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BBA - Molecular Cell Research (v.1823, #7)

Editorial Board (pp. i).

The relevance of the non-canonical PTS1 of peroxisomal catalase by Chris Williams; Eda Bener Aksam; Katja Gunkel; Marten Veenhuis; Ida J. van der Klei (pp. 1133-1141).

Catalase is sorted to peroxisomes via a C-terminal peroxisomal targeting signal 1 (PTS1), which binds to the receptor protein Pex5. Analysis of the C-terminal sequences of peroxisomal catalases from various species indicated that catalase never contains the typical C-terminal PTS1 tripeptide‐SKL, but invariably is sorted to peroxisomes via a non-canonical sorting sequence. We analyzed the relevance of the non-canonical PTS1 of catalase of the yeast Hansenula polymorpha (‐SKI). Using isothermal titration microcalorimetry, we show that the affinity of H. polymorpha Pex5 for a peptide containing ‐SKI at the C-terminus is 8-fold lower relative to a peptide that has a C-terminal ‐SKL. Fluorescence microscopy indicated that green fluorescent protein containing the ‐SKI tripeptide (GFP-SKI) has a prolonged residence time in the cytosol compared to GFP containing ‐SKL. Replacing the ‐SKI sequence of catalase into ‐SKL resulted in reduced levels of enzymatically active catalase in whole cell lysates together with the occurrence of catalase protein aggregates in the peroxisomal matrix. Moreover, the cultures showed a reduced growth yield in methanol-limited chemostats. Finally, we show that a mutant catalase variant that is unable to properly fold mislocalizes in protein aggregates in the cytosol. However, by replacing the PTS1 into ‐SKL the mutant variant accumulates in protein aggregates inside peroxisomes. Based on our findings we propose that the relatively weak PTS1 of catalase is important to allow proper folding of the enzyme prior to import into peroxisomes, thereby preventing the accumulation of catalase protein aggregates in the organelle matrix.► Peroxisomal catalases contain non-canonical peroxisomal sorting 1 sequences (PTS1). ► The catalase PTS1 has reduced affinity for Pex5 relative to -SKL. ► The catalase PTS1 results in prolonged residence times in the cytosol. ► Replacing ‐SKI by ‐SKL results in reduced catalase enzyme activity in vivo. ► Replacing ‐SKI by ‐SKL results in accumulation of catalase aggregates in peroxisomes.

Keywords: Catalase; Peroxisome; Yeast; Protein sorting; Protein assembly

The relevance of the non-canonical PTS1 of peroxisomal catalase by Chris Williams; Eda Bener Aksam; Katja Gunkel; Marten Veenhuis; Ida J. van der Klei (pp. 1133-1141).

Keywords: Catalase; Peroxisome; Yeast; Protein sorting; Protein assembly

The MIA pathway: A tight bond between protein transport and oxidative folding in mitochondria by Diana Stojanovski; Piotr Bragoszewski; Agnieszka Chacinska (pp. 1142-1150).

Many newly synthesized proteins obtain disulfide bonds in the bacterial periplasm, the endoplasmic reticulum (ER) and the mitochondrial intermembrane space. The acquisition of disulfide bonds is critical for the folding, assembly and activity of these proteins. Spontaneous oxidation of thiol groups is inefficient in vivo, therefore cells have developed machineries that catalyse the oxidation of substrate proteins. The identification of the machinery that mediates this process in the intermembrane space of mitochondria, known as MIA (mitochondrial intermembrane space assembly), provided a unique mechanism of protein transport. The MIA machinery introduces disulfide bonds into incoming intermembrane space precursors and thus tightly couples the process of precursor translocation to precursor oxidation. We discuss our current understanding of the MIA pathway and the mechanisms that oversee thiol-exchange reactions in mitochondria.► Assembly of the mitochondrial intermembrane space requires the MIA pathway. ► MIA drives the accumulation of proteins in the mitochondrial intermembrane space. ► MIA catalyses disulfide bond formation in the precursor proteins. ► The MINOS component regulates MIA-dependent mitochondrial protein biogenesis.

Keywords: Abbreviations; ERV1; essential for respiration and vegetative growth; MIA; mitochondrial intermembrane space assembly; PDI; protein disulfide isomerase; SAM; sorting and assembly machinery; TIM; translocase of the inner membrane; Δψ; membrane potential; TOM; translocase of the outer membraneDisulfide bond formation and transfer; Erv1; Mia40; Protein import; Protein translocation; Redox

The MIA pathway: A tight bond between protein transport and oxidative folding in mitochondria by Diana Stojanovski; Piotr Bragoszewski; Agnieszka Chacinska (pp. 1142-1150).

Neutrophils augment LPS-mediated pro-inflammatory signaling in human lung epithelial cells by Agnes W. Boots; Kirsten Gerloff; Bartholome Roger Bartholomé; Damien van Berlo; Kirstin Ledermann; Guido R.M.M. Haenen; Aalt Bast; Frederik-Jan van Schooten; Catrin Albrecht; Roel P.F. Schins (pp. 1151-1162).

The role of polymorphonuclear neutrophils in pulmonary host defense is well recognized. The influence of a pre-existing inflammation driven by neutrophils (neutrophilic inflammation) on the airway epithelial response toward pro-inflammatory exogenous triggers, however, is still poorly addressed. Therefore, the aim of the present study is to investigate the effect of neutrophils on lipopolysaccharide (LPS)-induced pro-inflammatory signaling in lung epithelial cells. Additionally, underlying signaling pathways are examined.Human bronchial epithelial cells (BEAS-2B) were co-incubated with human peripheral blood neutrophils or bone-marrow derived neutrophils from either C57BL/6J wild type or nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase deficient (p47^phox−/−) mice. Upon stimulation with LPS, interleukin (IL)-8 production and reactive oxygen species (ROS) generation were measured. Additionally, activation of the extracellular signal-regulated kinases (ERK) 1/2 and nuclear factor (NF)-κB signaling pathways was analyzed.Our studies show that the presence of neutrophils synergistically increases LPS-induced IL-8 and ROS production by BEAS-2B cells without inducing cytotoxicity. The observed IL-8 response to endotoxin increases in proportion to time, LPS-concentration and the number of neutrophils present. Moreover, this synergistic IL-8 production strongly correlated with the chemotactic properties of the co-incubations and significantly depended on a functional neutrophilic NADPH oxidase. The presence of neutrophils also augments LPS-induced phosphorylation of ERK1/2 and IκBα as well as NF-κB RelA DNA binding activity in BEAS-2B cells.Our results indicate that the pro-inflammatory effects of LPS toward lung epithelial cells are amplified during a pre-existing neutrophilic inflammation. These findings support the concept that patients suffering from pulmonary neutrophilic inflammation are more susceptible toward exogenous pro-inflammatory triggers.► We investigate the influence of a pre-existing neutrophilic inflammation on airway epithelial defense. ► Neutrophils increase IL-8 and ROS productions by human lung epithelial cells. ► The interaction involves ERK1/2 and NF-κB signaling in the epithelial cells. ► The interaction depends on a functional neutrophilic NADPH oxidase. ► Neutrophilic inflammation augments the response to inflammatory triggers.

Keywords: Abbreviations; BEAS-2B; human bronchial epithelial cultured cell line; CPH; 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine; DUOX1/2; dual oxidases 1 and 2; EPR; electron paramagnetic resonance; ERK1/2; extracellular signal-regulated kinases 1/2; H; ₂; O; ₂; hydrogen peroxide; IκBα; inhibitory part of NF-κB; IL; interleukin; KO; p47; ^phox−/−; knockout; LPS; lipopolysaccharide; NF-κB; nuclear factor-κB; NOX; NADPH oxidase; PMN; polymorphonuclear neutrophils; ROS; reactive oxygen species; TLR; toll-like receptor; WT; wild typeBEAS-2B cell; Interleukin 8; NADPH oxidase; Reactive oxygen species; Synergism

Insulin-like growth factor-1 (IGF-1) inhibits the basolateral Cl channels in the thick ascending limb of the rat kidney by Lijun Wang; Wennan Li; Shumin Kong; Peng Wu; Chengbiao Zhang; Li Gu; Mingxiao Wang; WenHui Wang; Ruimin Gu (pp. 1163-1169).

The aim of the present study is to test the hypothesis that insulin-like-growth factor-1 (IGF-1) plays a role in the regulation of basolateral Cl channels in the thick ascending limb (TAL). The patch-clamp experiments demonstrated that application of IGF-I or insulin inhibited the basolateral 10-pS Cl channels. However, the concentration of insulin required for the inhibition of the Cl channels by 50% (K_1/2) was ten times higher than those of IGF-1. The inhibitory effect of IGF-I on the 10-pS Cl channels was blocked by suppressing protein tyrosine kinase or by blocking phosphoinositide 3-kinase (PI3K). In contrast, inhibition of phospholipase C (PLC) failed to abolish the inhibitory effect of IGF-1 on the Cl channels in the TAL. Western blot analysis demonstrated that IGF-1 significantly increased the phosphorylation of phospholipid-dependent kinase (PDK) at serine residue 241 (Ser²⁴¹) and AKT at Ser⁴⁷³ in the isolated medullary TAL. Moreover, inhibition of PI3K with LY294002 abolished the effect of IGF-1 on the phosphorylation of PDK and AKT. The notion that the effect of IGF-1 on the 10-pS Cl channels was induced by stimulation of PDK–AKT–mTOR pathway was further suggested by the finding that rapamycin completely abolished the effect of IGF-1 on the 10-pS Cl channels in the TAL. We conclude that IGF-1 inhibits the basolateral Cl channels by activating PI3K–AKT–mTOR pathways. The inhibitory effect of IGF-1 on the Cl channels may play a role in ameliorating the ischemia-induced renal injury through IGF-1 administration.►The patch-clamp experiments were performed in the thick ascending limb (TAL). ►Application of 200nM IGF-1 inhibited the basolateral Cl channels in the TAL. ►The basolateral Cl channel is a rate limiting step for Cl exit in the TAL cells. ►Rising intracellular Cl level leads to inhibition of trans-cellular Cl transport. ►IGF-1 at high concentration may inhibit epithelial transport in the TAL.

Keywords: Phosphoinositide 3-kinase; PKD; AKT; mTOR; Cl transport

NDM29, a RNA polymerase III-dependent non coding RNA, promotes amyloidogenic processing of APP and amyloid β secretion by Sara Massone; Eleonora Ciarlo; Serena Vella; Mario Nizzari; Tullio Florio; Claudio Russo; Ranieri Cancedda; Aldo Pagano (pp. 1170-1177).

Neuroblastoma Differentiation Marker 29 (NDM29) is a RNA polymerase (pol) III-transcribed non-coding (nc) RNA whose synthesis drives neuroblastoma (NB) cell differentiation to a nonmalignant neuron-like phenotype. Since in this process a complex pattern of molecular changes is associated to plasma membrane protein repertoire we hypothesized that the expression of NDM29 might influence also key players of neurodegenerative pathways. In this work we show that the NDM29-dependent cell maturation induces amyloid precursor protein (APP) synthesis, leading to the increase of amyloid β peptide (Aβ) secretion and the concomitant increment of Aβ x-42/Aβ x-40 ratio. We also demonstrate that the expression of NDM29 RNA, and the consequent increase of Aβ formation, can be promoted by inflammatory stimuli (and repressed by anti-inflammatory drugs). Moreover, NDM29 expression was detected in normal human brains although an abnormal increased synthesis of this ncRNA is induced in patients affected by neurodegenerative diseases. Therefore, the complex of events triggered by NDM29 expression induces a condition that favors the formation of Aβ peptides in the extracellular space, as it may occur in Alzheimer's Disease (AD). In addition, these data unexpectedly show that a pol III-dependent small RNA can act as key regulator of brain physiology and/or pathology suggesting that a better knowledge of this portion of the human transcriptome might provide hints for neurodegeneration studies.

Keywords: RNA polymerase III; Alzheimer's disease; Beta amyloid; Neuroblastoma; Non-coding RNA

A role for the vesicle-associated tubulin binding protein ARL6 (BBS3) in flagellum extension in Trypanosoma brucei by Helen P. Price; Michael R. Hodgkinson; Megan H. Wright; Edward W. Tate; Barbara A. Smith; Mark Carrington; Meg Stark; Deborah F. Smith (pp. 1178-1191).

The small GTPase Arl6 is implicated in the ciliopathic human genetic disorder Bardet–Biedl syndrome, acting at primary cilia in recruitment of the octomeric BBSome complex, which is required for specific trafficking events to and from the cilium in eukaryotes. Here we describe functional characterisation of Arl6 in the flagellated model eukaryote Trypanosoma brucei, which requires motility for viability. Unlike human Arl6 which has a ciliary localisation, TbARL6 is associated with electron-dense vesicles throughout the cell body following co-translational modification by N-myristoylation. Similar to the related protein ARL-3A in T. brucei, modulation of expression of ARL6 by RNA interference does not prevent motility but causes a significant reduction in flagellum length. Tubulin is identified as an ARL6 interacting partner, suggesting that ARL6 may act as an anchor between vesicles and cytoplasmic microtubules. We provide evidence that the interaction between ARL6 and the BBSome is conserved in unicellular eukaryotes. Overexpression of BBS1 leads to translocation of endogenous ARL6 to the site of exogenous BBS1 at the flagellar pocket. Furthermore, a combination of BBS1 overexpression and ARL6 RNAi has a synergistic inhibitory effect on cell growth. Our findings indicate that ARL6 in trypanosomes contributes to flagellum biogenesis, most likely through an interaction with the BBSome.Display Omitted► The BBSome-associated protein ARL6 localises to vesicles in Trypanosoma brucei. ► T. brucei ARL6 is N-myristoylated. ► RNAi knockdown causes a decrease in flagellum length but does not affect motility. ► TbARL6 binds to tubulin and has a relatively low affinity for guanine nucleotides. ► The BBSome subunit BBS1 and ARL6 are functionally linked in trypanosomes.

Keywords: Abbreviations; Arf; ADP-ribosylation factor; Arl; ADP-ribosylation factor-like; Arl6ip; Arl6 interacting protein; BBS; Bardet–Biedl syndrome; BBS1; Bardet–Biedl syndrome 1 protein; BSF; bloodstream form; ConA; Concanavalin A; GEF; guanine nucleotide exchange factor; GPCR; G-protein coupled receptor; HRG4; human retinal gene 4; IFT; intraflagellar transport; ITC; isothermal titration calorimetry; MANT; N; -methylanthraniloyl; MAP2; microtubule associated protein 2; NES; nuclear export signal; NLS; nuclear localisation signal; NMT; myristoyl-CoA:protein; N; -myristoyltransferase; PCF; procyclic form; PCM1; pericentriolar material 1; PFR; paraflagellar rod; PM; plasma membrane; RNAi; RNA interference; RP2; retinitis pigmentosa protein 2; TAP; tandem affinity purification; TiEM; transmission immuno-electron microscopy Trypanosoma brucei; Arl6; BBSome; BBS1; Flagellum; Tubulin

Scavenger receptor A (SR-A) is required for LPS-induced TLR4 mediated NF-κB activation in macrophages by Honghui Yu; Tuanzhu Ha; Li Liu; Xiaohui Wang; Ming Gao; Jim Kelley; Race Kao; David Williams; Chuanfu Li (pp. 1192-1198).

Recent evidence suggests that the macrophage scavenger receptor class A (SR-A, aka, CD204) plays a role in the induction of innate immune and inflammatory responses. We investigated whether SR-A will cooperate with Toll-like receptors (TLRs) in response to TLR ligand stimulation. Macrophages (J774/a) were treated with Pam2CSK4, (TLR2 ligand), Polyinosinic:polycytidylic acid (Poly I:C) (TLR3 ligand), and Lipopolysaccharides (LPS) (TLR4 ligand) for 15min in the presence or absence of fucoidan (the SR-A ligand). The levels of phosphorylated IκBα (p-IκBα) were examined by Western blot. We observed that Poly I:C and LPS alone, but not Pam2CSK4 or fucoidan increased the levels of p-IκBα. However, LPS-induced increases in p-IκBα levels were further enhanced when presence of the fucoidan. Immunoprecipitation and double fluorescent staining showed that LPS stimulation promotes SR-A association with TLR4 in the presence of fucoidan. To further confirm our observation, we isolated peritoneal macrophages from SR-A deficient (SR-A^−/−), TLR4^−/− and wild type (WT) mice, respectively. The peritoneal macrophages were treated with LPS for 15min in the presence and absence of fucoidan. We observed that LPS-stimulated TNFα and IL-1β production was further enhanced in the WT macrophages, but did not in either TLR4^−/− or SR-A^−/− macrophages, when fucoidan was present. Similarly, in the presence of fucoidan, LPS-induced IκBα phosphorylation, NF-κB binding activity, and association between TLR4 and SR-A were significantly enhanced in WT macrophages compared with LPS stimulation alone. The data suggests that SR-A is needed for LPS-induced inflammatory responses in macrophages.► LPS-stimulated NF-κB activation and inflammatory cytokine production were further increased when SR-A ligand was present. ► Presence of SR-A ligand further enhanced LPS-promoted the association of SR-A with TLR4. ► Deficiency of either SR-A or TLR4 reduced the responses of macrophages to LPS challenge. ► Presence of SR-A is necessary for the amplification of LPS-induced response in macrophages.

Keywords: SR-A; TLR4; LPS; Macrophage; NF-κB

The chloroethylating anticancer drug ACNU induces FRA1 that is involved in drug resistance of glioma cells by Ruth Meise; Maja T. Tomicic; Bernd Kaina; Markus Christmann (pp. 1199-1207).

FRA1 belongs, together with c-Fos and FosB, to the family of Fos proteins that form with members of the ATF and Jun family the transcription factor AP-1 (activator protein 1). Previously we showed that c-Fos protects mouse embryonic fibroblasts against the cytotoxic effects of ultraviolet (UV) light by induction of the endonuclease XPF, leading to enhanced nucleotide excision repair (NER) activity. Here, we analyzed the regulation of FRA1 in glioma cells treated with the anticancer drug nimustine (ACNU) and its role in ACNU-induced toxicity. We show that FRA1 is upregulated in glioblastoma cells following ACNU on mRNA and protein levels. Knockdown of FRA1 by either siRNA or shRNA clearly sensitized glioma cells towards ACNU-induced cell death. Despite decreased AP-1 binding activity upon FRA1 knockdown, this effect is independent on regulation of the AP-1 target genes fasL, ercc1 and xpf. In addition, FRA1 knockdown does not affect DNA repair capacity. However, lack of FRA1 attenuated the ACNU-induced phosphorylation of CHK1 and led to a reduced arrest of cells in G2/M and, thereby, presumably leads to enhanced cell death in the subsequent cell cycle.► Fra1 is activated by the anticancer drug nimustine. ► Downmodulation of FRA1 sensitizes glioma cells to nimustine. ► Downmodulation of FRA1 does not alter the regulation of the AP-1 targets ercc1, fasL, and xpf and does not impact DNA repair. ► Downmodulation of FRA1 impacts the G2/M cell cycle checkpoint.

Keywords: Abbreviations; AP-1; activator protein 1; FRA1; Fos-related antigen 1; DSB; DNA double-strand breaks; SSB; DNA single-strand breaks; ACNU; nimustine, (1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochlorideFRA1; ACNU; Glioma; Sensitivity; AP-1

The chloroethylating anticancer drug ACNU induces FRA1 that is involved in drug resistance of glioma cells by Ruth Meise; Maja T. Tomicic; Bernd Kaina; Markus Christmann (pp. 1199-1207).

Localization of Ras signaling complex in budding yeast by Fiorella Belotti; Renata Tisi; Chiara Paiardi; Marco Rigamonti; Silvia Groppi; Enzo Martegani (pp. 1208-1216).

In Saccharomyces cerevisiae, cAMP/pKA pathway plays a major role in metabolism, stress resistance and proliferation control. cAMP is produced by adenylate cyclase, which is activated both by Gpr1/Gpa2 system and Ras proteins, regulated by Cdc25/Sdc25 guanine exchange factors and Ira GTPase activator proteins.Recently, both Ras2 and Cdc25 RasGEF were reported to localize not only in plasma membrane but also in internal membranes. Here, the subcellular localization of Ras signaling complex proteins was investigated both by fluorescent tagging and by biochemical cell membrane fractionation on sucrose gradients. Although a consistent minor fraction of Ras signaling complex components was found in plasma membrane during exponential growth on glucose, Cdc25 appears to localize mainly on ER membranes, while Ira2 and Cyr1 are also significantly present on mitochondria. Moreover, PKA Tpk1 catalytic subunit overexpression induces Ira2 protein to move from mitochondria to ER membranes. These data confirm the hypothesis that different branches of Ras signaling pathways could involve different subcellular compartments, and that relocalization of Ras signaling complex components is subject to PKA control.► Ras signaling complex is mainly localized on internal membranes during exponential growth on glucose. ► Ras signaling complex components are differentially localized. ► Cdc25 and Ras are present mainly on ER membranes, while Cyr1 and Ira2 are also localized on mitochondria.

Keywords: Abbreviations; cAMP; 3′-5′-cyclic adenosine monophosphate; GEF; guanine exchange factor; GAP; GTPase activating protein; ER; endoplasmic reticulum; PKA; protein kinase A; GPCR; G-protein coupled receptor Saccharomyces cerevisiae; Cdc25; Ira; Adenylate cyclase; Membrane fractionation; Mitochondria

Localization of Ras signaling complex in budding yeast by Fiorella Belotti; Renata Tisi; Chiara Paiardi; Marco Rigamonti; Silvia Groppi; Enzo Martegani (pp. 1208-1216).

Cap binding-independent recruitment of eIF4E to cytoplasmic foci by Paola V. Ferrero; Carla Layana; Ezequiel Paulucci; Gutierrez Pablo Gutiérrez; Hernandez Greco Hernández; Rolando V. Rivera-Pomar (pp. 1217-1224).

Eukaryotic translation initiation factor 4E (eIF4E) is required for cap-dependent initiation. In addition, eIF4E occurs in cytoplasmic foci such as processing bodies (PB) and stress granules (SG). We examined the role of key functional amino acid residues of eIF4E in the recruitment of this protein to cytoplasmic foci. We demonstrate that tryptophan residues required for mRNA cap recognition are not required for the recruitment of eIF4E to SG or PB. We show that a tryptophan residue required for protein–protein interactions is essential for the accumulation of eIF4E in granules. Moreover, we show, by the analysis of two Drosophila eIF4E isoforms, that the tryptophan residue is the common feature for eIF4E for the transfer of active mRNA from polysomes to other ribonucleoprotein particles in the cytoplasm. This residue resides in a putative interaction domain different than the eIF4E-BP domain. We conclude that protein–protein interactions rather than interactions with the mRNA are essential for the recruitment of eIF4E and for a putative nucleation function.► eIF4E is a translation factor detected in both active and inactive ribonucleoprotein complexes. ► Residues required for cap recognition are not required for targeting eIF4E to cytoplasmic granules. ► Inhibition of protein synthesis affects the localization of eIF4E mutants that does not recognize the mRNA cap. ► Localization in cytoplasmic granules requires a residue within eIF4E protein–protein interaction domain. ► The domain is different from the translation factor interaction domain.

Keywords: eIF4E; P-body; Cap-binding; Stress granule

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BBA - Molecular Cell Research (v.1823, #7)