BBA - Molecular Cell Research (v.1853, #3)
Editorial Board (i).
MAPK signaling triggers transcriptional induction of cFOS during amino acid limitation of HepG2 cells by Jixiu Shan; William Donelan; Jaclyn N. Hayner; Fan Zhang; Elizabeth E. Dudenhausen; Michael S. Kilberg (539-548).
Amino acid (AA) deprivation in mammalian cells activates a collection of signaling cascades known as the AA response (AAR), which is characterized by transcriptional induction of stress-related genes, including FBJ murine osteosarcoma viral oncogene homolog (cFOS). The present study established that the signaling mechanism underlying the AA-dependent transcriptional regulation of the cFOS gene in HepG2 human hepatocellular carcinoma cells is independent of the classic GCN2-eIF2-ATF4 pathway. Instead, a RAS-RAF-MEK-ERK cascade mediates AAR signaling to the cFOS gene. Increased cFOS transcription is observed from 4-24 h after AAR-activation, exhibiting little or no overlap with the rapid and transient increase triggered by the well-known serum response. Furthermore, serum is not required for the AA-responsiveness of the cFOS gene and no phosphorylation of promoter-bound serum response factor (SRF) is observed. The ERK-phosphorylated transcription factor E-twenty six-like (p-ELK1) is increased in its association with the cFOS promoter after activation of the AAR. This research identified cFOS as a target of the AAR and further highlights the importance of AA-responsive MAPK signaling in HepG2 cells.
Keywords: immediate early response genes; nutrient deprivation; hepatocellular carcinoma; GCN2; ATF4;
Onconase induces autophagy sensitizing pancreatic cancer cells to gemcitabine and activates Akt/mTOR pathway in a ROS-dependent manner by Claudia Fiorini; Marco Cordani; Giovanni Gotte; Delia Picone; Massimo Donadelli (549-560).
Onconase® (ONC) is a member of the RNase super-family that is secreted in oocytes and early embryos of Rana pipiens. Over the last years, research interest about this small and basic frog RNase, also called ranpirnase, constantly increased because of its high cytotoxicity and anticancer properties. Onconase is currently used in clinical trials for cancer therapy; however, the precise mechanisms determining cytotoxicity in cancer cells have not yet been fully investigated. In the present manuscript, we evaluate the antitumoral property of onconase in pancreatic adenocarcinoma cells and in non-tumorigenic cells as a control. We demonstrate that ONC stimulates a strong antiproliferative and proapoptotic effect in cancer cells by reporting for the first time that ONC triggers Beclin1-mediated autophagic cancer cell death. In addition, ONC inhibits the expression of mitochondrial uncoupling protein 2 (UCP2) and of manganese-dependent superoxide dismutase (MnSOD) triggering mitochondrial superoxide ion production. ONC-induced reactive oxygen species (ROS) are responsible for Akt/mTOR pathway stimulation determining the sensitivity of cancer cells to mTOR inhibitors and lessening autophagic stimulation. This indicates ROS/Akt/mTOR axis as a strategy adopted by cancer cells to reduce ONC-mediated cytotoxic autophagy stimulation. In addition, we demonstrate that ONC can sensitize pancreatic cancer cells to the standard chemotherapeutic agent gemcitabine allowing a reduction of drug concentration when used in combination settings, thus suggesting a lowering of chemotherapy-related side effects. Altogether, our results shed more light on the mechanisms lying at the basis of ONC antiproliferative effect in cancer cells and support its potential use to develop new anticancer strategies.
Keywords: autophagy; onconase; pancreatic cancer; reactive oxygen species (ROS); mammalian target of rapamycin (mTOR); gemcitabine;
Mitochondrial function contributes to oxysterol-induced osteogenic differentiation in mouse embryonic stem cells by Il Keun Kwon; Sang Cheon Lee; Yu-Shik Hwang; Jung Sun Heo (561-572).
Oxysterols, oxidized derivatives of cholesterol, are biologically active molecules. Specific oxysterols have potent osteogenic properties that act on osteoprogenitor cells. However, the molecular mechanisms underlying these osteoinductive effects on embryonic stem cells (ESCs) are unknown. This study investigated the effect of an oxysterol combination of 22(S)-hydroxycholesterol and 20(S)-hydroxycholesterol (SS) on osteogenic differentiation of ESCs and the alterations to mitochondrial activity during differentiation. Osteogenic differentiation was assessed by alkaline phosphatase (ALP) activity, matrix mineralization, mRNA expression of osteogenic factors, runt-related transcription factor 2, osterix, and osteocalcin, and protein levels of collagen type IA (COLIA) and osteopontin (OPN). Treatment of cells with SS increased osteoinductive activity compared to the control group. Intracellular reactive oxygen species production, intracellular ATP content, mitochondrial membrane potential, mitochondrial mass, mitochondrial DNA copy number, and mRNA expression of peroxisome proliferator-activated receptor-γ coactivators 1α and β, transcription factors involved in mitochondrial biogenesis, were significantly increased during osteogenesis, indicating upregulation of mitochondrial activity. Oxysterol combinations also increased protein levels of mitochondrial respiratory complexes I–V. We also found that SS treatment increased hedgehog signaling target genes, Smo and Gli1 expression. Inhibition of Hh signaling by cyclopamine suppressed mitochondrial biogenesis and ESC osteogenesis. Subsequently, oxysterol-induced Wnt/β-catenin pathways were inhibited by repression of Hh signaling and mitochondrial biogenesis. Transfection of β-catenin specific siRNA decreased the protein levels of COLIA and OPN, as well as ALP activity. Collectively, these data suggest that lipid-based oxysterols enhance differentiation of ESCs toward the osteogenic lineage by regulating mitochondrial activity, canonical Hh/Gli, and Wnt/β-catenin signaling.
Keywords: Embryonic stem cell; Oxysterol; Osteogenic differentiation; Mitochondrion; Hh signaling; Wnt/β-catenin;
Glucocorticoids enhance prolonged clearance of apoptotic cells by upregulating liver X receptor, peroxisome proliferator-activated receptor-δ and UCP2 by Éva Garabuczi; Zsolt Sarang; Zsuzsa Szondy (573-582).
Efficient phagocytic clearance of apoptotic cells (efferocytosis) is essential to prevent the development of chronic inflammation and autoimmunity. Glucocorticoids are widely used in the therapy of chronic inflammatory diseases, and increasing evidence suggests that they act partly via enhancing efferocytosis by macrophages. Glucocorticoids were previously shown to promote both protein S- and MFG-E8-dependent efferocytosis. Since previous studies in our laboratory have demonstrated that glucocorticoids induce the expression of retinaldehyde dehydrogenases in macrophages, in the present experiments the possible involvement of retinoids in the glucocorticoid-induced efferocytosis was studied in mouse bone marrow derived macrophages. Here we show that glucocorticoids promote not only short-term, but also long-term clearance of apoptotic cells. Glucocorticoids seem to directly induce the expression of the phagocytosis-related genes MERTK, C1q, UCP2, and the transcription factor C/EBPβ. C/EBPβ contributes to the further induction of the phagocytosis-related genes, and is required for the induction of lipid sensing receptors LXRs, PPARδ, RARα, RXRα and RALDH1, the latter one in an LXR- and RARα-dependent manner. Glucocorticoid-induced enhancement in long-term efferocytosis was dependent on the induction of lipid sensing receptors known to be triggered by the lipid content of the engulfed cells to enhance phagocytic capacity. Retinoids did not affect the glucocorticoid-induced short term phagocytosis of apoptotic cells, but were required for the glucocorticoid-induced enhancement of efferocytosis during prolonged clearance of apoptotic cells by promoting efficient LXR and PPARδ upregulation. Our data indicate that retinoids could be considered as potential promoters of the efficacy of glucocorticoid treatment in inflammatory diseases.
Keywords: Macrophage; Efferocytosis; Glucocorticoid; LXR; PPARδ; UCP2;
ANKHD1 silencing inhibits Stathmin 1 activity, cell proliferation and migration of leukemia cells by João Agostinho Machado-Neto; Mariana Lazarini; Patricia Favaro; Paula de Melo Campos; Renata Scopim-Ribeiro; Gilberto Carlos Franchi; Alexandre Eduardo Nowill; Paulo Roberto Moura Lima; Fernando Ferreira Costa; Serge Benichou; Sara Teresinha Olalla Saad; Fabiola Traina (583-593).
ANKHD1 is highly expressed in human acute leukemia cells and potentially regulates multiple cellular functions through its ankyrin-repeat domains. In order to identify interaction partners of the ANKHD1 protein and its role in leukemia cells, we performed a yeast two-hybrid system screen and identified SIVA, a cellular protein known to be involved in proapoptotic signaling pathways. The interaction between ANKHD1 and SIVA was confirmed by co-imunoprecipitation assays. Using human leukemia cell models and lentivirus-mediated shRNA approaches, we showed that ANKHD1 and SIVA proteins have opposing effects. While it is known that SIVA silencing promotes Stathmin 1 activation, increased cell migration and xenograft tumor growth, we showed that ANKHD1 silencing leads to Stathmin 1 inactivation, reduced cell migration and xenograft tumor growth, likely through the inhibition of SIVA/Stathmin 1 association. In addition, we observed that ANKHD1 knockdown decreases cell proliferation, without modulating apoptosis of leukemia cells, while SIVA has a proapoptotic function in U937 cells, but does not modulate proliferation in vitro. Results indicate that ANKHD1 binds to SIVA and has an important role in inducing leukemia cell proliferation and migration via the Stathmin 1 pathway. ANKHD1 may be an oncogene and participate in the leukemia cell phenotype.
Keywords: ANKHD1; SIVA1; Stathmin 1; Acute leukemia; Cell proliferation;
New insights into the model of dopamine D1 receptor and G-proteins interactions by Paweł Mystek; Magdalena Tworzydło; Marta Dziedzicka-Wasylewska; Agnieszka Polit (594-603).
The details of the interaction between G-proteins and the GPCRs have been subjected to extensive investigation with structural and functional assays, but still many fundamental questions regarding this macromolecular assembly and its mechanism remain unanswered. In the context of current structural data we investigated interactions of dopamine D1 receptor with cognate G-proteins (Gαs) in living cells, emphasizing the prevalence of preassembled D1-G-protein complexes. We also tested the effect of D1 receptor presence on the dynamics of Gαs and Gαi3 in the cellular plasma membrane. Using fluorescence resonance energy transfer (FRET) detected by fluorescence lifetime imaging microscopy (FLIM) or fluorescence recovery after photobleaching (FRAP) microscopy, we did not detect constitutive preassociated complex between D1 receptor and G-protein in the absence of receptor activation. Our work suggests that D1 receptor alters the distribution of Gαs and Gαi3 subunits inside the membrane. We also find that non-activated D1 receptor and Gαs or Gαi3 are present in the cell membrane within the same membrane microdomains in the proximity of about 9–10 nm.
Keywords: FRAP; FLIM–FRET; G-protein; GPCR; Dopamine receptor; G-protein coupling;
Monitoring of autophagy is complicated—salinomycin as an example by Jaganmohan Reddy Jangamreddy; Soumya Panigrahi; Marek J. Łos (604-610).
Monitoring of autophagy is challenging because of its multiple steps and lack of single befitting technique for a complete mechanistic understanding, which makes the task complicated. Here, we evaluate the functionality of autophagy triggered by salinomycin (anti-cancer stem cell agent) using flow cytometry and advanced microscopy. We show that salinomycin does induce functional autophagy at lower concentrations and such a dose is cell type-dependent. For example, PC3 cells show active autophagic flux at 10 μM concentration of salinomycin while murine embryonic fibroblasts already show an inhibition of flux at such doses. A higher concentration of salinomycin (i.e. 30 μM) inhibits autophagic flux in both cell types. The data confirms our previous findings that salinomycin is an inducer of autophagy, whereas autophagic flux inhibition is a secondary response.
Keywords: Autophagic flux; GFP-LC3; mTandem GFP-RFP LC3; p62/SQSTRM1; Vacuolization; Salinomycin;
Role of dynamin in elongated cell migration in a 3D matrix by Justin G. Lees; Nick N. Gorgani; Alaina J. Ammit; Adam McCluskey; Phillip J. Robinson; Geraldine M. O'Neill (611-618).
The use of 3-dimensional (3D) collagen gels has yielded new insights into the migratory behaviour of cancer cells. While the large GTPase dynamin has emerged as an important regulator of cancer cell migration and invasion under 2D conditions, its role in 3D migration is unclear. We have used a potent dynamin modulator, a bis-tyrphostin derivative, Ryngo® 1-23, to investigate the role of dynamin in 3D migration in 3 different cell lines. The compound specifically inhibits persistent, elongated 3D migration in U87MG and SMA-560 cells. Treated U87MG cells adopt a rounded morphology that is not due to apoptosis, loss of matrix metalloprotease activity or inhibition of clathrin-mediated endocytosis. Given that Ryngo 1-23 is known to regulate dynamin oligomerisation and actin dynamics at the leading edge, we analysed actin filament distribution. Ryngo 1-23 induced a switch in actin filament organization in 3D cultures resulting in the generation of multiple short actin-rich microspikes. Correlated with the change in actin filament distribution, cells displayed reduced collagen gel contraction. Since acto-myosin force transmission to the extra-cellular matrix underpins persistent, elongated migration, our results suggest that Ryngo 1-23 modulates this process in 3D migration via dynamin-mediated regulation of acto-myosin force transmission to the extra-cellular matrix.
Keywords: Cell migration; 3D matrix; Dynamin; Cancer biology; Invasion;
Cystamine induces AIF-mediated apoptosis through glutathione depletion by Sung-Yup Cho; Jin-Haeng Lee; Mi-kyeong Ju; Eui Man Jeong; Hyo-Jun Kim; Jisun Lim; Seungun Lee; Nam-Hyuk Cho; Hyun Ho Park; Kihang Choi; Ju-Hong Jeon; In-Gyu Kim (619-631).
Cystamine and its reduced form cysteamine showed protective effects in various models of neurodegenerative disease, including Huntington's disease and Parkinson's disease. Other lines of evidence demonstrated the cytotoxic effect of cysteamine on duodenal mucosa leading to ulcer development. However, the mechanism for cystamine cytotoxicity remains poorly understood. Here, we report a new pathway in which cystamine induces apoptosis by targeting apoptosis-inducing factor (AIF). By screening of various cell lines, we observed that cystamine and cysteamine induce cell death in a cell type-specific manner. Comparison between cystamine-sensitive and cystamine-resistant cell lines revealed that cystamine cytotoxicity is not associated with unfolded protein response, reactive oxygen species generation and transglutaminase or caspase activity; rather, it is associated with the ability of cystamine to trigger AIF nuclear translocation. In cystamine-sensitive cells, cystamine suppresses the levels of intracellular glutathione by inhibiting γ-glutamylcysteine synthetase expression that triggers AIF translocation. Conversely, glutathione supplementation completely prevents cystamine-induced AIF translocation and apoptosis. In rats, cysteamine administration induces glutathione depletion and AIF translocation leading to apoptosis of duodenal epithelium. These results indicate that AIF translocation through glutathione depletion is the molecular mechanism of cystamine toxicity, and provide important implications for cystamine in the neurodegenerative disease therapeutics as well as in the regulation of AIF-mediated cell death.
Keywords: Apoptosis-inducing factor; Nuclear translocation; Caspase-independent cell death; Glutathione; Cystamine; Cysteamine;
Characterization and modeling of Ca2 + oscillations in mouse primary mesothelial cells by László Pecze; Beat Schwaller (632-645).
Brief changes in the cytosolic and intra-organellar Ca2 + concentration serve as specific signals for various physiological processes. In mesothelial cells lining the surface of internal organs and the walls of body cavities, a re-entry in the cell cycle (G0–G1 transition) evoked by serum re-administration induces long-lasting Ca2 + oscillations with a slowly decreasing frequency. Individual mesothelial cells show a wide range of different oscillatory patterns within a single, supposedly homogenous cell population. Changes in the cytoplasmic Ca2 + concentration (ccyt) show baseline oscillatory patterns i.e., discrete Ca2 + transients starting from a constant basal ccyt level. The ER Ca2 + concentration (cER) displays a sawtooth wave at a semi-depleted ER state; the minimum level is reached just briefly after the maximal value for ccyt. These oscillations depend on plasmalemmal Ca2 + influx and on the inositol trisphosphate concentration [InsP3]; the Ca2 + influx is a crucial determinant of the oscillation frequency. Partial blocking of SERCA pumps modifies the oscillation frequency in both directions, i.e. increasing it in some cells and lowering it in others. Current mathematical models for Ca2 + oscillations mostly fail to reproduce two experimentally observed phenomena: the broad range of interspike intervals and constant basal ccyt levels between two Ca2 + spikes. Here we developed a new model based on – and fitted to – Ca2 + recordings of ccyt and cER recorded in primary mouse mesothelial cells. The model allowed for explaining many features of experimentally observed Ca2 + oscillations. We consider this model to be suitable to simulate various types of InsP3 receptor-based baseline Ca2 + oscillations.
Keywords: Calcium oscillation; Inositol trisphosphate; Mesothelial cell; G0–G1 transition;
The giardial ENTH protein participates in lysosomal protein trafficking and endocytosis by Constanza Feliziani; Nahuel Zamponi; Natalia Gottig; Andrea S. Rópolo; Adriana Lanfredi-Rangel; Maria C. Touz (646-659).
In the protozoa parasite Giardia lamblia, endocytosis and lysosomal protein trafficking are vital parasite-specific processes that involve the action of the adaptor complexes AP-1 and AP-2 and clathrin. In this work, we have identified a single gene in Giardia encoding a protein containing an ENTH domain that defines monomeric adaptor proteins of the epsin family. This domain is present in the epsin or epsin-related (epsinR) adaptor proteins, which are implicated in endocytosis and Golgi-to-endosome protein trafficking, respectively, in other eukaryotic cells. We found that GlENTHp (for G. lamblia ENTH protein) localized in the cytosol, strongly interacted with PI3,4,5P3, was associated with the alpha subunit of AP-2, clathrin and ubiquitin and was involved in receptor-mediated endocytosis. It also bonded PI4P, the gamma subunit of AP-1 and was implicated in ER-to-PV trafficking. Alteration of the GlENTHp function severely affected trophozoite growth showing an unusual accumulation of dense material in the lysosome-like peripheral vacuoles (PVs), indicating that GlENTHp might be implicated in the maintenance of PV homeostasis. In this study, we showed evidence suggesting that GlENTHp might function as a monomeric adaptor protein supporting the findings of other group indicating that GlENTHp might be placed at the beginning of the ENTH family.Display Omitted
Keywords: ENTH motif; Lysosome; Endosome; Giardia lamblia;
Retinoids induce Nur77-dependent apoptosis in mouse thymocytes by Beáta Kiss; Katalin Tóth; Zsolt Sarang; Éva Garabuczi; Zsuzsa Szondy (660-670).
Nur77 is a transcription factor, which plays a determinant role in mediating T cell receptor-induced cell death of thymocytes. In addition to regulation of transcription, Nur77 contributes to apoptosis induction by targeting mitochondria, where it can convert Bcl-2, an anti-apoptotic protein into a proapoptotic molecule. Previous studies have demonstrated that retinoids are actively produced in the mouse thymus and can induce a transcription-dependent apoptosis in mouse thymocytes. Here we show that retinoic acids induce the expression of Nur77, and retinoid-induced apoptosis is completely dependent on Nur77, as retinoids were unable to induce apoptosis in Nur77 null thymocytes. In wild-type thymocytes retinoids induced enhanced expression of the apoptosis-related genes FasL, TRAIL, NDG-1, Gpr65 and Bid, all of them in a Nur77-dependent manner. The combined action of these proteins led to Caspase 8-dependent Bid cleavage in the mitochondria. In addition, we could demonstrate the Nur77-dependent induction of STAT1 leading to enhanced Bim expression, and the mitochondrial translocation of Nur77 leading to the exposure of the Bcl-2/BH3 domain. The retinoid-induced apoptosis was dependent on both Caspase 8 and STAT1. Our data together indicate that retinoids induce a Nur77-dependent cell death program in thymocytes activating the mitochondrial pathway of apoptosis.
Keywords: Apoptosis; Nur77; Retinoid; Thymocyte;
Role of Ctf3 and COMA subcomplexes in meiosis: Implication in maintaining Cse4 at the centromere and numeric spindle poles by Meenakshi Agarwal; Gunjan Mehta; Santanu K. Ghosh (671-684).
During mitosis and meiosis, kinetochore, a conserved multi-protein complex, connects microtubule with the centromere and promotes segregation of the chromosomes. In budding yeast, central kinetochore complex named Ctf19 has been implicated in various functions and is believed to be made up of three biochemically distinct subcomplexes: COMA, Ctf3 and Iml3–Chl4. In this study, we aimed to identify whether Ctf3 and COMA subcomplexes have any unshared function at the kinetochore. Our data suggests that both these subcomplexes may work as a single functional unit without any unique functions, which we tested. Analysis of severity of the defects in the mutants suggests that COMA is epistatic to Ctf3 subcomplex. Interestingly, we noticed that these subcomplexes affect the organization of mitotic and meiotic kinetochores with subtle differences and they promote maintenance of Cse4 at the centromeres specifically during meiosis which is similar to the role of Mis6 (Ctf3 homolog) in fission yeast during mitosis. Interestingly, analysis of ctf3Δ and ctf19Δ mutants revealed a novel role of Ctf19 complex in regulation of SPB cohesion and duplication in meiosis.
Keywords: Kinetochore; Meiosis; Budding yeast; Ctf3; Ctf19; Spindle pole body;
Platinum-induced kidney damage: Unraveling the DNA damage response (DDR) of renal tubular epithelial and glomerular endothelial cells following platinum injury by Katharina Krüger; Jürgen Thomale; Nikolina Stojanović; Maja Osmak; Christian Henninger; Stefanie Bormann; Gerhard Fritz (685-698).
Platinum compounds are potent anticancer drugs but also evoke considerable normal tissue damage. Here, we elucidate the molecular mechanisms contributing to the nephrotoxic effects of cisplatin.We comparatively investigated the stress responses of rat kidney tubular (NRK-52E) and glomerular cells (RGE) following treatment with cisplatin (CisPt), oxaliplatin (OxaliPt) and carboplatin (CarboPt). To this end, cell viability, apoptosis, cell cycle progression, DNA damage response (DDR) and repair of DNA adducts were investigated.CisPt reduced the viability of tubular NRK-52E and glomerular RGE cells most efficiently. Cytotoxicity evoked by CarboPt occurred with a delay, which might be related to a retarded formation of Pt-(GpG) intrastrand crosslinks. RGE cells were more sensitive towards all platinum compounds than NRK-52E cells. Platinum drugs efficiently induced caspase-mediated apoptosis in tubular cells, while RGE cells favored G2/M arrest when treated with equitoxic platinum doses. Mitotic index of NKR-52E and RGE cells was worst affected by OxaliPt. Activation of the DDR was strikingly agent- and cell type-specific. Most comprehensive and substantial stimulation of DDR mechanisms was provoked by CisPt. Repair of Pt-(GpG) intrastrand crosslinks was best in RGE, which was reflected by high mRNA expression of nucleotide excision repair (NER) factors.There are substantial differences regarding the cause of sensitivity and mechanisms of DDR between tubular and glomerular cells following platinum injury. CisPt is the most potent stimulator of the DDR. We hypothesize that specific DNA adducts and thereby forcefully activated pro-toxic DDR mechanisms contribute to the exceptionally high acute nephrotoxicity of CisPt.
Keywords: Platinating anticancer drugs; Normal tissue damage; Nephrotoxicity; DNA damage response;
A role for adaptor protein complex 1 in protein targeting to rhoptry organelles in Plasmodium falciparum by K.M. Kaderi Kibria; Khushboo Rawat; Christen M. Klinger; Gaurav Datta; Manoj Panchal; Shailja Singh; Gayatri R. Iyer; Inderjeet Kaur; Veena Sharma; Joel B. Dacks; Asif Mohmmed; Pawan Malhotra (699-710).
The human malaria parasite Plasmodium falciparum possesses sophisticated systems of protein secretion to modulate host cell invasion and remodeling. In the present study, we provide insights into the function of the AP-1 complex in P. falciparum. We utilized GFP fusion constructs for live cell imaging, as well as fixed parasites in immunofluorescence analysis, to study adaptor protein mu1 (Pfμ1) mediated protein trafficking in P. falciparum. In trophozoites Pfμ1 showed similar dynamic localization to that of several Golgi/ER markers, indicating Golgi/ER localization. Treatment of transgenic parasites with Brefeldin A altered the localization of Golgi-associated Pfμ1, supporting the localization studies. Co-localization studies showed considerable overlap of Pfμ1 with the resident rhoptry proteins, rhoptry associated protein 1 (RAP1) and Cytoadherence linked asexual gene 3.1 (Clag3.1) in schizont stage. Immunoprecipitation experiments with Pfμ1 and PfRAP1 revealed an interaction, which may be mediated through an intermediate transmembrane cargo receptor. A specific role for Pfμ1 in trafficking was suggested by treatment with AlF4, which resulted in a shift to a predominantly ER-associated compartment and consequent decrease in co-localization with the Golgi marker GRASP. Together, these results suggest a role for the AP-1 complex in rhoptry protein trafficking in P. falciparum.
Keywords: Adaptor protein complex-1 (AP-1); Trafficking; Plasmodium falciparum; Vesicular trafficking; Clathrin; ER–Golgi network;
The APC tumor suppressor is required for epithelial cell polarization and three-dimensional morphogenesis by Alyssa C. Lesko; Kathleen H. Goss; Frank F. Yang; Adam Schwertner; Imge Hulur; Kenan Onel; Jenifer R. Prosperi (711-723).
The Adenomatous Polyposis Coli (APC) tumor suppressor has been previously implicated in the control of apical–basal polarity; yet, the consequence of APC loss-of-function in epithelial polarization and morphogenesis has not been characterized. To test the hypothesis that APC is required for the establishment of normal epithelial polarity and morphogenesis programs, we generated APC-knockdown epithelial cell lines. APC depletion resulted in loss of polarity and multi-layering on permeable supports, and enlarged, filled spheroids with disrupted polarity in 3D culture. Importantly, these effects of APC knockdown were independent of Wnt/β-catenin signaling, but were rescued with either full-length or a carboxy (c)-terminal segment of APC. Moreover, we identified a gene expression signature associated with APC knockdown that points to several candidates known to regulate cell–cell and cell–matrix communication. Analysis of epithelial tissues from mice and humans carrying heterozygous APC mutations further supports the importance of APC as a regulator of epithelial behavior and tissue architecture. These data also suggest that the initiation of epithelial-derived tumors as a result of APC mutation or gene silencing may be driven by loss of polarity and dysmorphogenesis.
Keywords: Adenomatous Polyposis Coli; Epithelial polarity; Madin–Darby Canine Kidney;
A novel mitochondrial carrier protein Mme1 acts as a yeast mitochondrial magnesium exporter by Yixian Cui; Shanke Zhao; Juan Wang; Xudong Wang; Bingquan Gao; Qiangwang Fan; Fei Sun; Bing Zhou (724-732).
The homeostasis of magnesium (Mg2 +), an abundant divalent cation indispensable for many biological processes including mitochondrial functions, is underexplored. Previously, two mitochondrial Mg2 + importers, Mrs2 and Lpe10, were characterized for mitochondrial Mg2 + uptake. We now show that mitochondrial Mg2 + homeostasis is accurately controlled through the combined effects of previously known importers and a novel exporter, Mme1 (mitochondrial magnesium exporter 1). Mme1 belongs to the mitochondrial carrier family and was isolated for its mutation that is able to suppress the mrs2Δ respiration defect. Deletion of MME1 significantly increased steady-state mitochondrial Mg2 + concentration, while overexpression decreased it. Measurements of Mg2 + exit from proteoliposomes reconstituted with purified Mme1 provided definite evidence for Mme1 as an Mg2 + exporter. Our studies identified, for the first time, a mitochondrial Mg2 + exporter that works together with mitochondrial importers to ensure the precise control of mitochondrial Mg2 + homeostasis.
Keywords: Lpe10; mitochondrial Mg2 + exporter; Mme1; Mrs2; proteoliposome;
Reticulon protein-1C is a key component of MAMs by Valentina Reali; Bisan Mehdawy; Roberta Nardacci; Giuseppe Filomeni; Anna Risuglia; Federica Rossin; Manuela Antonioli; Claudia Marsella; Gian Maria Fimia; Mauro Piacentini; Federica Di Sano (733-745).
The endoplasmic reticulum (ER) is a key organelle fundamental for the maintenance of cellular homeostasis and the determination of cell fate under stress conditions. Reticulon-1C (RTN-1C) is a member of the reticulon family proteins localized primarily on the ER membrane and known to regulate ER structure and function. Several cellular processes depend on the structural and functional crosstalk between different organelles, particularly on the endoplasmic reticulum and mitochondria. These dynamic contacts, called mitochondria-associated ER membranes (MAMs), are essential for the maintenance of mitochondrial structure and participate in lipid and calcium exchanges between the two organelles.In this study we investigated the impact of RTN-1C modulation on mitochondrial dynamics. We demonstrate that RTN-1C controls mitochondrial structure and function affecting intracellular Ca2 + homeostasis and lipid exchange between ER and mitochondria. We propose that these events depend on RTN-1C involvement in the regulation of ER–mitochondria cross-talk and define a role for RTN-1C in maintaining the function of contacts between the two organelles.
Keywords: RTN-1C; Endoplasmic reticulum; MAM; Mitochondria;
Hsf4 counteracts Hsf1 transcription activities and increases lens epithelial cell survival in vitro by Xiukun Cui; Pan Pan Xie; Pan Pan Jia; Qiang Lou; Guoqing Dun; Shulian Li; Guangchao Liu; Jun Zhang; Zheng Dong; Yuanfang Ma; Yanzhong Hu (746-755).
The interplay between Hsf4 and Hsf1 plays an important role in the regulation of lens homeostasis. However, the mechanism of the intermolecular association involved is still unclear. In this paper, we find that reconstitution of Hsf4b into Hsf4−/− lens epithelial (mLEC/Hsf4−/−) cells can simultaneously downregulate Hsp70 expression and upregulate the expression of small heat shock proteins Hsp25 and αB-crystallin at both RNA and protein levels. ChIP assay results indicate Hsf4b, which binds to the promoters of Hsp90α, Hsp70.3, Hsp25 and αB-crystallin but not Hsp70.1, can inhibit Hsf1 binding to Hsp70.3 promoter and the heat shock mediated Hsp70 promoter activity by reducing Hsf1 protein expression. Hsf4b N-terminal hydrophobic region can interact with Hsf1 N-terminal hydrophobic region. Their interaction impairs Hsf1's intramolecular interaction between the N- and C-terminal hydrophobic regions, leading to Hsf1's cytosolic retention and protein degradation. Both lysosome inhibitors (chloroquine, pepstatin A plus E64d) and proteasome inhibitor MG132 can inhibit Hsf4-mediated Hsf1 protein degradation, but MG132 can induce Hsf1 activation as well. Upregulation of Hsf4b can significantly inhibit cisplatin and staurosporine induced lens epithelial cell apoptosis through direct upregulation of Hsp25 and αB-crystallin expression. Taken together, our results imply that upregulation of Hsf4b modulates the expression pattern of heat shock proteins in lens tissue by either directly binding to their promoters or promoting Hsf1 protein degradation. Moreover, upregulation of Hsf4b protects lens cell survival by upregulating anti-apoptotic pathways. These studies reveal a novel regulatory mechanism between Hsf1 and Hsf4b in modulating lens epithelial cell homeostasis.
Keywords: Hsf4; Hsf1; Apoptosis; Lysosome and lens epithelial cell;
Efficient export of human growth hormone, interferon α2b and antibody fragments to the periplasm by the Escherichia coli Tat pathway in the absence of prior disulfide bond formation by Heli I. Alanen; Kelly L. Walker; M. Lourdes Velez Suberbie; Cristina F.R.O. Matos; Sarah Bönisch; Robert B. Freedman; Eli Keshavarz-Moore; Lloyd W. Ruddock; Colin Robinson (756-763).
Numerous therapeutic proteins are expressed in Escherichia coli and targeted to the periplasm in order to facilitate purification and enable disulfide bond formation. Export is normally achieved by the Sec pathway, which transports proteins through the plasma membrane in a reduced, unfolded state. The Tat pathway is a promising alternative means of export, because it preferentially exports correctly folded proteins; however, the reducing cytoplasm of standard strains has been predicted to preclude export by Tat of proteins that contain disulfide bonds in the native state because, in the reduced state, they are sensed as misfolded and rejected. Here, we have tested a series of disulfide-bond containing biopharmaceuticals for export by the Tat pathway in CyDisCo strains that do enable disulfide bond formation in the cytoplasm. We show that interferon α2b, human growth hormone (hGH) and two antibody fragments are exported with high efficiency; surprisingly, however, they are efficiently exported even in the absence of cytoplasmic disulfide formation. The exported proteins acquire disulfide bonds in the periplasm, indicating that the normal disulfide oxidation machinery is able to act on the proteins. Tat-dependent export of hGH proceeds even when the disulfide bonds are removed by substitution of the Cys residues involved, suggesting that these substrates adopt tertiary structures that are accepted as fully-folded by the Tat machinery.
Keywords: Tat pathway; Cell engineering; Biopharmaceuticals; E. coli; Protein export;