. PP2A is the key phosphatase involving in tau phosphorylation, which is localized in cholesterol-dependent caveola/raft lipid domains. Here, the PP2A activity was detected by western blot assay. Interestingly, the level of p-PP2Ac at Y307 (inactive) and p-GSK3β at Y216 (active) in the downstream of the PP2A signal pathway were both significantly increased in silencing DHCR24 SH-SY5Y cells, which denoted an inhibition of the PP2A and activation of GSK3β signaling. Conversely, overexpression of DHCR24 blunted the inhibition effect of PP2A and activation of GSK3β. Besides, in the SH-SY5Y cell lines we demonstrated that DHCR24 knockdown obviously induced hyperphosphorylation of tau at Thr181, Thr231, Ser262, Ser396, and Ser422 Sites. In contrast, DHCR24 overexpression protects neuronal SH-SY5Y cells against the hyperphosphorylation of tau at Thr181, Thr231, Ser262, Ser396, and Ser422 Sites. Furthermore, PP2A activator D-erythro-Sphingosine (DES) also obviously inhibited the hyperphosphorylation of tau induced by DHCR24 knockdown. Collectively, our findings firstly confirmed that DHCR24 knockdown obviously induced abnormal hyperphosphorylation of tau by a novel lipid raft-dependent PP2A signaling. We propose that DHCR24 downregulation led to altered cholesterol synthesis as a potential mechanism in the progression of tau hyperphosphorylation involving in AD and other tauopathies.

Off-target effects of protein tyrosine phosphatase inhibitors on oncostatin M-treated human epidermal keratinocytes: the phosphatase targeting STAT1 remains unknown.

Cytokine signaling in the epidermis has an important role in maintaining barrier function and is perturbed in pathological conditions. Environmental exposures, such as to metal compounds, are of interest for their potential contribution to skin disease. Present work explores the possibility that vanadate is a more effective protein tyrosine phosphatase inhibitor in human keratinocytes than previously observed in fibroblasts. It focuses on the state of phosphorylation of signal transducer and activator of transcription 1 (STAT1) on tyrosine 701 upon treatment of cultured human keratinocytes with the cytokine oncostatin M, a cutaneous inflammatory mediator that is highly effective in suppressing several differentiation markers and in preserving proliferative potential of keratinocytes. Exposure to sodium vanadate in the medium greatly prolonged the phosphorylation of STAT1, but only at high concentration (>30 µM). Inhibitors of protein tyrosine phosphatases known to dephosphorylate STAT1 (SHP2, TCPTP, PTP1B) were ineffective in mimicking the action of vanadate. The irreversible protein tyrosine phosphatase inhibitor phenyl vinyl sulfonate alone induced STAT1 phosphorylation and appeared to induce its limited cleavage. It also inhibited cross-linked envelope formation, a characteristic step of keratinocyte terminal differentiation, likely due to its reaction with the active site cysteine of keratinocyte transglutaminase. Thus, the key protein tyrosine phosphatase responsible for STAT1 dephosphorylation remains to be identified, and an off-target effect of a potential inhibitor was revealed.

Circular RNA-9119 protects IL-1β-treated chondrocytes from apoptosis in an osteoarthritis cell model by intercepting the microRNA-26a/PTEN axis

AimsOsteoarthritis (OA) is a common degenerative joint disease characterized by cartilage degeneration and joint inflammation. As its pathogenesis remains unclear, there are no effective treatments established. Circular RNA (circRNA), microRNA (miRNA), and other noncoding RNAs participate in OA development; however, the effects and mechanisms of circRNA and miRNA in OA remain unknown. Main methodsCartilage miRNA was examined in patients with and without OA. Key findingsCircRNA-9119 and phosphatase and tensin homolog (PTEN) expression decreased in OA-affected cartilage and interleukin (IL)-1β-induced chondrocytes, and miR-26a expression significantly decreased in normal cells and tissues. CircRNA-9119 overexpression restored chondrocyte growth, whereas IL-1β treatment impaired chondrocyte growth. Annexin V-FITC & PI flow cytometry and Bcl-2/Bax ratio measurement indicated that the apoptosis of IL-1β-treated articular chondrocytes was decreased by circRNA-9119 upregulation. Bioinformatic prediction and the dual-luciferase reporter assay indicated that circRNA-9119 served as a miR-26a sponge and that miR-26a targeted the 3′-UTR of PTEN. Transfection of chondrocytes with a circRNA-9119-overexpressing vector revealed downregulation of miR-26a expression. Furthermore, circRNA-9119 overexpression induced PTEN expression. In addition, a miR-26a mimic induced IL-1β-induced chondrocyte apoptosis, and circRNA-9119 overexpression inhibited IL-1β-induced chondrocyte apoptosis. SignificanceCircRNA-9119 is an important regulator of IL-1β-treated chondrocytes through the miR-26a/PTEN axis, possibly contributing to OA development.

Protopanaxadiol ginsenoside Rd protects against NMDA receptor-mediated excitotoxicity by attenuating calcineurin-regulated DAPK1 activity

Neuroprotective strategies in the treatment of stroke have been attracting a great deal of attentions. Our previous clinical and basic studies have demonstrated that protopanaxadiol ginsenoside-Rd (Rd), a monomer compound extracted from Panax ginseng or Panax notoginseng, has neuroprotective effects against ischemic stroke, probably due to its ability to block Ca2+ overload, an usual consequence of the overactivation of NMDA receptor (NMDAR). As an extending study, we explored here whether Rd exerted its neuroprotection as a novel NMDAR blocker. Our whole-cell patch-clamp results showed that Rd reduced NMDAR currents of cultured rat cortical neurons (EC50 = 7.7 μM) dose-dependently by acting on extrasynaptic NMDAR NR2b subunit. However, unexpectedly, cell transfection and radioligand binding assays revealed that Rd did not bind to the NMDAR channel directly. Alternatively, it inhibited the phosphorylation of NR2b at Ser-1303, a target of death associated protein kinase 1 (DAPK1). Moreover, cell-based and cell-free enzymatic assays showed that Rd did not inhibit the activity of DAPK1 directly, but blocked the activity of calcineurin, a key phosphatase for activating DAPK1. Importantly, other protopanaxadiol ginsenosides were also found to have potential inhibitory effects on calcineurin activity. Furthermore, as expected, calcineurin inhibition by cyclosporin A could mimic Rd’s effects and protect against NMDA-, oxygen glucose deprivation- or transient ischemic stroke-induced neuronal injury. Therefore, our present study provided the first evidence that Rd could exert an inhibitive effect on NMDAR-triggered currents and sequential excitotoxicity through mitigation of DAPK1-mediated NR2b phosphorylation by attenuating calcineurin activity.

Antagonism of PP2A is an independent and conserved function of HIV-1 Vif and causes cell cycle arrest.

The seminal description of the cellular restriction factor APOBEC3G and its antagonism by HIV-1 Vif has underpinned two decades of research on the host-virus interaction. We recently reported that HIV-1 Vif is also able to degrade the PPP2R5 family of regulatory subunits of key cellular phosphatase PP2A (PPP2R5A-E; Greenwood et al., 2016; Naamati et al., 2019). We now identify amino acid polymorphisms at positions 31 and 128 of HIV-1 Vif which selectively regulate the degradation of PPP2R5 family proteins. These residues covary across HIV-1 viruses in vivo, favouring depletion of PPP2R5A-E. Through analysis of point mutants and naturally occurring Vif variants, we further show that degradation of PPP2R5 family subunits is both necessary and sufficient for Vif-dependent G2/M cell cycle arrest. Antagonism of PP2A by HIV-1 Vif is therefore independent of APOBEC3 family proteins, and regulates cell cycle progression in HIV-infected cells.

A PP2A-B56-Centered View on Metaphase-to-Anaphase Transition in Mouse Oocyte Meiosis I.

Meiosis is required to reduce to haploid the diploid genome content of a cell, generating gametes—oocytes and sperm—with the correct number of chromosomes. To achieve this goal, two specialized cell divisions without intermediate S-phase are executed in a time-controlled manner. In mammalian female meiosis, these divisions are error-prone. Human oocytes have an exceptionally high error rate that further increases with age, with significant consequences for human fertility. To understand why errors in chromosome segregation occur at such high rates in oocytes, it is essential to understand the molecular players at work controlling these divisions. In this review, we look at the interplay of kinase and phosphatase activities at the transition from metaphase-to-anaphase for correct segregation of chromosomes. We focus on the activity of PP2A-B56, a key phosphatase for anaphase onset in both mitosis and meiosis. We start by introducing multiple roles PP2A-B56 occupies for progression through mitosis, before laying out whether or not the same principles may apply to the first meiotic division in oocytes, and describing the known meiosis-specific roles of PP2A-B56 and discrepancies with mitotic cell cycle regulation.

Transcriptomics Analysis of the Chinese Pear Pathotype of Alternaria alternata Gives Insights into Novel Mechanisms of HSAF Antifungal Activities.

Alternaria alternata (Fries) Keissler is a lethal pear pathogen that causes leaf black spot disease of pear in Southern China. Heat-stable activity factor (HSAF) is a polycyclic tetramate macrolactam (PTM) produced by Lysobacter enzymogenes and many other microbes with a broad-spectrum antifungal activity against many filamentous fungi. In this study, we evaluated the antifungal effect of HSAF against A. alternata and proposed its antifungal mechanism in A. alternata. We report that HSAF inhibited the mycelial growth of A. alternata in a dose-dependent manner. Transcriptomics analysis revealed that HSAF treatment resulted in an expression alteration of a wide range of genes, with 3729 genes being up-regulated, and 3640 genes being down-regulated. Furthermore, we observed that HSAF treatment disrupted multiple signaling networks and essential cellular metabolisms in A. alternata, including the AMPK signaling pathway, sphingolipid metabolism and signaling pathway, carbon metabolism and the TCA (tricarboxylic acid) cycle, cell cycle, nitrogen metabolism, cell wall synthesis and a key hub protein phosphatase 2A (PP2A). These observations suggest that HSAF breaches metabolism networks and ultimately induces increased thickness of the cell wall and apoptosis in A. alternata. The improved understanding of the antifungal mechanism of HSAF against filamentous fungi will aid in the future identification of the direct interaction target of HSAF and development of HSAF as a novel bio-fungicide.

Dephosphorylation of the HIV-1 restriction factor SAMHD1 is mediated by PP2A-B55\u03b1 holoenzymes during mitotic exit

SAMHD1 is a critical restriction factor for HIV-1 in non-cycling cells and its antiviral activity is regulated by T592 phosphorylation. Here, we show that SAMHD1 dephosphorylation at T592 is controlled during the cell cycle, occurring during M/G1 transition in proliferating cells. Using several complementary proteomics and biochemical approaches, we identify the phosphatase PP2A-B55α responsible for rendering SAMHD1 antivirally active. SAMHD1 is specifically targeted by PP2A-B55α holoenzymes during mitotic exit, in line with observations that PP2A-B55α is a key mitotic exit phosphatase in mammalian cells. Strikingly, as HeLa or activated primary CD4+ T cells enter the G1 phase, pronounced reduction of RT products is observed upon HIV-1 infection dependent on the presence of dephosphorylated SAMHD1. Moreover, PP2A controls SAMHD1 pT592 level in non-cycling monocyte-derived macrophages (MDMs). Thus, the PP2A-B55α holoenzyme is a key regulator to switch on the antiviral activity of SAMHD1.

The phosphatase PPM1A controls monocyte-to-macrophage differentiation

Differentiation of circulating monocytes into tissue-bound or tissue-resident macrophages is a critical regulatory process affecting host defense and inflammation. However, the regulatory signaling pathways that control the differentiation of monocytes into specific and distinct functional macrophage subsets are poorly understood. Herein, we demonstrate that monocyte-to-macrophage differentiation is controlled by the Protein Phosphatase, Mg2+/Mn2+-dependent 1A (PPM1A). Genetic manipulation experiments demonstrated that overexpression of PPM1A attenuated the macrophage differentiation program, while knockdown of PPM1A expression accelerated the ability of monocytes to differentiate into macrophages. We identify imiquimod and Pam3CSK4 as two Toll-like receptor agonists that induce PPM1A expression, and show that increased expression of PPM1A at the onset of differentiation impairs cellular adherence, reduces expression of inflammatory (M1) macrophage-specific markers, and inhibits the production of inflammatory cytokines. Our findings reveal PPM1A as a negative threshold regulator of M1-type monocyte-to-macrophage differentiation, establishing it as a key phosphatase that orchestrates this program.

Suppression of protein tyrosine phosphatase N23 predisposes to breast tumorigenesis via activation of FYN kinase.

Disruption of the balanced modulation of reversible tyrosine phosphorylation has been implicated in the etiology of various human cancers, including breast cancer. Protein Tyrosine Phosphatase N23 (PTPN23) resides in chromosomal region 3p21.3, which is hemizygously or homozygously lost in some breast cancer patients. In a loss-of-function PTPome screen, our laboratory identified PTPN23 as a suppressor of cell motility and invasion in mammary epithelial and breast cancer cells. Now, our TCGA (The Cancer Genome Atlas) database analyses illustrate a correlation between low PTPN23 expression and poor survival in breast cancers of various subtypes. Therefore, we investigated the tumor-suppressive function of PTPN23 in an orthotopic transplantation mouse model. Suppression of PTPN23 in Comma 1Dβ cells induced breast tumors within 56 wk. In PTPN23-depleted tumors, we detected hyperphosphorylation of the autophosphorylation site tyrosine in the SRC family kinase (SFK) FYN as well as Tyr142 in β-catenin. We validated the underlying mechanism of PTPN23 function in breast tumorigenesis as that of a key phosphatase that normally suppresses the activity of FYN in two different models. We demonstrated that tumor outgrowth from PTPN23-deficient BT474 cells was suppressed in a xenograft model in vivo upon treatment with AZD0530, an SFK inhibitor. Furthermore, double knockout of FYN and PTPN23 via CRISPR/CAS9 also attenuated tumor outgrowth from PTPN23 knockout Cal51 cells. Overall, this mechanistic analysis of the tumor-suppressive function of PTPN23 in breast cancer supports the identification of FYN as a therapeutic target for breast tumors with heterozygous or homozygous loss of PTPN23.