DJ-1 zymogen activation: implication for Parkinson disease Open Access
Chen, Jue (2011)
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
About this Dissertation
- Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.
School | |
---|---|
Department | |
Subfield / Discipline | |
Degree | |
Submission | |
Language |
|
Research Field | |
Keyword | |
Committee Chair / Thesis Advisor | |
Committee Members |
Primary PDF
Thumbnail | Title | Date Uploaded | Actions |
---|---|---|---|
|
DJ-1 zymogen activation: implication for Parkinson disease () | 2018-08-28 11:08:22 -0400 |
|
Supplemental Files
Thumbnail | Title | Date Uploaded | Actions |
---|