The pathogenic mechanism of the Mycobacterium ulcerans virulence factor, mycolactone, depends on blockade of protein translocation into the ER.

Infection with Mycobacterium ulcerans is characterised by tissue necrosis and immunosuppression due to mycolactone, the necessary and sufficient virulence factor for Buruli ulcer disease pathology. Many of its effects are known to involve down-regulation of specific proteins implicated in important cellular processes, such as immune responses and cell adhesion. We have previously shown mycolactone completely blocks the production of LPS-dependent proinflammatory mediators post-transcriptionally. Using polysome profiling we now demonstrate conclusively that mycolactone does not prevent translation of TNF, IL-6 and Cox-2 mRNAs in macrophages. Instead, it inhibits the production of these, along with nearly all other (induced and constitutive) proteins that transit through the ER. This is due to a blockade of protein translocation and subsequent degradation of aberrantly located protein. Several lines of evidence support this transformative explanation of mycolactone function. First, cellular TNF and Cox-2 can be once more detected if the action of the 26S proteasome is inhibited concurrently. Second, restored protein is found in the cytosol, indicating an inability to translocate. Third, in vitro translation assays show mycolactone prevents the translocation of TNF and other proteins into the ER. This is specific as the insertion of tail-anchored proteins into the ER is unaffected showing that the ER remains structurally intact. Fourth, metabolic labelling reveals a near-complete loss of glycosylated and secreted proteins from treated cells, whereas cytosolic proteins are unaffected. Notably, the profound lack of glycosylated and secreted protein production is apparent in a range of different disease-relevant cell types. These studies provide a new mechanism underlying mycolactone's observed pathological activities both in vitro and in vivo. Mycolactone-dependent inhibition of protein translocation into the ER not only explains the deficit of innate cytokines, but also the loss of membrane receptors, adhesion molecules and T-cell cytokines that drive the aetiology of Buruli ulcer.

Translational reprogramming following UVB irradiation is mediated by DNA-PKcs and allows selective recruitment to the polysomes of mRNAs encoding DNA repair enzymes.

UVB-induced lesions in mammalian cellular DNA can, through the process of mutagenesis, lead to carcinogenesis. However, eukaryotic cells have evolved complex mechanisms of genomic surveillance and DNA damage repair to counteract the effects of UVB radiation. We show that following UVB DNA damage, there is an overall inhibition of protein synthesis and translational reprogramming. This reprogramming allows selective synthesis of DDR proteins, such as ERCC1, ERCC5, DDB1, XPA, XPD, and OGG1 and relies on upstream ORFs in the 5' untranslated region of these mRNAs. Experiments with DNA-PKcs-deficient cell lines and a specific DNA-PKcs inhibitor demonstrate that both the general repression of mRNA translation and the preferential translation of specific mRNAs depend on DNA-PKcs activity, and therefore our data establish a link between a key DNA damage signaling component and protein synthesis.

The mechanism of micro-RNA-mediated translation repression is determined by the promoter of the target gene.

MicroRNAs (miRNAs) are noncoding RNAs that base pair imperfectly to homologous regions in target mRNAs and negatively influence the synthesis of the corresponding proteins. Repression is mediated by a number of mechanisms, one of which is the direct inhibition of protein synthesis. Surprisingly, previous studies have suggested that two mutually exclusive mechanisms exist, one acting at the initiation phase of protein synthesis and the other at a postinitiation event. Here, we resolve this apparent dichotomy by demonstrating that the promoter used to transcribe the mRNA influences the type of miRNA-mediated translational repression. Transcripts derived from the SV40 promoter that contain let-7 target sites in their 3' UTRs are repressed at the initiation stage of translation, whereas essentially identical mRNAs derived from the TK promoter are repressed at a postinitiation step. We also show that there is a miR-34 target site within the 3' UTR of c-myc mRNA and that promoter dependency is also true for this endogenous 3' UTR. Overall, these data establish a link between the nuclear history of an mRNA and the mechanism of miRNA-mediated translational regulation in the cytoplasm.

A novel method for poly(A) fractionation reveals a large population of mRNAs with a short poly(A) tail in mammalian cells.

The length of the poly(A) tail of an mRNA plays an important role in translational efficiency, mRNA stability and mRNA degradation. Regulated polyadenylation and deadenylation of specific mRNAs is involved in oogenesis, embryonic development, spermatogenesis, cell cycle progression and synaptic plasticity. Here we report a new technique to analyse the length of poly(A) tails and to separate a mixed population of mRNAs into fractions dependent on the length of their poly(A) tails. The method can be performed on crude lysate or total RNA, is fast, highly reproducible and minor changes in poly(A) tail length distribution are easily detected. We validated the method by analysing mRNAs known to undergo cytoplasmic polyadenylation during Xenopus laevis oocyte maturation. We then separated RNA from NIH3T3 cells into two fractions with short and long poly(A) tails and compared them by microarray analysis. In combination with the validation experiments, the results indicate that approximately 25% of the expressed genes have a poly(A) tail of less than 30 residues in a significant percentage of their transcripts.

Purification of P-Rex1 from neutrophils and nucleotide exchange assay.

The P-Rex family of guanine-nucleotide exchange factors (GEFs) are activators of the small GTPase Rac (Donald et al., 2004; Rosenfeldt et al., 2004; Welch et al., 2002). They are directly regulated in vitro and in vivo by the lipid second messenger phosphatidylinositol (3,4,5)-triphosphate (PtdIns(3,4,5)P3) and by the betagamma subunits of heterotrimeric G proteins (Donald et al., 2004; Rosenfeldt et al., 2004; Welch et al., 2002). Activation by PtdIns(3,4,5)P3 occurs by means of the PH domain of P-Rex1 and activation by Gbetagamma subunits by means of the catalytic DH domain (Hill et al., 2005). P-Rex1 and P-Rex2 also contain two DEP and two PDZ protein interaction domains, as well as homology over their COOH-terminal half to inositol polyphosphate 4-phosphatase (Donald et al., 2004; Welch et al., 2002). These domains, although not necessary for P-Rex1 activity in vitro, influence its basal and/or stimulated Rac-GEF activity, suggesting that their interaction with the DH/PH domain tandem is important for P-Rex1 function (Hill et al., 2005). P-Rex2B, a splice variant of P-Rex2, lacks the C-terminal half (Rosenfeldt et al., 2004). P-Rex1 was originally identified during a search for PtdIns(3,4,5)P3-dependent activators of Rac in neutrophils and purified to homogeneity from pig leukocyte cytosol, in which it is the major such activity (Welch et al., 2002). P-Rex1 is mainly expressed in neutrophils and regulates reactive oxygen species formation in these cells (Welch et al., 2002), whereas P-Rex2 is expressed in a wide variety of tissues but not in neutrophils (Donald et al., 2004), and P-Rex2B is expressed in the heart (Rosenfeldt et al., 2004). This Chapter describes our methods for (1) the purification of endogenous P-Rex1 from pig leukocyte cytosol, (2) the production and purification of recombinant P-Rex proteins and their substrate GTPase Rac from Sf9 cells, and (3) the in vitro assay for measuring the GEF activities of native or recombinant P-Rex proteins.

P-Rex1 regulates neutrophil function.

Rac GTPases regulate cytoskeletal structure, gene expression, and reactive oxygen species (ROS) production. Rac2-deficient neutrophils cannot chemotax, produce ROS, or degranulate upon G protein-coupled receptor (GPCR) activation. Deficiency in PI3Kgamma, an upstream regulator of Rac, causes a similar phenotype. P-Rex1, a guanine-nucleotide exchange factor (GEF) for Rac, is believed to link GPCRs and PI3Kgamma to Rac-dependent neutrophil responses. We have investigated the functional importance of P-Rex1 by generating a P-Rex1(-/-) mouse. P-Rex1(-/-) mice are viable and healthy, with apparently normal leukocyte development, but with mild neutrophilia. In neutrophils from P-Rex1(-/-) mice, GPCR-dependent Rac2 activation is impaired, whereas Rac1 activation is less compromised. GPCR-dependent ROS formation is absent in lipopolysaccharide (LPS)-primed P-Rex1(-/-) neutrophils, but less affected in unprimed or TNFalpha-primed cells. Recruitment of P-Rex1(-/-) neutrophils to inflammatory sites is impaired. Surprisingly, chemotaxis of isolated neutrophils is only slightly reduced, with a mild defect in cell speed, but normal polarization and directionality. Secretion of azurophil granules is unaffected. In conclusion, P-Rex1 is an important regulator of neutrophil function by mediating a subset of Rac-dependent neutrophil responses. However, P-Rex1 is not an essential regulator of neutrophil chemotaxis and degranulation.

Regulation of P-Rex1 by phosphatidylinositol (3,4,5)-trisphosphate and Gbetagamma subunits.

P-Rex1 is a guanine-nucleotide exchange factor (GEF) for the small GTPase Rac. We have investigated here the mechanisms of stimulation of P-Rex1 Rac-GEF activity by the lipid second messenger phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) and the Gbetagamma subunits of heterotrimeric G proteins. We show that a P-Rex1 mutant lacking the PH domain (DeltaPH) cannot be stimulated by PtdIns(3,4,5)P3, which implies that the PH domain confers PtdIns(3,4,5)P3 regulation of P-Rex1 Rac-GEF activity. Consistent with this, we found that PtdIns(3,4,5)P3 binds to the PH domain of P-Rex1 and that the DH/PH domain tandem is sufficient for PtdIns(3,4,5)P3-stimulated P-Rex1 activity. The Rac-GEF activities of the DeltaPH mutant and the DH/PH domain tandem can both be stimulated by Gbetagamma subunits, which infers that Gbetagamma subunits regulate P-Rex1 activity by binding to the catalytic DH domain. Deletion of the DEP, PDZ, or inositol polyphosphate 4-phosphatase homology domains has no major consequences on the abilities of either PtdIns(3,4,5)P3 or Gbetagamma subunits to stimulate P-Rex1 Rac-GEF activity. However, the presence of any of these domains impacts on the levels of basal and/or stimulated P-Rex1 Rac-GEF activity, suggesting that there are important functional interactions between the DH/PH domain tandem and the DEP, PDZ, and inositol polyphosphate 4-phosphatase homology domains of P-Rex1.

P-Rex2, a new guanine-nucleotide exchange factor for Rac.

We have identified a new guanine-nucleotide exchange factor, P-Rex2, and cloned it from human skeletal muscle and brain libraries. It has widespread tissue distribution but is not expressed in neutrophils. P-Rex2 is a 183 kDa protein that activates the small GTPase Rac and is regulated by phosphatidylinositol (3,4,5)-trisphosphate and the beta gamma subunits of heterotrimeric G proteins in vitro and in vivo. P-Rex2 has structure, activity and regulatory properties similar to P-Rex1 but has divergent tissue distribution, as P-Rex1 is mainly expressed in neutrophils. Together, they form an enzyme family capable of mediating Rac signalling downstream of G protein-coupled receptors and phosphoinositide 3-kinase.

Chemopreventive efficacy and pharmacokinetics of curcumin in the min/+ mouse, a model of familial adenomatous polyposis.

Curcumin, the major yellow pigment in turmeric, prevents the development of adenomas in the intestinal tract of the C57Bl/6J Min/+ mouse, a model of human familial APC. To aid the rational development of curcumin as a colorectal cancer-preventive agent, we explored the link between its chemopreventive potency in the Min/+ mouse and levels of drug and metabolites in target tissue and plasma. Mice received dietary curcumin for 15 weeks, after which adenomas were enumerated. Levels of curcumin and metabolites were determined by high-performance liquid chromatography in plasma, tissues, and feces of mice after either long-term ingestion of dietary curcumin or a single dose of [(14)C]curcumin (100 mg/kg) via the i.p. route. Whereas curcumin at 0.1% in the diet was without effect, at 0.2 and 0.5%, it reduced adenoma multiplicity by 39 and 40%, respectively, compared with untreated mice. Hematocrit values in untreated Min/+ mice were drastically reduced compared with those in wild-type C57Bl/6J mice. Dietary curcumin partially restored the suppressed hematocrit. Traces of curcumin were detected in the plasma. Its concentration in the small intestinal mucosa, between 39 and 240 nmol/g of tissue, reflects differences in dietary concentration. [(14)C]Curcumin disappeared rapidly from tissues and plasma within 2-8 h after dosing. Curcumin may be useful in the chemoprevention of human intestinal malignancies related to Apc mutations. The comparison of dose, resulting curcumin levels in the intestinal tract, and chemopreventive potency suggests tentatively that a daily dose of 1.6 g of curcumin is required for efficacy in humans. A clear advantage of curcumin over nonsteroidal anti-inflammatory drugs is its ability to decrease intestinal bleeding linked to adenoma maturation.