Wolfgang H. Fischer

A Novel CDK9-Associated C-Type Cyclin Interacts Directly with HIV-1 Tat and Mediates Its High-Affinity, Loop-Specific Binding to TAR RNA

The HIV-1 Tat protein regulates transcription elongation through binding to the viral TAR RNA stem-loop structure. We have isolated a novel 87 kDa cyclin C-related protein (cyclin T) that interacts specifically with the transactivation domain of Tat. Cyclin T is a partner for CDK9, an RNAPII transcription elongation factor. Remarkably, the interaction of Tat with cyclin T strongly enhances the affinity and specificity of the Tat:TAR RNA interaction, and confers a requirement for sequences in the loop of TAR that are not recognized by Tat alone. Moreover, overexpression of human cyclin T rescues Tat activity in nonpermissive rodent cells. We propose that Tat directs cyclin T-CDK9 to RNAPII through cooperative binding to TAR RNA.

Systematic mutagenesis of the Helicobacter pylori cag pathogenicity island: essential genes for CagA translocation in host cells and induction of interleukin-8

Helicobacter pylori (Hp) carries a type IV secretion system encoded by the cag pathogenicity island (cag-PAI), which is used to: (i) translocate the bacterial effector protein CagA into different types of eukaryotic cells; and (ii) induce the synthesis and secretion of chemokines, such as interleukin-8 (IL-8). The cag-PAI in Hp 26695 consists of 27 putative genes, six of which were identified as homologues to the basic type IV secretion system represented by the Agrobacterium tumefaciens virB operon. To define the role and contribution of each of the 27 genes, we applied a precise deletion/insertion mutagenesis procedure to knock out each individual gene without causing polar effects on the expression of downstream genes. Seventeen out of 27 genes were found to be absolutely essential for translocation of CagA into host cells and 14 out of 27 for the ability of Hp fully to induce transcription of IL-8. The products of hp0524 (virD4 homologue), hp0526 and hp0540 are absolutely essential for the translocation of CagA, but not for the induction of IL-8. In contrast, the products of hp0520, hp0521, hp0534, hp0535, hp0536 and hp0543 are not necessary for either translocation of CagA or for IL-8 induction. Our data argue against a translocated IL-8-inducing effector protein encoded by the cag-PAI. We isolated a variant of Hp 26695, which spontaneously switched off its capacity for IL-8 induction and translocation of CagA, but retained the complete cag-PAI. We identified a point mutation in gene hp0532, causing a premature translational stop in the corresponding polypeptide chain, providing a putative explanation for the defect in the type IV secretion system of the spontaneous mutant.

Protein disulfide bond formation in the cytoplasm during oxidative stress

The majority of disulfide-linked cytosolic proteins are thought to be enzymes that transiently form disulfide bonds while catalyzing oxidation-reduction (redox) processes. Recent evidence indicates that reactive oxygen species can act as signaling molecules by promoting the formation of disulfide bonds within or between select redox-sensitive proteins. However, few studies have attempted to examine global changes in disulfide bond formation following reactive oxygen species exposure. Here we isolate and identify disulfide-bonded proteins (DSBP) in a mammalian neuronal cell line (HT22) exposed to various oxidative insults by sequential nonreducing/reducing two-dimensional SDS-PAGE combined with mass spectrometry. By using this strategy, several known cytosolic DSBP, such as peroxiredoxins, thioredoxin reductase, nucleoside-diphosphate kinase, and ribonucleotide-diphosphate reductase, were identified. Unexpectedly, a large number of previously unknown DSBP were also found, including those involved in molecular chaperoning, translation, glycolysis, cytoskeletal structure, cell growth, and signal transduction. Treatment of cells with a wide range of hydrogen peroxide concentrations either promoted or inhibited disulfide bonding of select DSBP in a concentration-dependent manner. Decreasing the ratio of reduced to oxidized glutathione also promoted select disulfide bond formation within proteins from cytoplasmic extracts. In addition, an epitope-tagged version of the molecular chaperone HSP70 forms mixed disulfides with both β4-spectrin and adenomatous polyposis coli protein in the cytosol. Our findings indicate that disulfide bond formation within families of cytoplasmic proteins is dependent on the nature of the oxidative insult and may provide a common mechanism used to control multiple physiological processes.

FGF-2-Responsive Neural Stem Cell Proliferation Requires CCg, a Novel Autocrine/Paracrine Cofactor

We have purified and characterized a factor, from the conditioned medium of neural stem cell cultures, which is required for fibroblast growth factor 2s (FGF-2) mitogenic activity on neural stem cells. This autocrine/paracrine cofactor is a glycosylated form of cystatin C (CCg), whose N-glycosylation is required for its activity. We further demonstrated that, both in vitro and in vivo, neural stem cells undergoing cell division are immunopositive for cystatin C. Finally, we showed in vivo functional activity of CCg by demonstrating that the combined delivery of FGF-2 and CCg to the adult dentate gyrus stimulated neurogenesis. We propose that the process of neurogenesis is controlled by the cooperation between trophic factors and autocrine/paracrine cofactors, of which CCg is a prototype.

Characterization of motifs which are critical for activity of the cyclic AMP-responsive transcription factor CREB.

Cyclic AMP mediates the hormonal stimulation of a number of eukaryotic genes by directing the protein kinase A (PK-A)-dependent phosphorylation of transcription factor CREB. We have previously determined that although phosphorylation at Ser-133 is critical for induction, this site does not appear to participate directly in transactivation. To test the hypothesis that CREB ultimately activates transcription through domains that are distinct from the PK-A site, we constructed a series of CREB mutants and evaluated them by transient assays in F9 teratocarcinoma cells. Remarkably, a glutamine-rich region near the N terminus appeared to be important for PK-A-mediated induction of CREB since removal of this domain caused a marked reduction in CREB activity. A second region consisting of a short acidic motif (DLSSD) C terminal to the PK-A site also appeared to synergize with the phosphorylation motif to permit transcriptional activation. Biochemical experiments with purified recombinant CREB protein further demonstrate that the transactivation domain is more sensitive to trypsin digestion than are the DNA-binding and dimerization domains, suggesting that the activator region may be structured to permit interactions with other proteins in the RNA polymerase II complex.

The CREB coactivator CRTC2 links hepatic ER stress and fasting gluconeogenesis.

In fasted mammals, circulating pancreatic glucagon stimulates hepatic gluconeogenesis in part through the CREB regulated transcription coactivator 2 (CRTC2, also referred to as TORC2). Hepatic glucose production is increased in obesity, reflecting chronic increases in endoplasmic reticulum (ER) stress that promote insulin resistance. Whether ER stress also modulates the gluconeogenic program directly, however, is unclear. Here we show that CRTC2 functions as a dual sensor for ER stress and fasting signals. Acute increases in ER stress triggered the dephosphorylation and nuclear entry of CRTC2, which in turn promoted the expression of ER quality control genes through an association with activating transcription factor 6 alpha (ATF6α, also known as ATF6)—an integral branch of the unfolded protein response. In addition to mediating CRTC2 recruitment to ER stress inducible promoters, ATF6α also reduced hepatic glucose output by disrupting the CREB–CRTC2 interaction and thereby inhibiting CRTC2 occupancy over gluconeogenic genes. Conversely, hepatic glucose output was upregulated when hepatic ATF6α protein amounts were reduced, either by RNA interference (RNAi)-mediated knockdown or as a result of persistent stress in obesity. Because ATF6α overexpression in the livers of obese mice reversed CRTC2 effects on the gluconeogenic program and lowered hepatic glucose output, our results demonstrate how cross-talk between ER stress and fasting pathways at the level of a transcriptional coactivator contributes to glucose homeostasis.

Novel vertebrate nucleoporins Nup133 and Nup160 play a role in mRNA export.

RNA undergoing nuclear export first encounters the basket of the nuclear pore. Two basket proteins, Nup98 and Nup153, are essential for mRNA export, but their molecular partners within the pore are largely unknown. Because the mechanism of RNA export will be in question as long as significant vertebrate pore proteins remain undiscovered, we set out to find their partners. Fragments of Nup98 and Nup153 were used for pulldown experiments from Xenopus egg extracts, which contain abundant disassembled nuclear pores. Strikingly, Nup98 and Nup153 each bound the same four large proteins. Purification and sequence analysis revealed that two are the known vertebrate nucleoporins, Nup96 and Nup107, whereas two mapped to ORFs of unknown function. The genes encoding the novel proteins were cloned, and antibodies were produced. Immunofluorescence reveals them to be new nucleoporins, designated Nup160 and Nup133, which are accessible on the basket side of the pore. Nucleoporins Nup160, Nup133, Nup107, and Nup96 exist as a complex in Xenopus egg extracts and in assembled pores, now termed the Nup160 complex. Sec13 is prominent in Nup98 and Nup153 pulldowns, and we find it to be a member of the Nup160 complex. We have mapped the sites that are required for binding the Nup160 subcomplex, and have found that in Nup98, the binding site is used to tether Nup98 to the nucleus; in Nup153, the binding site targets Nup153 to the nuclear pore. With transfection and in vivo transport assays, we find that specific Nup160 and Nup133 fragments block poly[A]+ RNA export, but not protein import or export. These results demonstrate that two novel vertebrate nucleoporins, Nup160 and Nup133, not only interact with Nup98 and Nup153, but themselves play a role in mRNA export.

A Hormone-Dependent Module Regulating Energy Balance

Under fasting conditions, metazoans maintain energy balance by shifting from glucose to fat burning. In the fasted state, SIRT1 promotes catabolic gene expression by deacetylating the forkhead factor FOXO in response to stress and nutrient deprivation. The mechanisms by which hormonal signals regulate FOXO deacetylation remain unclear, however. We identified a hormone-dependent module, consisting of the Ser/Thr kinase SIK3 and the class IIa deacetylase HDAC4, which regulates FOXO activity in Drosophila. During feeding, HDAC4 is phosphorylated and sequestered in the cytoplasm by SIK3, whose activity is upregulated in response to insulin. SIK3 is inactivated during fasting, leading to the dephosphorylation and nuclear translocation of HDAC4 and to FOXO deacetylation. SIK3 mutant flies are starvation sensitive, reflecting FOXO-dependent increases in lipolysis that deplete triglyceride stores; reducing HDAC4 expression restored lipid accumulation. Our results reveal a hormone-regulated pathway that functions in parallel with the nutrient-sensing SIRT1 pathway to maintain energy balance.

Three-finger toxin fold for the extracellular ligand-binding domain of the type II activin receptor serine kinase.

The transforming growth factor β (TGFβ) superfamily of cytokines elicit diverse biological responses by interacting with two distinct, but structurally related transmembrane receptor serine kinases (type I and type II). The binding of these dimeric ligands to the type II receptor is the first event in transmembrane signaling for this family. Here we report the 1.5 Å resolution crystal structure of the extracellular ligand-binding domain of the type II activin receptor (ActRII-ECD), which reveals a fold similar to that of a class of toxins known as three-finger toxins. This fold is primarily dictated by disulfide bonds formed by eight conserved cysteines, with a characteristic spacing, and thus is likely to be shared by most of the type I and II receptors for the TGFβ family. Sequence comparison with an evolutionarily distant activin binding-protein identifies several conserved residues, including two hydrophobic clusters that may form binding surfaces for activin and the type I receptor.

Autocrine/paracrine regulation of pituitary function by activin, inhibin and follistatin.

The precise regulation of the anterior pituitary is achieved by the cell-specific and combined actions of central, peripheral and local factors. Activins, inhibins, and follistatins were first discovered as gonadal factors with actions on FSH production from pituitary gonadotropes. With the realization that these factors are expressed in a wide array of tissues, including the pituitary, it became apparent that the functional importance of activins, inhibins, and follistatins extends beyond the reproductive axis and that they often exert their effects by autocrine/paracrine mechanisms. As members of the TGF-beta superfamily, activins and inhibins control and orchestrate many physiological processes and are vital for the development, the growth, and the functional integrity of most tissues, including the pituitary. Activins exert effects on multiple pituitary cell types but the best-characterized pituitary targets of the autocrine/paracrine function of activins are the gonadotropes. The autocrine/paracrine function of the activin-binding proteins, follistatins, constitutes an important local mechanism to modulate activin bioactivity while the restricted actions of gonadal inhibins to betaglycan-expressing gonadotropes provides a secondary mode of regulation of cell-specific actions of activins. The aim of this review is to highlight and evaluate experimental evidence that supports the roles of activins, inhibins, and follistatins as autocrine, paracrine, and/or endocrine modulators of the pituitary.