microRNA‐independent recruitment of Argonaute 1 to nanos mRNA through the Smaug RNA‐binding protein

Argonaute (Ago) proteins are typically recruited to target messenger RNAs via an associated small RNA such as a microRNA (miRNA). Here, we describe a new mechanism of Ago recruitment through the Drosophila Smaug RNA‐binding protein. We show that Smaug interacts with the Ago1 protein, and that Ago1 interacts with and is required for the translational repression of the Smaug target, nanos mRNA. The Ago1/nanos mRNA interaction does not require a miRNA, but it does require Smaug. Taken together, our data suggest a model whereby Smaug directly recruits Ago1 to nanos mRNA in a miRNA‐independent manner, thereby repressing translation.     

Oxysterol‐binding protein recruitment and activity at the endoplasmic reticulum‐Golgi interface are independent of Sac1

Oxysterol‐binding protein (OSBP) localizes to endoplasmic reticulum (ER)‐Golgi contact sites where it transports cholesterol and phosphatidylinositol 4‐phosphate (PI‐4P), and activates lipid transport and biosynthetic activities. The PI‐4P phosphatase Sac1 cycles between the ER and Golgi apparatus where it potentially regulates OSBP activity. Here we examined whether the ER‐Golgi distribution of endogenous or ectopically expressed Sac1 influences OSBP activity. OSBP and Sac1 co‐localized at apparent ER‐Golgi contact sites in response to 25‐hydroxycholesterol (25OH), cholesterol depletion and p38 MAPK inhibitors. A Sac1 mutant that is unable to exit the ER did not localize with OSBP, suggesting that sterol perturbations cause Sac1 transport to the Golgi apparatus. Ectopic expression of Sac1 in the ER or Golgi apparatus, or Sac1 silencing, did not affect OSBP localization to ER‐Golgi contact sites, OSBP‐dependent activation of sphingomyelin synthesis, or cholesterol esterification in the ER. p38 MAPK inhibition and retention of Sac1 in the Golgi apparatus also caused OSBP phosphorylation and OSBP‐dependent activation of sphingomyelin synthesis at ER‐Golgi contacts. These results demonstrate that Sac1 expression in either the ER or Golgi apparatus has a minimal impact on the PI‐4P that regulates OSBP activity or recruitment to contact sites.      

Inhibition of NF‐κB activity by HIV‐1 Vpr is dependent on Vpr binding protein

Numerous studies have reported that Vpr alters NF‐κB signaling in various cell types, however, the findings have been largely conflicting with reports of both stimulatory and inhibitory effects of Vpr. Our aim was to investigate the role of Vpr signaling in myeloid cells using an adenovirus based expression and indicator system. Our results show that Vpr is inhibitory to NF‐κB, however, this effect is dependent on the particular manner of NF‐κB stimulation. Consistent with this notion, we report that Vpr has inhibitory effects that are specific to the TNF‐α pathway, but not affecting the LPS pathway, suggesting that differential targets of Vpr may exist for NF‐κB regulation. Further, we identify VprBP as one possible cellular component of Vpr's regulation of IκBα in response to TNF‐α stimulation. We did not identify such a role for HSP27, which instead seems to inhibit Vpr functions. Chronically HIV‐1 infected U1 cells with knockdown constructs for Vpr were unexpectedly less responsive to TNF‐α mediated viral replication, perhaps suggesting that other HIV‐1 components may antagonize these anti‐NF‐κB effects in infected cells. We hypothesize that Vpr may serve an important role in the context of viral infection and immune function in vivo, through its selective inhibition of NF‐κB pathways. J. Cell. Physiol. 228: 781–790, 2013. © 2012 Wiley Periodicals, Inc.     

Inhibition of viral protein translation by indomethacin in vesicular stomatitis virus infection: role of eIF2α kinase PKR

Indomethacin, a cyclooxygenase‐1 and ‐2 inhibitor widely used in the clinic for its potent anti‐inflammatory/analgesic properties, possesses antiviral activity against several viral pathogens; however, the mechanism of antiviral action remains elusive. We have recently shown that indomethacin activates the double‐stranded RNA (dsRNA)‐dependent protein kinase R (PKR) in human colon cancer cells. Because of the important role of PKR in the cellular defence response against viral infection, herein we investigated the effect of indomethacin on PKR activity during infection with the prototype rhabdovirus vesicular stomatitis virus. Indomethacin was found to activate PKR in an interferon‐ and dsRNA‐independent manner, causing rapid (< 5 min) phosphorylation of eukaryotic initiation factor‐2 α‐subunit (eIF2α). These events resulted in shutting off viral protein translation and blocking viral replication (IC₅₀ = 2 μM) while protecting host cells from virus‐induced damage. Indomethacin did not affect eIF2α kinases PKR‐like endoplasmic reticulum‐resident protein kinase (PERK) and general control non‐derepressible‐2 (GCN2) kinase, and was unable to trigger eIF2α phosphorylation in the presence of PKR inhibitor 2‐aminopurine. In addition, small‐interfering RNA‐mediated PKR gene silencing dampened the antiviral effect in indomethacin‐treated cells. The results identify PKR as a critical target for the antiviral activity of indomethacin and indicate that eIF2α phosphorylation could be a key element in the broad spectrum antiviral activity of the drug.     

Interferon‐inducible protein,P56, inhibits HPV DNA replication by binding to the viral protein E1

Type I interferon (IFN) inhibits, by an unknown mechanism, the replication of human papillomaviruses (HPV), which are major human pathogens, Here, we present evidence that P56 (a protein), the expression of which is strongly induced by IFN, double‐stranded RNA and viruses, mediates the anti‐HPV effect of IFN. Ectopic expression of P56 inhibited HPV DNA replication and its ablation in IFN‐treated cells alleviated the inhibitory effect of IFN on HPV DNA replication. Protein–protein interaction and mutational analyses established that the antiviral effect of P56 was mediated by its direct interaction with the DNA replication origin‐binding protein E1 of several strains of HPV, through the tetratricopeptide repeat 2 in the N‐terminal region of P56 and the C‐terminal region of E1. In vivo, the interaction with P56, a cytoplasmic protein, caused translocation of E1 from the nucleus to the cytoplasm. In vitro, recombinant P56, or a small fragment derived from it, inhibited the DNA helicase activity of E1 and E1‐mediated HPV DNA replication. These observations delineate the molecular mechanism of IFN's antiviral action against HPV.     

Inhibition of protein synthesis by the β-subunit of spectrin

The 220 kDa β-subunit of erythroid cell spectrin is a potent inhibitor of protein synthesis in lysates from rabbit reticulocytes. On the basis of weight of protein added to a lysate reaction mixture, it has about half the inhibitory activity of highly purified heme-regulated eIF-2 kinase. Inhibition appears to be at the level of peptide initiation but does not involve a kinase that phosphorylates eIF-2 on its -subunit.     

New Cdc2 Tyr 4 phosphorylation by dsRNA‐activated protein kinase triggers Cdc2 polyubiquitination and G2 arrest under genotoxic stresses

Cell division cycle 2 (Cdc2) protein is an essential subunit of M‐phase kinase (MPK), which has a key role in G2/M transition. Even though the control of MPK activity has been well established with regard to the phosphorylation of Cdc2 at Thr 14 and/or Tyr 15 and Thr 161, little is known about the proteolytic control of Cdc2. In this study, we observed that Cdc2 was downregulated under genotoxic stresses and that double‐stranded RNA‐activated protein kinase (PKR) was involved in the process. The PKR‐mediated Tyr4 phosphorylation triggered Cdc2 ubiquitination. Phospho‐mimic mutations at the Tyr 4 residue (Y4D or Y4E) caused significant ubiquitination of Cdc2 even in the absence of PKR. Our findings demonstrate that (i) PKR, Ser/Thr kinase, phosphorylates its new substrate Cdc2 at the Tyr 4 residue, (ii) PKR‐mediated Tyr 4‐phosphorylation facilitates Cdc2 ubiquitination and proteosomal degradation, (iii) unphosphorylated Tyr 4 prevents Cdc2 ubiquitination, and (iv) downstream from p53, PKR has a crucial role in G2 arrest and triggers Cdc2 downregulation under genotoxic conditions.     

Inhibition of x‐box binding protein 1 reduces tunicamycin‐induced apoptosis in aged murine macrophages

Endoplasmic reticulum (ER) stress is induced by the accumulation of unfolded and misfolded proteins in the ER. Although apoptosis induced by ER stress has been implicated in several aging‐associated diseases, such as atherosclerosis, it is unclear how aging modifies ER stress response in macrophages. To decipher this relationship, we assessed apoptosis in macrophages isolated from young (1.5–2 months) and aged (16–18 months) mice and exposed the cells to the ER stress inducer tunicamycin. We found that aged macrophages exhibited more apoptosis than young macrophages, which was accompanied by reduced activation of phosphorylated inositol‐requiring enzyme‐1 (p‐IRE1α), one of the three key ER stress signal transducers. Reduced gene expression of x‐box binding protein 1 (XBP1), a downstream effector of IRE1α, enhanced p‐IRE1α levels and reduced apoptosis in aged, but not young macrophages treated with tunicamycin. These findings delineate a novel, age‐dependent interaction by which macrophages undergo apoptosis upon ER stress, and suggest an important protective role of IRE1α in aging‐associated ER stress‐induced apoptosis. This novel pathway may not only be important in our understanding of longevity, but may also have important implications for pathogenesis and potential treatment of aging‐associated diseases in general.     

Inhibition of mitogen-activated protein kinase phosphatase 3 activity by interdomain binding

Mitogen-activated protein (MAP) kinase phosphatase 3 (MKP3) is a cytoplasmic dual specificity phosphatase that functions to attenuate signaling via dephosphorylation and subsequent deactivation of its substrate and allosteric regulator, extracellular signal-regulated protein kinase 2 (ERK2). Expression of MKP3 has been shown to be under the control of ERK2, thus providing an elegant feedback mechanism for regulating the rate and duration of proliferative signals. Previously published studies suggest that MKP3 might serve as a tumor suppressor; however, significantly elevated, rather than reduced, levels of this protein have been reported in early lesions. Because overexpression of this phosphatase is counterintuitive to a proposed tumor suppressor function, the observed cellular tolerance suggested a self-inactivation mechanism. Using surface plasmon resonance, we have provided direct evidence of physical interaction between the N- and C-terminal domains. Kinetic analysis using dimethyl sulfoxide to activate the C-terminal fragment in the absence of ERK2 showed that the isolated C-terminal domain had higher catalytic efficiency than the similarly activated full-length protein. Furthermore, when the isolated N-terminal domain was added to the activated C-terminal domain, a dose-dependant inhibition of catalytic activity was observed. The similarity between the K(I) and K(D) values obtained indicate that interdomain binding stabilizes the inactive conformation of the catalytic site and implies that the N-terminal domain functions as an allosteric inhibitor of phosphatase activity. Finally, we have provided evidence for oligomerization of MKP3 in pancreatic cancer cells expressing elevated levels of this phosphatase.     

Inhibition of α‐glucosidase activity by N‐deoxynojirimycin analogs in several insect phloem sap feeders

Secondary metabolites and synthetic iminosugars that structurally resemble monosaccharides are potent inhibitors of α‐glucosidase activity. The enzyme is core in cleaving sucrose in phloem feeding insects and it also plays a crucial role of reducing osmotic stress via the formation of oligosaccharides. Inhibition of hydrolysis by iminosugars should result in nutritional deficiencies and/or disruption of normal osmoregulation. Deoxynojirimycin (DNJ) and 2 N‐alkylated analogs [N‐butyl DNJ (NB‐DNJ) and N‐nonyl DNJ (NN‐DNJ)] were the major iminosugars used throughout the study. The extensive experiments conducted with α‐glucosidase of the whitefly Bemisia tabaci indicated the competitive nature of inhibition and that the hydrophilic DNJ is a potent inhibitor in comparison to the more hydrophobic NB‐DNJ and NN‐DNJ compounds. The same inhibitory pattern was observed with the psyllid Cacopsylla bidens α‐glucosidase. In contrast to the above pattern, enzymes of the aphids, Myzus persicae and Aphis gossypii were more sensitive to the hydrophobic iminosugars as compared to DNJ. In vivo experiments in which adult B. tabaci were fed dietary iminosugars, show that the hydrophilic DNJ was far less toxic than the lipophilic NB‐DNJ and NN‐DNJ. It is proposed that this pattern is attributed to the better accessibility of the hydrophobic NN‐DNJ to the α‐glucosidase membrane‐bound compartment in the midgut. Based on the inhibitory effects of certain polyhydroxy N‐alkylated iminosugars, α‐glucosidase of phloem feeding hemipterans could serve as an attractive target site for developing novel pest control agents.