Protein Kinase C-α Regulates Insulin Action and Degradation by Interacting with Insulin Receptor Substrate-1 and 14-3-3ϵ

Protein kinase C (PKC)-alpha exerts a regulatory function on insulin action. We showed by overlay blot that PKC alpha directly binds a 180-kDa protein, corresponding to IRS-1, and a 30-kDa molecular species, identified as 14-3-3 epsilon. In intact NIH-3T3 cells overexpressing insulin receptors (3T3-hIR), insulin selectively increased PKC alpha coprecipitation with IRS-1, but not with IRS-2, and with 14-3-3 epsilon, but not with other 14-3-3 isoforms. Overexpression of 14-3-3 epsilon in 3T3-hIR cells significantly reduced IRS-1-bound PKC alpha activity, without altering IRS-1/PKC alpha co-precipitation. 14-3-3 epsilon overexpression also increased insulin-stimulated insulin receptor and IRS-1 tyrosine phosphorylation, followed by increased activation of Raf1, ERK1/2, and Akt/protein kinase B. Insulin-induced glycogen synthase activity and thymidine incorporation were also augmented. Consistently, selective depletion of 14-3-3 epsilon by antisense oligonucleotides caused a 3-fold increase of IRS-1-bound PKC alpha activity and a similarly sized reduction of insulin receptor and IRS-1 tyrosine phosphorylation and signaling. In turn, selective inhibition of PKC alpha expression by antisense oligonucleotides reverted the negative effect of 14-3-3 epsilon depletion on insulin signaling. Moreover, PKC alpha inhibition was accompanied by a > 2-fold decrease of insulin degradation. Similar results were also obtained by overexpressing 14-3-3 epsilon. Thus, in NIH-3T3 cells, insulin induces the formation of multimolecular complexes, including IRS-1, PKC alpha, and 14-3-3 epsilon. The presence of 14-3-3 epsilon in the complex is not necessary for IRS-1/PKC alpha interaction but modulates PKC alpha activity, thereby regulating insulin signaling and degradation

Zoonotic Risk of Encephalitozoon cuniculi in Animal-Assisted Interventions: Laboratory Strategies for the Diagnosis of Infections in Humans and Animals

The involvement of animals for therapeutic purposes has very ancient roots. To date, it is clear that animal-assisted interventions (AAIs), in addition to ensuring the replacement of missing or deficient affects, improves psychophysiological parameters connected to human health. However, AAI could potentially present risks related to the transmission of infectious agents from animals to humans. Among these microorganisms, E. cuniculi is a microspore which induces pathological effects (fever, headache, nausea, vomiting, diarrhea, breathlessness, respiratory symptoms, and weakness) in both humans and animals. Consequently, an accurate and fast diagnosis of E. cuniculi infection, as well as the identification of new diagnostic approaches, is of fundamental importance. This literature review was carried out to provide an extensive and comprehensive analysis of the most recent diagnostic techniques to prevent and care for E. cuniculi-associated risks in the AAI field

Glucose regulates diacylglycerol intracellular levels and protein kinase C activity by modulating diacylglycerol kinase subcellular localization.

Although chronic hyperglycemia reduces insulin sensitivity and leads to impaired glucose utilization, short term exposure to high glucose causes cellular responses positively regulating its own metabolism. We show that exposure of L6 myotubes overexpressing human insulin receptors to 25 mm glucose for 5 min decreased the intracellular levels of diacylglycerol (DAG). This was paralleled by transient activation of diacylglycerol kinase (DGK) and of insulin receptor signaling. Following 30-min exposure, however, both DAG levels and DGK activity returned close to basal levels. Moreover, the acute effect of glucose on DAG removal was inhibited by >85% by the DGK inhibitor R59949. DGK inhibition was also accompanied by increased protein kinase C-alpha (PKCalpha) activity, reduced glucose-induced insulin receptor activation, and GLUT4 translocation. Glucose exposure transiently redistributed DGK isoforms alpha and delta, from the prevalent cytosolic localization to the plasma membrane fraction. However, antisense silencing of DGKdelta, but not of DGKalpha expression, was sufficient to prevent the effect of high glucose on PKCalpha activity, insulin receptor signaling, and glucose uptake. Thus, the short term exposure of skeletal muscle cells to glucose causes a rapid induction of DGK, followed by a reduction of PKCalpha activity and transactivation of the insulin receptor signaling. The latter may mediate, at least in part, glucose induction of its own metabolism

Protein Kinase C-ζ and Protein Kinase B Regulate Distinct Steps of Insulin Endocytosis and Intracellular Sorting

We have investigated the molecular mechanisms regulating insulin internalization and intracellular sorting. Insulin internalization was decreased by 50% upon incubation of the cells with the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002. PI3K inhibition also reduced insulin degradation and intact insulin release by 50 and 75%, respectively. Insulin internalization was reduced by antisense inhibition of protein kinase C-zeta (PKCzeta) expression and by overexpression of a dominant negative PKCzeta mutant (DN-PKCzeta). Conversely, overexpression of PKCzeta increased insulin internalization as a function of the PKCzeta levels achieved in the cells. Expression of wild-type protein kinase B (PKB)-alpha or of a constitutively active form (myr-PKB) did not significantly alter insulin internalization and degradation but produced a 100% increase of intact insulin release. Inhibition of PKB by a dominant negative mutant (DN-PKB) or by the pharmacological inhibitor ML-9 reduced intact insulin release by 75% with no effect on internalization and degradation. In addition, overexpression of Rab5 completely rescued the effect of PKCzeta inhibition on insulin internalization but not that of PKB inhibition on intact insulin recycling. Indeed, PKCzeta bound to and activated Rab5. Thus, PI3K controls different steps within the insulin endocytic itinerary. PKCzeta appears to mediate the PI3K effect on insulin internalization in a Rab5-dependent manner, whereas PKB directs intracellular sorting toward intact insulin release

The meningococcal vaccine antigen GNA2091 is an analogue of YraP and plays key roles in outer membrane stability and virulence

K.L.S. was supported by the Australian National Health and Medical Research Council (NHMRC) C. J. Martin Fellowship and Career Development Fellowship. A.F.H. was supported by a Marie Curie Fellowship (PIEF-GA-2012-328377). F.O., L.F., and S.B. were recipients of Novartis fellowships from the Ph.D. program of the University of Siena (Siena, Italy) and University of Bologna (Bologna, Italy), respectively.GNA2091 is one of the components of the 4-component meningococcal serogroup B vaccine (4CMenB) vaccine and is highly conserved in all meningococcal strains. However, its functional role has not been fully characterized. Here we show that nmb2091 is part of an operon and is cotranscribed with the nmb2089, nmb2090, and nmb2092 adjacent genes, and a similar but reduced operon arrangement is conserved in many other gram-negative bacteria. Deletion of the nmb2091 gene causes an aggregative phenotype with a mild defect in cell separation; differences in the outer membrane composition and phospholipid profile, in particular in the phosphoethanolamine levels; an increased level of outer membrane vesicles; and deregulation of the zinc-responsive genes such as znuD. Finally, the Δ2091 strain is attenuated with respect to the wild-type strain in competitive index experiments in the infant rat model of meningococcal infection. Altogether these data suggest that GNA2091 plays important roles in outer membrane architecture, biogenesis, homeostasis, and in meningococcal survival in vivo, and amodel for its role is discussed. These findings highlight the importance of GNA2091 as a vaccine component.PostprintPeer reviewe

Phorbol Esters Induce Intracellular Accumulation of the Anti-apoptotic Protein PED/PEA-15 by Preventing Ubiquitinylation and Proteasomal Degradation

Phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA)-15 is an anti-apoptotic protein whose expression is increased in several cancer cells and following experimental skin carcinogenesis. Exposure of untransfected C5N keratinocytes and transfected HEK293 cells to phorbol esters (12-O-tetradecanoylphorbol-13-acetate (TPA)) increased PED/PEA-15 cellular content and enhanced its phosphorylation at serine 116 in a time-dependent fashion. Ser-116 --> Gly (PED(S116G)) but not Ser-104 --> Gly (PED(S104G)) substitution almost completely abolished TPA regulation of PED/PEA-15 expression. TPA effect was also prevented by antisense inhibition of protein kinase C (PKC)-zeta and by the expression of a dominant-negative PKC-zeta mutant cDNA in HEK293 cells. Similar to long term TPA treatment, overexpression of wild-type PKC-zeta increased cellular content and phosphorylation of WT-PED/PEA-15 and PED(S104G) but not of PED(S116G). These events were accompanied by the activation of Ca2+-calmodulin kinase (CaMK) II and prevented by the CaMK blocker, KN-93. At variance, the proteasome inhibitor lactacystin mimicked TPA action on PED/PEA-15 intracellular accumulation and reverted the effects of PKC-zeta and CaMK inhibition. Moreover, we show that PED/PEA-15 bound ubiquitin in intact cells. PED/PEA-15 ubiquitinylation was reduced by TPA and PKC-zeta overexpression and increased by KN-93 and PKC-zeta block. Furthermore, in HEK293 cells expressing PED(S116G), TPA failed to prevent ubiquitin-dependent degradation of the protein. Accordingly, in the same cells, TPA-mediated protection from apoptosis was blunted. Taken together, our results indicate that TPA increases PED/PEA-15 expression at the post-translational level by inducing phosphorylation at serine 116 and preventing ubiquitinylation and proteosomal degradation

Omi/HtrA2 promotes cell death by binding and degrading the anti-apoptotic protein ped/pea-15

ped/pea-15 is a ubiquitously expressed 15-kDa protein featuring a broad anti-apoptotic function. In a yeast two-hybrid screen, the pro-apoptotic Omi/HtrA2 mitochondrial serine protease was identified as a specific interactor of the ped/pea-15 death effector domain. Omi/HtrA2 also bound recombinant ped/pea-15 in vitro and co-precipitated with ped/pea-15 in 293 and HeLa cell extracts. In these cells, the binding of Omi/HtrA2 to ped/pea-15 was induced by UVC exposure and followed the mitochondrial release of Omi/HtrA2 into the cytoplasm. Upon UVC exposure, cellular ped/pea-15 protein expression levels decreased. This effect was prevented by the ucf-101 specific inhibitor of the Omi/HtrA2 proteolytic activity, in a dose-dependent fashion. In vitro incubation of ped/pea-15 with Omi/HtrA2 resulted in ped/pea-15 degradation. In intact cells, the inhibitory action of ped/pea-15 on UVC-induced apoptosis progressively declined at increasing Omi/HtrA2 expression. This further effect of Omi/HtrA2 was also inhibited by ucf-101. In addition, ped/pea-15 expression blocked Omi/HtrA2 co-precipitation with the caspase inhibitor protein XIAP and caspase 3 activation. Thus, in part, apoptosis following Omi/HtrA2 mitochondrial release is mediated by reduction in ped/pea-15 cellular levels. The ability of Omi/HtrA2 to relieve XIAP inhibition on caspases is modulated by the relative levels of Omi/HtrA2 and ped/pea-15

Omi/HtrA2 promotes cell death by binding and degrading the anti-apoptotic protein ped/pea-15

ped/pea-15 is a ubiquitously expressed 15-kDa protein featuring a broad anti-apoptotic function. In a yeast two-hybrid screen, the pro-apoptotic Omi/HtrA2 mitochondrial serine protease was identified as a specific interactor of the ped/pea-15 death effector domain. Omi/HtrA2 also bound recombinant ped/pea-15 in vitro and co-precipitated with ped/pea-15 in 293 and HeLa cell extracts. In these cells, the binding of Omi/HtrA2 to ped/pea-15 was induced by UVC exposure and followed the mitochondrial release of Omi/HtrA2 into the cytoplasm. Upon UVC exposure, cellular ped/pea-15 protein expression levels decreased. This effect was prevented by the ucf-101 specific inhibitor of the Omi/HtrA2 proteolytic activity, in a dose-dependent fashion. In vitro incubation of ped/pea-15 with Omi/HtrA2 resulted in ped/pea-15 degradation. In intact cells, the inhibitory action of ped/pea-15 on UVC-induced apoptosis progressively declined at increasing Omi/HtrA2 expression. This further effect of Omi/HtrA2 was also inhibited by ucf-101. In addition, ped/pea-15 expression blocked Omi/HtrA2 co-precipitation with the caspase inhibitor protein XIAP and caspase 3 activation. Thus, in part, apoptosis following Omi/HtrA2 mitochondrial release is mediated by reduction in ped/pea-15 cellular levels. The ability of Omi/HtrA2 to relieve XIAP inhibition on caspases is modulated by the relative levels of Omi/HtrA2 and ped/pea-15

Expression of Factor H Binding Protein of Meningococcus Responds to Oxygen Limitation through a Dedicated FNR-Regulated Promoter▿

Factor H binding protein (fHBP) is a surface-exposed lipoprotein in Neisseria meningitidis, which is a component of several investigational vaccines against serogroup B meningococcus (MenB) currently in development. fHBP enables the bacterium to evade complement-mediated killing by binding factor H, a key downregulator of the complement alternative pathway, and, in addition, fHBP is important for meningococcal survival in the presence of the antimicrobial peptide LL-37. In this study, we investigate the molecular mechanisms involved in transcription and regulation of the fHBP-encoding gene, fhbp. We show that the fHBP protein is expressed from two independent transcripts: one bicistronic transcript that includes the upstream gene and a second shorter monocistronic transcript from its own dedicated promoter, Pfhbp. Transcription from the promoter Pfhbp responds to oxygen limitation in an FNR-dependent manner, and, accordingly, the FNR protein binds to a Pfhbp probe in vitro. Furthermore, expression in meningococci of a constitutively active FNR mutant results in the overexpression of the fHBP protein. Finally, the analysis of fHBP regulation was extended to a panel of strains expressing different fHBP allelic variants at different levels, and we demonstrate that FNR is involved in the regulation of this antigen in all but one of the strains tested. Our data suggest that oxygen limitation may play an important role in inducing the expression of fHBP from a dedicated FNR-regulated promoter. This implies a role for this protein in microenvironments lacking oxygen, for instance in the submucosa or intracellularly, in addition to its demonstrated role in serum resistance in the blood

The RNA Chaperone Hfq Is Involved in Stress Response and Virulence in Neisseria meningitidis and Is a Pleiotropic Regulator of Protein Expression▿

The well-conserved protein Hfq has emerged as the key modulator of riboregulation in bacteria. This protein is thought to function as an RNA chaperone and to facilitate base pairing between small regulatory RNA (sRNA) and mRNA targets, and many sRNAs are dependent on the Hfq protein for their regulatory functions. To address the possible role of Hfq in riboregulated circuits in Neisseria meningitidis, we generated an Hfq mutant of the MC58 strain, and the knockout mutant has pleiotropic phenotypes; it has a general growth phenotype in vitro in culture media, and it is sensitive to a wide range of stresses, including those that it may encounter in the host. Furthermore, the expression profile of a vast number of proteins is clearly altered in the mutant, and we have identified 27 proteins by proteomics. All of the phenotypes tested to date are also restored by complementation of Hfq expression in the mutant strain. Importantly, in ex vivo and in vivo models of infection the Hfq mutant is attenuated. These data indicate that Hfq plays a key role in stress response and virulence, and we propose a major role for Hfq in regulation of gene expression. Moreover, this study suggests that in meningococcus there is a large Hfq-mediated sRNA network which so far is largely unexplored