Microglia-derived TNFα induces apoptosis in neural precursor cells via transcriptional activation of the Bcl-2 family member Puma

Neuroinflammation is a common feature of acute neurological conditions such as stroke and spinal cord injury, as well as neurodegenerative conditions such as Parkinson''s disease, Alzheimer''s disease, and amyotrophic lateral sclerosis. Previous studies have demonstrated that acute neuroinflammation can adversely affect the survival of neural precursor cells (NPCs) and thereby limit the capacity for regeneration and repair. However, the mechanisms by which neuroinflammatory processes induce NPC death remain unclear. Microglia are key mediators of neuroinflammation and when activated to induce a pro-inflammatory state produce a number of factors that could affect NPC survival. Importantly, in the present study we demonstrate that tumor necrosis factor α (TNFα) produced by lipopolysaccharide-activated microglia is necessary and sufficient to trigger apoptosis in mouse NPCs in vitro. Furthermore, we demonstrate that microglia-derived TNFα induces NPC apoptosis via a mitochondrial pathway regulated by the Bcl-2 family protein Bax. BH3-only proteins are known to play a key role in regulating Bax activation and we demonstrate that microglia-derived TNFα induces the expression of the BH3-only family member Puma in NPCs via an NF-κB-dependent mechanism. Specifically, we show that NF-κB is activated in NPCs treated with conditioned media from activated microglia and that Puma induction and NPC apoptosis is blocked by the NF-κB inhibitor BAY-117082. Importantly, we have determined that NPC apoptosis induced by activated microglia-derived TNFα is attenuated in Puma-deficient NPCs, indicating that Puma induction is required for NPC death. Consistent with this, we demonstrate that Puma-deficient NPCs exhibit an ∼13-fold increase in survival as compared with wild-type NPCs following transplantation into the inflammatory environment of the injured spinal cord in vivo. In summary, we have identified a key signaling pathway that regulates neuroinflammation induced apoptosis in NPCs in vitro and in vivo that could be targeted to promote regeneration and repair in diverse neurological conditions.     

SUMOylation of the C-terminal domain of DNA topoisomerase IIα regulates the centromeric localization of Claspin

DNA topoisomerase II (TopoII) regulates DNA topology by its strand passaging reaction, which is required for genome maintenance by resolving tangled genomic DNA. In addition, TopoII contributes to the structural integrity of mitotic chromosomes and to the activation of cell cycle checkpoints in mitosis. Post-translational modification of TopoII is one of the key mechanisms by which its broad functions are regulated during mitosis. SUMOylation of TopoII is conserved in eukaryotes and plays a critical role in chromosome segregation. Using Xenopus laevis egg extract, we demonstrated previously that TopoIIα is modified by SUMO on mitotic chromosomes and that its activity is modulated via SUMOylation of its lysine at 660. However, both biochemical and genetic analyses indicated that TopoII has multiple SUMOylation sites in addition to Lys660, and the functions of the other SUMOylation sites were not clearly determined. In this study, we identified the SUMOylation sites on the C-terminal domain (CTD) of TopoIIα. CTD SUMOylation did not affect TopoIIα activity, indicating that its function is distinct from that of Lys660 SUMOylation. We found that CTD SUMOylation promotes protein binding and that Claspin, a well-established cell cycle checkpoint mediator, is one of the SUMOylation-dependent binding proteins. Claspin harbors 2 SUMO-interacting motifs (SIMs), and its robust association to mitotic chromosomes requires both the SIMs and TopoIIα-CTD SUMOylation. Claspin localizes to the mitotic centromeres depending on mitotic SUMOylation, suggesting that TopoIIα-CTD SUMOylation regulates the centromeric localization of Claspin. Our findings provide a novel mechanistic insight regarding how TopoIIα-CTD SUMOylation contributes to mitotic centromere activity.     

Thermodynamic analysis of protein kinase A Iα activation

Thermodynamic analysis of protein kinase A (PKA) Iα activation was performed using Quantum 3.3.0 docking software and a Gaussian 03W quantum mechanical computational package. Expected stacking interactions between adenine of 3′:5′-AMP and aromatic moieties of amino acids were taken into account by means of MP2/6-31G(d) IPCM (iso-density polarizable continuum model) computations (ɛ = 4.0). It is demonstrated that thermodynamically favorable agonist-induced PKA Iα activation is mediated by two processes. First, 3′:5′-AMP binding is accompanied by structural changes leading to a thermodynamically favorable regulatory subunit conformation, which is hardly realized in the absence of the ligand (ΔG_R^o = −23.9 ± 8.2 kJ/mol). Second, 3′:5′-AMP affinity to the regulatory subunit conformation observed after agonist-induced PKA Iα activation is higher than that to inactive holoenzyme complex (ΔG_{3′:5′−AMP}^o = −28.1 ± 9.7 kJ/mol). ATP is capable of docking into the 3′:5′-AMP-binding site B of the regulatory subunit complexed with the catalytic one, resulting in inhibition of kinase activation. True constants of 3′:5′-AMP binding to PKA Iα holoenzyme were found to be 60 and 57 μM for the regulatory subunit domains A and B, respectively. These constants, unlike the binding equilibrium constant determined using established experimental techniques and ranging from 15 nM to 2.9 μM, are proved to be direct measures of 3′:5′-AMP-PKA Iα binding affinity. Their values are in a reasonable agreement with the changes in 3′:5′-AMP concentration in the cell (2-55 μM) and account for PKA Iα activation in response to adequate stimuli.     

Protein kinase C-α activation promotes recovery of mitochondrial function and cell survival following oxidant injury in renal cells

We demonstrated that nonselective PKC activation promotes mitochondrial function in renal proximal tubular cells (RPTC) following toxicant injury. However, the specific PKC isozyme mediating this effect is unknown. This study investigated the role of PKC-α in the recovery of mitochondrial functions in oxidant-injured RPTC. Wild-type PKC-α (wtPKC-α) and inactive PKC-α mutants were overexpressed in RPTC to selectively increase or block PKC-α activation. Oxidant (tert-butyl hydroperoxidel; TBHP) exposure activated PKC-α in RPTC but decreased PKC-α levels in mitochondria following treatment. Uncoupled and state 3 respirations and activities of complexes I and IV in TBHP-injured cells decreased to 55, 44, 49, and 65% of controls, respectively. F(0)F(1)-ATPase activity and ATP content in injured RPTC decreased to 59 and 60% of controls, respectively. Oxidant exposure increased reactive oxygen species (ROS) production by 210% and induced mitochondrial fragmentation and 52% RPTC lysis. Overexpressing wtPKC-α did not block TBHP-induced ROS production but improved respiration and complex I activity, restored complex IV and F(0)F(1)-ATPase activities, promoted recovery of ATP content, blocked mitochondrial fragmentation, and reduced RPTC lysis to 14%. In contrast, inhibiting PKC-α 1) induced mitochondrial hyperpolarization and fragmentation; 2) blocked increases in ROS production; 3) prevented recovery of respiratory complexes and F(0)F(1)-ATPase activities, respiration, and ATP content; and 4) exacerbated TBHP-induced RPTC lysis. We conclude that 1) activation of PKC-α prevents mitochondrial hyperpolarization and fragmentation, decreases cell death, and promotes recovery of mitochondrial respiration and ATP content following oxidant injury in RPTC; and 2) respiratory complexes I and IV and F(0)F(1)-ATPase are targets of active PKC-α.     

Extra cytoplasmic function σ factor activation

The bacterial cell envelope is essential for cell viability and is a target for numerous antibiotics and host immune defenses. Thus bacteria must sense and respond to damage to the cell envelope. Many bacteria utilize alternative σ factors such as extracytoplasmic function (ECF) σ factors to respond to cell envelope stress. Although ECF σ factors are utilized by both Gram negative and Gram positive bacteria to respond to cell envelope stress, the mechanisms of sensing differ. In this review, we examine the events and proteins that are required for activation of two model extracytoplasmic function σ factors, σ(E) in E. coli and σ(W) in B. subtilis.     

Development of an HTS-compatible assay for discovery of RORα modulators using AlphaScreen® technology

The retinoid acid receptor-related orphan receptors (RORs) represent important targets for the treatment of metabolic and immune disorders. Here the authors describe the application of AlphaScreen(?) technology to develop a high-throughput screening (HTS)-compatible assay to facilitate the discovery of RORα modulators. Using the ligand binding domain (LBD) of RORα and a peptide derived from the NR1 box of the nuclear receptor coactivator PGC-1α, a 384-well format assay was developed exhibiting high sensitivity, requiring only low nanomolar concentration of reagents. Recently, it was shown that oxysterols such as 7α-hydroxycholesterol (7α-OHC) function as modulators of the RORs. In this assay, 7α-OHC produced a concentration-response curve with an EC(50) of 162 nM, a Z' factor of 0.6, and a signal-to-background (S/B) ratio of 4.2, demonstrating that the assay is HTS compatible. Validation of the assay was afforded by screening against the Sigma LOPAC1280? library in a 384-well format. In summary, the results presented here demonstrate that this assay can be used to screen large chemical libraries to discover novel modulators of RORα.