BBA - Molecular Cell Research (v.1793, #10)

Biological cells maintain a myriad of nanopores which, although relying on the same basic small-hole principle, serve a large variety of functions. Here we consider how the nuclear pore complex (NPC), a large nanopore mediating the traffic between genetic material and protein synthesizing apparatus, is functionalized to carry out a set of transport functions. A major parameter of NPC functionalization is a lining of it external and internal surfaces with so-called phenylalanine glycine (FG) proteins. FG proteins integrate a multitude of transport factor binding sites into intrinsically disordered domains. This surprising finding has given rise to a number of transport models assigning direct gating functions to FG proteins. However, recent data suggest that the properties of FG proteins cannot be properly assessed by considering only the purified, transport-factor-stripped NPC. At physiological conditions transport factors may shape FG proteins in a way allotting an essential role to surface diffusion, reconciling tight binding with efficient transport. Thus, NPC studies are revealing both general traits and novel aspects of nanopore functionalization. In addition, they inspire artificial molecule sorters for proteomic and pharmaceutical applications.
Keywords: Intrinsically disordered protein; Membrane transport; Nanopore; Nuclear pore complex; Reduction of dimensionality;

There has been increasing evidence pointing to the mitochondrial respiratory chain (MRC) as a novel and important target for the actions of 17β-estradiol (E2) and estrogen receptors (ER) in a number of cell types and tissues that have high demands for mitochondrial energy metabolism. This novel E2-mediated mitochondrial pathway involves the cooperation of both nuclear and mitochondrial ERα and ERβ and their co-activators on the coordinate regulation of both nuclear DNA- and mitochondrial DNA-encoded genes for MRC proteins. In this paper, we have: 1) comprehensively reviewed studies that reveal a novel role of estrogens and ERs in the regulation of MRC biogenesis; 2) discussed their physiological, pathological and pharmacological implications in the control of cell proliferation and apoptosis in relation to estrogen-mediated carcinogenesis, anti-cancer drug resistance in human breast cancer cells, neuroprotection for Alzheimer's disease and Parkinson's disease in brain, cardiovascular protection in human heart and their beneficial effects in lens physiology related to cataract in the eye; and 3) pointed out new research directions to address the key questions in this important and newly emerging area. We also suggest a novel conceptual approach that will contribute to innovative regimens for the prevention or treatment of a wide variety of medical complications based on E2/ER-mediated MRC biogenesis pathway.
Keywords: Anti-cancer drug resistance; Alzheimer's disease; Cataract; Estrogen; Estrogen carcinogenesis in breast cancer; Estrogen protection of cardiovascular disease; Estrogen receptor; Mitochondrial DNA replication; Transcription and translation; Mitochondrial estrogen receptor; Respiratory chain biogenesis; Mitochondrial transcription factor A; Nuclear respiratory factor; Parkinson's disease;

Identification of a nuclear localization signal in the polo box domain of Plk1 by Moon-Sing Lee; Yi-Han Huang; Shu-Ping Huang; Ru-Inn Lin; Shu-Fen Wu; Chin Li (1571-1578).
Polo-like kinase 1 plays an essential role in mitosis and cytokinesis. Expression and nuclear localization of Plk1 during the S phase are necessary for its functions. Although it was reported that a bipartite nuclear localization signal located at the N-terminal kinase domain is required for nuclear import of Plk1, Plk1 carrying mutations in the polo box I of the polo box domain exhibited increased cytoplasmic accumulation. We further showed that the polo box domain was able to confer nuclear import of β-galactosidase in vivo and GST–EGFP in vitro. The import carriers transportin and importin α were found to interact with the polo box domain directly in a Ran–GTP sensitive manner. These results indicate the presence of a nuclear localization signal in the polo box domain. A 38 amino acid sequence with the function of nuclear localization signal was identified to interact with transportin. Our findings demonstrated that a transportin-dependent nuclear localization signal is present in the polo box domain of Plk1, possibly required for efficient nuclear import. Showing little similarity to the M9 sequence, the 38 amino acid sequence identified here likely represents a novel nuclear localization signal.
Keywords: Polo-like kinase 1; Polo box domain; Nuclear localization signal; Transportin; Importin α;

Functional analysis of the posttranslational modifications of the death receptor 6 by Martin Klíma; Jitka Zájedová; Lenka Doubravská; Ladislav Anděra (1579-1587).
Death receptor 6 (DR6/TNFRSF21) is a death domain-containing receptor of the TNFR superfamily with an apparent regulatory function in hematopoietic and neuronal cells. In this study we document that DR6 is an extensively posttranslationally modified transmembrane protein and that N- and O-glycosylations of amino acids in its extracellular part are mainly responsible for its approximately 40 kDa mobility shift in SDS polyacrylamide gels. Site-directed mutagenesis confirmed that all six extracellular asparagines are N-glycosylated and that the Ser/Thr/Pro cluster in the “stalk” domain juxtaposed to the cysteine-rich domains (CRDs) is a major site for the likely mucine-type of O-glycosylation. Deletion of the entire linker region between CRDs and the transmembrane domain, spanning over 130 amino acids, severely compromises the plasma membrane localization of DR6 and leads to its intracellular retention. Biosynthetic labeling with radiolabeled palmitate and side-directed mutagenesis also revealed that the membrane-proximal Cys368 in the intracellular part of DR6 is, similarly as cysteines in Fas/CD95 or DR4 ICPs, S-palmitoylated. However, palmitoylation of Cys368 is apparently not required for DR6 targeting into Brij-98 insoluble lipid rafts. In contrast, we show that N-glycosylation of the extracellular part might participate in directing DR6 into these membrane microdomains.
Keywords: Death receptor; TNFRSF21; Glycosylation; Palmitoylation; Lipid rafts;

The human thioredoxin reductase-1 splice variant TXNRD1_v3 is an atypical inducer of cytoplasmic filaments and cell membrane filopodia by Pauliina E. Damdimopoulou; Antonio Miranda-Vizuete; Elias S.J. Arnér; Jan-Åke Gustafsson; Anastasios E. Damdimopoulos (1588-1596).
Thioredoxin reductases are important selenoproteins maintaining cellular redox balance and regulating several redox dependent processes in apoptosis, cell proliferation and differentiation. Specific functions of dedicated splice variants may add further complexity to the functions of these proteins. We show here that a splice variant of human thioredoxin reductase 1, TXNRD1_v3, forms both dynamic cytoplasmic filaments and provokes instantaneous formation of dynamic cell membrane protrusions identified as filopodia. Using truncated versions of the protein we found that both the cytoplasmic filaments and the filopodia formation were exclusively dependent on the glutaredoxin domain of the protein. Interestingly, actin polymerization was required for filopodia formation triggered by TXNRD1_v3, but not for generation of cytoplasmic filaments. We conclude that the glutaredoxin domain of TXNRD1_v3 is an atypical regulator of the cell cytoskeleton that potently induces formation of highly ordered cytoplasmic filaments and cell membrane filopodia.
Keywords: Thioredoxin reductase; Cytoskeleton; Actin; Filopodia;

Hsp90 transcriptionally and post-translationally regulates the expression of NDRG1 and maintains the stability of its modifying kinase GSK3β by Vanessa M. Banz; Michaela Medová; Adrian Keogh; Cynthia Furer; Yitzhak Zimmer; Daniel Candinas; Deborah Stroka (1597-1603).
N-myc downstream-regulated gene 1 (NRDG1) is a stress-induced protein whose putative function is suppression of tumor metastasis. A recent proteonomic study showed NDRG1 interacts with the molecular chaperone heat shock protein 90 (Hsp90). From their reported association, we investigated if NDRG1 is dependent on Hsp90 for its stability and is therefore a yet unidentified Hsp90 client protein. Here, we demonstrate that endogenous NDRG1 and Hsp90 physically associate in hepatocellular cancer cell lines. However, geldanamycin (GA)-mediated inhibition of Hsp90 did not disrupt their interaction or result in NDRG1 protein destabilization. On the contrary, inhibition of Hsp90 led to a transcriptional increase of NDRG1 protein which was associated with cell growth arrest. We also observed that GA inhibited the phosphorylation of NDRG1 by targeting its regulating kinases, serum- and glucocorticoid-induced kinase 1 (SGK1) and glycogen synthase kinase 3 β (GSK3β). We demonstrate that in the presence of GA, GSK3β protein and activity were decreased thus indicating that Hsp90 is necessary for GSK3β stability. Taken together, our data demonstrate that NDRG1 is not a classic client protein but interacts with Hsp90 and is still dually regulated by Hsp90 at a transcriptional and post-translational level. Finally, we suggest for the first time GSK3β as a new client protein of Hsp90.
Keywords: NDRG1; HSP90; SKG1; GSK3β; Geldanamycin; Phosphorylation;

Neuronal activity is highly dependent on energy metabolism; yet, the two processes have traditionally been regarded as independently regulated at the transcriptional level. Recently, we found that the same transcription factor, nuclear respiratory factor 1 (NRF-1) co-regulates an important energy-generating enzyme, cytochrome c oxidase, as well as critical subunits of glutamatergic receptors. The present study tests our hypothesis that the co-regulation extends to the next level of glutamatergic synapses, namely, neuronal nitric oxide synthase, which generates nitric oxide as a downstream signaling molecule. Using in silico analysis, electrophoretic mobility shift assay, chromatin immunoprecipitation, promoter mutations, and NRF-1 silencing, we documented that NRF-1 functionally bound to Nos1, but not Nos2 (inducible) and Nos3 (endothelial) gene promoters. Both COX and Nos1 transcripts were up-regulated by depolarizing KCl treatment and down-regulated by TTX-mediated impulse blockade in neurons. However, NRF-1 silencing blocked the up-regulation of both Nos1 and COX induced by KCl depolarization, and over-expression of NRF-1 rescued both Nos1 and COX transcripts down-regulated by TTX. These findings are consistent with our hypothesis that synaptic neuronal transmission and energy metabolism are tightly coupled at the molecular level.
Keywords: Gene regulation; EMSA; NRF-1 over-expression; Rat primary neuron; Transcription factor; TTX;

Store-operated Ca2+ entry is sensitive to the extracellular Ca2+ concentration through plasma membrane STIM1 by Isaac Jardín; José J. López; Pedro C. Redondo; Ginés M. Salido; Juan A. Rosado (1614-1622).
Store-operated Ca2+ entry (SOCE) is a major mechanism for Ca2+ influx in platelets and other cells activated by a reduction in Ca2+ concentration in the intracellular stores. SOCE has been reported to be regulated by extracellular Ca2+, although the underlying mechanism remains unclear. Here we have examined the involvement of plasma membrane-located STIM1 (PM-STIM1) in the regulation of SOCE by extracellular Ca2+. Treatment of platelets with the SERCA inhibitor thapsigargin (TG) induced Mn2+ entry, which was inhibited by extracellular Ca2+ in a concentration-dependent manner. Incubation of platelets with a specific antibody, which recognizes the extracellular amino acid sequence 25–139 of PM-STIM1 that contains the Ca2+-binding domain, prevented the inactivation of Ca2+ entry induced by extracellular Ca2+. TG induced translocation of STIM1 to the plasma membrane (PM), an event that was found to be Ca2+-dependent. In addition, TG stimulated association of PM-STIM1 with Orai1, an event that was not prevented by stabilization of the membrane cytoskeleton using jasplakinolide. These findings suggest that PM-STIM1 is important for the inactivation of SOCE by extracellular Ca2+, an event that is likely to be mediated by interaction with Orai1.
Keywords: STIM1; Ca2+ entry; Orai1; Platelet; Manganese;