111123 Altun, Zhao et al

15 Pages·2011·1.34 MB·English

by  Kristina Lindsten

JBC Papers in Press. Published on November 29, 2011 as Manuscript M111.305615
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M111.305615
USP19 is a regulator of the hypoxic response

The ubiquitin specific protease 19 (USP19) regulates the hypoxia inducible factor 1α (HIF-
1α) during hypoxia*

Mikael Altun1,2,3, Bin Zhao1,3, Kelly Velasco1, Haiyin Liu1, Gerco Hassink1, Julia
Paschke1, Teresa Pereira1, Kristina Lindsten1

1 Department of Cell and Molecular Biology, Karolinska Institutet, Box 285, 171 77
Stockholm, Sweden. 2 Department of Neuroscience, Karolinska Institutet, Box 285, 171 77
Stockholm, Sweden.

*Running title: USP19 is a regulator of the hypoxic response

3 These authors contributed equally to this work

To whom the correspondence should be addressed: Kristina Lindsten, Department of Cell and
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Molecular Biology, Karolinska Institutet, Box 285, 171 77 Stockholm, Sweden, Tel: +46-8- o
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52487467. E-mail: [email protected] lo
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Keywords: deubiquitylating enzyme, HIF-1α, hypoxia, non-catalytic, USP19 fro
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Background: The highly regulated hypoxia Here we show that the ubiquitin ttp
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inducible factor-1α (HIF-1α) is a key player in specific protease-19 (USP19) interacts with w
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the cellular response to hypoxia. components of the hypoxia pathway including .jb
Results: The ubiquitin specific protease 19 HIF-1α and rescues it from degradation c.o
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(USP19) rescues HIF-1α from degradation in a independent of its catalytic activity. In the b/
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non-catalytic manner. absence of USP19, cells fail to mount an g
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Conclusion: USP19 is required for cells to appropriate response to hypoxia, indicating es
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mount an appropriate response to hypoxia. an important role for this enzyme in normal n
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Significance: Learning about HIF-1α regulation or pathological conditions. ec
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is essential for understanding the physiological m
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and pathophysiological conditions of the Cells have evolved sophisticated r 1
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hypoxic response. mechanisms to sense and adapt to the natural , 2
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fluctuations of oxygen levels. This adaptation is 18
SUMMARY crucial for normal physiology such as adaptation
A proper cellular adaptation to low to high altitude or proper embryogenesis but is
oxygen levels is essential for processes such as also involved in numerous pathophysiological
development, growth, metabolism and conditions such as inflammation, cardiovascular
angiogenesis. The response to decrease in diseases, stroke and cancer (1,2). Limitation in
oxygen supply, referred to as hypoxia, is also oxygen triggers a chain of events that leads to
involved in numerous human diseases the activation of hypoxia inducible factors
including cancer, inflammatory conditions (HIF). HIFs are transcription factors formed by a
and vascular disease. The hypoxia inducible dimer consisting of an unstable α-subunit and a
factor 1-α (HIF-1α), a key player in the stable β subunit, also referred to as aryl
hypoxic response, is kept under stringent hydrocarbon receptor nuclear translocator
regulation. At normoxia the levels are kept (ARNT). Human HIF-α has three isoforms, HIF-
low as a consequence of the efficient 1α, HIF-2α and HIF-3α, of which the first two
degradation by the ubiquitin-proteasome are closely related and have been extensively
system and in response to hypoxia the studied while HIF-3α is subject to extensive
degradation is blocked and the accumulating splicing and the role of its different forms
HIF-1α promotes a transcriptional response remain largely unknown (3,4) HIF-1α plays a
essential for proper adaptation and survival. role in the acute hypoxic response and is known
1
Copyright 2011 by The American Society for Biochemistry and Molecular Biology, Inc.
USP19 is a regulator of the hypoxic response
to promote the expression of more than 60 genes hypoxic conditions, the role for Siah1 is less
associated with processes such as erythropoiesis, clear (15,16). Upon oxygen deprivation HIF-1α
angiogenesis, cell growth, differentiation, rapidly accumulates and dimerizes with ARNT
survival or apoptosis (5). HIF-1α is kept under to form an active transcription factor complex in
tight regulation and in normoxia it is one of the the nucleus. The activity of the HIF-1α/ARNT
most short-lived proteins known (6). The steady heterodimer can be further regulated by the
state levels are kept low due to its degradation oxygen dependent factor inhibiting HIF (FIH),
by the ubiquitin-proteasome system. The an asparaginyl hydroxylase, acting on nuclear
detailed mechanisms by which HIF-1α stability HIF-1α inhibiting the recruitment of
and activity is regulated are under intense transcriptional co-activators such as CREB-
investigation and may withhold yet unidentified binding protein and p300 (17,18).
players and therapeutic targets (7). Key players in essential pathways are
Protein modifications by ubiquitin often subject to ubiquitin regulation mediated by
regulate numerous cellular processes by one or several ubiquitin ligases or DUBs. The
affecting protein stability or function (8). prime example is probably the tumor suppressor
Covalent linkage of ubiquitin to target proteins p53, which is subject for regulation by more
is directed by the orchestrated activity of a than ten ubiquitin ligases and three DUBs (19).
ubiquitin activating enzyme (E1), a ubiquitin The VHL interacting deubiquitylating enzyme
conjugating enzyme (E2) and a substrate- (VDU)-2, is to our knowledge so far the only
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specific ubiquitin ligase (E3) that mediates the reported DUB in the hypoxia pathway (20), o
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transfer of ubiquitin to the target. Like most rendering it possible that there are unidentified lo
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postranslational modifications, ubiquitination is DUBs in this pathway still to be discovered. de
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also reversible. This process is performed by the Here we show that USP19 interacts with fro
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family of approximately 100 deubiquitylating HIF-1α and describe a non-catalytic role for this h
enzymes (DUBs), which are cystein or metallo enzyme in stabilizing HIF-1α after cellular ttp
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proteases emerging as important regulators in exposure to hypoxia. The presence of USP19 is w
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numerous molecular signaling pathways (9). required to mount a proper hypoxic response and .jb
DUBs are categorized into five sub-classses we therefore suggest that USP19 is a previously c.o
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based on homology between their catalytic unknown regulator of HIF-1α stability. b/
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domains; ubiquitin specific proteases (USP), g
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ubiquitin C-terminal hydrolases, Otubain es
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proteases, Machado Joseph disease proteases EXPERIMENTAL PROCEDURES n
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and JAMM metallo-proteases (9). ec
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The functional outcome of Plasmids and yeast-two-hybrid screen- For the m
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ubiquitylation depends on the type of ubiquitin yeast-two hybrid bait construct 1-1485 nt of r 1
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chain formed. For HIF-1α it typically triggers USP19 were cloned in frame with the GAL4 , 2
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degradation by the proteasome (10). The DNA binding domain (DBD) and MYC tag, in 18
instability of HIF-1α at normoxia is mainly due the yeast expression vector pGBKT7 (Clontech,
to the activity of prolyl hydroxylases (PHDs) Mountain View, CA, USA), generating
that use molecular oxygen as a co-substrate for pGBKT7-GAL4(DBD)-USP19(1-495aa)-Myc.
catalysis to hydroxylate HIF-1α (11). This The yeast-two-hybrid screen was performed
increases the affinity for the von Hippel-Lindau using the MatchmakerTM pretransformed HeLa
(VHL) ubiquitin ligase, which promotes HIF-1α library (Clontech, Mountain View, CA, USA)
ubiquitylation and subsequent degradation according to manufacturer’s protocol. The short
(10,12). Three PHDs have been identified, and hairpin RNA (shRNA) expressing plasmids
their abundance varies greatly between cell pRETRO-SUPER-USP19A and –D were
types. While the role of PHD1 is still unclear, generated by cloning the target sequences
PHD2 has been reported as the major regulator GAGACAGGGTCTCGATATG and
of HIF-1α hydroxylation during normoxia and GATCAATGACTTGGTGGAG of USP19
PHD3 has been appointed a function in mild or mRNA in the pRETRO-SUPER vector (21).
prolonged hypoxic conditions (13,14). The Plasmids overexpressing Myc-USP19, Myc-
PHDs are also regulated by their interaction with USP19(C506S), USP19ΔTM-Myc and FLAG-
the family of Siah ubiquitin ligases (for Seven in tagged HIF-1α, HIF-2α, HIF-3α (splice form:
absentia homologue). While Siah2 controls HIF-3α1), PHD1, PHD2, PHD3 and VHL have
PHD1 and PHD3 ubiquitination during mild been previously described (4,22,23).
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USP19 is a regulator of the hypoxic response
Tissue culture, transfections and The antibodies used were: FLAG(M2)
immunostainings- The human cervical cancer (Sigma, St Louis, MO, USA), β-actin(AC-15),
cell line HeLa, the human embryonic kidney cell Myc(9E10) or Myc(A14) (Santa Cruz
line HEK293T, the human melanoma cell line Biotechnology, Santa Cruz, CA, USA),
M2 and the human osteosarcoma cell line U2OS USP19(A301-586A), USP19(A301-587A)
were maintained in Dulbecco’s Modified Eagle (Bethyl Laboratories, Montgomery, TX, USA),
Medium supplemented with 10% fetal calf HIF-1α (BD Biosciences, Franklin Lakes, NJ,
serum (v/v), 2 mM glutamine, and 100 U/ml USA). Secondary antibodies for
penicillin 100 µg/ml streptomycin (SIGMA, St immunostainings: donkey anti-rabbit Alexa
Louis, MO, USA). All exposures to hypoxia Fluor 488 and donkey anti-mouse Alexa Fluor
were performed with 1% O for indicated time. 555 (Invitrogen, Carlsbad, CA, USA).
2
Transfections were performed using JetPEI Secondary antibodies for western blot: donkey
(Polyplus Transfection, NY, USA) according to anti-rabbit, sheep anti-mouse (Zymed, San
manufacturer’s protocol or by the calcium Francisco, CA, USA).
phosphate method. To achieve an efficient Quantitative PCR (qPCR)- Total RNA
knock-down of USP19 in HeLa cells transfected was obtained using RNeasy Mini kit (Qiagen,
with the pRETRO-SUPER plasmids, the cells Valencia, CA, USA) and 1µg RNA was used for
were treated with 0.5 µg/ml puromicin to kill reverse transcription using M-MuLV Reverse
untransfected cells. Transcriptase (New England Biolabs) according
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For immunostainings the cells were to the manufacturer’s protocol. q-PCR was ow
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grown and transfected on glass coverslips and performed with Power SYBR Green PCR lo
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fixed in 4% formaldehyde in PBS (w/v) and Master Mix (Applied Biosystems) using the e
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stained with appropriate antibodies diluted in 7500 Real-Time PCR System (Applied fro
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50mM Tris, pH 7.5, 0.9% NaCl (w/v), 0.1% Biosystems). The primer sequences used are; β- h
gelatin (w/v) and 0.5% Triton X-100 (v/v). actin, 5'-CCTGGCACCCAGCACAAT-3’ and ttp://w
Immunoprecipitations- Co- 5'-GGGCCGGACTCGTCATACT-3'; USP19, w
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immunoprecipitations were performed in a 5’-CGGCACAAGATGAGGGA-3’ and 5’- .jb
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buffer containing 50 mM Tris (pH 7.4), 150 mM GGCACCGGCAGATAAAGAAA-3’; GLUT1, .o
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NaCl, 0.05 mM EDTA and 1% Igepal CA-630 5’-CAGCAGCAAGAAGCTGAC-3’ and 5’- b/
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(Sigma, St Louis, MO, USA) (v/v) GGGCATTGATGACTCCAG-3’; VEGFα, 5’- gu
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supplemented with protease inhibitor cocktail ATTATGCGGATCAAACCTCAC-3’ and 5’ st o
(Roche, Indianapolis, IN, USA). TCTTTCTTTGGTCTGCATTCAC-3. The n D
Immunoprecipitations with USP19 antibodies expression of β-actin was used as internal ece
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were performed over-night followed by binding control. be
to GammaBind sepharose (GE Healthcare, r 12
Uppsala, Sweden) and subsequent washings in RESULTS , 20
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lysis buffer. Immunoprecipitations of FLAG- 8
tagged proteins was performed with FLAG(M2) USP19 interacts with HIF-1α - We have
affinity gel (Sigma, St Louis, MO, USA) previously shown that USP19 is a DUB with a
according to manufacturer’s protocol. C-terminal transmembrane domain (TMD)
Western blot and antibodies- Proteins anchoring it to the endoplasmic reticulum (ER).
were fractionated in precast polyacrylamide Bis- At the ER, USP19 can affect the degradation of
Tris 4–12% gradient gels (Invitrogen, Carlsbad, ER-associated degradation (ERAD) substrates
CA, USA). After transfer to polyvinylidene and appears upregulated in response to ER-stress
fluoride membranes (Millipore, Billerica, MA, (22). USP19 is also upregulated during catabolic
USA), the filters were blocked in phosphate- stress causing skeletal muscle atrophy (24) and
buffered saline (PBS) containing 5% fat-free affects cell cycle progression (25). This
milk (w/v) and 0.1% Tween 20 (v/v) for 1 h. The motivated us to further investigate USP19
membranes were incubated with primary function and identify interacting partners by
antibodies for 1 h at room temperature or performing a yeast-two-hybrid screen. The bait
overnight at 4 °C followed by washing steps and was restricted to the N-terminal part of USP19
1 h incubation with the appropriate horseradish (1-495 aa) harboring a bipartite CS domain
peroxidase-conjugated secondary antibodies. named after CHORD-containing proteins (for
The results were revealed by enhanced cystein- and histidin-rich domain) and SGT1 (for
chemiluminescence (ECL; GE Healthcare).
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USP19 is a regulator of the hypoxic response
suppressor of G-two allele of SKP1). This region HIF-2α is suggested to play a more prominent
shares high homology to the p23 protein and is role in the chronic adaptation to hypoxia (30)
therefore occasionally also referred to as a p23 which may imply a function for USP19 in the
domain (26) (Figure 1A). CS-domains are acute hypoxic response.
frequently found in co-chaperones of Hsp90,
however the impact of its presence in USP19 is Mapping interaction domains of HIF-
still unclear. 1α- HIF-1α belongs to the family of basic-loop-
In the yeast-two-hybrid screen Siah1 helix (bHLH) and PER-ARNT-SIM (PAS)
and Siah2, the vertebrate homologs of the domain containing transcription factors. The
Drosophila ‘seven in absentia’ gene (Sina) (27), bHLH domain near the N-terminus is required
appeared as interacting partners of USP19. Apart for the binding to HRE sequences present in HIF
from their role in hypoxia they have been target genes, and the PAS domains mediate
ascribed functions in diverse cellular processes dimerization to ARNT. The transactivation
including cell proliferation, apoptosis and tumor domains (TADs) and the oxygen-dependent
suppression (16,28,29). Among these vast degradation (ODD) domain, which is the target
functions of Siah we chose to investigate any of hydroxylation-dependent ubiquitination,
potential involvement of USP19 in the hypoxia reside in the C-terminal region (Figure 2A). To
pathway. pinpoint which region of HIF-1α is responsible
To test if USP19 could interact with for the interaction with USP19, co-
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additional components within the hypoxia immunoprecipitations with different FLAG-HIF- o
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pathway we performed co-immunoprecipitation 1α fragments were performed. Interaction was lo
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experiments from HEK293T cells transiently detected between USP19 and the N-terminal part de
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overexpressing HIF-1α, the hydroxylases PHD1 of HIF-1α containing the PAS- and bHLH fro
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PHD2, PHD3 and the ubiquitin ligase VHL. The domains (Figure 2B). h
hypoxia components were FLAG-tagged and ttp
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FLAG-Siah2ΔRING was included as a positive USP19 stabilizes HIF-1α independent of w
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control. A deletion mutant lacking the RING DUB-activity - Previous studies on USP19 have .jb
domain was used to avoid the inherent instability shown that it can rescue proteins such as Kip1 c.o
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brought to these ligases by the RING. ubiquitylation-promoting complex (KPC1) and b/
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Immunoprecipitations using FLAG-affinity gel ERAD substrates from proteasomal degradation g
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confirmed the interaction between USP19 and (22,25). For this reason we tested whether es
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Siah2 as expected, although it appeared with low USP19 activity was able to rescue also HIF-1α n
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efficiency. More striking was a solid interaction from degradation. First we performed labeling ec
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between USP19 and FLAG-HIF-1α (Figure 1B). experiments using the active site-directed DUB mb
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As illustrated with arrows, the endogenous probe; HA-ubiquitin-VME, which gives a r 1
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USP19 appears as multiple bands in western measure of the DUB activity (31), and certified , 2
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blot, most dominant are bands around 100, 130 that the catalytic mutant Myc-USP19(C506S) 18
and 150 kDa (Figure 1B). The 100 and 150 kDa indeed was inactive (Figure 3A). The interaction
forms repeatedly co-immunoprecipitated with between USP19 and HIF-1α remained, or
FLAG-HIF-1α, however with a clear preference occurred even more efficiently, with the
for the 100 kDa variant. catalytic mutant USP19 (Figure S2). Next we
The interaction was validated by co- tested if overexpression of Myc-USP19 and the
immunoprecipitation of endogenous proteins inactive Myc-USP19(C506S) influenced the
using an anti-USP19 antibody. In order to levels of co-transfected FLAG-HIF-1α.
accumulate detectable HIF-1α levels, HeLa cells Interestingly both the wild-type and the inactive
were exposed to hypoxia for 2 hours prior to the USP19 stabilized FLAG-HIF-1α to similar
immunoprecipitation (Figure 1C). This result extent (Figure 3B). This non-catalytic rescue of
was reproduced with two different antibodies HIF-1α was confirmed looking also at
against USP19 (Figure S1). The interaction was endogenous HIF-1α levels (Figure 3C). To test if
specific to HIF-1α and neither to a long splice the stabilizing effect was limited to HIF-1α and
form of HIF-3α containing all the major domains not to other short-lived proteins in general, the
(bHLH, PASa, PASb, ODDD/NTAD and a same samples were probed against p53 which
leucine zipper), nor to HIF-2α (Figure 1D). HIF- did not appear specifically regulated under these
1α and HIF-2α are highly homologous and bind conditions (Figure 3C). Immunostainings of
to similar HIF response elements (HRE) but HeLa cells transiently transfected with Myc-
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USP19 is a regulator of the hypoxic response
USP19 and Myc-USP19(C506S) confirmed the with treatment with the proteasome inhibitor
rescue of endogenous HIF-1α (Figure 3D). MG132 (10 µM). Indeed HIF-1α accumulated in
Scoring revealed that approximately 80% of the response to the treatment with MG132 in USP19
USP19 overexpressing cells accumulated knock-down cells, illustrating that USP19 is
endogenous HIF-1α in normoxia (Figure 3E). important for the natural rescue of HIF-1α from
This effect of USP19 was reproduced in proteasomal degradation in response to hypoxia
additional cell lines suggesting that it is not cell (Figure 5D).
type specific, but a rather general effect (Figure To test if the lack of HIF-1α
4). The HIF-1α accumulating in response to accumulation in response to hypoxia was of
USP19 overexpression however appeared functional significance we performed
transcriptionally inactive under these conditions quantitative PCR (qPCR) in USP19 knock-down
suggesting that additional events priming its cells assaying the expression of the well-
activity were absent. established HIF-1α target genes; the glucose
transporter 1 (GLUT1) and the vascular
USP19 rescues HIF-1α from endothelial growth factor (VEGF). Since loss of
degradation independent on ER localization - USP19 delays progression of cell cycle (25) and
Our previous study spatially and functionally appears slightly toxic to cells we performed the
placed USP19 to the ER (22). This raises the qPCR with limited levels of USP19 knock-
question if ER localization is of significance for down. An approximate 50% reduction of USP19
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stabilizing HIF-1α. We therefore tested if mRNA was used expecting to provide a good ow
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deletion of the C-terminal anchor of USP19, representation of a physiological condition lo
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which is required for ER localization and ability (Figure S3), Indeed the HIF-1α transcriptional de
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to stabilize ERAD substrates, influenced the response was significantly reduced in USP19 fro
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stabilizing effect on HIF-1α in normoxia. knock-down cells during hypoxia (Figure 5E). h
However deletion of the TM domain behaved Taken together our data strongly supports a role ttp://w
similar to full-length USP19 suggesting that ER for USP19 in stabilizing HIF-1α and promoting w
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localization is not required for the rescue of HIF- a proper transcriptional response during hypoxia. .jb
1α from degradation (Figure 4A,B). c.o
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DISCUSSION b/
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Loss of USP19 impairs the cellular g
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response to hypoxia - Our findings that USP19 During the last years, substantial progress has es
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can stabilize and interact with HIF-1α and not been made to delineate the molecular n
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HIF-2α suggest that it may be involved in the mechanisms that resolve reduced oxygen levels ec
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regulation of the acute cellular response to into an adjusting cellular response (33). Here we mb
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hypoxia. To address this possibility we analyzed show that USP19 and HIF-1α interact with each r 1
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the hypoxic response in HeLa cells with other and that USP19 regulates HIF-1α stability , 2
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suppressed USP19 expression. The knock-down in a non-catalytic manner. In the absence of 18
was performed by transfection of plasmids USP19 cells fail to mount an appropriate
expressing shRNAs targeting two different response to hypoxia.
regions within the USP19 mRNA (Figure 5A). USP19 is a DUB reported to protect
Three days after transfection of the shRNA certain proteasomal substrates from degradation
expressing plasmid pRETRO-SUPER and by virtue of its enzymatic activity (22,25).
pRETRO-SUPER-USP19A, the cells were However our findings suggest that USP19 exerts
exposed to hypoxia for the indicated time. a non-catalytic mode of regulation since
Interestingly cells with low USP19 expression overexpression of the inactive USP19 stabilized
dramatically failed to accumulate HIF-1α after HIF-1α to the same extent as the wild-type.
exposure to hypoxia (Figure 5B,C). Since the Similar observations for USP19 were made
main regulation of HIF-1α steady-state levels is studying its effect on the turnover of particular
mediated by proteasomal degradation rather than ERAD substrates (22) and the cellular inhibitors
at transcriptional level (32), our data suggest that of apoptosis (c-IAPs), c-IAP-1 and c-IAP2 (34).
in the absence of USP19 HIF-1α is continuously Non-catalytic functions of DUBs are not
degraded by the proteasome, disregarding the unprecedented and have recently emerged as an
hypoxic conditions. To test this, USP19 was important means for these enzymes to increase
knocked-down in HeLa cells and exposed to their functions (35,36). Hence it seems as if
hypoxia or exposed to hypoxia in combination USP19 belongs to these DUBs that have
5
USP19 is a regulator of the hypoxic response
developed this non-canonical way of regulation, stabilization of ERAD substrates. However here
possibly by their ability to recognize ubiquitin or we found that the TMD was perfectly
by mere protein interactions or competitive dispensable for the stabilization of HIF-1α
bindings with additional partners. (Figure 4). In agreement with this is also our
HIF-1α and HIF-2α are highly observation that the preferred form of USP19
homologous and bind similar HRE motifs (100 kDa form) interacting with HIF-1α (Figure
however they exert different functions. This is 1B), is likely lacking the TMD since this is only
best illustrated by their dissimilar embryonic present in 3 of the 12 documented splice forms,
deletion phenotypes, their role in tumor all expected to migrate around 150 kDa
angiogenesis or in adaptation to chronic hypoxia (www.ensembl.org). Thus, ER localization is not
(37-40). Although HIF-1α and HIF-2α share a likely criterion for USP19 regulation of HIF-
some common interacting partners we found that 1α and may represent a multi-functionality of
USP19 interacts specifically with HIF-1α. It is this protein directed by its subcellular
likely that the differences between HIF-1α and localization. However it should be noted that the
HIF-2α, in part is mediated through their regulation of HIF-1α is complex and conditions
selective protein interactions, and possibly of severe hypoxia (<0.01% O ) induces ER-
2
USP19 by this mean contributes to their stress. This is due to defects in the protein-
functional differences. folding capacity of the ER emerging at these O
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HIF-1α interacts with USP19 via its N- levels (46). Thus, possibly USP19 is induced
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terminal region harboring the PAS- and bHLH after such ER-stress stimulation and plays an o
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domains. Although this region is typically integrated role in the hypoxic and ER-stress lo
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involved in DNA binding and dimerization to response under conditions of extreme hypoxia. de
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ARNT, other interactions taking place here By summarizing USP19 functions, a fro
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include binding to the molecular chaperone general impression of stress-involvement arises. h
Hsp90 and RACK1 associated with O - Here we show that USP19 is involved in ttp
2 ://w
independent regulation of HIF-1α (41) or to the hypoxic stress and we previously showed w
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minichromosome maintenance protein 7 involvement in ER-stress (22). Other studies .jb
(MCM7) involved in the O2-dependent have showed an upregulation of USP19 in rat c.org
regulation (42). The region within USP19 skeletal muscle in response to different stress b/
y
important for interacting or stabilizing HIF-1α including streptozotocin-induced diabetes, g
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remains to be determined and may withhold dexamethasone treatment, and cancer (24). es
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interesting mechanistic insight. p23/CS domains Considering also the ability of USP19 to rescue n
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have been proposed to play a role in Hsp90 c-IAPs from degradation after apoptotic ec
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binding (26) or suggested to function as a stimulation (34), it is tempting to speculate that m
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general binding module recruiting heat shock the general biological function of USP19 is of a r 1
2
proteins to multi-protein complexes (43). This cytoprotective nature intended to adapt cells to , 2
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raises the question if USP19 is part of such a different types of stress. 18
stabilizing complex and if it in addition could be In conclusion we have found that USP19
relevant for O independent regulation of HIF- is important for regulating HIF-1α and that loss
2
1α. MYND domains are believed to mediate of USP19 expression dramatically impaired the
protein-protein interactions and interestingly cellular response to hypoxia. Hence, we suggest
USP19 shares this domain with numerous other that the role of USP19 in the hypoxia pathway
proteins, including PHD2 (44,45). Any may have important implications for normal
significance of this for the role of USP19 in physiology or pathophysiology.
hypoxia remains to be discovered.
We have previously identified the TMD
of USP19 to be indispensable for USP19

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ttp
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FOOTNOTES c.o
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b/
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Acknowledgements - We thank Ze’ev Ronai (Burnham Institute for Medical Research, USA) for g
u
kindly providing the Siah2 plasmid, René Bernards (The Netherlands Cancer Institute) for the es
t o
pRetroSuper plasmid and Johanna Myllyharju (University of Oulu, Finland) for the FLAG-HIF-3α n D
plasmid. This research was supported by the Swedish Research council (KL, TP and MA), Karolinska ec
e
m
Institutet (KL, BZ and MA), Magn Bergvalls stiftelse (KL), Åke Wibergs Stiftelse (KL) and ERACOL b
e
(KV). r 1
2
, 2
0
1
The abbreviations used are: Aryl hydrocarbon receptor nuclear translocator (ARNT), Basic Helix- 8
Loop-Helix (bHLH), CHORD and SGT1-domain (CS), deubiquitylation enzyme (DUB), endoplasmic
reticulum (ER), ER-associated degradation (ERAD), glucose transporter 1 (GLUT1), hypoxia
inducible factor (HIF), inhibitor of apoptosis (IAP), Myeloid translocation protein 8, Nervy protein,
Deaf-1 (MYND), N/C-terminal transactivation domain (N/C-TAD), Nuclear export signal (NES),
Nuclear localization signal (NLS), oxygen-dependent domain (ODD), PER-ARNT-SIM domain
(PAS), prolyl hydroxylase (PHD), quantitative PCR (qPCR), transmembrane domain (TMD), ubiquitin
specific protease 19 (USP19), vascular endothelial growth factor (VEGF), von Hippel-Lindau (VHL).

FIGURE LEGENDS

FIGURE 1. USP19 interacts with HIF-1α.
A) Schematic representation of the full-length, and the 1-495 amino acids long, USP19, which was
used as bait in a yeast-two-hybrid screen. CHORD and SGT1-domain (CS), p23 protein domain (p23),
ubiquitin specific protease domain (USP), ubiquitin like domain (UBL), Myeloid translocation protein
8, Nervy protein, Deaf-1 (MYND/Zinc-finger), transmembrane domain (TMD) and the position of the
amino acids Cys, His and Asp in the catalytic triad are indicated. B) Immunoprecipitations using
FLAG(M2) affinity gel from HEK293T cells transfected with FLAG-tagged components of the
8
USP19 is a regulator of the hypoxic response
hypoxia pathway as indicated. The co-immunoprecipitated endogenous USP19 was detected with the
anti-USP19(A301-587A) antibody (Bethyl Laboratories) as indicated. Note that USP19 appears in
multiple forms, indicated by arrows, likely representing splice variants or processed forms. C) Co-
immunoprecipitation of endogenous proteins from HeLa cells in normoxia and hypoxia using the anti-
USP19(A301-587A) antibody as indicated. D) Co-immunoprecipitation using anti-FLAG(M2) affinity
gel showing selective interaction between Myc-USP19 and FLAG-HIF-1α but not FLAG-HIF-2α and
FLAG-HIF-3α.

FIGURE 2. Mapping the HIF-1α interaction domain.
A) Schematic illustration of truncated, FLAG-tagged, HIF-1α constructs. Basic Helix-Loop-Helix
(bHLH), PER-ARNT-SIM (PAS) domain, oxygen-dependent domain (ODD), N/C-terminal
transactivation domain (N-TAD) (C-TAD), nuclear localization signal (NLS), nuclear export signal
(NES) are indicated. B) Co-immunoprecipitations using a FLAG(M2)-affinity gel from lysates of HEK
293T cells co-transfected with the truncated forms of HIF-1α or FLAG-GAL4 as control, together with
Myc-USP19.

FIGURE 3. USP19 stabilizes HIF-1α independent of catalytic activity.
A) Active site labeling with the HA-ubiquitin-VME probe in lysates from cells expressing Myc-
USP19 and the mutant Myc-USP19(C506S). The upper blot illustrates the enzymatically active Myc-
D
USP19 covalently linked to the probe as detected using an anti-HA antibody. The lower blot illustrates ow
n
the expression of both Myc-USP19 and Myc-USP19(C506S) using anti-Myc(9E10) antibody. B) lo
a
d
Western blot to test the effect of overexpressed USP19 on co-transfected FLAG-HIF-1α steady-state e
d
levels as indicated. GFP was included as a cotransfection control. C) Western blot analysis of U2OS fro
m
cells overexpressing Myc-USP19 and Myc-USP19(C506S), probed as indicated. D) Micrographs of h
cells transfected with Myc-USP19, Myc-USP19(C506S) or GFP as control. Immunostainings with ttp://w
anti-HIF-1α (red) and anti-Myc(A14) (green) and nuclear counterstaining using DAPI as indicated. E) w
w
Quantification of results in (D) where 100 USP19 positive cells were scored for positive HIF-1α co- .jb
c
staining. Values show mean ± SD of triplicates. .o
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b/
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FIGURE 4. USP19 stabilizes HIF-1α independent of ER localization. A) Graphs illustrating HeLa g
u
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cells, the melanoma cell line M2 and HEK293T cells overexpressing Myc-USP19, Myc- st o
USP19(C506S) and Myc-USP19ΔTM, which lacks ER localization. The number of cells accumulating n D
e
HIF-1α in USP19 overexpressing cells were scored by counting cells positive for HIF-1α ce
m
immunostaining B). Western blot of HeLa cells transiently overexpressing Myc-USP19, Myc- b
e
USP19(C506S) and Myc-USP19ΔTM in normoxia. The blots were probed against -Myc, -HIF-1α and r 12
-β-actin as indicated. , 20
1
8

FIGURE 5. Loss of USP19 impairs the hypoxic response.
A) Western blot experiment assessing the efficiency of two different shRNA expressing vectors,
pRETRO-SUPER-USP19A and -D, in suppressing USP19 protein expression. B) Three days post-
transfection HeLa cells transfected with the empty control plasmid pRETRO-SUPER or pRETRO-
SUPER-USP19A were exposed to hypoxia or kept in normoxia as indicated. Western blots were
probed anti-HIF-1α (short and long exposure) to investigate the effect by USP19 knock-down on HIF-
1α accumulation. β-actin was included as control. C) Quantification by densitometry from short
exposure western blots of three independent experiments performed as in 4B. Values represent relative
induction of HIF-1α during hypoxia compared to normoxia, mean ± SD. D) Same experimental setup
as in 4B but cells exposed to hypoxia for 8 hrs were treated in parallel with the proteasome inhibitor
MG132 (10 µM). The results illustrate a continuous proteasomal degradation of HIF-1α during
hypoxia in cells with suppressed USP19 expression E) Knock-down of USP19 impairs the HIF-1α
transcriptional response during hypoxia. Relative mRNA expression assessed by qPCR of the HIF-1α
target genes GLUT1 and VEGF in cells with or without USP19 knock-down ± hypoxia. Values
represent expression levels relative to β-actin mRNA ± SD from four independent experiments. *p
values < 0.05 for indicated comparisons.

9
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