The subcellular trafficking of the prion protein [Elektronische Ressource] : characterisation of the function of the PrP_1hnc N-terminus / vorgelegt von Maximilian Nunziante

Nunziante, Maximilian

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2003
• Nombre de lectures: 18
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Aus dem Max-von-Pettenkofer Institut für Virologie

der Ludwig-Maximilians-Universität

Vorstand: Prof. Dr. med. U. Koszinowski







The subcellular trafficking

of the prion protein:

Characterisation of the function of the

cPrP N-terminus






Dissertation

zum Erwerb des Doktorgrades der Humanbiologie

an der Medizinischen Fakultät der

Ludwig-Maximilians-Universität zu München





vorgelegt von

Maximilian Nunziante
aus
Catania (Italien)

Jahr
2003




Mit Genehmigung der medizinischen Fakultät

der Universität München














Berichterstatter: Prof. Dr. rer. nat. K. Conzelmann

Mitberichterstatter: Priv. Doz. Dr. H. Steiner

Prof. Dr. J. Herms

Mitbetreuung durch den
promovierten Mitarbeiter: Prof. Dr. med. H. M. Schätzl


Dekan: Prof. Dr. med. Dr. h. c. K. Peter


Tag der mündlichen Prüfung: 26. 09. 2003 Table of contents
___________________________________________________________________________
Table of contents


1 Introduction…………………………………………………………………………. 1

1.1 Historical Background…………………………………………………….….. 1
1.2 Animal prion diseases……………………………………………………....… 2
1.3 Human Prion Diseases…………………………………………………....……4
1.3.1 Variant CJD…………………………………………………..………..7
1.4 Therapeutic and prophylactic approaches…………………………………… .8
1.5 PrP gene structure……………………………………………………...……....9
1.6 The function of the prion protein…………………………………..…………10
1.7 The structural properties of the prion protein…………………….…….…….11
c Sc1.7.1 The conformation of PrP and PrP ………………………….………13
1.7.2 Mechanisms of prion conversion……………………………….…….14
1.8 Prion strains and the species barrier…………………………………….……15
1.9 The N-terminus of the prion protein……………………………….…………17
1.10 The Xenopus laevis prion protein………………………………….…………18
1.11 The subcellular trafficking of proteins……………………………….………18
1.11.1 Subcellular trafficking of the prion protein…………………….…….20
1.12 Aim of this work………………………………………………………….….21


2 Material and methods………………………………………………………………22

2.1 Antibodies……………………………………………………………………22
2.2 DNA cloning…………………………………………………………………22
2.2.1 Polymerase chain reaction (PCR)……………………………………22
2.2.2 Agarose gel electrophoresis………………………………………….25
2.2.3 Isolation and elution of DNA fragments from agarose gel…………..25
2.2.4 Enzymatic digestion of DNA………………………….……………..25
2.2.5 DNA dephopsphorylation……………………………..…………......26
2.2.6 Ligation……………………………………………………………... 26
I Table of contents
___________________________________________________________________________
2.2.7 DNA sequencing……………………………………………………....26
2.2.8 Preparation of heat shock competent bacteria…………………..…… 27
2.2.9 Heat shock transformation…………………………………..………...28
2.3 DNA extraction and purification……………………………… ………...…..28
2.3.1 Small scale preparation of plasmid DNA from bacteria
(Minipreparation) ……………………………………….………...…. 28
2.3.2 Large scale preparation of plasmid DNA from bacteria
(Maxipreparation) ………………………………………..…….……..29
2.3.3 Spectrophotometric determination of amount of DNA………..……. 29
2.4 Culture of bacterial cells…………………………………………………..… 30
2.4.1 Bacterial cells used in this work……………………………....………30
2.4.2 Growing of bacterial cells……………………………………………..30
2.4.3 Determination of cell density…………………………………………31
2.4.4 Storage of bacteria…………………………………………………….31
2.5 Working with mammalian cells……………………………………………….31
2.5.1 Mammalian cell lines used in this work………………………………31
2.5.2 Culture Media…………………………………………………………31
2.5.3 Culture of mammalian cells……………………………………….…..32
2.5.4 Storage of eukaryotic cells…………………………………….….…..32
2.5.5 Transient transfection of mammalian cells…………………………...32
2.6 Internalisation assay with cell surface biotinylation…………………….……33
2.7 Metabolic labelling and radio-immunoprecipitation………………………….34
2.7.1 Trypsin digestion……………………………………………….……..36
2.7.2 Treatment with Endoglycosidase-H (Endo-H)………………….…….36
2.7.3 Digestion with N-Glycosidase F (PNGase F)………………….….…..36
2.8 Preparation of postnuclear lysates…….………………………………...…….37
2.8.1 Detergent solubility assay…………………………………….…..……38
2.8.2 Proteinase K (PK) digestion………….……………………………….38
2.8.3 SDS-PAGE………………………………………………...………….38
2.8.4 Western blot………………………………………………..………….40
2.9 Detection and evaluation of radioactive signals………………..……………..41
2.10 Detection and evaluation of non-radioactive signals
(densitometric analysis) ………………………………...……...……………..41
II Table of contents
___________________________________________________________________________
2.11 Treatment of cells with lysosomal inhibitor Leupeptin…………………..….41
2.12 FACS analysis……………………………………………………………….42
2.12.1 Cell surface analysis…………………………………………………42
2.12.2 Intracellular analysis…………………………………………………43

3 Results……………………………………………………………………...…….... 44
c3.1 Biochemical characterisation of PrP constructs showing progressive
N-terminal deletions………………………………………………………...45
3.2 Effect of progressive N-terminal deletion on the internalisation of
cmurine PrP ………………………………………………………………… 47
c3.3 Equal detection of wtPrP and PrP ∆(23-90) in the culture medium……..…50
3.4 Lysosomal accumulation of PrP ∆ (23-90) upon treatment of the cells
with leupeptin……………………………………………………………….51
c3.5 Effect of N-terminal deletion on the half-life of PrP ……………………….52
3.6 Modulation of the transport through the secretory pathway by
the N-terminal segment of the prion protein……………………………...…54
3.7 N-terminal deletions and PrP glycosylation………………………………...56
3.8 Biochemical characterisation of a chimeric prion protein…………………..59
3.9 Restoration of the wild type phenotype by the Xenopus laevis N-terminus...62

4 Discussion…………………………………………………………………………..64
4.1 N-terminal deletion does not affect the biochemical properties
of the prion protein……………………………………………………….....64
c 4.2 Progressive deletions within the N-terminus of PrP result
in reduced endocytosis…………………………………………………...…65
4.3 The N-terminal truncated form of the prion protein is not
exceedingly released into the culture medium and accumulates
in lysosomes upon protease inhibition………………………………….......68
c4.4 Deletion of the N-terminal part significantly affects PrP turnover…….......69
c 4.5 The N-terminus of PrP and the secretory pathway…………………………70
4.6 Targeting function of the PrP N-terminus is conserved in evolution….……73



III Table of contents
___________________________________________________________________________
5 Summary I (English version)………………………………………………………76
6 Summary II (German version)…………………………………………………….78
7 Reference list………………………………………………………………………. 80
8 Abbreviations……………………………………………………………………… 94
9 Publications…………………………………………………………………...…… 96
10 Acknowledgements………………………………………………………………... 97
11 Curriculum vitae………………………………………………………………...…98

IV Introduction
__________________________________________________________________________________________

1 Introduction

1.1 Historical Background
Although at least some of the closely related neurodegenerative conditions known as
transmissible spongiform encephalopathies (TSEs) or prion diseases have been recognised for
many years, only in the last two decades decisive steps have been made in the characterisation
of their underlying causes. These rapidly progressing fatal syndromes can affect both humans
and animals and present scientists and public health with distinctive phenomena and
challenges. This is because of the unique biology of the transmissible agent and because of
the fear that the epidemic of the bovine prion disease BSE, with more the 180,000 cases in the
U.K. and numerous confirmed cases in other European countries, could represent a threat to
public health through dietary exposure. In 1996 a direct link between BSE and a novel form
of the human Creutzfeldt-Jakob disease was argued for (Will et al., 1996; Collinge et al.,
1996b;Will, 1998).
The nature of the agent in these disorders has been a matter of debate for many years. The
naturally occurring scrapie disease affecting sheep and goat present in many countries world
wide was first described as early as 1732. In 1936 inoculation between sheep (and goat)
showed that scrapie could be transmitted after prolonged incubation. Some kind of virus was
therefore assumed to be the causative agent and in 1954 Sigurdson referred to it as a “slow
virus”, due to incubation times as long as 20 years. Five years later Hadlow drew attention to
the similarities between scrapie and kuru, a disease of New Guinea highlanders, at a
neuropathological and clinical level. Intracerebral inoculation of chimpanzees with brain
homogenates a few years later established the transmissibility of kuru (Gajdusek et a