Regulation of parkin and PINK1 by neddylation
Yeun Su Choo1, Georg Vogler1, Danling Wang1,2, Sreehari Kalvakuri1, Anton Iliuk3,
W. Andy Tao3, Rolf Bodmer1and Zhuohua Zhang1,2,∗
1Sanford-Burnham Medical Research Institute, 10901 N. Torrey Pines Road, La Jolla, CA 92037, USA,2State Key
Laboratory of Medical Genetics, Xiangya Medical School, Central South University, Changsha, Hunan 410078, China
and3Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA
Received December 8, 2011; Revised and Accepted February 21, 2012
Neddylation is a posttranslational modification that plays important roles in regulating protein structure and
function by covalently conjugating NEDD8, an ubiquitin-like small molecule, to the substrate. Here, we report
that Parkinson’s disease (PD)-related parkin and PINK1 are NEDD8 conjugated. Neddylation of parkin and
PINK1 results in increased E3 ligase activity of parkin and selective stabilization of the 55 kDa PINK1 frag-
ment. Expression of dAPP-BP1, a NEDD8 activation enzyme subunit, in Drosophila suppresses abnormalities
induced by dPINK1 RNAi. PD neurotoxin MPP1inhibits neddylation of both parkin and PINK1. NEDD8 immu-
noreactivity is associated with Lewy bodies in midbrain dopaminergic neurons of PD patients. Together,
these results suggest that parkin and PINK1 are regulated by neddylation and that impaired NEDD8 modifi-
cation of these proteins likely contributes to PD pathogenesis.
Parkinson’s disease (PD) is the most frequent neurodegenera-
tive movement disorder affecting ?1% of the population over
age 65 (1). Most PD cases are sporadic. Mutations in several
genes, however, are associated with the familial form of PD.
These genes include SNCA (2), parkin (3), UCHL1 (4),
PINK1 (5), DJ-1 (6), LRRK2 (7,8), ATP13A2 (9), GIGYF2
(10), Omi/HTRA2 (11), PLA2G6 (12) and FBXO7 (13).
Recent linkage analysis and genome-wide association studies
have identified additional genetic loci as PD risk factors,
such as MAPT, BST1, GAK and HLA-DRB5 (1,14). Under-
standing the pathophysiological functions of these genes will
help define PD etiology and design novel strategies for early
diagnosis and treatment. Among these proteins, parkin is a
RING/HECK hybrid ubiquitin E3 ligase with an ubiquitin-like
domain at its N-terminus and a RING-IBR-RING domain at its
C-terminus (3,15). It mediates ubiquitination and degradation
of multiple proteins (16). In addition, parkin has an important
role in protecting neurons against various insults and maintain-
ing mitochondrial integrity (17–24). Recent studies suggest
that parkin also functions as a tumor suppressor in multiple
cancers (25,26). PINK1 is a putative kinase with an N-terminal
mitochondrial targeting signal (5). In addition to the full-
length protein, a 55 kDa PINK1 fragment with a truncated
N-terminal segment is detected in the cytosol, suggesting the
compartmental functions for PINK1 (5,16). Several potential
substrates of PINK1 have been identified despite the unclear
biological consequence of PINK1-mediated protein phosphor-
ylation (27–29). PINK1 functions in common pathways with
parkin to maintain mitochondrial integrity, quality control
and transport (17,21,24,30). Moreover, parkin, PINK1 and
DJ-1 form an E3 ligase complex (the PPD complex) to
promote the degradation of mis/unfolded proteins (16). Never-
theless, the cellular mechanism for the regulation of parkin
and PINK1 is largely unknown.
NEDD8 isan ubiquitin-like small moleculethat is covalently
conjugated to proteins to regulate protein functions. Cullins
are the first protein groups known to be NEDD8 conjugated.
The NEDD8 conjugation of cullins activates the cullin-ring
ligase complex. The conjugation process, also known as ned-
dylation, is catalyzed by an enzymatic pathway similar to ubi-
quitination with distinct enzymes, such as APP-BP1/Uba3
heterodimer (E1), Ubc12 or UbeF2 (E2), and Dcn1 or
Dcn1-like proteins (E3) (31–33). Several ubiquitin E3
ligases such as c-Cbl, mdm2 and mammalian IAPs can act
as NEDD8 E3 ligases (34–36), suggesting the presence of
diverse NEDD8 substrates besides cullin proteins and a
potential crosstalk between ubiquitination and neddylation.
An increasing number of non-cullin proteins is found to be
neddylation regulated, including transcriptional factor p53
ofcellularPINK1 processingand multi-
∗To whom correspondence should be addressed. Tel: +86 73184805358; Fax: +86 73184474152; Email: firstname.lastname@example.org
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Human Molecular Genetics, 2012, Vol. 21, No. 11
Advance Access published on March 2, 2012
and BCA3 (36,37). Neddylation of the epidermal growth
factor receptor (EGFR) and some ribosomal proteins modu-
lates the stability of these proteins (35,38). In PD, specific
immunoreactivity to NEDD8 is detected in Lewy bodies, sug-
gesting that protein neddylation is involved in PD pathogen-
esis (39,40). Little is known, however, about the molecular
role of neddylation in PD development.
In the present study, we identified that PD-associated parkin
and PINK1 are NEDD8 modified. Neddylation results in
increased parkin E3 ligase activity and stabilization of
PINK1 55 kDa fragment. Expression of APP-BP1 in Drosoph-
ila suppresses dPINK1 RNAi-induced ommatidial degener-
ation, abnormal wing phenotype and male sterility.
Neddylation of parkin and PINK1
To study regulation of the parkin/PINK1/DJ-1 E3 ligase
complex(16), we expressed
SH-SY5Y cells, immunoprecipitated exogenous PINK1 and
analyzed PINK1 interactome with mass spectrometry. The
resulting PINK1 interactors include several essential compo-
nents for NEDD8 conjugation: NEDD8, APP-BP1, UBC12
and COP9 signalosome proteins (Table 1). This result suggests
that PINK1 or its interacting proteins are NEDD8 modified. To
examine neddylation of parkin and PINK1 in the cell, we
expressed parkin and PINK1 in HEK293 cells individually
with NEDD8. Immunoprecipitation followed by immunoblot-
ting for NEDD8 showed that immunoprecipitated parkin pro-
duced NEDD8-positive smear bands above the molecular
weight of parkin (Fig. 1A). Likewise, PINK1 immunoprecipi-
tation resulted in a NEDD8-positive smear band above the mo-
lecular weight of PINK1 (Fig. 1D). In contrast, in the absence
of NEDD8, the NEDD8-positive band was not detected with
either parkin or PINK1 immunoprecipitation (Fig. 1A and
D). In addition, immunoprecipitation of a-synuclein in the
presence of NEDD8 did not result in the NEDD8-positive
band (Fig. 1A), demonstrating the specificity of NEDD8 con-
jugation of parkin and PINK1.
To verify that the observed smear bands were NEDD8
modification of parkin and PINK1, we treated parkin and
PINK1 immunoprecipitates with a recombinant NEDD8-
specific deneddylation enzyme NEDP1 in vitro. NEDP1 treat-
high-molecular-weight smear bands of parkin and PINK1
(Fig. 1B). In addition, the expression of wild-type NEDP1,
but not the isopeptidase-deficient NEDP1 mutant, abolished
neddylation of parkin (Fig. 1C) and PINK1 (Fig. 1D). These
results suggest that both parkin and PINK1 are specifically
modified by NEDD8 conjugation.
Previous reports indicate that cullins are mononeddylated
and appear as a single modified band. However, neddylated
parkin and PINK1 appear as smear bands, indicating hyper-
neddylation. To exclude the possible contribution of polyubi-
quitination to the observed smear bands, we treated parkin and
PINK1 immunoprecipitates with a recombinant ubiquitin-
specific deubiquitinating enzyme Usp2 that non-specifically
cleaves all ubiquitin conjugates to ubiquitin monomers
(41,42). We observed little change to the NEDD8 smear
bands associated with high-molecular-weight parkin or
PINK1. The Usp2 activity was demonstrated by abolishing
parkin polyubiquitination with Usp2-cc treatment. These
results suggest that parkin and PINK1 are likely polyneddy-
lated or multiple-mononeddylated.
To further confirm the neddylation of parkin and PINK1, we
performed in vitro neddylation analysis using purified parkin
and PINK1. NEDD8-modified parkin (Fig. 1E) and PINK1
(Fig. 1F) were detected only with the presence of the com-
pleted neddylation components. Together, the results support
the notion that parkin and PINK1 are modified by NEDD8
Increased parkin E3 ligase activity by NEDD8
To determine the functional consequence of neddylation, we
analyzed the E3 ligase activity of parkin in the presence of
NEDD8. The results revealed that parkin ubiquitination was
dose-dependently upregulated with increased neddylation
(Fig. 2A). Likewise, ubiquitination of synphilin-1, another
parkin substrate, was also upregulated with increased
NEDD8 in a dose-dependent manner (Fig. 2B). This observa-
tion is likely specific to parkin substrates because the total
ubquitination is inhibited with increased NEDD8. One explan-
ation of this observation is that ubiquitination and neddylation
compete for target proteins other than parkin in the cell.
NEDD8 and ubiquitin are known to modify overlapping sets
of lysine residues of certain substrates (35,36). The results
suggest that NEDD8 modification potentiates parkin E3
Stabilization of the 55 kDa fragment of PINK1 by
Ectopic expression of NEDD8 regulates the stability of EGFR
and ribosomal protein L11 (35,38). We next examined the
effect of PINK1 neddylation. With the expression of
NEDD8, the steady-state level of a 55 kDa PINK1 fragment
was increased (Fig. 1D). Cycloheximide-mediated chase ana-
lyses showed that the levels of both full-length PINK1 and
its 55 kDa fragment were decreased rapidly with a half-life
of less than 2 h (Fig. 3A and B). With the expression of
NEDD8, the half-life of the full-length PINK1 remained as
2 h. Nevertheless, half-life of the PINK1 55 kDa fragment
was increased to more than 6 h (Fig. 3A and B). The results
indicate that PINK1 neddylation selectively increases the sta-
bility of the PINK1 55 kDa fragment.
Table 1. Interaction of PINK1 with neddylation machinery
Protein IDTotal spectra Unique spectraCoverage (%)
Human Molecular Genetics, 2012, Vol. 21, No. 112515
Expression of neddylation E1 component dAPP-BP1 in
Drosophila suppresses abnormalities induced by dPINK1
The NEDD8 system is conserved across species including Dros-
ophila. Previously, we reported that inhibition of Drosophila
PINK1 (dPINK1) via RNAi in Drosophila eyes, using
GMR-GAL4 driver induces ommatidial degeneration resulting
in black speckles, patches or lesions in eyes (Fig. 3C, bottom)
(43). We next examined whether the dPINK1-RNAi-induced
eyephenotypes areaffected byneddylation.Expression of Dros-
regulatory subunit, shows little effect on external eyes of wild-
type flies.Nevertheless, expression
dPINK1-RNAi flies significantly suppressed eye phenotypes, in-
cludingspeckles,patchesorlesions(Chi-squaretest,P , 0.0001
between dPINK1-RNAi+dAPP-BP1 and dPINK1-RNAi flies)
(Fig. 3C). Stabilization of dPINK1 expected from dAPP-BP1
overexpression may compensate the dPINK1 reduction.
We further examined the effect of dAPP-BP1 on another
dPINK1-RNAi fly line showing abnormal wing phenotypes
(44). At 7 days of age, 63% of the dPINK1-RNAi flies with
muscle-specific knockdown using Mhc-GAL4 (n ¼ 232) dis-
played a drooped wing posture (Fig. 3D). With dAPP-BP1
overexpression, only 50% of dPINK1-RNAi flies (n ¼ 231)
showed the drooped wing phenotypes (Fig. 3D). Therefore,
overexpression of dAPP-BP1 significantly inhibits the abnor-
mal wing phenotype induced by dPINK1 RNAi (P ¼ 0.0058,
Chi-square test). It is well documented that flies with either
PINK1 null alleles or dPINK1 knockdown show reduced
male fertility (17,22,45). Overexpression of dAPP-BP1 under
tubulin-GAL4 driver greatly improves the male fertility of
dPINK1-RNAi flies from 12 to 57% (Chi-square test, P ¼
0.0009, Fig. 3E).
Consistent with the results, knockdown of dAPP-BP1 using
a different mesoderm-specific driver, how24B-GAL4.dcr-2,
results in droopy wing phenotype in 86% of males (n ¼ 78,
Fig. 3F). No wing phenotype was observed in control male
flies with genotypes of how24B-GAL4.dcr-2 (0%, n ¼ 59)
or dAPP-BP1-RNAi (0%, n ¼ 134). The results provide add-
itional evidence to support regulation of PINK1 by neddyla-
Together, results from two independent dPINK1-RNAi fly
lines suggest that Drosophila PINK1 function is likely regu-
lated by protein neddylation.
Detection of parkin neddylation in human brain
Next, we analyzed the neddylation of endogenous parkin in
human brain tissues. Endogenous parkin was immunoprecipi-
tated from the brain lysates of PD patients (Fig. 4A). Bands
that were immunopositive both for parkin and NEDD8 were
excised and subjected to analysis with mass spectrometry.
The results identified a 13-amino acid-length parkin fragment
with lysine 76 residue glycine–glycine modified (Fig. 4B).
We were unable to analyze neddylation of PINK1 because
anti-PINK1 antibody for immunoprecipitation is not available.
The result provides supporting evidence of neddylation of en-
dogenous parkin in human brain.
Regulation of parkin and PINK1 neddylation by MPP1
We also determined whether neddylation of parkin or PINK1
is regulated by PD-related neurotoxin. HEK293 cells were
co-transfected NEDD8 with either parkin or PINK1 followed
PD-relevant toxic cation, at sub-lethal concentrations. The
levels of both neddylated parkin and PINK1 were obviously
reduced starting at 10 mM with dose dependence (Fig. 5).
The results suggest that treatment of PD-related MPP+alters
the neddylation of parkin and PINK1. We further examined
the distribution of NEDD8 in midbrain dopaminergic
neurons of PD patients and their sex- and age-matched
normal individuals using an anti-NEDD8 antibody. The
NEDD8 immunoreactivity was visualized in cytoplasmic com-
partment in both PD and controls (Fig. 6A). NEDD8-
immunopositive Lewy bodies in the dopaminergic neurons
from the PD patient substantia nigra were also identified
(Fig. 6B). Thus, NEDD8 or NEDD8-modified proteins are
associated with PD pathology.
Neddylation is an important posttranslational modification
affecting protein structure and function (46,47). In this study,
we demonstrate that two PD-related proteins, parkin and
PINK1, are neddylated. Neddylation modification results in
increased ubiquitin E3 ligase activity of parkin and stabilization
of a 55 kDa PINK1 proteolytic fragment. In vivo, enhanced ned-
dylation by dAPP-BP1 overexpression rescues abnormal pheno-
types induced by dPINK1 reduction in two independent
dPINK1-RNAi Drosophila lines. NEDD8 or its conjugates are
associated with the PD pathological hallmark Lewy bodies.
PD-related neurotoxin MPP+treatment inhibits neddylation of
both parkin and PINK1. These findings identify a new posttran-
dylation abnormalities likely contribute to PD pathogenesis.
Several lines of in vitro and in vivo evidence indicate NEDD8
modification of parkin and PINK1. Parkin and PINK1 are
specifically NEDD8 conjugated in transfected cells and in an
in vitro neddylation assay using recombinant proteins. The ned-
dylation of parkin and PINK1 is sensitive to a deneddylation
enzyme but not a deubiquitination enzyme. In vivo, endogenous
parkin purified from human brain is found modified at K76.
Unlike ubiquitination, neddylation traditionally refers to
theless, recentstudies identify
high-molecular-weight smear bands similar to hyperubiquitina-
tion with non-cullin targets, such as EGFR (35). Our results
suggest that parkin and PINK1 are likely hyperneddylated
based on the high-molecular-weight smears. Deletion experi-
(polyneddylation versus multiple mononeddylation) of NEDD8
modification on parkin and PINK1 remain to be determined.
The best-studied posttranslational NEDD8 modification is
the neddylation of cullins. Cullins are a component of the
SCF ubiquitin E3 ligase complex. Neddylation of cullins
plays an essential role in regulating the E3 ligase activity of
2516 Human Molecular Genetics, 2012, Vol. 21, No. 11
Human Molecular Genetics, 2012, Vol. 21, No. 11 2517
the SCF complex (48). Our results suggest that the neddylation
of parkin potentiates its E3 ligase activity based on the obser-
vation of the increased ubiquitination of two parkin substrates,
parkin and synphilin-1, in cells transfected with NEDD8. One
of the biological consequences of neddylation is to alter the
stability of target proteins. The ribosomal protein L11
becomes stabilized after NEDD8 modification, whereas Cul1
or Cul5 and EGFR were destabilized with NEDD8 modifica-
tion (35,38,49). Consistent with the previous reports, PINK1
neddylation results in selected stabilization of a 55 kDa
PINK1 fragment, leading to its accumulation in the cell. Inter-
estingly, the 55 kDa PINK1 fragment is mainly detected in the
cytosolic fraction and forms a complex with parkin (16). In
contrast, the full-length PINK1 is mainly detected and likely
functions in the mitochondrial fraction. Therefore, the sum
of both parkin and PINK1 neddylation is to regulate the E3
ligase activity of the parkin-PINK1-DJ-1 complex. Regulation
of PINK1 function by neddylation is further supported by an
observation that neddylation E1 dAPP-BP1 subunit rescues
the ommatidial degeneration, abnormal wing posture and
Figure 1. Neddylation of parkin and PINK1. (A) Neddylation of parkin in transfected cells. Cells were transfected with control empty vector, parkin-VSVG,
a-synuclein-VSVG with/without NEDD8 as shown on top of the panel. Parkin and a-synuclein were immunoprecipitated (IP) followed by immunoblotting
(IB) with an anti-NEDD8 antibody (top panel). Immunoprecipitated parkin, a-synuclein and expression of NEDD8 were shown. For parkin-expressing cells,
half of the protein amount was used for immunoprecipitation and immunoblotting compared with the other cells. (B) In vitro deneddylation by NEDP1. Immu-
noprecipitated parkin (left panel) and PINK1 (right panel) were treated with recombinant NEDP1 followed by detection of NEDD8 (top panel), parkin or PINK1
(middle panel), and NEDP1 (bottom panel). (C) In vivo deneddylation of parkin by NEDP1. Cells were cotransfected wild-type (W) or mutant (M) NEDP1 with
parkin. Parkin was immunoprecipitated followed by detection of NEDD8 (top panel), NEDP1 (second panel to the top), parkin (third panel to the top) and total
NEDD8 (bottom panel). (D) In vivo deneddylation of PINK1 by NEDP1. Cells were cotransfected wild-type (W) or mutant (M) NEDP1 with PINK1. PINK1 was
immunoprecipitated followed by detection of NEDD8 (top panel), NEDP1 (second panel to the top), PINK1 (third panel to the top) and total NEDD8 (second
panel to the bottom). Bip was detected as a loading control (bottom panel). Note that NEDD8 coexpression results in accumulation of the 55 kDa PINK1 frag-
ment (lanes 8 and 10). (E and F) In vitro neddylation of parkin and PINK1. Purified recombinant parkin (E) and PINK1 (F) were mixed with different com-
bination of neddylation components (indicated on the top of the figure). The reaction mixtures were detected for parkin (E, top panel) or PINK1 (F, top
panel), APP-BP1 (second panel to the top), Ubc12 (third panel to the top) and NEDD8 (bottom panel). Arrows indicate NEDD8-modified proteins. Note
that higher molecular weight parkin or PINK1 is detected only in reaction containing parkin/PINK1 and all neddylation components.
Figure 2. Ectopic expression of NEDD8 increases parkin E3 ligase activity. (A) Cells expressing parkin, ubiquitin and NEDD8 in different combination shown
on the top of the figure. Parkin ubiquitination (top panel), total ubiquitination (second panel to the top), parkin (third panel to the top), parkin neddylation (forth
panel to the top) and total neddylation (bottom panel) were shown. Number of ‘+’ represents the quantity of NEDD8. Note that increased detection of parkin
ubiquitination is seen with the increase in NEDD8. (B) Cells expressing synphilin-1, parkin, ubiquitin and NEDD8 in different combinations shown on the top of
the figure. Synphilin-1 ubiquitination (top panel), total neddylation (middle panel) and expression of synphilin-1 were shown. Number of ‘+’ represents the
quantity of NEDD8. Note that increased detection of synphilin-1 is seen with the increase in NEDD8.
2518 Human Molecular Genetics, 2012, Vol. 21, No. 11
Human Molecular Genetics, 2012, Vol. 21, No. 112519
male sterility induced by dPINK1 reduction. Moreover,
knockdown of dAPP-BP1 results in the droopy wings resem-
bling those found in flies with either parkin or PINK1 knock-
down. These results are further supported by the findings from
a recent genome-wide study identifying the lethal interaction
of heterozygous deletion of a cytological region Df(3L)vin5
with both parkin knockdown and dPINK1 knockdown (45).
The cytological region of Df(3L)vin5 contains dAPP-BP1.
These findings suggest that neddylation regulates parkin and
PINK1 in vivo.
Mutations of parkin and PINK1 are associated with the
recessive familial form of PD. The pathogenic mutants of
parkin and PINK1 have been shown to reduce the ubiquitin
E3 ligase activity of parkin. The parkin and PINK1 complex
promotes degradation of unfolded proteins via ubiquitin pro-
teasomal pathway (16). Neddylation of parkin and PINK1
increases the ubiquitin E3 ligase activity of parkin. Impair-
ment of neddylation likely results in reduced E3 ligase activity
of the complex and leads to accumulation of mis/unfolded
proteins in the cells. Consistent with this hypothesis, PD
treatment at sub-lethal concentrations
results in reduced neddylation of both parkin and PINK1.
Moreover, Lewy bodies are NEDD8 positive. These findings
suggest that NEDD8 modification of parkin and PINK1 are
involved in PD pathogenesis. The study also opens a new
avenue to design potential treatment of PD via modulating
neddylation of parkin and PINK1. Parkin and PINK1 play im-
portant roles in regulating the structure and function of mito-
chondria, including maintaining integrity, dynamics, quality
control and transport (17,18,21,22,24). Further studies to
address the effects NEDD8 modification of parkin and
PINK1 on mitochondria are clearly warranted.
MATERIALS AND METHODS
SH-SY5Y and HEK293 cells were purchased from ATCC and
maintained as suggested. Human tissues were obtained from the
NICHD Brain and Tissue Band for Developmental Disorders at
the University of Maryland, Baltimore, MD, USA. Plasmids en-
coding FLAG-tagged, VSVG-tagged, myc-tagged parkin or
PINK1 and HA-tagged ubiquitin were described previously
(16). cDNAs encoding RH-NEDD8, FLAG-tagged synphilin-1
and NEDP1 were generated by polymerase chain reaction
and subcloned into pcDNA3.1(-) (Invitrogen). Mutant NEDP1
was generated by site-directed mutagenesis (Stratagene) using
mutagenic oligonucleotides as described previously (50). All
plasmids are confirmed by sequencing. Rabbit monoclonal anti-
NEDD8 antibody, rabbit polyclonal anti-His tag antibody,
rabbit monoclonal anti-myc antibody and rabbit monoclonal
anti-Bip antibody were from Cell Signaling Technology.
Rabbit polyclonal anti-VSVG antibody was from QED Bio-
science, Inc. Sheep polyclonal anti-NEDP1 antibody and goat
polyclonal anti-APP-BP1 antibody were from Biomol Inter-
national and Santa Cruz Biotechnology, Inc., respectively.
All other primary antibodies were from Sigma-Aldrich. The
secondary antibodies were from Jackson ImmunoResearch
Laboratories. 1-methyl-4-phenyl-pyridinium iodide is from
Protein neddylation or ubiquitination in transfected cells
Neddylation or ubiquitination was analyzed as previously
described (16). Briefly, HEK293 cells were transfected with
various plasmid combinations using calcium phosphate.
Cells were lysed in 2% SDS buffer (2% SDS, 150 mM NaCl,
10 mM Tris–HCl, pH 8.0, 2 mM sodium orthovanadate, 5 mM
sodium fluoride, 1× protease inhibitors) and boiled for
10 min followed by sonication. Lysates were diluted 1:10 in
dilution buffer (10 mM Tris–HCl, pH 8.0, 150 mM NaCl,
2 mM EDTA, 1% Triton X-100), incubated at 48C for 1 h
with rotation and centrifuged at 20 800g for 30 min. The
protein concentration of the resulting supernatants was deter-
mined by a modified Lowry assay (Dc Protein Assay,
BioRad) and 500 mg of protein was used for immunoprecipita-
tion unless otherwise stated. Immunoprecipitated proteins were
washed with washing buffer (10 mM Tris–HCl, pH 8.0, 1 M
NaCl, 1 mM EDTA, 1% NP-40), boiled in SDS sample buffer
and separated on sodium dodecyl sulfate–polyacrylamide gel
(SDS–PAGE). NEDD8, ubiquitin and precipitated proteins
were immunodetected with respective antibodies.
In vitro deneddylation and neddylation assay
For the deneddylation assay, parkin-VSVG or PINK1-FLAG
was coexpressed with NEDD8 in HEK293 cells. Immuno-
precipitated parkin or PINK1 and recombinant NEDP1
(1 mg, Biomol) were added to 15 ml of deneddylation
buffer (50 mM Tris–HCl, pH 7.5, 50 mM NaCl, 5 mM
Figure 3. Enhanced neddylation stabilizes the 55 kDa PINK1 cleavage product in cells and rescues dPINK1-RNAi-induced abnormalities in Drosophila. (A and
B) Stabilization of the 55 kDa PINK1 cleavage product by NEDD8. (A) Representative immunoblotting results for PINK1 and NEDD8 upon cycloheximide
treatment. Duration of treatment is shown on the top of the figure. (B) Quantification of PINK1 and its cleavage product after cycloheximide treatment. The
results are from two independent experiments. Values represent mean+standard error. 55 kDa PINK1: the 55 kDa PINK1 fragment; Control: an empty
vector.∗P ¼ 0.0314 at 6 h treatment time point between 55 kDa PINK1 + NEDD8 and 55 kDa PINK1 + Control by an unpaired, two-tailed t-test. (C)
dAPP-BP1 overexpression rescues dPINK1-RNAi-induced ommatidial degeneration. Quantification of flies in different eye phenotypic categories. The observed
eye phenotypes are categorized into different phenotypic groups ranging in levels of severity shown at the bottom: including wild-type , single spot (least
severe) , speckles , patches , lesion (most severe). Arrowheads indicate affected ommatidia of the eye. Representative external eye phenotypes of
dPINK1-RNAi flies without (left) and with dAPP-BP1 overexpression (right) are inserted into the upper right of the graph. Note that the Chi-square test indicates
significantly different eye phenotype distribution (P , 0.0001) between two groups of flies. (D) dAPP-BP1 overexpression rescues dPINK1-RNAi-induced ab-
normal wing posture. Note that the Chi-square test indicates significantly different wing phenotype distribution (P ¼ 0.0058) between two groups of flies. (E)
dAPP-BP1 overexpression rescues tubulin-GAL4 driven, dPINK1-RNAi-induced male sterility. Note that the Chi-square test indicates significant improved male
fertility (P ¼ 0.0009) with coexpression of dAPP-BP1. (F) APP-BP1 knockdown results in the droopy wing phenotype. ‘+’ and ‘APP-BP1 RNAi’ indicate
how24B-GAL4.dcr-2/+ flies and how24B-GAL4.dcr-2, dAPP-BP1 RNAi flies, respectively. ‘APP-BP1 RNAi without driver’ indicates UAS-dAPP-BP1
RNAi flies expressing no GAL4 driver.
2520 Human Molecular Genetics, 2012, Vol. 21, No. 11
b-mercaptoethanol). Reactions proceeded overnight at 378C
and were stopped by addition of SDS–PAGE sample buffer.
Proteins were separated on a 4–20% SDS–PAGE gel and
immunoblotted with an anti-NEDD8 antibody. For the neddy-
lation assay, recombinant parkin-FLAG-myc and PINK1-
FLAG-VSVG were generated as described previously using
a Bac-to-Bac baculovirus expressison system (Invitrogen)
(16). Affinity-purified parkin or PINK1 was mixed with
Ubc12 (0.5 mg, Biomol) and His6-NEDD8 (1 mg, Biomol) in
25 ml of neddylation buffer (20 mM Tris–HCl, pH 7.5, 2 mM
ATP, 1 mM DTT, 5 mM MgCl2) in various combinations. E3
ligase is not necessarily required for NEDD8 conjugation in
vitro (36,39). Neddylation reactions proceeded overnight at
378C and were stopped by addition of SDS–PAGE sample
(0.165 mg, Biomol),His6-
buffer. Proteins were separated on a 4–20% SDS–PAGE
gel and immunoblotted with respective antibodies.
Detection of neddylated parkin in human brain
Anti-parkin antibody and control mouse IgG were immobi-
lized according to the manufacturer’s protocol (Pierce, prod
# 26147). Two micrograms lysates were prepared using 2%
SDS buffer followed by diluting SDS as described above.
Endogenous parkin was immunoprecipitated using the immo-
bilized anti-parkin antibody. Samples were divided into two
parts and resolved on 4–20% SDS–PAGE gels, separately.
One part was used to detect NEDD8 and parkin, and the
other part was used for silver staining and mass spectrometric
Figure 5. MPP+suppresses neddylation of parkin and PINK1. Cells trans-
fected with the combination of parkin (A) or PINK1 (B) and NEDD8
(shown on top of the figures) were incubated with MPP+at various concentra-
tions (shown on top of the figures) for 18 h. Arrow indicates the 55 kDa
PINK1 fragments. Note that neddylation of both parkin and PINK1 is sup-
pressed by MPP+at 10 mM.
Figure 6. Detection of NEDD8-immunopositive Lewy bodies. (A) NEDD8
immunoreactivity was detected in the pigmented dopaminergic neurons in
control and PD substantia nigra. The pictures are representative NEDD8 stain-
ing images from a control (male, 76years) and a PD patient (male, 78years). In
total, four pairs of control and PD cases were examined. (B) Lewy bodies in
pigmented dopaminergic neurons were stained with an anti-NEDD8 antibody
(top panel) and eosin (bottom panel). Lewy bodies are indicated by arrows.
Scale bar, 10 mm.
Figure 4. Neddylation of endogenous parkin in human brain. (A) Parkin is immunopurified from human brain lysates. The purified parkin was verified by im-
munoblotting with parkin (IB: Parkin) and NEDD8 (IB: NEDD8) specific antibodies. On the top of the figure, mIgG indicates immobilized control mouse IgG,
while Parkin indicates immobilized anti-parkin antibody. Arrowhead and arrow indicate unmodified and modified parkin, respectively. (B) The sequence and
tandem mass spectrum of a neddylated peptide derived from parkin. K76 in small letter (k) is Gly–Gly modified.
Human Molecular Genetics, 2012, Vol. 21, No. 112521
analysis using a high-resolution hybrid LTQ-Orbitrap Velos
(Thermo Fisher Scientific). Identification of neddylation sites
was carried out with differential modification of lysine resi-
dues (GG, +114.1 kDa) in the database search.
Cycloheximide chase experiments
To determine the half-life of the PINK1 protein, HEK293 cells
were cotransfected with PINK1-FLAG and NEDD8 followed
by cycloheximide treatment (50 mg/ml) for 0, 2, 4 and 6 h.
PINK1 protein levels were detected by immunoprecipitation.
Cells cotransfected with PINK1 and an empty vector were
included as a control.
Flies expressing dPINK1 RNAi and GMR-GAL4 were
described previously (43). Flies expressing UAS-dAPP-BP1
were a gift from J. Yim (51). Flies expressing how24B-GAL4
were obtained from the Bloomington Drosophila Stock
Center. Flies expressing UAS-dcr-2, UAS-dAPP-BP1 RNAi
tubulin-GAL4 were from
RNAi Center. External eye phenotypes were examined
and photographed as described (43). An additional fly line
expressing Mhc-GAL4 and dPINK1 RNAi was a gift from
Bingwei Lu (44). For the analysis of wing phenotypes, the
crosses were performed at room temperature and the
F1 adults were kept at 298C immediately after eclosion. After
7 days, flies displaying drooped wing postures were counted.
To examine the fertility of males, tubulin-GAL4/dPINK1-RNAi
flies were crossed with either UAS-dAPP-BP1 flies, w1118flies
or UAS-lacZ flies. The progenies were incubated at 298C to op-
timize RNAi-mediated knockdown. Each individual F1 male
was crossed to three w1118virgin females. After 10 days, the
number of vials with larvae or pupae was counted.
the Vienna Drosophila
We examined NEDD8 distributions in dopaminergic neurons
using midbrain tissues from four PD patients and four
sex- and age-matched normal individuals. Ten percent
formalin-fixed midbrain tissues were paraffin embedded.
Five-micrometer-thin sections were prepared and deparaffi-
nized/rehydrated prior to being microwaved for antigen re-
trieval for 10 min, three times in a 10 mM citrate buffer (pH
6.0). Sections were pretreated with avidin/biotin blocking so-
lution (Vector laboratories, Inc.) to mask endogenous biotin
prior to being treated with hydrogen peroxide and blocked
with 5% goat serum. Further, sections were stained with anti-
NEDD8 antibody according to the antibody manufacturer’s
instruction. The NEDD8 immunoreactivity was visualized
using the avidin–biotin–peroxidase
(ABC Elite, Vector laboratories, Inc.) with VIP (Vector labora-
tories, Inc.) as the substrate. To verify NEDD8-specific
immunoreactivity, non-immunized rabbit IgG was used. Sec-
tions were scanned at a magnification of ×20 using the
Aperio ScanScope XT system (Aperio Technologies). After
saving the scanned images, the sections were further stained
with eosin to verify Lewy bodies.
The unpaired t-test and the Chi-square test in Figure 3 were
performed using GraphPad Prism 5 (GraphPad Software, Inc.).
We thank Drs Sung-Il Yoon, Toshiya Tsuji and Rena Baek for
technical advice and Christopher Wu for manuscript proof-
Conflict of Interest statement. None declared.
This work was supported by National Institutes of Health
(RS1-00331-1 and RL1-00682-1 to Z.Z.); grants from Nation-
al Natural Science Foundation of China (to Z.Z. and D.W. ); a
973 project from Ministry of Science and Technology of
China (to Z.Z.); and fellowships from the Parkinson’s
Disease Foundation (to G.V.); and from American Parkinson
Disease Association (to Y.S.C.).
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