BBA - Molecular Cell Research (v.1864, #5)
Editorial Board (i).
Cellular vacuolization caused by overexpression of the PIKfyve-binding deficient Vac14L156R is rescued by starvation and inhibition of vacuolar-ATPase by Ulf Schulze; Beate Vollenbröker; Alexander Kühnl; Daniel Granado; Samet Bayraktar; Ursula Rescher; Hermann Pavenstädt; Thomas Weide (749-759).
Phosphoinositides (PI) and converting enzymes are crucial determinants of organelle identity and morphology. One important endolysosomal specific PI is PI(3,5)P2, generated by the PIKfyve kinase, which orchestrates in combination with Vac14 and Fig4. Dysfunction of this complex leads to large intracellular vacuoles in various cell types and is linked to neurological diseases. Here, we characterize the vacuolization phenotype caused by overexpression of the PIKfyve binding deficient mutant Vac14L156R in podocytes, which represent specialized cells of the kidney. Vacuolization of podocytes, which was associated with strong maturation defects in the endolysosomal system, could be completely rescued by starvation or treatment of cells with the v-ATPase inhibitor Bafilomycin A1. Moreover, we elucidated a strong and reversible de-vacuolization effect of the cholesterol export inhibitor U18666A, which was accompanied by increased basification of the lysosomal pH values. Taken together, our data give new hints to potential therapeutic targets in the treatment of disease linked to intracellular vacuolization.
SMN1 functions as a novel inhibitor for TRAF6-mediated NF-κB signaling by Eun Kyung Kim; Eui-Ju Choi (760-770).
Survival motor neuron (SMN) is a 38-kDa protein, whose deficiency in humans develops spinal muscular atrophy (SMA), an autosomal recessive neurodegenerative disease with muscular atrophy due to motor neuron death in the spinal cord. We now report that SMN prevents the activation of TRAF6 and IκB kinase (IKK) and thereby negatively regulates the NF-κB signaling processes. SMN physically interacted with TRAF6 and with each component of the IKK complex, IKK-α, IKK-β, and IKK-γ in BV2 microglia cells. Moreover, SMN1 inhibited the E3 ubiquitin ligase activity of TRAF6 as well as the kinase activity of IKK. Furthermore, depletion of endogenous SMN by RNA interference enhanced the IL-1β-induced activation of IKK and production of inflammatory mediators such as TNF-α and nitric oxide in BV2 cells. Consistently, the potentiation of IL-1β-induced IKK activity was also found in SMA patient fibroblasts, compared with that of normal ones. Our results thus suggest that SMN functions as a natural inhibitor for IL-1β-induced NF-κB signaling by targeting TRAF6 and the IKK complex.Display Omitted
Keywords: NF-κB signaling; Spinal muscular atrophy; Survival motor neuron; TRAF6;
The effect and mechanism of inhibiting glucose-6-phosphate dehydrogenase activity on the proliferation of Plasmodium falciparum by Zhiqiang Zhang; Xiaodan Chen; Chengrui Jiang; Zishui Fang; Yi Feng; Weiying Jiang (771-781).
We screened > 40,000 patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency and found that the G6PD Kaiping allele was under the most positive selection for fighting against malaria in the Chinese population. However, the mechanism is unknown. The current study was designed to investigate the anti-malarial effect and mechanism of G6PD deficiency. Dehydroepiandrosterone (DHEA) was utilised for inhibiting the G6PD activity of erythrocytes. Giemsa staining of blood smears and quantitative real-time PCR were used for the detection and quantification of Plasmodium falciparum infection. A transmission electron microscope was used to observe the structural changes of P. falciparum. An atomic force microscopy was used for the analyses of morphology, roughness and Young's Modulus of the infective erythrocyte membrane. When G6PD activity was inhibited by DHEA, the infection rate of P. falciparum decreased, its cell nucleus shrank, the cell organelles and metabolites were reduced gradually and the Young's Modulus of the erythrocyte membrane increased with increasing DHEA concentrations. These data indicated that Plasmodium multiplication would be inhibited in G6PD deficient erythrocytes because the Plasmodium organelles could not obtain enough nutrients, including ribose-5-phosphate and the reducing equivalent, NADPH. Moreover, the Young's Modulus of the erythrocyte membrane increased, which resulted in an increased membrane stiffness and decreased deformation. It was difficult for the merozoites to invade erythrocytes through endocytosis. Understanding these points will have a major effect on searching for new anti-malarial drug targets.
Keywords: Glucose-6-phosphate dehydrogenase deficiency; Plasmodium falciparum; Dehydroepiandrosterone; Malaria; Mechanical properties;
Role of CCL20 mediated immune cell recruitment in NF-κB mediated TRAIL resistance of pancreatic cancer by Claudia Geismann; Frauke Grohmann; Anita Dreher; Robert Häsler; Philip Rosenstiel; Karen Legler; Charlotte Hauser; Jan Hendrik Egberts; Bence Sipos; Stefan Schreiber; Andreas Linkermann; Zonera Hassan; Günter Schneider; Heiner Schäfer; Alexander Arlt (782-796).
Pancreatic ductal adenocarcinoma (PDAC) represents one of the deadliest cancers. From a clinical view, the transcription factor NF-κB is of particular importance, since this pathway confers apoptosis resistance and limits drug efficacy. Whereas the role of the most abundant NF-κB subunit p65/RelA in therapeutic resistance is well documented, only little knowledge of the RelA downstream targets and their functional relevance in TRAIL mediated apoptosis in PDAC is available.In the present study TRAIL resistant and sensitive PDAC cell lines were analyzed for differentially expressed RelA target genes, to define RelA downstream targets mediating TRAIL resistance. The most upregulated target gene was then further functionally characterized.Unbiased genome-wide expression analysis demonstrated that the chemokine CCL20 represents the strongest TRAIL inducible direct RelA target gene in resistant PDAC cells. Unexpectedly, targeting CCL20 by siRNA, blocking antibodies or by downregulation of the sole CCL20 receptor CCR6 had no effect on PDAC cell death or cancer cell migration, arguing against an autocrine role of CCL20 in PDAC. However, by using an ex vivo indirect co-culture system we were able to show that CCL20 acts paracrine to recruit immune cells. Importantly, CCL20-recruited immune cells further increase TRAIL resistance of CCL20-producing PDAC cells.In conclusion, our data show a functional role of a RelA-CCL20 pathway in PDAC TRAIL resistance. We demonstrate how the therapy-induced cross-talk of cancer cells with immune cells affects treatment responses, knowledge needed to tailor novel bi-specific treatments, which target tumor cell as well as immune cells.Display Omitted
Keywords: Apoptosis resistance; TRAIL; Pancreatic cancer; NF-κB; CCL20;
Tight junction protein ZO-1 controls organic cation/carnitine transporter OCTN2 (SLC22A5) in a protein kinase C-dependent way by Dominika Jurkiewicz; Katarzyna Michalec; Krzysztof Skowronek; Katarzyna A. Nałęcz (797-805).
OCTN2 (SLC22A5) is an organic cation/carnitine transporter belonging to the solute carrier transporters (SLC) family. OCTN2 is ubiquitously expressed and its presence was shown in various brain cells, including the endothelial cells forming blood-brain barrier, where it was mainly detected at abluminal membrane and in proximity of tight junctions (TJ). Since OCTN2 contains a PDZ-binding domain, the present study was focused on a possible role of transporter interaction with a TJ-associated protein ZO-1, containing PDZ domains and detected in rat Octn2 proteome. We showed previously that activation of protein kinase C (PKC) in rat astrocytes regulates Octn2 surface presence and activity. Regulation of a wild type Octn2 and its deletion mutant without a PDZ binding motif were studied in heterologous expression system in HEK293 cells. Plasma membrane presence of overexpressed Octn2 did not depend on either PKC activation or presence of PDZ-binding motif, anyhow, as assayed in proximity ligation assay, the truncation of PDZ binding motif resulted in a strongly diminished Octn2/ZO-1 interaction and in a decreased transporter activity. The same effects on Octn2 activity were detected upon PKC activation, what correlated with ZO-1 phosphorylation. It is postulated that ZO-1, when not phosphorylated by PKC, keeps Octn2 in an active state, while elimination of this binding in ΔPDZ mutant or after ZO-1 phosphorylation leads to diminution of Octn2 activity.Display Omitted
Keywords: OCTN2 (SLC22A5); ZO-1, tight junction protein; Protein kinase C;
TRPC1 and TRPC4 channels functionally interact with STIM1L to promote myogenesis and maintain fast repetitive Ca2 + release in human myotubes by Fabrice Antigny; Jessica Sabourin; Sophie Saüc; Laurent Bernheim; Stéphane Koenig; Maud Frieden (806-813).
STIM1 and Orai1 are essential players of store-operated Ca2 + entry (SOCE) in human skeletal muscle cells and are required for adult muscle differentiation. Besides these two proteins, TRPC (transient receptor potential canonical) channels and STIM1L (a longer STIM1 isoform) are also present on muscle cells. In the present study, we assessed the role of TRPC1, TRPC4 and STIM1L in SOCE, in the maintenance of repetitive Ca2 + transients and in muscle differentiation. Knockdown of TRPC1 and TRPC4 reduced SOCE by about 50% and significantly delayed the onset of Ca2 + entry, both effects similar to STIM1L invalidation. Upon store depletion, TRPC1 and TRPC4 appeared to interact preferentially with STIM1L compared to STIM1. STIM1L invalidation affected myoblast differentiation, with the formation of smaller myotubes, an effect similar to what we reported for TRPC1 and TRPC4 knockdown. On the contrary, the overexpression of STIM1L leads to the formation of larger myotubes. All together, these data strongly suggest that STIM1L and TRPC1/4 are working together in myotubes to ensure efficient store refilling and a proper differentiation program.Display Omitted
Keywords: Skeletal muscle; STIM1L; TRPC; Calcium channel; SOCE;
Sphingosine 1-phosphate signaling axis mediates fibroblast growth factor 2-induced proliferation and survival of murine auditory neuroblasts by Marina Bruno; Ilaria Maria Rizzo; Ricardo Romero-Guevara; Caterina Bernacchioni; Francesca Cencetti; Chiara Donati; Paola Bruni (814-824).
Hearing loss affects millions of people in the world. In mammals the auditory system comprises diverse cell types which are terminally differentiated and with no regenerative potential. There is a tremendous research interest aimed at identifying cell therapy based solutions or pharmacological approaches that could be applied therapeutically alongside auditory devices to prevent hair cell and neuron loss.Sphingosine 1-phosphate (S1P) is a pleiotropic bioactive sphingolipid that plays key role in the regulation of many physiological and pathological functions. S1P is intracellularly produced by sphingosine kinase (SK) 1 and SK2 and exerts many of its action consequently to its ligation to S1P specific receptors (S1PR), S1P1–5. In this study, murine auditory neuroblasts named US/VOT-N33 have been used as progenitors of neurons of the spiral ganglion. We demonstrated that the fibroblast growth factor 2 (FGF2)-induced proliferative action was dependent on SK1, SK2 as well as S1P1 and S1P2. Moreover, the pro-survival effect of FGF2 from apoptotic cell death induced by staurosporine treatment was dependent on SK but not on S1PR. Additionally, ERK1/2 and Akt signaling pathways were found to mediate the mitogenic and survival action of FGF2, respectively.Taken together, these findings demonstrate a crucial role for S1P signaling axis in the proliferation and the survival of otic vesicle neuroprogenitors, highlighting the identification of possible novel therapeutical approaches to prevent neuronal degeneration during hearing loss.
Keywords: Auditory neuroblasts; Proliferation; Survival; Sphingosine 1-phosphate; Fibroblast growth factor 2; Sphingosine 1-phosphate receptor;
An insight into the orphan nucleotide sugar transporter SLC35A4 by Paulina Sosicka; Dorota Maszczak-Seneczko; Bożena Bazan; Yauhen Shauchuk; Beata Kaczmarek; Mariusz Olczak (825-838).
SLC35A4 has been classified in the SLC35A subfamily based on amino acid sequence homology. Most of the proteins belonging to the SLC35 family act as transporters of nucleotide sugars. In this study, the subcellular localization of endogenous SLC35A4 was determined via immunofluorescence staining, and it was demonstrated that SLC35A4 localizes mainly to the Golgi apparatus. In silico topology prediction suggests that SLC35A4 has an uneven number of transmembrane domains and its N-terminus is directed towards the Golgi lumen. However, an experimental assay refuted this prediction: SLC35A4 has an even number of transmembrane regions with both termini facing the cytosol. In vivo interaction analysis using the FLIM–FRET approach revealed that SLC35A4 neither forms homomers nor associates with other members of the SLC35A subfamily except SLC35A5. Additional assays demonstrated that endogenous SLC35A4 is 10 to 40 nm proximal to SLC35A2 and SLC35A3. To determine SLC35A4 function SLC35A4 knock-out cells were generated with the CRISPR-Cas9 approach. Although no significant changes in glycosylation were observed, the introduced mutation influenced the subcellular distribution of the SLC35A2/SLC35A3 complexes. Additional FLIM–FRET experiments revealed that overexpression of SLC35A4-BFP together with SLC35A3 and the SLC35A2-Golgi splice variant negatively affects the interaction between the two latter proteins. The results presented here strongly indicate a modulatory role for SLC35A4 in intracellular trafficking of SLC35A2/SLC35A3 complexes.Display Omitted
Keywords: Nucleotide sugar transporters; SLC35A subfamily; Golgi apparatus; Endoplasmic reticulum; Glycosylation;