PI3K and PTEN Regulatory Modules

<div><p>(A) Elements controlling the activity of PI3K and upstream components. In addition to the basic, “excitatory,” signaling elements like the cAMP receptor, Gβγ, and Ras, we introduced further elements controlling the activity of PI3K and upstream components. “PI3Ktp” (module 1) stands for a tyrosine phosphatase that deactivates PI3K. This phosphatase becomes enzymatically activated and is recruited to the membrane after interaction with Gβγ. “RasGAP” (module 2) translocates to the membrane and deactivates Ras after activation by Gβγ. RAK blocks Gβγ, Gα, and the receptor, thereby reducing all signals (module 3).</p><p>(B) Elements controlling the activity and localization of PTEN. In our model, PTEN is phosphorylated by a Src-like kinase, here simply called “Src” (module 1). Src is activated by Gα and deactivated by Csk, which in turn is recruited by phosphoPaxillin (“pPaxillin”) (module 2). SHP2, which is membrane recruited by pGab1 bound to PIP₃, dephosphorylates pPaxillin (module 3), thereby leading to increased activation of PTEN.</p></div

Correspondence in Time and Space between the Predicted and Measured Changes in PIP₃ at the Front and Back of Cells Exposed cAMP Gradients

<p>Experimental data from exposure of <i>Dictyostelium</i> to a 2:1 gradient with a mean cAMP concentration of 100 nmol were used to adjust model parameters. The other two responses are predictions of the model. (A), (B), and (C) are simulated responses. (D), (E), and (F) are experimental measurements, using PH-GFP to monitor PIP₃ levels in single cells exposed to gradients with a mean cAMP concentration of 1 μmol, 100 nmol, and 10 nmol. See <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020082#pcbi-0020082-sg006" target="_blank">Figure S6</a> for details on the full dataset of experimental replicates.</p

Standard Model of the Extracellular and Intracellular Distribution of Key Components of <i>Dictyostelium</i> Chemotactic Signaling

<div><p>(A) In an unstimulated cell PTEN is homogeneously distributed at the membrane. The cell membrane contains very little PIP₃.</p><p>(B) Stimulation of the cell leads to the membrane recruitment and activation of PI3K, as indicated by the arrows (1) leading from inactive, mainly cytosolic PI3K (yellow) to membrane-proximal, active PI3K (orange). Activated PI3K transforms PIP₂ into PIP₃. PTEN is deactivated following cAMP stimulation and leaves the membrane. This process is indicated by arrows (2) connecting active PTEN (dark green) and the mainly cytosolic inactive PTEN (light green). Regulatory processes lead to reactivation of PTEN (3). Differences in the speed and degree of cAMP receptor ligation between front and back of the cell lead to preferential accumulation of PTEN at the back of the cell. As a result, the front experiences a higher concentration of PI3K and a lower concentration of PTEN than the back and accumulates PIP₃. Note: To emphasize the changes in PIP₃ content, the amount of PIP₃ relative to that of PIP₂ has been overstated. Even after cAMP stimulation, the actual amount of PIP₂ will be much higher than that of PIP₃.</p></div

Comparison of the Simulated Activities of PI3K, Membrane-Bound PTEN, and the Resulting Behavior of PIP₃ (Composite Screenshot)

<p>Stimulation of a cell in a 2:1 cAMP gradient (mean concentration 500 nmol) leads to a rapid 3-fold increase in the membrane proximal activity of PI3K (green) and to a loss of membrane-bound PTEN (blue; tracked as GFP-PTEN in real cells). This results in a rapid accumulation of PIP₃ (red; reported by the location of PH-GFP in real cells). Subsequently, the PI3K activity is strongly quenched by the recruitment of regulatory components to the membrane and falls below its prestimulus level in less than 20 s. PTEN returns more slowly to the membrane. During the phase of downregulation of PI3K activity and reattachment of PTEN to the membrane, the concentration of PIP₃ decays. In the front of the cell (which experiences a high cAMP concentration), membrane-associated PTEN only returns to a fraction of its prestimulus level and then enters a second phase of decline. After approximately 50 s, the low level of membrane-bound PTEN that is reached in the front of the cell allows PIP₃ to increase again, even though the amount of active PI3K in this region is modest. In the back of the cell (low cAMP concentration), membrane-bound PTEN increases beyond its prestimulus level, resulting in a decrease of PIP₃ below its resting state concentration. The circular inset shows a two-dimensional representation of the dynamics of membrane-bound PTEN and PIP₃ in different regions of the three-dimensional simulation of a cell.</p

Correspondence in Time and Space between the Predicted and Measured Changes in Membrane-Bound PTEN at the Front and Back of the Cells Exposed to cAMP Gradients

<p>These dose-dependent dynamics of PTEN were produced by the simulation after the dynamics of PIP₃ for one cAMP concentration (100 nM) had been used to adjust model parameters. (A), (B), and (C) are simulated responses. (D), (E), and (F) are experimental measurements based on GFP-PTEN analysis in single cells exposed to gradients with a mean cAMP concentration of 1 μmol, 100 nmol, and 10 nmol. See <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020082#pcbi-0020082-sg007" target="_blank">Figure S7</a> for details on the full dataset of experimental replicates.</p

Table_1_Human adenovirus infection induces pulmonary inflammatory damage by triggering noncanonical inflammasomes activation and macrophage pyroptosis.docx

IntroductionHuman adenovirus (HAdV) is a common respiratory virus, which can lead to severe pneumonia in children and immunocompromised persons, and canonical inflammasomes are reported to be involved in anti-HAdV defense. However, whether HAdV induced noncanonical inflammasome activation has not been explored. This study aims to explore the broad roles of noncanonical inflammasomes during HAdV infection to investigate the regulatory mechanism of HAdV-induced pulmonary inflammatory damage. MethodsWe mined available data on GEO database and collected clinical samples from adenovirus pneumonia pediatric patients to investigate the expression of noncanonical inflammasome and its clinical relevance. An in vitro cell model was employed to investigate the roles of noncanonical inflammasomes in macrophages in response to HAdV infection.ResultsBioinformatics analysis showed that inflammasome-related genes, including caspase-4 and caspase-5, were enriched in adenovirus pneumonia. Moreover, caspase-4 and caspase-5 expression levels were significantly increased in the cells isolated from peripheral blood and broncho-alveolar lavage fluid (BALF) of pediatric patients with adenovirus pneumonia, and positively correlated with clinical parameters of inflammatory damage. In vitro experiments revealed that HAdV infection promoted caspase-4/5 expression, activation and pyroptosis in differentiated THP-1 (dTHP-1) human macrophages via NF-κB, rather than STING signaling pathway. Interestingly, silencing of caspase-4 and caspase-5 in dTHP-1 cells suppressed HAdV-induced noncanonical inflammasome activation and macrophage pyroptosis, and dramatically decreased the HAdV titer in cell supernatants, by influencing virus release rather than other stages of virus life cycle.DiscussionIn conclusion, our study demonstrated that HAdV infection induced macrophage pyroptosis by triggering noncanonical inflammasome activation via a NF-kB-dependent manner, which may explore new perspectives on the pathogenesis of HAdV-induced inflammatory damage. And high expression levels of caspase-4 and caspase-5 may be a biomarker for predicting the severity of adenovirus pneumonia.</p

Image_1_Human adenovirus infection induces pulmonary inflammatory damage by triggering noncanonical inflammasomes activation and macrophage pyroptosis.pdf

IntroductionHuman adenovirus (HAdV) is a common respiratory virus, which can lead to severe pneumonia in children and immunocompromised persons, and canonical inflammasomes are reported to be involved in anti-HAdV defense. However, whether HAdV induced noncanonical inflammasome activation has not been explored. This study aims to explore the broad roles of noncanonical inflammasomes during HAdV infection to investigate the regulatory mechanism of HAdV-induced pulmonary inflammatory damage. MethodsWe mined available data on GEO database and collected clinical samples from adenovirus pneumonia pediatric patients to investigate the expression of noncanonical inflammasome and its clinical relevance. An in vitro cell model was employed to investigate the roles of noncanonical inflammasomes in macrophages in response to HAdV infection.ResultsBioinformatics analysis showed that inflammasome-related genes, including caspase-4 and caspase-5, were enriched in adenovirus pneumonia. Moreover, caspase-4 and caspase-5 expression levels were significantly increased in the cells isolated from peripheral blood and broncho-alveolar lavage fluid (BALF) of pediatric patients with adenovirus pneumonia, and positively correlated with clinical parameters of inflammatory damage. In vitro experiments revealed that HAdV infection promoted caspase-4/5 expression, activation and pyroptosis in differentiated THP-1 (dTHP-1) human macrophages via NF-κB, rather than STING signaling pathway. Interestingly, silencing of caspase-4 and caspase-5 in dTHP-1 cells suppressed HAdV-induced noncanonical inflammasome activation and macrophage pyroptosis, and dramatically decreased the HAdV titer in cell supernatants, by influencing virus release rather than other stages of virus life cycle.DiscussionIn conclusion, our study demonstrated that HAdV infection induced macrophage pyroptosis by triggering noncanonical inflammasome activation via a NF-kB-dependent manner, which may explore new perspectives on the pathogenesis of HAdV-induced inflammatory damage. And high expression levels of caspase-4 and caspase-5 may be a biomarker for predicting the severity of adenovirus pneumonia.</p

DataSheet_2_C2GAP2 is a common regulator of Ras signaling for chemotaxis, phagocytosis, and macropinocytosis.docx

Phagocytosis, macropinocytosis, and G protein coupled receptor-mediated chemotaxis are Ras-regulated and actin-driven processes. The common regulator for Ras activity in these three processes remains unknown. Here, we show that C2GAP2, a Ras GTPase activating protein, highly expressed in the vegetative growth state in model organism Dictyostelium. C2GAP2 localizes at the leading edge of chemotaxing cells, phagosomes during phagocytosis, and macropinosomes during micropinocytosis. c2gapB− cells lacking C2GAP2 displayed increased Ras activation upon folic acid stimulation and subsequent impaired chemotaxis in the folic acid gradient. In addition, c2gaB- cells have elevated phagocytosis and macropinocytosis, which subsequently results in faster cell growth. C2GAP2 binds multiple phospholipids on the plasma membrane and the membrane recruitment of C2GAP2 requires calcium. Taken together, we show a shared negative regulator of Ras signaling that mediates Ras signaling for chemotaxis, phagocytosis, and macropinocytosis.</p

DataSheet_1_C2GAP2 is a common regulator of Ras signaling for chemotaxis, phagocytosis, and macropinocytosis.pdf

Phagocytosis, macropinocytosis, and G protein coupled receptor-mediated chemotaxis are Ras-regulated and actin-driven processes. The common regulator for Ras activity in these three processes remains unknown. Here, we show that C2GAP2, a Ras GTPase activating protein, highly expressed in the vegetative growth state in model organism Dictyostelium. C2GAP2 localizes at the leading edge of chemotaxing cells, phagosomes during phagocytosis, and macropinosomes during micropinocytosis. c2gapB− cells lacking C2GAP2 displayed increased Ras activation upon folic acid stimulation and subsequent impaired chemotaxis in the folic acid gradient. In addition, c2gaB- cells have elevated phagocytosis and macropinocytosis, which subsequently results in faster cell growth. C2GAP2 binds multiple phospholipids on the plasma membrane and the membrane recruitment of C2GAP2 requires calcium. Taken together, we show a shared negative regulator of Ras signaling that mediates Ras signaling for chemotaxis, phagocytosis, and macropinocytosis.</p

Comparison of SFTS IgG in different occupational groups of healthy volunteers in Laizhou.

<p>Comparison of SFTS IgG in different occupational groups of healthy volunteers in Laizhou.</p