Mechanism for fetal globin gene expression: Role of the soluble guanylate cyclase–cGMP-dependent protein kinase pathway

Despite considerable concerns with pharmacological stimulation of fetal hemoglobin (Hb F) as a therapeutic option for the β-globin disorders, the molecular basis of action of Hb F-inducing agents remains unclear. Here we show that an intracellular pathway including soluble guanylate cyclase (sGC) and cGMP-dependent protein kinase (PKG) plays a role in induced expression of the γ-globin gene. sGC, an obligate heterodimer of α- and β-subunits, participates in a variety of physiological processes by converting GTP to cGMP. Northern blot analyses with erythroid cell lines expressing different β-like globin genes showed that, whereas the β-subunit is expressed at similar levels, high-level expression of the α-subunit is preferentially observed in erythroid cells expressing γ-globin but not those expressing β-globin. Also, the levels of expression of the γ-globin gene correlate to those of the α-subunit. sGC activators or cGMP analogs increased expression of the γ-globin gene in erythroleukemic cells as well as in primary erythroblasts from normal subjects and patients with β-thalassemia. Nuclear run-off assays showed that the sGC activator protoporphyrin IX stimulates transcription of the γ-globin gene. Furthermore, increased expression of the γ-globin gene by well known Hb F-inducers such as hemin and butyrate was abolished by inhibiting sGC or PKG activity. Taken together, these results strongly suggest that the sGC–PKG pathway constitutes a mechanism that regulates expression of the γ-globin gene. Further characterization of this pathway should permit us to develop new therapeutics for the β-globin disorders.

Activity-dependent CREB phosphorylation: Convergence of a fast, sensitive calmodulin kinase pathway and a slow, less sensitive mitogen-activated protein kinase pathway

The cAMP-responsive element binding protein (CREB), a key regulator of gene expression, is activated by phosphorylation on Ser-133. Several different protein kinases possess the capability of driving this phosphorylation, making it a point of potential convergence for multiple intracellular signaling cascades. Previous work in neurons has indicated that physiologic synaptic stimulation recruits a fast calmodulin kinase IV (CaMKIV)-dependent pathway that dominates early signaling to CREB. Here we show in hippocampal neurons that the fast, CaMK-dependent pathway can be followed by a slower pathway that depends on Ras/mitogen-activated protein kinase (MAPK), along with CaMK. This pathway was blocked by dominant-negative Ras and was specifically recruited by depolarizations that produced strong intracellular Ca2+ transients. When both pathways were recruited, phosphorylated CREB (pCREB) formation was overwhelmingly dominated by the CaMK pathway between 0 and 10 min, and by the MAPK pathway at 60 min, whereas the two pathways acted in concert at 30 min. The Ca2+ signals that produced only rapid CaMK signaling to pCREB or both rapid CaMK and slow MAPK signaling deviated significantly for only ≈1 min, yet their differential impact on pCREB extended over a much longer period, between 20 and 60 min and beyond, which is of likely significance for gene expression. The CaMK-dependent MAPK pathway may inform the nucleus about stimulus amplitude. In contrast, the CaMKIV pathway may be well suited to conveying information on the precise timing of localized synaptic stimuli, befitting its greater speed and sensitivity, whereas the previously described calcineurin pathway may carry information about stimulus duration.

Peroxide Sensors for the Fission Yeast Stress-activated Mitogen-activated Protein Kinase Pathway

The Schizosaccharomyces pombe stress-activated Sty1p/Spc1p mitogen-activated protein (MAP) kinase regulates gene expression through the Atf1p and Pap1p transcription factors, homologs of human ATF2 and c-Jun, respectively. Mcs4p, a response regulator protein, acts upstream of Sty1p by binding the Wak1p/Wis4p MAP kinase kinase kinase. We show that phosphorylation of Mcs4p on a conserved aspartic acid residue is required for activation of Sty1p only in response to peroxide stress. Mcs4p acts in a conserved phospho-relay system initiated by two PAS/PAC domain-containing histidine kinases, Mak2p and Mak3p. In the absence of Mak2p or Mak3p, Sty1p fails to phosphorylate the Atf1p transcription factor or induce Atf1p-dependent gene expression. As a consequence, cells lacking Mak2p and Mak3p are sensitive to peroxide attack in the absence of Prr1p, a distinct response regulator protein that functions in association with Pap1p. The Mak1p histidine kinase, which also contains PAS/PAC repeats, does not regulate Sty1p or Atf1p but is partially required for Pap1p- and Prr1p-dependent transcription. We conclude that the transcriptional response to free radical attack is initiated by at least two distinct phospho-relay pathways in fission yeast.

Stress signaling in plants: a mitogen-activated protein kinase pathway is activated by cold and drought.

Yeast and animals use mitogen-activated protein (MAP) kinase cascades to mediate stress and extracellular signals. We have tested whether MAP kinases are involved in mediating environmental stress responses in plants. Using specific peptide antibodies that were raised against different alfalfa MAP kinases, we found exclusive activation of p44MMK4 kinase in drought- and cold-treated plants. p44MMK4 kinase was transiently activated by these treatments and was correlated with a shift in the electrophoretic mobility of the p44MMK4 protein. Although transcript levels of the MMK4 gene accumulated after drought and cold treatment, no changes in p44MMK4 steady state protein levels were observed, indicating a posttranslational activation mechanism. Extreme temperatures, drought, and salt stress are considered to be different forms of osmotic stress. However, high salt concentrations or heat shock did not induce activation of p44MMK4, indicating the existence of distinct mechanisms to mediate different stresses in alfalfa. Stress adaptation in plants is mediated by abscisic acid (ABA)-dependent and ABA-independent processes. Although ABA rapidly induced the transcription of an ABA-inducible marker gene, MMK4 transcript levels did not increase and p44MMK4 kinase was not activated. These data indicate that the MMK4 kinase pathway mediates drought and cold signaling independently of ABA.

Protein export from the nucleus requires the GTPase Ran and GTP hydrolysis.

Nuclei of digitonin-permeabilized cells that had been preloaded with a model transport substrate in a cytosol-dependent import reaction were subsequently incubated to investigate which conditions would result in export of transport substrate. We found that up to 80% of the imported substrate was exported when recombinant human Ran and GTP were present in the export reaction. Ran-mediated export was inhibited by nonhydrolyzable GTP analogs and also by wheat germ agglutinin but was unaffected by a nonhydrolyzable ATP analog. Moreover, a recombinant human Ran mutant that was deficient in its GTPase activity inhibited export. These data indicate that export of proteins from the nucleus requires Ran and GTP hydrolysis but not ATP hydrolysis. We also found that digitonin-permeabilized cells were depleted of their endogenous nuclear Ran, thus allowing detection of Ran as a limiting factor for export. In contrast, most endogenous karyopherin alpha was retained in nuclei of digitonin-permeabilized cells. Unexpectedly, exogenously added, fluorescently labeled Ran, although it accessed the nuclear interior, was found to dock at the nuclear rim in a punctate pattern, suggesting the existence of Ran-binding sites at the nuclear pore complex.

The DMSO reductase family of microbial molybdenum enzymes; Molecular properties and role in the dissimilatory reduction of toxic elements

The dimethylsulfoxide (DMSO) reductase family of molybdenum enzymes is a large and diverse group that is found in bacteria and archaea. These enzymes are characterised by a bis(molybdopterin guanine dinucleotide)Mo form of the molybdenum cofactor, and they are particularly important in anaerobic respiration including the dissimilatory reduction of certain toxic oxoanions. The structural and phylogenetic relationship between the proteins of this family is discussed. High-resolution crystal structures of enzymes of the DMSO reductase family have revealed a high degree of similarity in tertiary structure. However, there is considerable variation in the structure of the molybdenum active site and it seems likely that these subtle but important differences lead to the great diversity of function seen in this family of enzymes. This diversity of catalytic capability is associated with several distinct pathways of electron transport.

A new pathway for the secretion of virulence factors by bacteria: The flagellar export apparatus functions as a protein-secretion system

Biogenesis of the flagellum, a motive organelle of many bacterial species, is best understood for members of the Enterobacteriaceae. The flagellum is a heterooligomeric structure that protrudes from the surface of the cell. Its assembly initially involves the synthesis of a dedicated protein export apparatus that subsequently transports other flagellar proteins by a type III mechanism from the cytoplasm to the outer surface of the cell, where oligomerization occurs. In this study, the flagellum export apparatus was shown to function also as a secretion system for the transport of several extracellular proteins in the pathogenic bacterium Yersinia enterocolitica. One of the proteins exported by the flagellar secretion system was the virulence-associated phospholipase, YplA. These results suggest type III protein secretion by the flagellar system may be a general mechanism for the transport of proteins that influence bacterial–host interactions.

A novel alternate secretory pathway for the export of Plasmodium proteins into the host erythrocyte

The malarial parasite dramatically alters its host cell by exporting and targeting proteins to specific locations within the erythrocyte. Little is known about the mechanisms by which the parasite is able to carry out this extraparasite transport. The fungal metabolite brefeldin A (BFA) has been used to study the secretory pathway in eukaryotes. BFA treatment of infected erythrocytes inhibits protein export and results in the accumulation of exported Plasmodium proteins into a compartment that is at the parasite periphery. Parasite proteins that are normally localized to the erythrocyte membrane, to nonmembrane bound inclusions in the erythrocyte cytoplasm, or to the parasitophorous vacuolar membrane accumulate in this BFA-induced compartment. A single BFA-induced compartment is detected per parasite and the various exported proteins colocalize to this compartment regardless of their final destinations. Parasite membrane proteins do not accumulate in this novel compartment, but accumulate in the endoplasmic reticulum (ER), suggesting that the parasite has two secretory pathways. This alternate secretory pathway is established immediately after merozoite invasion and at least some dense granule proteins also use the alternate pathway. The BFA-induced compartment exhibits properties that are similar to the ER, but it is clearly distinct from the ER. We propose to call this new organelle the secondary ER of apicomplexa. This ER-like organelle is an early, if not the first, step in the export of Plasmodium proteins into the host erythrocyte.

Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli

We cloned the streptokinase (STK) gene of Streptococcus equisimilis in an expression vector of Escherichia coli to overexpress the profibrinolytic protein under the control of a tac promoter. Almost all the recombinant STK was exported to the periplasmic space and recovered after gentle lysozyme digestion of induced cells. The periplasmic fraction was chromatographed on DEAE Sepharose followed by chromatography on phenyl-agarose. Active proteins eluted between 4.5 and 0% ammonium sulfate, when a linear gradient was applied. Three major STK derivatives of 47.5 kDa, 45 kDa and 32 kDa were detected by Western blot analysis with a polyclonal antibody. The 32-kDa protein formed a complex with human plasminogen but did not exhibit Glu-plasminogen activator activity, as revealed by a zymographic assay, whereas the 45-kDa protein showed a Km = 0.70 µM and kcat = 0.82 s-1, when assayed with a chromogen-coupled substrate. These results suggest that these proteins are putative fragments of STK, possibly derived from partial degradation during the export pathway or the purification steps. The 47.5-kDa band corresponded to the native STK, as revealed by peptide sequencing

Genetic interactions of yeast eukaryotic translation initiation factor 5a (eIF5A) reveal connections to poly(A)-binding protein and protein kinase C signaling

The highly conserved eukaryotic translation initiation factor eIF5A has been proposed to have various roles in the cell, from translation to mRNA decay to nuclear protein export. To further our understanding of this essential protein, three temperature-sensitive alleles of the yeast TIF51A gene have been characterized. Two mutant eIF5A proteins contain mutations in a proline residue at the junction between the two eIFSA domains and the third, strongest allele encodes a protein with a single mutation in each domain, both of which are required for the growth defect. The stronger tif51A alleles cause defects in degradation of short-lived mRNAs, supporting a role for this protein in mRNA decay. A multicopy suppressor screen revealed six genes, the overexpression of which allows growth of a tif51A-1 strain at high temperature; these genes include PAB1, PKC1, and PKC1 regulators WSC1, WSC2, and WSC3. Further results suggest that eIFSA may also be involved in ribosomal synthesis and the WSC/PKC1 signaling pathway for cell wall integrity or related processes.

The human homologue of Saccharomyces cerevisiae Gle1p is required for poly(A)+ RNA export

The mechanism of mRNA export is a complex issue central to cellular physiology. We characterized previously yeast Gle1p, a protein with a leucine-rich (LR) nuclear export sequence (NES) that is essential for poly(A)+ RNA export in Saccharomyces cerevisiae. To characterize elements of the vertebrate mRNA export pathway, we identified a human homologue of yeast Gle1p and analyzed its function in mammalian cells. hGLE1 encodes a predicted 75-kDa polypeptide with high sequence homology to yeast Gle1p, but hGle1p does not contain a sequence motif matching any of the previously characterized NESs. hGLE1 can complement a yeast gle1 temperature-sensitive export mutant only if a LR-NES is inserted into it. To determine whether hGle1p played a role in nuclear export, anti-hGle1p antibodies were microinjected into HeLa cells. In situ hybridization of injected cells showed that poly(A)+ RNA export was inhibited. In contrast, there was no effect on the nuclear import of a glucocorticoid receptor reporter. We conclude that hGle1p functions in poly(A)+ RNA export, and that human cells facilitate such export with a factor similar to yeast but without a recognizable LR-NES. With hGle1p localized at the nuclear pore complexes, hGle1p is positioned to act at a terminal step in the export of mature RNA messages to the cytoplasm.

Reconstitution of HIV-1 Rev nuclear export: Independent requirements for nuclear import and export

The Rev protein of HIV-1 actively shuttles between nucleus and cytoplasm and mediates the export of unspliced retroviral RNAs. The localization of shuttling proteins such as Rev is controlled by the relative rates of nuclear import and export. To study nuclear export in isolation, we generated cell lines expressing a green fluorescent protein-labeled chimeric protein consisting of HIV-1 Rev and a hormone-inducible nuclear localization sequence. Steroid removal switches off import thus allowing direct visualization of the Rev export pathway in living cells. After digitonin permeabilization of these cells, we found that a functional nuclear export sequence (NES), ATP, and fractionated cytosol were sufficient for nuclear export in vitro. Nuclear pore-specific lectins and leptomycin B were potent export inhibitors. Nuclear export was not inhibited by antagonists of calcium metabolism that block nuclear import. These data further suggest that nuclear pores do not functionally close when luminal calcium stores are depleted. The distinct requirements for nuclear import and export argue that these competing processes may be regulated independently. This system should have wide applicability for the analysis of nuclear import and export.

Role of Nuclear Pools of Aminoacyl-tRNA Synthetases in tRNA Nuclear Export

Reports of nuclear tRNA aminoacylation and its role in tRNA nuclear export (Lund and Dahlberg, 1998; Sarkar et al., 1999; Grosshans et al., 2000a) have led to the prediction that there should be nuclear pools of aminoacyl-tRNA synthetases. We report that in budding yeast there are nuclear pools of tyrosyl-tRNA synthetase, Tys1p. By sequence alignments we predicted a Tys1p nuclear localization sequence and showed it to be sufficient for nuclear location of a passenger protein. Mutations of this nuclear localization sequence in endogenous Tys1p reduce nuclear Tys1p pools, indicating that the motif is also important for nucleus location. The mutations do not significantly affect catalytic activity, but they do cause defects in export of tRNAs to the cytosol. Despite export defects, the cells are viable, indicating that nuclear tRNA aminoacylation is not required for all tRNA nuclear export paths. Because the tRNA nuclear exportin, Los1p, is also unessential, we tested whether tRNA aminoacylation and Los1p operate in alternative tRNA nuclear export paths. No genetic interactions between aminoacyl-tRNA synthetases and Los1p were detected, indicating that tRNA nuclear aminoacylation and Los1p operate in the same export pathway or there are more than two pathways for tRNA nuclear export.

Expression of p16 protein in acral lentiginous melanoma

Background Acral lentiginous melanoma (ALM) is a clinicopathologic subtype of cutaneous malignant melanoma. ALM is the most common type of melanoma amongst Asians, Africans, and patients with mixed ancestry. In Brazil, ALM comprises 10% of cutaneous melanoma. ALM develops on palmar, plantar, and subungual skin, and its biology is different from that of other cutaneous melanomas, where sunlight is the major known environmental determinant. Alterations and inactivation of the p16INK4 gene that encodes a specific inhibitor of cyclin-dependent kinase have been related to melanoma genesis and progression. Few studies, however, have addressed p16 expression in ALM. Methods In order to verify and compare p16 protein expression, 32 paraffin-embedded ALM specimens were subjected to a immunohistochemical technique using a monoclonal anti-p16 antibody. The tumors were classified according to thickness (up to 1.0 mm and thicker than 1.0 mm) and the presence of ulceration. Results Twenty-five (78%) ALMs displayed positive p16 protein expression: 21 of the 25 (84%) with a thickness of more than 1.0 mm, and four of the seven (57%) with a thickness of 1.0 mm or less. Thirteen of the 17 (76%) nonulcerated lesions and 12 of the 15 (80%) ulcerated lesions displayed positive p16 protein expression. Conclusion The data obtained suggest that p16 protein expression per se may not represent a marker of retinoblastoma protein (pRb) pathway disturbance in ALM tumorigenesis.