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BBA - Molecular Cell Research (v.1745, #2)

Editorial Board (pp. ii).

Bridging advanced glycation end product, receptor for advanced glycation end product and nitric oxide with hormonal replacement/estrogen therapy in healthy versus diabetic postmenopausal women: A perspective by Srirupa Mukhopadhyay; Tapan K. Mukherjee (pp. 145-155).

Cardiovascular diseases (CVD) are the most significant cause of death in postmenopausal women. The loss of estrogen biosynthesis with advanced age is suggested as one of the major causes of higher CVD in postmenopausal women. While some studies show beneficial effects of estrogen therapy (ET)/hormonal replacement therapy (HRT) in the cardiovascular system of healthy postmenopausal women, similar studies in diabetic counterparts contradict these findings. In particular, ET/HRT in diabetic postmenopausal women results in a seemingly detrimental effect on the cardiovascular system. In this review, the comparative role of estrogens is discussed in the context of CVD in both healthy and diabetic postmenopausal women in regard to the synthesis or expression of proinflammatory molecules like advanced glycation end products (AGEs), receptor for advanced glycation end products (RAGEs), inducible nitric oxide synthases (iNOS) and the anti-inflammatory endothelial nitric oxide synthases (eNOS). The interaction of AGE–RAGE signaling with molecular nitric oxide (NO) may determine the level of reactive oxygen species (ROS) and influence the overall redox status of the vascular microenvironment that may further determine the ultimate outcome of the effects of estrogens on the CVD in healthy versus diabetic women.

Keywords: AGE; RAGE; Estrogen; HRT; ET; Diabetes; NO; ROS; Postmenopausal women

Immediate up-regulation of the calcium-binding protein S100P and its involvement in the cytokinin-induced differentiation of human myeloid leukemia cells by Yuki Ishii; Takashi Kasukabe; Yoshio Honma (pp. 156-165).

Cytokinins are important purine derivatives that act as redifferentiation-inducing hormones to control many processes in plants. Cytokinins such as isopentenyladenine (IPA) and kinetin are very effective at inducing the granulocytic differentiation of human myeloid leukemia HL-60 cells. We examined the gene expression profiles associated with exposure to IPA using cDNA microarrays and compared the results with those obtained with other inducers of differentiation, such as all- trans retinoic acid (ATRA), 1α,25-dihydroxyvitamin D₃ (VD3) and cotylenin A (CN-A). Many genes were up-regulated, and only a small fraction were down-regulated, upon exposure to the inducers. IPA and CN-A, but not ATRA or VD3, immediately induced the expression of mRNA for the calcium-binding protein S100P. The up-regulation of S100P was confirmed at the protein expression level. We also examined the expression of other S100 proteins, including S100A8, S100A9 and S100A12, and found that IPA preferentially up-regulated S100P at the early stages of differentiation. IPA-induced differentiation of HL-60 cells was suppressed by treatment with antisense oligonucleotides against S100P, suggesting that S100P plays an important role in cell differentiation.

Keywords: Abbreviations; IPA; isopentenyladenine; ATRA; all-; trans; retinoic acid; VD3; 1α,25-dihydroxyvitamin D; ₃; CN-A; cotylenin A; MAPK; mitogen-activated protein kinase; NBT; nitroblue tetrazoliumMyeloid leukemia; Differentiation; S100P; Calcium-binding protein

Redox regulation of platelet-derived-growth-factor-receptor: Role of NADPH-oxidase and c-Src tyrosine kinase by Serena Catarzi; Chiara Biagioni; Elisa Giannoni; Fabio Favilli; Tommaso Marcucci; Teresa Iantomasi; Maria Teresa Vincenzini (pp. 166-175).

This study identifies some early events contributing to the redox regulation of platelet-derived growth factor receptor (PDGFr) activation and its signalling in NIH3T3 fibroblasts. We demonstrate for the first time that the redox regulation of PDGFr tyrosine autophosphorylation and its signalling are related to NADPH oxidase activity through protein kinase C (PKC) and phosphoinositide-3-kinase (PI3K) activation and H₂O₂ production. This event is also essential for complete PDGF-induced activation of c-Src kinase by Tyr⁴¹⁶ phosphorylation, and the involvement of c-Src kinase on H₂O₂-induced PDGFr tyrosine phosphorylation is demonstrated, suggesting a role of this kinase on the redox regulation of PDGFr activation. Finally, it has been determined that not only PI3K activity, but also PKC activity, are related to NADPH oxidase activation due to PDGF stimulation in NIH3T3 cells, as it occurs in non-phagocyte cells. Therefore, we suggest a redox circuit whereby, upon PDGF stimulation, PKC, PI3K and NADPH oxidase activity contribute to complete c-Src kinase activation, thus promoting maximal phosphorylation and activation of PDGFr tyrosine phosphorylation.

Keywords: Platelet-derived growth factor receptor; Redox regulation; c-Src kinase; NADPH oxidase; Tyrosine phosphorylation

Obstruction of polyubiquitination affects PTS1 peroxisomal matrix protein import by Jan A.K.W. Kiel; Marleen Otzen; Marten Veenhuis; Ida J. van der Klei (pp. 176-186).

Pex4p is an ubiquitin-conjugating enzyme that functions at a late stage of peroxisomal matrix protein import. Here we show that in the methylotrophic yeast Hansenula polymorpha production of a mutant form of ubiquitin (Ub^K48R) has a dramatic effect on PTS1 matrix protein import. This effect was not observed in cells lacking Pex4p, in which the peroxisome biogenesis defect was largely suppressed. These findings provide the first indication that the function of Pex4p in matrix protein import involves polyubiquitination. We also demonstrate that the production of Ub^K48R in H. polymorpha results in enhanced Pex5p degradation. A similar observation was made in cells in which the PEX4 gene was deleted. We demonstrate that in both strains Pex5p degradation was due to ubiquitination and subsequent degradation by the proteasome. This process appeared to be dependent on a conserved lysine residue in the N-terminus of Pex5p (Lys21) and was prevented in a Pex5p^K21R mutant. We speculate that the degradation of Pex5p by the proteasome is important to remove receptor molecules that are stuck at a late stage of the Pex5p-mediated protein import pathway.

Keywords: Peroxisome; Protein translocation; PTS1 protein import; Ubiquitin; Yeast; Hansenula polymorpha

Accumulation and nuclear import of HIF1 alpha during high and low oxygen concentration in skeletal muscle cells in primary culture by Hans-Peter Kubis; Nina Hanke; Renate J. Scheibe; Gerolf Gros (pp. 187-195).

The hypoxia-inducible-factor-1 (HIF1) mediates the transcriptional upregulation of several target genes during hypoxia. HIF1 itself is known to be regulated essentially by ubiquitinylation and proteolytic degradation of the subunit HIF1α of the dimeric transcription factor HIF1. In contrast to other tissues, skeletal muscle expresses high amounts of HIF1α in normoxia as well as in hypoxia. In view of this, we aimed to investigate HIF1α accumulation and subcellular localization as well as the transcriptional activity of the HIF1α-regulated gene of glyceraldehyde dehydrogenase (GAPDH) in skeletal muscle cells exposed to low oxygen concentration (3% O₂), normoxia (20% O₂) or high oxygen concentration (42% O₂). Immunofluorescence analysis reveals that under normoxic and high oxygen conditions, significant amounts of HIF1α can be found exclusively in the cytoplasm of the myotubes. Muscle cells treated with CoCl₂, a known inhibitor of HIF1α degradation, show even higher levels of HIF1α, again exclusively in the cytoplasm. Under conditions of low oxygen, HIF1α in controls as well as in CoCl₂-treated cells is found in the nuclei. CdCl₂ inhibits nuclear import of HIF1α at low oxygen concentration and leads to a transcriptional downregulation of the marker enzyme of anaerobic glycolysis GAPDH. Immunoprecipitation with anti-HIF1α antibody co-precipitates HSP90 in an oxygen-dependent manner, more at high pO₂ than at low pO₂. Cadmium-treated samples also show high amounts of co-immunoprecipitated HSP90, independent of oxygen concentration. We conclude that in skeletal muscle cells, HIF1α, in contrast to other tissues, may, in addition to its regulation by degradation, also be regulated by binding to HSP90 and subsequent inhibition of its import into the nuclei.

Keywords: Hypoxia-inducible-factor-1 alpha; Heat shock protein 90; Skeletal muscle; Hypoxia

Interaction of casein kinase 1 delta (CK1δ) with the light chain LC2 of microtubule associated protein 1A (MAP1A) by Sonja Wolff; Zhenyu Xiao; Mathias Wittau; Nadine S�ssner; Martin St�ter; Uwe Knippschild (pp. 196-206).

CK1δ, a member of the casein kinase 1 family of serine/threonine specific kinases, has been shown to be involved in the regulation of microtubule dynamics. We have now identified a 176 aa fragment of the light chain LC2 of MAP1A (termed LC2-P16) specifically interacting with CK1δ. Two CK1δ interacting domains of LC2 were identified, located between aa 2629 and 2753 close to aa 2683 and between aa 2712 and 2805 of LC2. The two regions necessary for the interaction of LC2 with CK1δ have been mapped between aa 76–103 and aa 351–375 of CK1δ. Furthermore, LC2 has been identified as a new substrate of CK1δ. We therefore propose a model in which CK1δ could modulate microtubule dynamics by changing the phosphorylation status of the light chain LC2 of MAP1A.

Keywords: CK1δ; MAP1A; Microtubule; Yeast two-hybrid screen; Phosphorylation

Intracellular translocation of the decapeptide carboxyl terminal of G_i3α induces the dual phosphorylation of p42/p44 MAP kinases by Sarah Jones; Michelle Farquhar; Ashley Martin; John Howl (pp. 207-214).

The carboxyl terminal of heterotrimeric G protein alpha subunits binds both G protein-coupled receptors and mastoparan (MP), a tetradecapeptide allostere. Moreover, peptides corresponding to the carboxyl domains of G_i3α and G_t display intrinsic biological activities in cell-free systems. Thus, the purpose of this study was to develop a cell penetrant delivery system to further investigate the biological properties of a peptide mimetic of the G_i3α carboxyl terminal (G_i3α^346–355; H-KNNLKECGLY-NH₂). Kinetic studies, using a CFDA-conjugated analogue of G_i3α^346–355, confirmed the rapid and efficient intracellular translocation of TP10-G_i3α^346–355 ( t_0.5=3 min). Translocated G_i3α^346–355, but not other bioactive cargoes derived from PKC and the CB₁ cannabinoid receptor, promoted the dual phosphorylation of p42/p44 MAPK without adverse changes in cellular viability. The relative specificity of this novel biological activity was further confirmed by the observation that translocated G_i3α^346–355 did not influence the exocytosis of β-hexoseaminidase from RBL-2H3, a secretory event stimulated by other cell penetrant peptide cargoes and MP. We conclude that TP10-G_i3α^346–355 is a valuable, non-toxic research tool with which to study and modulate signal transduction pathways mediated by heterotrimeric G proteins and MAPK.

Keywords: Abbreviations; CFDA; 5(-6)-carboxyfluorescein diacetate; G; _i; 3α; type 3 isoform of the α-subunit of the inhibitory guanine nucleotide binding protein; MP; mastoparan; TP10; transportan-10Mastoparan; Secretion; p42/p44 MAP kinase; G protein; Cell penetrating peptide; Signalling

Characterization of nuclear localization signals and cytoplasmic retention region in the nuclear receptor CAR by Yuichiro Kanno; Motoyoshi Suzuki; Takayuki Nakahama; Yoshio Inouye (pp. 215-222).

The constitutive androstane receptor (CAR) is a ligand/activator-dependent transactivation factor that resides in the cytoplasm and forms part of an as yet unidentified protein complex. Upon stimulation, CAR translocates into the nucleus where it modulates the transactivation of target genes. However, CAR exogenously expressed in rat liver RL-34 cells is located in the nucleus even in the absence of activators. By transiently transfecting RL-34 cells with various mutated rat CAR segments, we identified two nuclear localization signals: a basic amino acid-rich sequence (RRARQARRR) between amino acids 100 and 108; and an assembly of noncontiguous residues widely spread over amino acid residues 111 to 320 within the ligand binding domain. A C-terminal leucine-rich segment corresponding to a previously reported murine xenochemical response signal was not found to exhibit nuclear import activity in cultured cells. Using rat primary hepatocytes transfected with various CAR segments, we identified the region required for the cytoplasmic retention of CAR. Based on these results, the intracellular localization of CAR would be determined by the combined effects of nuclear localization signals, the xenochemical response signal, and the cytoplasmic retention region.

Keywords: Constitutive androstane receptor; Nuclear localization signal; RL-34 cell

Genomic analysis of the protein secretion systems in Clostridium acetobutylicum ATCC 824 by Micka�l Desvaux; Arshad Khan; Anthony Scott-Tucker; Roy R. Chaudhuri; Mark J. Pallen; Ian R. Henderson (pp. 223-253).

Consistent information about protein secretion in Gram-positive bacteria is essentially restricted to the model organism Bacillus subtilis. Among genome-sequenced clostridia, Clostridium acetobutylicum has been the most extensively studied from a physiological point of view and is the organism for which the largest variety of molecular biology tools have been developed. Following in silico analyses, both secreted proteins and protein secretion systems were identified. The Tat (Twin arginine translocation; TC #2.A.64) pathway and ABC (ATP binding cassette) protein exporters (TC #3.A.1.) could not be identified, but the Sec (secretion) pathway (TC #3.A.5) appears to be used prevalently. Similarly, a flagella export apparatus (FEA; TC #3.A.6.), holins (TC #1.E.), and an ESAT-6/WXG100 (early secreted antigen target of 6 kDa/proteins with a WXG motif of ∼100 residues) secretion system were identified. Here, we report for the first time the identification of a fimbrilin protein exporter (FPE; TC #3.A.14) and a Tad (tight adherence) export apparatus in C. acetobutylicum. This investigation highlights the potential use of this saprophytic bacterium in biotechnological and biomedical applications as well as a model organism for studying protein secretion in pathogenic Gram-positive bacteria.

Keywords: Clostridium; Protein secretion system; Virulence factor; Gram-positive bacteria; Secretome

Gα selectivity and inhibitor function of the multiple GoLoco motif protein GPSM2/LGN by Christopher R. McCudden; Francis S. Willard; Randall J. Kimple; Christopher A. Johnston; Melinda D. Hains; Miller B. Jones; David P. Siderovski (pp. 254-264).

GPSM2 (G-protein signalling modulator 2; also known as LGN or mammalian Pins) is a protein that regulates mitotic spindle organization and cell division. GPSM2 contains seven tetratricopeptide repeats (TPR) and four Gα_i/o–Loco (GoLoco) motifs. GPSM2 has guanine nucleotide dissociation inhibitor (GDI) activity towards both Gα_o- and Gα_i-subunits; however, a systematic analysis of its individual GoLoco motifs has not been described. We analyzed each of the four individual GoLoco motifs from GPSM2, assessing their relative binding affinities and GDI potencies for Gα_i1, Gα_i2, and Gα_i3 and Gα_o. Each of the four GPSM2 GoLoco motifs (36–43 amino acids in length) was expressed in bacteria as a GST-fusion protein and purified to homogeneity. The binding of each of the four GST–GoLoco motifs to Gα_i1-, Gα_o-, and Gα_s-subunits was assessed by surface plasmon resonance; all of the motifs bound Gα_i1, but exhibited low affinity towards Gα_o. GDI activity was assessed by a fluorescence-based nucleotide-binding assay, revealing that all four GoLoco motifs are functional as GDIs for Gα_i1, Gα_i2, and Gα_i3. Consistent with our binding studies, the GDI activity of GPSM2 GoLoco motifs on Gα_o was significantly lower than that toward Gα_i1, suggesting that the in vivo targets of GPSM2 are most likely to be Gα_i-subunits.

Keywords: Abbreviations; BODIPY®; 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; GDI; guanine nucleotide dissociation inhibitor; GL1, GL2, GL3, GL4; individual GPSM2 GoLoco motifs; GoLoco; Gα; _i/o; –Loco interaction; GPSM2; G-protein signalling modulator 2; GST; glutathione; S; -transferase; GTPγS; guanosine 5′-[γ-thio]triphosphate; RFU; relative fluorescence units; RGS; regulator of G-protein signalling; RU; resonance units; SPR; surface plasmon resonanceGoLoco; G-protein; GPSM2; LGN

Differential expression of EDF-1 and endothelial nitric oxide synthase by proliferating, quiescent and senescent microvascular endothelial cells by Daniela Bernardini; Erica Ballabio; Massimo Mariotti; Jeanette A.M. Maier (pp. 265-272).

Endothelial Differentiation-related Factor (EDF)-1 is a low molecular weight polypeptide downregulated in endothelial cells exposed to HIV-1-Tat or the phorbol ester TPA. EDF-1 acts in the cytosol as a calmodulin binding protein, and in the nucleus as a transcriptional coactivator. Here, we show that EDF-1 is downregulated in non-proliferating microvascular endothelial cells. Indeed, both quiescence and senescence reduce the levels of EDF-1 and this is due to protein degradation through the proteasome. We also describe a different subcellular localization of EDF-1 which is mainly nuclear in senescent 1G11 cells. Since (i) endothelial nitric oxide (NO) seems to play a role in endothelial proliferation and (ii) NO is an important mediator involved in the control of vascular tone, inflammatory responses and angiogenesis, it is noteworthy that senescence downregulates the expression and the activity of endothelial nitric oxide synthase (eNOS) in microvascular endothelial cells. On the contrary, quiescence does not affect NOS expression and activity. The modulation of EDF-1 in microvascular endothelial cells might offer new insights into the molecular events involved in angiogenesis and in microvascular dysfunctions in the elderly.

Keywords: Abbreviations; EDF; Endothelial differentiation-related Factor; NO; nitric oxide; eNOS; endothelial nitric oxide synthase; TAF; TATA binding protein-associated factor; PD; population doublings; HUVEC; human umbilical vein endothelial cells; TBP; TATA Binding ProteinEDF-1; Endothelial cell; Quiescence; Senescence; Nitric oxide; Angiogenesis

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BBA - Molecular Cell Research (v.1745, #2)