DJ-1 zymogen activation: implication for Parkinson disease Pubblico

Chen, Jue (2011)

Permanent URL: https://etd.library.emory.edu/concern/etds/xk81jk591?locale=it
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Abstract

DJ-1 zymogen activation: implication for Parkinson disease
By Jue Chen
Loss-of-function mutations in DJ-1 have been linked to early onset familial PD but the
exact cellular roles of DJ-1 remain unknown. DJ-1 shares structural homology with a
bacterial and archea cysteine protease family but it has an extra α-helix (H8). The H8 of
DJ-1 is predicted to prevent substrate entry and formation of a mature catalytic pocket.
To understand the biological functions of DJ-1, we investigated whether DJ-1 is a
cysteine protease and further studied the regulation of DJ-1 zymogen activation and the
role of DJ-1 protease in anti-oxidative stress response.
Our data demonstrate that removal of H8 region enhances DJ-1 protease activity
by increasing substrate affinity and improving catalytic efficiency. Further, DJ-1
functions as a cysteine protease because protease activity of DJ-1 is dependent on the
Cys-His catalytic diad and is inhibited by oxidation. We also find that endogenous DJ-1
undergoes C-terminal cleavage in response to mild oxidation and protease activity
contributes to DJ-1 cytoprotective function.
To further understand the mechanisms by which DJ-1 zymogen is activated, we
investigated whether methionine residues regulate DJ-1 zymogen activation and
cytoprotective function. Methionine residues can be reversibly oxidized, serving as the
basis for redox-dependent regulation of cell signaling. Our findings demonstrate that
selective methionine residues are required for zymogen activation and are critical for DJ-
1 cytoprotective function. Moreover, the PD linked M26I mutation alters DJ-1 secondary
structure and destabilizes DJ-1 protein.
Together the findings presented in this dissertation reveal that DJ-1 functions as a
cysteine protease that requires activation in response to mild oxidation and DJ-1 zymogen
activation is regulated by oxidation of selective methionine residues. Furthermore, our
data suggest that the pathologic effects of PD linked M26I mutation are a combination of
loss of enzymatic function and mild unfolding of DJ-1 protein.

Table of Contents

TABLE OF CONTENTS
CHAPTER I. INTRODUCTION AND BACKGROUND
OPENING REMARKS

........................................................................................................ 2
PARKINSON DISEASE ....................................................................................................... 3
Clinical presentations .................................................................................................. 3
Epidemiology .............................................................................................................. 4
Pathology .................................................................................................................... 5
Treatment .................................................................................................................... 7
TOXICOLOGICAL MODELS OF PD ................................................................................ 10
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) ........................................... 10
Rotenone ................................................................................................................... 12
6-hydroxyldopamine (6-OHDA) .............................................................................. 13
Paraquat..................................................................................................................... 13
GENETIC MODELS OF PD ............................................................................................. 13
PARK1/4 ................................................................................................................... 14
PARK2 ...................................................................................................................... 15
PARK3 ...................................................................................................................... 16
PARK5 ...................................................................................................................... 16
PARK6 ...................................................................................................................... 19
PARK7 ...................................................................................................................... 20
PARK8 ...................................................................................................................... 20
PARK9 ...................................................................................................................... 21

PARK10 .................................................................................................................... 22
PARK11 .................................................................................................................... 22
PARK12 .................................................................................................................... 22
PARK13 .................................................................................................................... 22
PARK14 .................................................................................................................... 23
PARK15 .................................................................................................................... 24
PARK16 .................................................................................................................... 25
MOLECULAR MECHANISMS OF PD............................................................................... 26
DJ-1 PROTEIN ............................................................................................................... 32
DJ-1 is oncogenic ...................................................................................................... 32
Loss-of-function mutations in DJ-1 linked to PD ..................................................... 33
Phenotypes of DJ-1 knockout mice .......................................................................... 39
Structure of DJ-1 protein .......................................................................................... 41
DJ-1 as a protease zymogen ...................................................................................... 43
DJ-1 is a redox-sensitive protein............................................................................... 44
The mechanisms of DJ-1 mediated antioxidative function are unknown ................. 47
PROTEASES ................................................................................................................... 48
Catalytic strategy ...................................................................................................... 48
Substrate specificity .................................................................................................. 49
Activation of zymogens ............................................................................................ 50
Physiological and pathological roles of proteases .................................................... 51
Cysteine proteases are inhibited by oxidation .......................................................... 52
Dual function proteins with protease and chaperone activity ................................... 52

REDOX REGULATION OF PROTEIN FUNCTIONS

............................................................ 54
Generation of ROS and the antioxidant systems ...................................................... 54
Oxidation of the protein backbone ............................................................................ 55
Oxidation of the amino acid side chain..................................................................... 56
Oxidation of cysteine ................................................................................................ 56
Oxidation of methionines .......................................................................................... 57
Reversible methionine oxidation can regulate protein functions .............................. 58
Increased methionine oxidation has been implicated in
neurodegenerative disorders ..................................................................................... 58
HYPOTHESES AND ORGANIZATIONAL OVERVIEW ...................................................... 61
CHAPTER II. PARKINSIN DISEASE PROTEIN DJ-1 CONVERTS FROM A
ZYMOGEN TO A PROTEASE BY CARBOXYL-TERMINAL CLEAVAGE
ABSTRAT ....................................................................................................................... 74
INTRODUCTION ............................................................................................................. 75
EXPERIMENTAL PROCEDURES ..................................................................................... 77
Expression constructs and antibodies ....................................................................... 77
DJ-1 knockout mice and primary culture .................................................................. 77
Protein expression and purification .......................................................................... 78
Protease activity assay .............................................................................................. 78
DJ-1 cleavage assay .................................................................................................. 79
Cell transfection, immunoprecipitation and immunoblot analysis ........................... 79
Immunofluorescent confocal microscopy ................................................................. 80
Cell viability and apoptosis assay ............................................................................. 80

Statistical analysis ..................................................................................................... 81
RESULTS ....................................................................................................................... 81
Cleavage of the C-terminal 15-amino acid peptide activates DJ-1 protease function
without affecting its dimerization ............................................................................. 81
The C-terminally cleaved form of DJ-1 is an active cysteine protease with a
functional Cys-His catalytic diad .............................................................................. 83
DJ-1 cysteine protease is susceptible to inactivation by oxidation .......................... 84
C-terminal cleavage activates DJ-1 protease by enhancing substrate binding as well
as catalysis ................................................................................................................ 85
Endogeous DJ-1 in dopaminergic cells undergoes C-terminal cleavage in response to
mild oxidative stress ................................................................................................ 86
DJ-1 protease activation by C-terminal cleavage enhances its cytoprotective function
................................................................................................................................... 87
Activated DJ-1 protease exerts enhanced neuroprotection against oxidative stress-
induced apoptosis ...................................................................................................... 89
DISCUSSION ................................................................................................................... 90
ACKNOWLEDGEMENTS ................................................................................................. 93
CHAPTER III. MET-17 AND MET-26 ARE CRITICALLY INVOLVED IN DJ-1
ZYMOGEN ACTIVATION AND CYTOPROTECTIVE FUNCTION
ABSTRAT ..................................................................................................................... 115
INTRODUCTION ........................................................................................................... 116
EXPERIMENTAL PROCEDURES ................................................................................... 118
Expression constructs and reagents ........................................................................ 118

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