Browsing by Subject "endoplasmic reticulum"

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  • Anwar, Tahira; Liu, Xiaonan; Suntio, Taina; Marjamäki, Annika; Biazik, Joanna; Chan, Edmond Y. W.; Varjosalo, Markku; Eskelinen, Eeva-Liisa (2019)
    Autophagy transports cytoplasmic material and organelles to lysosomes for degradation and recycling. Beclin 1 forms a complex with several other autophagy proteins and functions in the initiation phase of autophagy, but the exact role of Beclin 1 subcellular localization in autophagy initiation is still unclear. In order to elucidate the role of Beclin 1 localization in autophagosome biogenesis, we generated constructs that target Beclin 1 to the endoplasmic reticulum (ER) or mitochondria. Our results confirmed the proper organelle-specific targeting of the engineered Beclin 1 constructs, and the proper formation of autophagy-regulatory Beclin 1 complexes. The ULK kinases are required for autophagy initiation upstream of Beclin 1, and autophagosome biogenesis is severely impaired in ULK1/ULK2 double knockout cells. We tested whether Beclin 1 targeting facilitated its ability to rescue autophagosome formation in ULK1/ULK2 double knockout cells. ER-targeted Beclin 1 was most effective in the rescue experiments, while mitochondria-targeted and non-targeted Beclin 1 also showed an ability to rescue, but with lower activity. However, none of the constructs was able to increase autophagic flux in the knockout cells. We also showed that wild type Beclin 1 was enriched on the ER during autophagy induction, and that ULK1/ULK2 facilitated the ER-enrichment of Beclin 1 under basal conditions. The results suggest that one of the functions of ULK kinases may be to enhance Beclin 1 recruitment to the ER to drive autophagosome formation.
  • Tuovinen, Elina A.; Grönholm, Juha; Öhman, Tiina; Pöysti, Sakari; Toivonen, Raine; Kreutzman, Anna; Heiskanen, Kaarina; Trotta, Luca; Toiviainen-Salo, Sanna; Routes, John M.; Verbsky, James; Mustjoki, Satu; Saarela, Janna; Kere, Juha; Varjosalo, Markku; Hänninen, Arno; Seppänen, Mikko R. J. (2020)
    Hypomorphic IL2RG mutations may lead to milder phenotypes than X-SCID, named variably as atypical X-SCID or X-CID. We report an 11-year-old boy with a novel c. 172C>T;p.(Pro58Ser) mutation in IL2RG, presenting with atypical X-SCID phenotype. We also review the growing number of hypomorphic IL2RG mutations causing atypical X-SCID. We studied the patient's clinical phenotype, B, T, NK, and dendritic cell phenotypes, IL2RG and CD25 cell surface expression, and IL-2 target gene expression, STAT tyrosine phosphorylation, PBMC proliferation, and blast formation in response to IL-2 stimulation, as well as protein-protein interactions of the mutated IL2RG by BioID proximity labeling. The patient suffered from recurrent upper and lower respiratory tract infections, bronchiectasis, and reactive arthritis. His total lymphocyte counts have remained normal despite skewed T and B cells subpopulations, with very low numbers of plasmacytoid dendritic cells. Surface expression of IL2RG was reduced on his lymphocytes. This led to impaired STAT tyrosine phosphorylation in response to IL-2 and IL-21, reduced expression of IL-2 target genes in patient CD4+ T cells, and reduced cell proliferation in response to IL-2 stimulation. BioID proximity labeling showed aberrant interactions between mutated IL2RG and ER/Golgi proteins causing mislocalization of the mutated IL2RG to the ER/Golgi interface. In conclusion, IL2RG p.(Pro58Ser) causes X-CID. Failure of IL2RG plasma membrane targeting may lead to atypical X-SCID. We further identified another carrier of this mutation from newborn SCID screening, lost to closer scrutiny.
  • Tuovinen, Elina (Helsingin yliopisto, 2020)
    Interleukiini (IL)-2 reseptorin gammaketjun (IL2RG) mutaatiot aiheuttavat X-kromosomissa periytyvää vaikeaa kombinoitua immuunipuutosta (X-SCID). IL-2 -reseptorin lisäksi IL2RG on olennainen osa myös IL-4, -7, -9, -15 ja -21 -reseptoreita. IL2RG-mutaatiot aiheuttavatkin usean signaalireitin häiriintymiseen johtaen edelleen sekä T- ja NK- solujen alhaisiin määriin, että B-solujen toiminnan häiriöihin. T-solujen alentuneen määrän vuoksi tila on fataali, ja ilman hematopoieettista kantasolusiirtoa potilaat menehtyvät tyypillisesti ensimmäisen elinvuotensa aikana. Tutkimuspotilaamme on 11-vuotias poika, jolta löytyi eksomisekvensoinnissa c.172C>T, p.(Pro58Ser) mutaatio IL2RG -geenissään. Potilaan lymfosyyttimäärät olivat normaalit, mutta T-, B- ja NK-solujen ala-luokat epänormaalisti jakautuneet. B-solujen luokanvaihto oli häiriintynyt ja B-muistisolujen määrä alen-tunut. CD4- ja CD8- effector memory -solujen ja plasmasytoidisten dendriittisolujen määrät olivat poik-keavan matalat. γδ-solujen osuus T-soluista oli poikkeavan korkea. Kahden vuoden iästä alkaen potilas sairasti toistuvia ylä- ja alahengitystulehduksia, ja molemminpuoliset bronkiektasiat havaittiin kuuden vuoden iässä. Potilasta on hoidettu immunoglobuliinikorvaushoidolla, eikä hän toistaiseksi ole tarvinnut kantasolusiirtoa. IL2RG:n pintaekspressio potilaan lymfosyyteissä oli alentunut ja IL-2 ja -21 stimulaation indusoima STAT-fosforylaatio potilaalla kontrolleja heikompaa. Proteomiikkatutkimukset BioID proximity labeling -menetelmällä osoittivat mutatoituneen IL2RG:n epänormaalit interaktiot ER/Golgi -proteiinien kanssa johtaen sen häiriintyneeseen ohjautumiseen solukalvolle. Artikkelissa kuvaamme aiemmin tuntemattoman epätyypillistä X-SCID -tautia aiheuttavan IL2RG-geenin mutaation. Vastaavia, fenotyypiltään hypomorfisia IL2RG-mutaatioita on raportoitu viime vuosina enene-vissä määrin. Näiden potilaiden rekisteröiminen on jatkossakin tärkeää, sillä tällä hetkellä epätyypillisen X-SCID -taudin pitkäaikaisennuste on tuntematon ja hoidon valinta vaikeaa.
  • Battista, Theo; Fiorillo, Annarita; Chiarini, Valerio; Genovese, Ilaria; Ilari, Andrea; Colotti, Gianni (2020)
    The development of drug resistance is one of the main causes of failure in anti-cancer treatments. Tumor cells adopt many strategies to counteract the action of chemotherapeutic agents, e.g., enhanced DNA damage repair, inactivation of apoptotic pathways, alteration of drug targets, drug inactivation, and overexpression of ABC (Adenosine triphosphate-binding cassette, or ATP-binding cassette) transporters. These are broad substrate-specificity ATP-dependent efflux pumps able to export toxins or drugs out of cells; for instance, ABCB1 (MDR1, or P-glycoprotein 1), overexpressed in most cancer cells, confers them multidrug resistance (MDR). The gene coding for sorcin (SOluble Resistance-related Calcium-binding proteIN) is highly conserved among mammals and is located in the same chromosomal locus and amplicon as the ABC transporters ABCB1 and ABCB4, both in human and rodent genomes (two variants of ABCB1, i.e., ABCB1a and ABCB1b, are in rodent amplicon). Sorcin was initially characterized as a soluble protein overexpressed in multidrug (MD) resistant cells and named "resistance-related" because of its co-amplification with ABCB1. Although for years sorcin overexpression was thought to be only a by-product of the co-amplification with ABC transporter genes, many papers have recently demonstrated that sorcin plays an important part in MDR, indicating a possible role of sorcin as an oncoprotein. The present review illustrates sorcin roles in the generation of MDR via many mechanisms and points to sorcin as a novel potential target of different anticancer molecules.
  • Zachari, Maria; Gudmundsson, Sigurdur R.; Li, Ziyue; Manifava, Maria; Shah, Ronak; Smith, Matthew; Stronge, James; Karanasios, Eleftherios; Piunti, Caterina; Kishi-Itakura, Chieko; Vihinen, Helena; Jokitalo, Eija; Guan, Jun-Lin; Buss, Folma; Smith, Andrew M.; Walker, Simon A.; Eskelinen, Eeva-Liisa; Ktistakis, Nicholas T. (2019)
    The dynamics and co-ordination between autophagy machinery and selective receptors during mitophagy are unknown. Also unknown is whether mitophagy depends on pre-existing membranes, or is triggered on the surface of damaged mitochondria. Using a ubiquitin-dependent mitophagy inducer, the lactone ivermectin, we have combined genetic and imaging experiments to address these questions. Ubiquitination of mitochondrial fragments is required earliest followed by autophosphorylation of TBK1. Next, early essential autophagy proteins FIP200 and ATG13 act at different steps whereas ULK1/2 are dispensable. Receptors act temporally and mechanistically upstream of ATG13 but downstream of FIP200. The VPS34 complex functions at the omegasome step. ATG13 and optineurin target mitochondria in a discontinuous oscillatory way suggesting multiple initiation events. Targeted ubiquitinated mitochondrial are cradled by endoplasmic reticulum strands even without functional autophagy machinery and mitophagy adaptors. We propose that damaged mitochondria are ubiquitinated and dynamically encased in ER strands providing platforms for formation of the mitophagosomes.
  • Jämsä, Eija (University of Helsinki, 1994)