BBA - Molecular Cell Research (v.1803, #5)

Quinolinate phosphoribosyl transferase, a key enzyme in de novo NAD+ synthesis, suppresses spontaneous cell death by inhibiting overproduction of active-caspase-3 by Kazumi Ishidoh; Norio Kamemura; Takahito Imagawa; Masataka Oda; Jun Sakurai; Nobuhiko Katunuma (527-533).
Quinolinate phosphoribosyl transferase (QPRT) is a key enzyme in de novo NAD+ synthesis. QPRT enzyme activity has a restricted tissue distribution, although QPRT mRNA is expressed ubiquitously. This study was designed to elucidate the functions of QPRT protein in addition to NAD+ synthesis. QPRT was identified as a caspase-3 binding protein using double layer fluorescent zymography, but was not a substrate for caspase-3. Surface plasmon resonance analysis using recombinant proteins showed interaction of QPRT with active-caspase-3 in a dose dependent manner at 55 nM of the dissociation constant. The interaction was also confirmed by immunoprecipitation analysis of actinomycin D-treated QPRT-FLAG expressing cells using anti-FLAG-agarose. QPRT-depleted cells showed increased sensitivity to spontaneous cell death, upregulated caspase-3 activity and strong active-caspase-3 signals. Considered together, the results suggested that QPRT protein acts as an inhibitor of spontaneous cell death by suppressing overproduction of active-caspase-3.
Keywords: Quinolinate phosphoribosyl transferase; NAD+ synthesis; Active-caspase-3; Spontaneous cell death;

Gonadotropin-regulated testicular RNA helicase (GRTH/DDX25), a multifunctional protein and a component of ribonucleoprotein complexes, is essential for the completion of spermatogenesis. We investigated the nuclear/cytoplasmic shuttling of GRTH in germ cells and its impact on the chromatoid body (CB)—a perinuclear organelle viewed as a storage/processing site of mRNAs. GRTH resides in the nucleus, cytoplasm and CB of round spermatids. Treatment of these cells with inhibitors of nuclear export or RNA synthesis caused nuclear retention of GRTH and its absence in the cytoplasm and CB. The nuclear levels of GRTH bound RNA messages were significantly enhanced and major reduction was observed in the cytoplasm. This indicated GRTH main transport function of mRNAs to the cytoplasm and CB. MVH, a germ cell helicase, and MIWI, a component of the RNA-induced-silencing complex (RISC), confined to the CB/cytoplasm, were absent in the CB and accumulated in the cytoplasm upon treatment. This also occurred in spermatids of GRTH-KO mice. The CB changed from lobular-filamentous to a small condensed structure after treatment resembling the CB of GRTH-KO. No interaction of GRTH with MVH or RISC members in both protein and RNA were observed. Besides of participating in the transport of messages of relevant spermatogenic genes, GRTH was found to transport its own message to cytoplasmic sites. Our studies suggest that GRTH through its export/transport function as a component of mRNP is essential to govern the CB structure in spermatids and to maintain systems that may participate in mRNA storage and their processing during spermatogenesis.
Keywords: GRTH/DDX25; DEAD-box RNA helicase; Chromatoid body; Round spermatid; GRTH-mRNP; Nuclear export; Spermatogenesis;

Suppression of MHC class I surface expression by calreticulin's P-domain in a calreticulin deficient cell line by Changzhen Liu; Hongmei Fu; Barry Flutter; Simon J. Powis; Bin Gao (544-552).
Calreticulin (CRT) is an important chaperone protein, comprising an N-domain, P-domain and C-domain. It is involved in the folding and assembly of multi-component protein complexes in the endoplasmic reticulum, and plays a critical role in MHC class I antigen processing and presentation. To dissect the functional role and molecular basis of individual domains of the protein, we have utilized individual domains to rescue impaired protein assembly in a CRT deficient cell line. Unexpectedly, both P-domain fragment and NP domain of CRT not only failed to rescue defective cell surface expression of MHC class I molecules but further inhibited their appearance on the surface of cells. Formation of the TAP-associated peptide-loading complex and trafficking of the few detectable MHC class I molecules were not significantly impaired. Instead, this further suppression of MHC class I molecules on the cell surface appears due to the complex missing antigenic peptides, the third member of fully assembled MHC class I molecules. Therefore the P-domain of calreticulin appears to play a significant role in antigen presentation by MHC class I molecules.
Keywords: MHC class I; Calreticulin; P-domain;

The scavenger receptor FEEL-1/stabilin-1 is known as the marker of alternatively activated macrophage and sinusoidal endothelial cell. FEEL-1/stabilin-1 is a multifunctional transmembrane glycoprotein that is implicated in bacterial infection, diabetes, atherosclerosis, wound healing, and innate immunity. In the current study, we have identified the phox-homology domain containing protein SNX17 as a novel interaction partner of FEEL-1/stabilin-1 in endothelial cells. SNX17 directly interacts with FEEL-1/stabilin-1 and regulates its trafficking. Studies using the cytoplasmic domain of truncated or mutant FEEL-1/stabilin-1 suggest that the NPxF motif of the FEEL-1/stabilin-1 cytoplasmic tail is required for its interaction with SNX17. By transfecting cells with small interfering RNA targeting SNX17, total cellular FEEL-1/stabilin-1 expression and FEEL-1/stabilin-1-mediated ligand uptake were significantly decreased due to the enhancement of FEEL-1/stabilin-1 protein degradation. Our results identify SNX17 as a novel interaction partner of FEEL-1/stabilin-1 in endothelial cells.
Keywords: FEEL-1/stabilin-1; SNX17; Endothelial cell;

Human Miltons associate with mitochondria and induce microtubule-dependent remodeling of mitochondrial networks by Olga S. Koutsopoulos; David Laine; Laura Osellame; Dmitriy M. Chudakov; Robert G. Parton; Ann E. Frazier; Michael T. Ryan (564-574).
Proper mitochondrial distribution is crucial for cell function. In Drosophila, mitochondrial transport is facilitated by Miro and Milton, which regulate mitochondrial attachment to microtubules via kinesin heavy chain. Mammals contain two sequence orthologs of Milton however, they have been ascribed various functions in intracellular transport. In this report, we show that the human Miltons target to mitochondria irrespective of whether they are linked to GFP at their C- or N-termini. Their ectopic expression induces the formation of extended mitochondrial tubules as well as large bulbous-like mitochondria with narrow tubular membrane necks that connect them to the mitochondrial mass. The mitochondrial extensions appear highly dynamic and their formation relies on the presence of microtubules. Using the photoswitchable fluorescent protein Dendra2 targeted to the mitochondrial matrix, we found that the mitochondrial extensions and bulbous mitochondria are fused with neighboring regions of the network. Truncation analysis of huMilton1 revealed that the N-terminal region, inclusive of the coiled-coil segment could localize to microtubules, suggesting that Milton attachment to kinesin occurs independent of Miro or mitochondrial attachment. In addition, we show that the huMiltons have the capacity to self-interact and can also facilitate mitochondrial recruitment of a cytosolic Miro mutant. We conclude that the human Miltons are important mediators of the mitochondrial trafficking machinery.
Keywords: Mitochondria; Morphology; Distribution; Trafficking; Milton;

Mutations that increase both Hsp90 ATPase activity in vitro and Hsp90 drug resistance in vivo by Anna Zurawska; Jakub Urbanski; Jurgita Matulienė; Janina Baraniak; Marcin P. Klejman; Slawomir Filipek; Daumantas Matulis; Pawel Bieganowski (575-583).
Hsp90 inhibitors are currently tested in clinical trials as anticancer agents. We investigated whether inhibitor resistance may arise as a result of a point mutation in Hsp90. We used yeast cells that expressed human Hsp90β to select inhibitor-resistant mutants from the randomly mutagenized library. Single amino acid substitution, I123T, in a selected mutant was sufficient to confer inhibitor resistance. Transfection of human cells with the HSP90β I123T and the corresponding HSP90α I128T yielded cell lines resistant to inhibitors of the Hsp90 ATPase. Unexpectedly, mutations did not result in diminished inhibitor binding in vitro. Similarly resistant cells were obtained after transfection with previously described A116N and T31I mutants of HSP90β that cause increase in ATPase activity in vitro. Inhibitor-resistant phenotypes of the I123T and A116N mutants depended on their increased affinity for Aha1, whereas T31I mutation did not result in increased Aha1 binding. These results show possible scenario by which resistance may arise in patients treated with Hsp90 inhibitors. Additionally, our results show that each isoform of Hsp90 can alone sustain cellular functions.
Keywords: Hsp90; Geldanamycin; 17-AAG; Radicicol; Inhibitor; Aha1; ATPase;

Colocalization of mineralocorticoid and EGF receptor at the plasma membrane by Claudia Grossmann; Britta Husse; Sigrid Mildenberger; Barbara Schreier; Katja Schuman; Michael Gekle (584-590).
The mineralocorticoid receptor (MR), a ligand-activated transcription factor expressed in various cell types (e.g. epithelial cells, neurons, smooth muscle cells, immune cells), plays important roles in neurohumoral, neuronal, cardiovascular, renal and intestinal function. Pathophysiological relevant signaling mechanisms include nongenomic pathways involving the EGF receptor (EGFR). We investigated whether a MR–EGFR colocalization may underlie the functional MR–EGFR interaction by coimmunoprecipitation, fluorescence resonance energy transfer (FRET) and confocal microscopy in a heterologous expression system. EGFR and a small fraction of MR colocalize at the cell membrane, independently of short time exposure (≤ 60 min) to receptor ligands. Twenty-four-hour-exposure to saturating concentrations of aldosterone (10 nmol/l) resulted in an almost complete nuclear translocation of MR and disappearance of MR–EGFR colocalization. EGFR transactivation is enhanced only after MR stimulation. Inhibition of HSP90 by geldanamycin did not reduce the fraction of MR interacting with EGFR. Disruption of cholesterol-rich membrane domains by cyclodextrin reduced MR–EGFR interaction. In conclusion, a subfraction of MR interacts with EGFR at the plasma membrane in our heterologous expression system, possibly at cholesterol-rich domains, to form a steroid receptor/growth factor receptor signaling module.
Keywords: Mineralocorticoid receptor; Epidermal growth factor receptor; Aldosterone; FRET;

Luteinizing hormone receptor (LHR) mRNA binding protein (LRBP), identified as mevalonate kinase, has been shown to be a trans factor mediating the post-transcriptional regulation of LHR mRNA expression in ovaries. LRBP binds to the coding region of LHR mRNA and accelerates its degradation. Our previous studies in an in vitro system showed that LRBP represses the translation of LHR mRNA by forming an untranslatable ribonucleoprotein (mRNP) complex, further suggesting that the untranslatable mRNP complex is directed to the mRNA repression/decay machinery for subsequent mRNA turnover. In the present studies, we used yeast two-hybrid system to screen a cDNA library which was constructed from LHR down-regulated ovaries. Two proteins were identified interacting with LRBP: ribosomal protein S20 (RP S20) and ubiquitin conjugating enzyme 2i (UBCE2i). Their interactions with LRBP were confirmed by the mating assay, co-immunoprecipitation analyses and in vitro sumoylation assays. Furthermore, we show that LRBP is a target for modification by SUMO2/3 but not by SUMO1, at K256 and/or K345. Mutation of both lysine residues is sufficient to abrogate the sumoylation of LRBP. These findings suggest that the direct interaction of LRBP with the translation machinery, through RP S20, may be responsible for the transition of LHR mRNA to an untranslatable complex, and that sumoylation of LRBP may play a role in targeting the untranslatable mRNP complex to the mRNA decay machinery in specific cytoplasmic foci.
Keywords: Luteinizing hormone receptor; LRBP; RNA decay; Sumoylation; UBCE2i;

Tartrate-resistant acid phosphatase, although encoded by a single gene, exists as two isoforms in human serum, TRAP 5a and 5b, differing in post-translational modifications such as proteolytic processing and kinetic properties including pH optimum and specific activity. The biogenetic relationship between the TRAP isoforms was assessed in a stably transfected breast cancer epithelial MDA-MB-231 cell subline overexpressing 5a- and 5b-like TRAP isoforms intracellularly, with only the monomeric 5a-like isoform being secreted. As judged by immunolocalization and comparative N-glycan profiling by Con A lectin chromatography and glycanase analysis, the majority of the intracellular monomeric TRAP was destined for secretion, while a minor portion provided the putative precursor for the intracellular 5b-like isoform. Brefeldin A blocked secretion of 5a-like TRAP isoform as well as appearance of its putative intracellular precursor, and augmented the intracellular level of proteolytically processed 5b-like isoform, indicating a common early biosynthetic precursor for TRAP isoforms 5a and 5b. The cysteine proteinase inhibitor E64 partially blocked formation of the 5b-like isoform while augmenting the level of its putative monomeric precursor, but did not alter the levels of secreted TRAP or its intracellular precursor, suggesting that distinct precursors for secreted TRAP 5a and intracellular 5b-like isoform are segregated in the ER or Golgi prior to proteolytic processing. In conclusion, these data provide evidence that distinct monomeric TRAP populations are diverted early in the secretory pathway either giving rise to a secreted, monomeric 5a-like TRAP isoform or to an intracellular, proteolytically processed 5b-like TRAP isoform.
Keywords: Tartrate-resistant acid phosphatase; Purple acid phosphatase; Acp 5; Uteroferrin; Cysteine proteinase; Osteoclast; Macrophage; Breast cancer;

Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is a tumor suppressor and the suppression of RECK is induced by Ras or Her-2/neu oncogenes. However, regulation of RECK under hypoxic microenvironment is largely unknown. Here, we identified that hypoxia significantly downregulates RECK mRNA and protein expression using semiquantitative RT-PCR, real-time RT-PCR and western blot analysis. This repression was reversed by the HDAC inhibitor, trichostatin A (TSA) and HIF-1 inhibitor, YC-1. Hypoxia-induced downregulation of RECK was abolished by knockdown of HDAC1 and HIF-1α with respective small interfering RNAs (siRNAs), whereas overexpression of HDAC1 and HIF-1α suppressed RECK expression similar to the level under hypoxic conditions. Transfection of a deletion mutant of the second reverse HRE (rHRE2, − 2345 to − 2333) site of RECK promoter completely removed RECK suppression under hypoxia, indicating that the rHRE2 site is responsible for the inhibition of RECK. Chromatin immunoprecipitation and DNA affinity precipitation assays demonstrated that HDAC1 and HIF-1α were recruited to the rHRE2 region of RECK promoter under hypoxic conditions, but the treatment of TSA or YC-1 inhibited their binding to the rHRE2 site. Moreover, TSA and YC-1 inhibited hypoxia-induced cancer cell migration, invasion and MMPs secretion. Taken together, we can conclude that hypoxia induces RECK downregulation through the recruitment of HDAC1 and HIF-1α to the rHRE2 site in the promoter and the inhibition of hypoxic RECK silencing would be a therapeutic and preventive target for early tumorigenesis.
Keywords: Hypoxia; RECK; Reverse HRE; HDAC1; HIF-1α; Tumor suppressor silencing;

A conserved function for Inp2 in peroxisome inheritance by Ruchi Saraya; Małgorzata N. Cepińska; Jan A.K.W. Kiel; Marten Veenhuis; Ida J. van der Klei (617-622).
In budding yeast Saccharomyces cerevisiae, the peroxisomal protein Inp2 is required for inheritance of peroxisomes to the bud, by connecting the organelles to the motor protein Myo2 and the actin cytoskeleton. Recent data suggested that the function of Inp2 may not be conserved in other yeast species. Using in silico analyses we have identified a weakly conserved Inp2-related protein in 18 species of budding yeast and analyzed the role of the identified protein in the methylotrophic yeast Hansenula polymorpha in peroxisome inheritance. Our data show that H. polymorpha Inp2 locates to peroxisomes, interacts with Myo2, and is essential for peroxisome inheritance.
Keywords: Budding yeast; Cytoskeleton; Organelle inheritance; Peroxisome; Protein–protein interaction;

Identification of two distinct cell binding sequences in the vitamin D binding protein by Jianhua Zhang; David M. Habiel; Mahalakshmi Ramadass; Richard R. Kew (623-629).
The vitamin D binding protein (DBP) is a multifunctional, albumin-like plasma protein that often requires cell surface binding to mediate some of its diverse functions. DBP binds to several different molecules on the external face of the plasma membrane indicating that it may possess distinct cell binding sequences. In this report, surface plasmon resonance was utilized to evaluate the relative binding of the human myeloid cell line U937 to immobilized recombinant expressed DBP in order to identify cell localization sequences. U937 cells showed robust binding to immobilized native DBP, but essentially no interaction when sensor chips were coated with β2-microglobulin or BSA. The cell–DBP interaction was completely eliminated if cells were pretreated with soluble DBP. Recombinant DBP domains and truncated domains were next evaluated to determine the location of cell binding regions. Domains I (amino acids 1–191) and III (379–458), but not domain II (192–378), could support cell binding. Further evaluation of domain I, using truncated proteins and overlapping peptides, demonstrated that a single amino acid sequence, residues 150–172 (NYGQAPLSLLVSYTKSYLSMVGS), mediated cell binding. The domain III cell binding region was investigated using truncated versions of domain III fused to full-length domain II that served as a scaffold. These experiments indicated that the cell binding sequence is located in the first portion of that domain (379–402: ELSSFIDKGQELCADYSENTFTEY). Overlapping peptides spanning this sequence could partially block cell binding only when used in combination. We conclude that DBP contains two cell localization sequences that may be required for some of the multiple functions of this protein.
Keywords: Gc-globulin; Vitamin D binding protein; Cell binding; Plasma membrane;

Sir2-dependent asymmetric segregation of damaged proteins in ubp10 null mutants is independent of genomic silencing by Ivan Orlandi; Maurizio Bettiga; Lilia Alberghina; Thomas Nyström; Marina Vai (630-638).
Carbonylation of proteins is an irreversible oxidative damage that increases during both chronological and replicative yeast aging. In the latter, a spatial protein quality control system that relies on Sir2 is responsible for the asymmetrical damage segregation in the mother cells. Proper localization of Sir2 on chromatin depends on the deubiquitinating enzyme Ubp10, whose loss of function deeply affects the recombination and gene-silencing activities specific to Sir2. Here, we have analyzed the effects of SIR2 and UBP10 inactivations on carbonylated protein patterns obtained in two aging models such as stationary phase cells and size-selected old mother ones. In line with the endogenous situation of higher oxidative stress resulting from UBP10 inactivation, an increase of protein carbonylation has been found in the ubp10Δ stationary phase cells compared with sir2Δ ones. Moreover, Calorie Restriction had a salutary effect for both mutants by reducing carbonylated proteins accumulation. Remarkably, in the replicative aging model, whereas SIR2 inactivation resulted in a failure to establish damage asymmetry, the Sir2-dependent damage inheritance is maintained in the ubp10Δ mutant which copes with the increased oxidative damage by retaining it in the mother cells. This indicates that both Ubp10 and a correct association of Sir2 with the silenced chromatin are not necessary in such a process but also suggests that additional Sir2 activities on non-chromatin substrates are involved in the establishment of damage asymmetry.
Keywords: Aging; Protein carbonylation; Sir2; Ubp10; Oxidative damage; Calorie Restriction;