The Type I NADH Dehydrogenase of Mycobacterium tuberculosis Counters Phagosomal NOX2 Activity to Inhibit TNF-α-Mediated Host Cell Apoptosis

The capacity of infected cells to undergo apoptosis upon insult with a pathogen is an ancient innate immune defense mechanism. Consequently, the ability of persisting, intracellular pathogens such as the human pathogen Mycobacterium tuberculosis (Mtb) to inhibit infection-induced apoptosis of macrophages is important for virulence. The nuoG gene of Mtb, which encodes the NuoG subunit of the type I NADH dehydrogenase, NDH-1, is important in Mtb-mediated inhibition of host macrophage apoptosis, but the molecular mechanism of this host pathogen interaction remains elusive. Here we show that the apoptogenic phenotype of MtbΔnuoG was significantly reduced in human macrophages treated with caspase-3 and -8 inhibitors, TNF-α-neutralizing antibodies, and also after infection of murine TNF^−/− macrophages. Interestingly, incubation of macrophages with inhibitors of reactive oxygen species (ROS) reduced not only the apoptosis induced by the nuoG mutant, but also its capacity to increase macrophage TNF-α secretion. The MtbΔnuoG phagosomes showed increased ROS levels compared to Mtb phagosomes in primary murine and human alveolar macrophages. The increase in MtbΔnuoG induced ROS and apoptosis was abolished in NOX-2 deficient (gp91^−/−) macrophages. These results suggest that Mtb, via a NuoG-dependent mechanism, can neutralize NOX2-derived ROS in order to inhibit TNF-α-mediated host cell apoptosis. Consistently, an Mtb mutant deficient in secreted catalase induced increases in phagosomal ROS and host cell apoptosis, both of which were dependent upon macrophage NOX-2 activity. In conclusion, these results serendipitously reveal a novel connection between NOX2 activity, phagosomal ROS, and TNF-α signaling during infection-induced apoptosis in macrophages. Furthermore, our study reveals a novel function of NOX2 activity in innate immunity beyond the initial respiratory burst, which is the sensing of persistent intracellular pathogens and subsequent induction of host cell apoptosis as a second line of defense.     

Metabolic Regulator βKlotho Interacts with Fibroblast Growth Factor Receptor 4 (FGFR4) to Induce Apoptosis and Inhibit Tumor Cell Proliferation

In organs involved in metabolic homeostasis, transmembrane α and βklothos direct FGFR signaling to control of metabolic pathways. Coordinate expression of βklotho and FGFR4 is a property of mature hepatocytes. Genetic deletion of FGFR4 or βklotho in mice disrupts hepatic cholesterol/bile acid and lipid metabolism. The deletion of FGFR4 has no effect on the proliferative response of hepatocytes after liver injury. However, its absence results in accelerated progression of dimethynitrosamine-initiated hepatocellular carcinomas, indicating that FGFR4 suppresses hepatoma proliferation. The mechanism underlying the FGFR4-mediated hepatoma suppression has not been addressed. Here we show that βklotho expression is more consistently down-regulated in human and mouse hepatomas than FGFR4. Co-expression and activation by either endocrine FGF19 or cellular FGF1 of the FGFR4 kinase in a complex with βklotho restricts cell population growth through induction of apoptotic cell death in both hepatic and nonhepatic cells. The βklotho-FGFR4 partnership caused a depression of activated AKT and mammalian target of rapamycin while activating ERK1/2 that may underlie the pro-apoptotic effect. Our results show that βklotho not only interacts with heparan sulfate-FGFR4 to form a complex with high affinity for endocrine FGF19 but also impacts the quality of downstream signaling and biological end points activated by either FGF19 or canonical FGF1. Thus the same βklotho-heparan sulfate-FGFR4 partnership that mediates endocrine control of hepatic metabolism plays a role in cellular homeostasis and hepatoma suppression through negative control of cell population growth mediated by pro-apoptotic signaling.     

Junctional Music that the Nucleus Hears: Cell–Cell Contact Signaling and the Modulation of Gene Activity

Cell–cell junctions continue to capture the interest of cell and developmental biologists, with an emerging area being the molecular means by which junctional signals relate to gene activity in the nucleus. Although complexities often arise in determining the direct versus indirect nature of such signal transduction, it is clear that such pathways are essential for the function of tissues and that alterations may contribute to many pathological outcomes. This review assesses a variety of cell–cell junction-to-nuclear signaling pathways, and outlines interesting areas for further study.The evolution of multicellular life forms has to a significant extent involved refinements of each cell''s capacity to sense the state of its directly contacting neighbors. This exchange of information often occurs within tissues, with the result that gene activity in the nucleus is altered or maintained accordingly. In this article, we focus on how signals arise at cell–cell junctions and are transduced to the nucleus; we do not include discussion of mechanical/cytoskeletal signals influencing nuclear decisions, and the reader is directed to a recent review of this topic (Ingber 2008).An issue that arises when addressing cell–cell junction(s), referred to as CCJ(s), -to-nuclear signals, is that homotypic or heterotypic junctional proteins responsible for conferring adhesive activity are often in a much larger complex of proteins. These interactions may be either in cis (interacting within the plasma membrane of the cell) or trans orientations (interacting through ectodomain contacts extended between cells). Most of these transmembrane proteins are likely to have the potential to contribute to downstream signaling events, and many may associate with one another only under specific physiological conditions. For example, certain receptor tyrosine kinases (RTKs) associate with particular cadherins, and when associated are relevant to that cadherin''s functions (Wheelock and Johnson 2003; Andl and Rustgi 2005). In this article, we discuss relationships such as these in the context of CCJ-nuclear signaling. A topic not represented here is the CCJ signaling of immune surveillance cells, for example, pathways activated following leukocyte–endothelia contact. This area is of great basic and biomedical interest, but is addressed elsewhere (Dustin 2007).We focus on signaling by a select number of junction types, including adherens, desmosomal, and tight junctions, and to a lesser extent, gap junctions. Details of the structure and function of each of these junctions are presented in other articles (see Meng and Takeichi 2009, Delva et al. 2009, Furuse 2009, and Goodenough and Paul 2009, respectively). These junctions are often represented in textbooks as distinct entities in the context of epithelial tissues, but their structures and how they respond to or generate signaling cues vary according to cellular context. Select components within these junctions may be shared, for example between desmosomal, adherens, and tight junctions, and in some instances, intimate physical proximities are likely to advance these junctions'' functional interrelation. Further, different cell types show less common junctional organizations (Straub et al. 2003; Wuchter et al. 2007), such that the total spectrum of CCJ signals is likely to be impressive, and far beyond what is currently known or understood. Given the interdependence of cell neighbors in forming and maintaining cell groupings, high diversity and sophistication arose in complex organisms, both in CCJ structures themselves and their associated nuclear signaling pathways. Compared with the knowledge accumulated over the past two decades on cell–extracellular matrix signaling via integrins (Abram and Lowell 2009), we know less about signals initiated from forming or mature cell–cell contacts in epithelial, neural, or endothelial tissues. Thus, as the field moves forward, there is the potential to achieve a deepened understanding of how the cell–extracellular matrix and cell–cell adhesion systems are coupled in a signaling context, and how they collectively relate to the adhesion, motility, and differentiation of cells and tissues.     

NSC666715 and Its Analogs Inhibit Strand-Displacement Activity of DNA Polymerase β and Potentiate Temozolomide-Induced DNA Damage,Senescence and Apoptosis in Colorectal Cancer Cells

Recently approved chemotherapeutic agents to treat colorectal cancer (CRC) have made some impact; however, there is an urgent need for newer targeted agents and strategies to circumvent CRC growth and metastasis. CRC frequently exhibits natural resistance to chemotherapy and those who do respond initially later acquire drug resistance. A mechanism to potentially sensitize CRC cells is by blocking the DNA polymerase β (Pol-β) activity. Temozolomide (TMZ), an alkylating agent, and other DNA-interacting agents exert DNA damage primarily repaired by a Pol-β-directed base excision repair (BER) pathway. In previous studies, we used structure-based molecular docking of Pol-β and identified a potent small molecule inhibitor (NSC666715). In the present study, we have determined the mechanism by which NSC666715 and its analogs block Fen1-induced strand-displacement activity of Pol-β-directed LP-BER, cause apurinic/apyrimidinic (AP) site accumulation and induce S-phase cell cycle arrest. Induction of S-phase cell cycle arrest leads to senescence and apoptosis of CRC cells through the p53/p21 pathway. Our initial findings also show a 10-fold reduction of the IC₅₀ of TMZ when combined with NSC666715. These results provide a guide for the development of a target-defined strategy for CRC chemotherapy that will be based on the mechanisms of action of NSC666715 and TMZ. This combination strategy can be used as a framework to further reduce the TMZ dosages and resistance in CRC patients.     

Are Rod Outer Segment ATP-ase and ATP-Synthase Activity Expression of the Same Protein?

Vertebrate retinal rod outer segments (OS) consist of a stack of disks surrounded by the plasma membrane, where phototransduction takes place. Energetic metabolism in rod OS remains obscure. Literature described a so-called Mg²⁺-dependent ATPase activity, while our previous results demonstrated the presence of oxidative phosphorylation (OXPHOS) in OS, sustained by an ATP synthetic activity. Here we propose that the OS ATPase and ATP synthase are the expression of the same protein, i.e., of F₁F_o-ATP synthase. Imaging on bovine retinal sections showed that some OXPHOS proteins are expressed in the OS. Biochemical data on bovine purified rod OS, characterized for purity, show an ATP synthase activity, inhibited by classical F₁F_o-ATP synthase inhibitors. Moreover, OS possess a pH-dependent ATP hydrolysis, inhibited by pH values below 7, suggestive of the functioning of the inhibitor of F1 (IF1) protein. WB confirmed the presence of IF1 in OS, substantiating the expression of F₁F_o ATP synthase in OS. Data suggest that the OS F₁Fo ATP synthase is able to hydrolyze or synthesize ATP, depending on in vitro or in vivo conditions and that the role of IF1 would be pivotal in the prevention of the reversal of ATP synthase in OS, for example during hypoxia, granting photoreceptor survival.     

Antiproliferative Activity and Mechanism of Action of Fatty Acid Derivatives of Arabinosylcytosine (ara‐C) in Leukemia and Solid Tumor Cell Lines

Resistance to, the hydrophilic drug ara‐C, might be meditated by decreased membrane transport. Lipophillic prodrugs were synthesized to facilitate uptake. These compounds were equally active as ara‐C, while the compounds with the shortest fatty‐acid group and highest number of double bonds were the more active. These compounds also show a better retention profile, their effect is retained longer than for ara‐C.     

Mutant p53 Promotes Tumor Cell Malignancy by Both Positive and Negative Regulation of the Transforming Growth Factor β (TGF-β) Pathway

Specific p53 mutations abrogate tumor-suppressive functions by gaining new abilities to promote tumorigenesis. Inactivation of p53 is known to distort TGF-β signaling, which paradoxically displays both tumor-suppressive and pro-oncogenic functions. The molecular mechanisms of how mutant p53 simultaneously antagonizes the tumor-suppressive and synergizes the tumor-promoting function of the TGF-β pathway remain elusive. Here we demonstrate that mutant p53 differentially regulates subsets of TGF-β target genes by enhanced binding to the MH2 domain in Smad3 upon the integration of ERK signaling, therefore disrupting Smad3/Smad4 complex formation. Silencing Smad2, inhibition of ERK, or introducing a phosphorylation-defective mutation at Ser-392 in p53 abrogates the R175H mutant p53-dependent regulation of these TGF-β target genes. Our study shows a mechanism to reconcile the seemingly contradictory observations that mutant p53 can both attenuate and cooperate with the TGF-β pathway to promote cancer cell malignancy in the same cell type.     

Physicochemical Properties and Biological Activity of the Peptide–Protein Complex from Bovine Scleral Tissue

The peptide–protein complex from bovine sclera was studied. It is shown that it contained a protein with a molecular weight of 66387 Da with the partial N-terminal amino acid sequence DTHKSEIAHRFKDLG-, which is homologous to the mature molecule of bovine serum albumin, and polypeptides with molecular weights of 1300–5080 Da. With a model of the organotypic cultivation of posterior eye tissues of the newt Pleurodeles waltl in vitro, it was shown that the effect of this peptide–protein complex in low doses increased the viability of scleral fibroblasts.     

Models of Electrical Activity: Calibration and Prediction Testing on?the?Same Cell

Mathematical models are increasingly important in biology, and testability is becoming a critical issue. One limitation is that one model simulation tests a parameter set representing one instance of the biological counterpart, whereas biological systems are heterogeneous in their properties and behavior, and a model often is fitted to represent an ideal average. This is also true for models of a cell’s electrical activity; even within a narrowly defined population there can be considerable variation in electrophysiological phenotype. Here, we describe a computational experimental approach for parameterizing a model of the electrical activity of a cell in real time. We combine the inexpensive parallel computational power of a programmable graphics processing unit with the flexibility of the dynamic clamp method. The approach involves 1), recording a cell’s electrical activity, 2), parameterizing a model to the recording, 3), generating predictions, and 4), testing the predictions on the same cell used for the calibration. We demonstrate the experimental feasibility of our approach using a cell line (GH4C1). These cells are electrically active, and they display tonic spiking or bursting. We use our approach to predict parameter changes that can convert one pattern to the other.     

Comparison Between the Influences of Phosphatidylcholine and Triton X-100 on the Protein Secondary Structures and Oxygen-evolving Activity of Photosystem ￠ò

The structural and functional alterations within the PSⅡ membrane from phosphatidylcholine reconstitution and Triton X-100 (TX-100) treatment were studied by using Fourier transform-infrared (FT-IR) spectroscopic technique and oxygen electrode. Phosphatidylcholine reconstitution showed no significant effect on the protein secondary structures of PSⅡ membrane but an increase of the rate of PSⅡ-mediated oxygen-evolution. The phosphatidylcholine lipids with different length of acyl chains displayed different capabilities to stimulate oxygen-evolution. In contrast, perturbation of the bilayer lipids by TX-100 resulted in obvious changes of the protein secondary structures within the PSⅡ membrane and in the loss of the PSⅡ-mediated oxygen-evolving activity. The results indicate the importance of membrane integrity in maintaining the stability of the photosynthetic membrane proteins.     

Regulation of Prostate Cancer Cell Survival by Protein Kinase C? Involves Bad Phosphorylation and Modulation of the TNFα/JNK Pathway

Protein kinase Cϵ (PKCϵ), a diacyglycerol- and phorbol ester-responsive serine-threonine kinase, has been implicated in mitogenic and survival control, and it is markedly overexpressed in human tumors, including in prostate cancer. Although prostate cancer cells undergo apoptosis in response to phorbol ester stimulation via PKCδ-mediated release of death factors, the involvement of PKCϵ in this response is not known. PKCϵ depletion by RNAi or expression of a dominant negative kinase-dead PKCϵ mutant potentiated the apoptotic response of PMA and sensitized LNCaP cells to the death receptor ligand TNFα. On the other hand, overexpression of PKCϵ by adenoviral means protected LNCaP cells against apoptotic stimuli. Interestingly, PKCϵ RNAi depletion significantly enhanced the release of TNFα in response to PMA and greatly potentiated JNK activation by this cytokine. Further mechanistic analysis revealed that PMA fails to promote phosphorylation of Bad in Ser¹¹² in PKCϵ-depleted LNCaP cells, whereas PKCϵ overexpression greatly enhanced Bad phosphorylation. This effect was independent of Akt, ERK, or p90Rsk, well established kinases for Ser¹¹² in Bad. Moreover, expression of a S112A-Bad mutant potentiated PMA-induced apoptosis. Finally, we found that upon activation PKCϵ accumulated in mitochondrial fractions in LNCaP cells and that Bad was a substrate of PKCϵ in vitro. Our results established that PKCϵ modulates survival in prostate cancer cells via multiple pathways.