The intracellular localization and traffic of Shadoo protein

Transcription

1 The intracellular localization and traffic of Shadoo protein Ph.D. Thesis Eszter Tóth Eötvös Loránd University, Faculty of Science, Doctoral School of Biology, head: Prof. ANNA ERDEI, Ph.D. D.Sc. Molecular cell- and neurobiology program, head: Prof. MIKLÓS SASS, Ph.D. D.Sc. Habil. Supervisor ERVIN WELKER, Ph.D. D.Sc. LAJOS LÁSZLÓ, Ph.D. C.Sc. Budapest, 2014

2 INTRODUCTION Neurodegenerative disorders are a group of protein misfolding diseases, such as Alzheimer disease, Parkinson s disease, transmissible spongiform encephalopathies (TSE) or Huntington disease. By increasing the average age of the human population, the number of patients suffering from neurodegenerative disorders are continuously growing. Besides the temporary treatment of symptoms, the therapy of these neurodegenerative diseases is impossible with usually fatal outcome. The disease pathogenesis is characterized by the altered conformation of one or more neural proteins resulting in susceptibility to aggregation. TSE diseases are a group of rare, infectious, lethal neurodegenerative disorders in mammals, including Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker syndrome or kuru in human, bovine spongiform encephalopathy in cattle, scrapie in sheep and goat, and chronic wasting disease in mule deer and elk [1,2]. The main causatives are generally unknown (~80%, sporadic); however, genetic abnormalities (~19%) or infection (~1%) can result in TSE disease in some cases [3]. The infectious agent contains an improperly folded form of the host-encoded cellular prion protein (PrP c ). Conversion of PrP c into the diseaseassociated isoform, termed PrP sc, is thought to be the primary pathogenic event, although the mechanisms by which the conversion to PrP sc causes disease are poorly understood. The fact that PrP c knock out animals are resistant to infection [4] provided the evidence for the requirement of PrP c expression for pathogenesis. The prion protein is a glycophosphatydil-inositol-anchored plasma membrane glycoprotein with an unstructured N-terminal and a globular C-terminal. Its synthesis and processing take place through the secretory pathway [5,6]. PrP is expressed throughout the body, but in varying amounts; the highest levels occur in the central nervous system (CNS) and the heart [5,7]. The prion protein is associated to many cellular processes [8,9,10,11]; however, its physiological function and its pathologically relevant function is elusive. Moreover, an as-yetunidentified hypothetical π protein is suggested to replace the critical function of the N- terminal part of PrP c in PrP knock out mice. Two more members belong to the prion protein family, the doppel and the Shadoo proteins [12]. Shadoo protein has similar structural motifs (repeat region and hydrophobic domain) [13] to the antiapoptotic and cytoprotective N-terminal part of the prion protein

3 [9,10] displaying similar functions to those of the prion protein under defined circumstances [14]. Shadoo protein is expressed at the highest levels in the central nervous system. Studying the physiological function of an analogous protein may help us to better understand the physiological role of prion protein and to identify the critical functions of prion protein in physiological and pathological processes. We attempted to collect the similarities and differences between Shadoo and prion proteins to reveal the main functions of the protein family and their role in TSE diseases. The first task to achieve this aim was to reveal the intracellular localization of Shadoo protein. AIMS OF THE STUDY The main aim of the study was to monitor the intracellular localization of the newest member of the prion protein family (the Shadoo protein) by antibodies and fluorescent fusion constructs. We primarily focused on the similarities and differences of the localization of the prion protein. Based on our experiments, Shadoo protein is a dual localization protein, similarly to prion protein, both are detected in the plazma membrane and the nucleus. Therefore we aimed to study the dual localization of Shadoo protein using various fluorescent fusion constructs. We investigated the following questions: 1.) Which part of the Shadoo protein is responsible for the nuclear accumulation, or does any nuclear localization signal sequence exist in Shadoo protein similarly to the prion protein? 2.) Do active processes contribute to the nuclear transport of Shadoo and prion proteins? 3.) Does any nuclear binding partner (e.g. nucleic acids) of Shadoo and prion proteins exist? 4.) Does endoproteolytic cleavage occur in Shadoo protein, and is it associated with the dual localization? 5.) Which pathway of the intracellular traffic is followed by the Shadoo, or the part of the Shadoo protein present in the nucleus? At which point of the secretory pathway (endoplasmic reticulum or Golgi cisterns) does the Shadoo protein leave?

4 METHODS For the experiments, more than fifty plasmid constructs have been designed according to the classical cloning protocols. We developed a novel genetic engineering method using Type IIS restriction endonucleases to facilitate the cloning in special cases. The plasmid constructs were transformed into mammalian cell lines, and the efficient transfection and the intracellular localization were followed by EGFP/EYFP fluorescence analyzed by laser scanning confocal microscope. The intracellular localization of endogenous proteins was monitored by immunocytochemistry. The normal synthesis and processing of the fusion proteins were monitored by Western blot analysis. We treated the transiently transfected mammalian cells by chilling, deoxy-glucose, Brefeldin-A or Eeyarestatin I to follow the intracellular traffic and the nuclear transport of Shadoo protein. The potential nuclear localization signal was selected by various bioinformatic tools, available on the internet. The conservation of the Shadoo protein and its different structural motifs were analized using bioinformatic softwares. The recombinant proteins were expressed in bacterial cell cultures and were purified on nickel beads. The nucleic acid binding ability of Shadoo was tested by agarose gelshift assay.

5 RESULTS 1.) Using the novel genetic engineering method developed in our laboratory can solve a few frequently arising problems during general cloning experiments. The use of Body Double Type IIS restriction endonucleases is recommended. To facilitate their use, we have provided an online, freely available Body Double enzyme finder and primer designer software. 2.) It has been established that the YFP fused variant of Shadoo protein is a dual localization protein, similarly to prion protein. The YFP fused variant of Shadoo protein can be detected in the plazma membrane, in the nucleus and in the nucleolus. 3.) We have identified a conserved motif at the N-terminal of Shadoo protein, which is characterized by periodically occurring arginins [(RXXX) 8 motif]. Using deletion and fusion constructs we have showed that the (RXXX) 8 motif is responsible for the nuclear targeting of Shadoo. The arginines of the (RXXX) 8 motif may play a key role in the nuclear enhancement of Shadoo protein. 4.) Our results suggest that the (RXXX) 8 motif of Shadoo does not mediate active transport through the nuclear pore complexes. 5.) The YFP-fused variant of Shadoo seems to bind an as-yet-unidentified binding partner in the nucleolus. 6.) The nuclear accumulation of Shadoo requires active processes. 7.) Using fusion constructs we have displayed that the prion protein enters the nucleus by active transport; however, in contrast to Shadoo protein, it is excluded from the nucleolii. 8.) Our data suggest that the secretory pathway independent appearance of Shadoo protein and its nuclear accumulation could not be explained by the ineffective cotranslational translocation into the ER lumen. 9.) The exit of Shadoo protein from the secretory pathway has been proved to occur after the endoplasmic reticulum and the cis-golgi network. 10.) A non-radioactive and non-carcinogenic agarose gelshift assay was established for detection of the protein-nucleic acid binding based on the mobility changes of the complex. 11.) We have confirmed that the Shadoo protein and the (RXXX) 8 motif are able to bind single or double stranded DNA oligonucleotides and RNA molecules.

6 CONCLUSIONS We have confirmed that Shadoo protein is a dual localization protein, similarly to the TSE disease associated prion protein [15]. The Shadoo protein has been proved to exit from the secretory pathway somewhere after the cis-golgi network, it enters the nucleus from the cytosol by passive transport. The conserved and positively charged N-terminal part of Shadoo protein is responsible for the nuclear and nucleolar accumulation of the protein. In this region, the positions of the arginines are special, between two arginines, three, usually small amino acids hold the distance [(RXXX) 8 motif]. The prion protein was reported to possess two nuclear localization signals [16] and to occur in the nucleus in several cases (infected cells [17], dividing cells of the Lieberkuhn crypts [18], different STOP mutants [19], etc). The nuclear localization signals of Shadoo and prion proteins differ in type, in nuclear transport mediated (prion protein: active transport, Shadoo protein: active process dependent passive transport) and in nucleolar localization. The Shadoo and prion proteins may enter the nucleus under physiological conditions as the compartmentalization is never an all-or-none process. Their nuclear interactive partners are not known; however, they are likely to bind nucleic acids. Further analysis is required for the investigation of the secretory pathway exit mechanism and the consequences of the nuclear appearance.

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8 PUBLICATIONS RELATED TO THE PH.D. THESIS E. Tóth, E. Welker, Comparison of anti-shadoo antibodies Where is the endogenous Shadoo protein? WASET (2012) 71: E. Tóth, P. I. Kulcsár, E. Fodor, F. Ayaydin, L. Kalmár, A. É. Borsy, L. László, E. Welker, The highly conserved, N-terminal (RXXX) 8 motif of mouse Shadoo mediates nuclear accumulation BBA Mol Cell Res (2013) 1833: E. Tóth, K. Huszár, P. Bencsura, P. I. Kulcsár, B. Vodicska, A. Nyeste, Zs. Welker, Sz. Tóth, E. Welker, Restriction enzyme body doubles and PCR cloning: On the general use of Type IIS restriction enzymes for cloning PlosONE (2014) 9(3): e90896 Conference abstracts: E. Tóth, P. I. Kulcsár, E. Fodor, F. Ayaydin, L. László, E. Welker, Prion Proteins in health and disease: the (RXXX) 8 motif of Shadoo is a NLS Prion 5(2011):90 Prion 2011: New World Congress (2011, Montreal, Canada) A. Nyeste, F. Ayaydin, E. Fodor, E. Tóth, G. E. Tusnády, I. Simon, E. Welker, Alternative translation initiation may alter the cellular distribution of GFP-tagged proteins Prion 5(2011):70 Prion 2011: New World Congress (2011, Montreal, Canada)

9 E. Tóth, P. I. Kulcsár, E. Fodor, F. Ayaydin, L. László, E. Welker, Intracellular localization of SHadoo protein XVI. Sejt- és Fejlődésbiológiai Napok (2011, Siófok, Hungary) E. Tóth, P. I. Kulcsár, L. László, E. Welker, Nuclear localization and transport of Shadoo and Prion Proteins Prion 6(2012):119 Prion 2012 Conference (2012, Amsterdam, The Netherlands) I. Vida, A. Borsy, E. Tóth, E. Welker, Examination of nucleic acid binding of the newest prion protein, Shadoo, using Agarose Gel Shift Assay Hungarian Molecular Life Sciences 2013 (2013, Siófok, Hungary) B. Vodicska, A. Nyeste, E. Tóth, E. Welker, A membrane protein in the nucleus Hungarian Molecular Life Sciences 2013 (2013, Siófok, Hungary) E. Tóth, P. I. Kulcsár, E. Fodor, F. Ayaydin, L. Kalmár, A. É. Borsy, L. László, E. Welker, Dual localization of the prion protein family members: the highly conserved, N- terminal (RXXX) 8 motif of mouse Shadoo mediates nuclear accumulation Hungarian Molecular Life Sciences 2013 (2013, Siófok, Hungary) E. Tóth, P. I. Kulcsár, E. Fodor, F. Ayaydin, L. Kalmár, A. É. Borsy, L. László, E. Welker, Dual localization of the prion protein family members: the highly conserved, N- terminal (RXXX) 8 motif of mouse Shadoo mediates nuclear accumulation Prion 7(2013):92 Prion 2013: Conquering frontiers (2014, Banff, Canada) E. Tóth, K. Huszár, P. Bencsura, P. I. Kulcsár, B. Vodicska, A. Nyeste, Zs. Welker, Sz. Tóth, E. Welker, Restriction enzyme body doubles: the general use of Type IIS restriction endonucleases

10 44. Membrán-Transzport Konferencia (2014, Sümeg, Hungary) E. Tóth, K. Huszár, P. Bencsura, P. I. Kulcsár, B. Vodicska, A. Nyeste, Zs. Welker, Sz. Tóth, E. Welker, Cloning the Shadoo and Prion proteins: Restriction Enzyme Body Doubles and PCR cloning Prion 8(2014): 43 Prion 2014: Shaping the future of prion research (2014, Trieste, Italy)