Analysis of dehydrogenase-independent functions of HSD17B10 in humans and animal models [Elektronische Ressource] / presented by Katharina Rauschenberger

Xenopus

Sujets

• Biologie

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2011
• Nombre de lectures: 19
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Analysis of dehydrogenase-independent
functions of HSD17B10 in humans
and animal models



















presented by
Dipl.-Biol. Katharina Rauschenberger


Analysis of dehydrogenase-independent
functions of HSD17B10 in humans
and animal models



Dissertation

submitted to the
Combined Faculties for the Natural Sciences
and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany

for the degree of

Doctor of Natural Sciences




presented by
Dipl.-Biol. Katharina Rauschenberger
born in Friedrichshafen, Germany





Date of oral examination: 25 February 2011



Analysis of dehydrogenase-independent
functions of HSD17B10 in humans
and animal models





prepared at the

Institute of Human Genetics, Heidelberg
Division of Developmental Genetics










Referees:
Prof. Dr. rer. nat. Herbert Steinbeisser
Prof. Dr. Dr. med. Johannes Zschocke



The following publication originated from this work:

Rauschenberger K, Scholer K, Sass JO, Sauer S, Djuric Z, Rumig C, Wolf NI, Okun JG,
Kolker S, Schwarz H, Fischer C, Grziwa B, Runz H, Numann A, Shafqat N, Kavanagh KL,
Hammerling G, Wanders RJ, Shield JP, Wendel U, Stern D, Nawroth P, Hoffmann GF,
Bartram CR, Arnold B, Bierhaus A, Oppermann U, Steinbeisser H, Zschocke J (2010)
A non-enzymatic function of 17beta-hydroxysteroid dehydrogenase type 10 is
required for mitochondrial integrity and cell survival.
EMBO Mol Med 2(2): 51-62
Table of contents

1 SUMMARY................................................................................................. 1
2 ZUSAMMENFASSUNG ................................................................................ 2
3 INTRODUCTION........................................................................................ 3
3.1. 17 β-hydroxysteroid-dehydrogenase type 10 – an evolutionary
conserved multifunctional protein....................................................... 3
3.2. Function of HSD10 in fatty acid metabolism and isoleucine
breakdown .......................................................................................... 4
3.3. HSD10 in steroid metabolism .............................................................. 6
3.4. HSD10 deficiency in humans ............................................................... 7
3.5. Implication of HSD10 in Parkinson’s, Alzheimer’s and other
clinical symptoms.............................................................................. 10
3.6. HSD10 – cytoprotective or cytotoxic?................................................ 12
3.7. Intrinsic and extrinsic regulation of apoptosis .................................. 13
3.8. HSD10 function in the model organisms Drosophila and
mouse................................................................................................ 15
3.9. HSD10 in the development of Xenopus laevis.................................... 17
3.10. Interaction partners of HSD10 .......................................................... 18
3.11. HSD10 as a component of human mitochondrial RNaseP................... 18
3.12. Purpose of this study......................................................................... 22
4 RESULTS................................................................................................. 23
4.1. Localisation and amount of HSD10 in human cells under
various conditions ............................................................................. 23
4.1.1. Mitochondrial localisation of HSD10 in HSD10
deficiency patient fibroblasts ............................................. 23
4.1.2. Mitochondrial and HSD10 content in HSD10 broblasts 25
4.1.3. HSD10 translocates from the mitochondrial matrix
to the membrane under oxidative stress ............................ 26
4.2. Mitochondrial morphology and function after HSD10 loss-of-
function............................................................................................. 28
4.2.1. Mutations D86G and R130C cause severe disruption
of mitochondrial morphology.............................................. 28
4.2.2. Mitochondrial disintegration after conditional
HSD17B10 knock-out in mice ............................................. 29
4.2.3. HSD10 knock-down in Xenopus impairs
mitochondrial integrity....................................................... 32
4.3. Induction of apoptosis after HSD10 gain- and loss-of-function ......... 35
4.3.1. HSD10 gain-of-function in Xenopus induces
apoptosis............................................................................ 35
4.3.2. Apoptosis induced by HSD10 gain-of-function is not
due to the unfolded protein response................................. 36
4.3.3. Analysis of the apoptotic pathway induced by HSD10
loss-of-function .................................................................. 38
4.4. HSD10 function and characterisation of mutations under
cellular stress conditions................................................................... 43
4.5. Identification of interaction partners of HSD10................................. 47
4.5.1. Homology based BLAST in yeast......................................... 47
4.5.2. Functional interaction of human HSD10 and UXT in
vivo .................................................................................... 50
4.5.3. Pull-down/IMAC approach to identify binding
partners of HSD10 .............................................................. 52
4.6. Function of HSD10 as a component of human mitochondrial
RNaseP.............................................................................................. 59
4.6.1. Accumulation of tRNA precursors after HSD10 loss-
of-function.......................................................................... 59
4.6.2. Reconstitution of RNaseP activity using mutated
HSD10 after HSD10 loss-of-function................................... 61
4.6.3. RNaseP activity in patient fibroblasts under
physiological and oxidative stress conditions..................... 63
4.6.4. Effect of impaired RNaseP activity on mitochondrial
translation 66
4.6.5. RNaseP dependency of apoptosis induced by HSD10
loss-of-function .................................................................. 68
5 DISCUSSION........................................................................................... 73
5.1. A dehydrogenase-independent function of HSD10 is essential
for mitochondrial integrity ................................................................ 73
5.2. HSD10 function in apoptosis, RNaseP activity and cellular
stress ................................................................................................ 75
5.2.1. HSD10 function in apoptosis............................................... 75
5.2.2. HSD10 function under cellular stress.................................. 77
5.2.3. Function of HSD10 as a component of human
mitochondrial RNaseP ........................................................ 79
5.3. Dehydrogenase- and RNaseP-independent function of HSD10
outside of mitochondria?................................................................... 82