Annegret Nath

Multiple importins function as nuclear transport receptors for the Rev protein of human immunodeficiency virus type 1.

The Rev protein of human immunodeficiency virus type 1 is an RNA-binding protein that is required for nuclear export of unspliced and partially spliced viral mRNAs. Nuclear import of human immunodeficiency virus type 1 Rev has been suggested to depend on the classic nuclear transport receptor importin β, but not on the adapter protein importin α. We now show that, similar to importin α, Rev is able to dissociate RanGTP from recycling importin β, a reaction that leads to the formation of a novel import complex. Besides importin β, the transport receptors transportin, importin 5, and importin 7 specifically interact with Rev and promote its nuclear import in digitonin-permeabilized cells. A single arginine-rich nuclear localization sequence of Rev is required for interaction with all importins tested so far. In contrast to the importin β-binding domain of importin α, Rev interacts with an N-terminal fragment of importin β. Transportin contains two independent binding sites for Rev. Hence, the mode of interaction of importin β and transportin with Rev is clearly distinct from that with their classic import cargoes. Taken together, the viral protein takes advantage of multiple cellular transport pathways for its nuclear accumulation.

The Anti-inflammatory Prostaglandin 15-Deoxy-Δ12,14-PGJ2 Inhibits CRM1-dependent Nuclear Protein Export

The signaling molecule 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) has been described as the “anti-inflammatory prostaglandin.” Here we show that substrates of the nuclear export receptor CRM1 accumulate in the nucleus in the presence of 15d-PGJ2, identifying this prostaglandin as a regulator of CRM1-dependent nuclear protein export that can be produced endogenously. Like leptomycin B (LMB), an established fungal CRM1-inhibitor, 15d-PGJ2 reacts with a conserved cysteine residue in the CRM1 sequence. This covalent modification prevents the formation of nuclear export complexes. Cells that are transfected with mutant CRM1 (C528S) are resistant to the inhibitory effects of LMB and 15d-PGJ2, demonstrating that the same single amino acid is targeted by the two compounds. Inhibition of the CRM1 pathway by endogenously produced prostaglandin and/or exogenously applied 15d-PGJ2 may contribute to its anti-inflammatory, anti-proliferative, and anti-viral effects.

The Nucleoporin Nup358/RanBP2 Promotes Nuclear Import in a Cargo- and Transport Receptor-Specific Manner

In vertebrates, the nuclear pore complex (NPC), the gate for transport of macromolecules between the nucleus and the cytoplasm, consists of approximately 30 different nucleoporins (Nups). The Nup and SUMO E3‐ligase Nup358/RanBP2 are the major components of the cytoplasmic filaments of the NPC. In this study, we perform a structure–function analysis of Nup358 and describe its role in nuclear import of specific proteins. In a screen for nuclear proteins that accumulate in the cytoplasm upon Nup358 depletion, we identified proteins that were able to interact with Nup358 in a receptor‐independent manner. These included the importin α/β‐cargo DBC‐1 (deleted in breast cancer 1) and DMAP‐1 (DNA methyltransferase 1 associated protein 1). Strikingly, a short N‐terminal fragment of Nup358 was sufficient to promote import of DBC‐1, whereas DMAP‐1 required a larger portion of Nup358 for stimulated import. Neither the interaction of RanGAP with Nup358 nor its SUMO‐E3 ligase activity was required for nuclear import of all tested cargos. Together, Nup358 functions as a cargo‐ and receptor‐specific assembly platform, increasing the efficiency of nuclear import of proteins through various mechanisms.

Transportin Is a Major Nuclear Import Receptor for c-Fos A NOVEL MODE OF CARGO INTERACTION

c-Fos, a component of the transcription factor AP-1, is rapidly imported into the nucleus after translation. We established an in vitro system using digitonin-permeabilized cells to analyze nuclear import of c-Fos in detail. Two import receptors of the importin β superfamily, importin β itself and transportin, promote import of c-Fos in vitro. Under conditions where importin β-dependent transport was blocked, c-Fos still accumulated in the nucleus in the presence of cytosol. Inhibition of the transportin-dependent pathway, in contrast, abolished import of c-Fos. Furthermore, c-Fos mutants that interact with transportin but not with importin β were efficiently imported in the presence of cytosol. Hence, transportin appears to be the predominant import receptor for c-Fos. A detailed biochemical characterization revealed that the interaction of transportin with c-Fos is distinct from the interaction with its established import cargoes, the M9 sequence of heterogeneous nuclear ribonucleoprotein A1 or the nuclear localization sequence of some basic proteins. Likewise, the binding sites on importin β for its classic import cargo and for c-Fos can be separated. In summary, c-Fos employs a novel mode of receptor-cargo interaction. Hence, transportin may be as versatile as importin β in recognizing different nuclear import cargoes.

Increase of urate formation by stimulation of sympathetic hepatic nerves, circulating noradrenaline and glucagon in the perfused rat liver

In the isolated rat liver perfused in situ stimulation of the nerve bundles around the portal vein and the hepatic artery caused an increase of urate formation that was inhibited by the α1‐blocker prazosine and the xanthine oxidase inhibitor allopurinol. Moreover, nerve stimulation increased glucose and lactate output and decreased perfusion flow. Infusion of noradrenaline had similar effects. Compared to nerve stimulation infusion of glucagon led to a less pronounced increase of urate formation and a twice as large increase in glucose output but a decrease in lactate release without affecting the flow rate. Insulin had no effect on any of the parameters studied.

Gluconeogenic-glycolytic capacities and metabolic zonation in liver of rats with streptozotocin, non-ketotic as compared to alloxan, ketotic diabetes

SummaryActivities (μmol x min−1 x g liver) and zonal distributions of key enzymes of carbohydrate metabolism were studied in livers of streptozotocin-diabetic rats and compared to the values in alloxan-diabetes.1.Streptozotocin led to a non-ketotic diabetes with blood glucose being increased by more than fivefold but ketone bodies being in the normal range, while alloxan produced a ketotic diabetes with blood glucose, acetoacetate and β-hydroxybutyrate being elevated by more than fivefold.2.Portal insulin was decreased to about 20% in streptozotocin- and more drastically to about 7% in alloxan-diabetes. Conversely, portal glucagon was increased in the two states to about 250% and 180%, respectively.3.The glucogenic key enzyme phosphoenolpyruvate carboxykinase (PEPCK) was enhanced in streptozotocinand alloxan-diabetes to over 300%, while the glycolytic pyruvate kinase L (PKL) was lowered to 65% and 80%, respectively. The normal periportal to perivenous gradient of PEPCK of about 3:1, as measured in microdissected tissue samples, was maintained with elevated activities in the two zones. The normal periportal to perivenous gradient of PKL of 1:1.7 was diminished with lowered activities in the two zones.4.The glucogenic glucose-6-phosphatase (G6Pase) was increased in streptozotocin- and alloxan-diabetes to 130% and 140%, respectively, while the glucose utilizing glucokinase (GK) was decreased to 60% and 50%, respectively. The normal periportal to perivenous gradient of G6Pase, demonstrated histochemically, remained unaffected.5.Carnitine palmitoyltransferase (CPT) was increased to over 190% and acetyl-CoA carboxylase (ACC) was decreased to 60% in streptozotocin, non-ketotic diabetes, while the two enzymes were altered more drastically to 400% and 50%, respectively, in alloxan, ketotic diabetes. The results indicate that both in the ketotic and nonketotic diabetic state the gluconeogenic capacity of the liver was increased mainly in the periportal zone and the glycolytic capacity was decreased mainly in the perivenous area. In both types of diabetes the glucostat function of the liver with its typical reciprocal zonal distribution of glucogenic and glycolytic enzymes was not lost, but only impaired owing to shifts of the enzyme levels in the periportal and perivenous zone. This quantitative rather than qualitative alteration would be in accord with the requirement for the liver persisting in both non-ketotic and ketotic diabetes to handle excess nutritional glucose at least in part.

Importin 7 and Nup358 Promote Nuclear Import of the Protein Component of Human Telomerase

In actively dividing eukaryotic cells, chromosome ends (telomeres) are subject to progressive shortening, unless they are maintained by the action of telomerase, a dedicated enzyme that adds DNA sequence repeats to chromosomal 3′end. For its enzymatic function on telomeres, telomerase requires nuclear import of its protein component (hTERT in human cells) and assembly with the RNA component, TERC. We now confirm a major nuclear localization signal (NLS) in the N-terminal region of hTERT and describe a novel one in the C-terminal part. Using an siRNA approach to deplete several import receptors, we identify importin 7 as a soluble nuclear transport factor that is required for efficient import. At the level of the nuclear pore complex (NPC), Nup358, a nucleoporin that forms the cytoplasmic filaments of the NPC, plays an important role in nuclear import of hTERT. A structure-function analysis of Nup358 revealed that the zinc finger region of the nucleoporin is of particular importance for transport of hTERT. Together, our study sheds light on the nuclear import pathway of hTERT.

Purification of a RNA-binding Protein from Rat Liver IDENTIFICATION AS FERRITIN L CHAIN AND DETERMINATION OF THE RNA/PROTEIN BINDING CHARACTERISTICS

In cultured rat hepatocytes the degradation of phosphoenolpyruvate carboxykinase mRNA might be regulated by protein(s), which by binding to the mRNA alter its stability. The 3′-untranslated region of phosphoenolpyruvate carboxykinase mRNA as a potential target was used to select RNA-binding protein(s) from rat liver by the use of gel retardation assays. A cytosolic protein was isolated, which bound to the phosphoenolpyruvate carboxykinase mRNA 3′-untranslated region and other in vitro synthesized RNAs. The protein was purified to homogeneity; it had an apparent molecular mass of 400 kDa and consisted of identical subunits with an apparent size of 24.5 kDa. Sequence analysis of a tryptic peptide from the 24.5-kDa protein revealed its identity with rat ferritin light chain. Binding of ferritin to RNA was abolished after phosphorylation with cAMP-dependent protein kinase and was augmented after dephosphorylation with alkaline phosphatase. Weak binding was observed in extracts from okadaic acid-treated cultured hepatocytes compared with untreated cells. Preincubation of ferritin with an anti-phosphoserine or an anti-phosphothreonine antibody attenuated binding to RNA, while an anti-phosphotyrosine antibody generated a supershift indicating that phosphoserine and phosphothreonine but not phosphotyrosine residues were in close proximity to the RNA-binding region. Ferritin is the iron storage protein in the liver. Binding of ferritin to RNA was diminished in the presence of increasing iron concentrations, whereas the iron chelator desferal was without effect. It is concluded that ferritin might function as RNA-binding protein and that it may have important functions in the general regulation of cellular RNA metabolism.

Interactions of nuclear protein from cultured rat hepatocytes with the cyclic AMP responsive elements and the NF1-CTF site in the promotor of the rat phosphoenolpyruvate carboxykinase gene

Nuclear extracts from cultured rat hepatocytes were analyzed by gel mobility shift assay for protein binding to the cyclic AMP responsive elements CRE1 (-96/-77) and CRE2 (-152/-132) and the NF1-CTF binding site (-121/-99) of the phosphoenolpyruvate carboxykinase (PCK) promotor. Binding was very weak to the CRE2 and CRE1. The NF1-CTF site formed two complexes with nuclear protein. Protein binding was increased, when the NF1-CTF site was coupled to the CRE1, and further, when it was coupled to both the CRE1 and the CRE2. Complex formation was not altered by treatment of the hepatocytes with glucagon or with glucagon and insulin. Thus, protein binding was most efficient when all three elements were in context, which might be necessary for full transcriptional activation of the PCK gene.

Inhibition of prostaglandin D2 clearance in rat hepatocytes by the thromboxane receptor antagonists daltroban and ifetroban and the thromboxane synthase inhibitor furegrelate

Prostanoids, i.e. prostaglandins and thromboxane, regulate liver-specific functions both in homeostasis and during defense reactions. For example, prostanoids are released from Kupffer cells, the resident liver macrophages, in response to the inflammatory mediator anaphylatoxin C5a, and mediate an enhanced glucose output from hepatocytes as energy supply. In perfused rat livers, the thromboxane receptor antagonist daltroban enhanced C5a-induced prostanoid overflow and reduced glucose output. It was the aim of this study to elucidate whether daltroban interfered with prostanoid release from Kupffer cells or prostanoid clearance by hepatocytes, and/or whether it directly influenced prostanoid-dependent glucose metabolism in these cells. In perfused rat livers, daltroban enhanced prostaglandin (PG)D(2) overflow not only after infusion of C5a (15-fold), but also after PGD(2) (10-fold). Neither daltroban nor another receptor antagonist, ifetroban, or the thromboxane synthase inhibitor furegrelate enhanced prostanoid release from Kupffer cells. In contrast, all inhibitors reduced clearance, i.e. uptake and degradation, of PGD(2) by hepatocytes: within 5 min uptake of 1 nmol/L PGD(2) was reduced from 43+/-5 fmol (controls) to 22+/-6 fmol (daltroban), 24+/-6 fmol (ifetroban) and 21+/-6 fmol (furegrelate). PGD(2) in the medium was reduced to 39+/-7% in the controls, but remained at 93+/-9%, 93+/-11% and 60+/-3% in the presence of the inhibitors. PGD(2)-dependent glucose output in the perfused liver or activation of glycogen phosphorylase in isolated hepatocytes remained unaffected by daltroban. These data clearly demonstrate that the thromboxane-inhibitors reduced PGD(2) clearance by hepatocytes, presumably by inhibition of prostanoid transport into the cells. In contrast, they did not interfere with PGD(2)-dependent glucose metabolism, suggesting an independent mechanism for the inhibition of glucose output from the liver.