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We study
posttranscriptional mechanisms of cellular control in the fission yeast
Schizosaccharomyces pombe and in mammalian
cancer cells.
Our work focuses
on three major areas:
(1.) Function and
control
of fission yeast cullin/RING ubiquitin ligases (CRLs)
(2.) Mechanisms
controlling
mRNA translation in fission yeast
(3.) The role of
ubiquitin-dependent proteolysis of tumor suppressors in prostate cancer
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1.1. CRL Control by the CSN
CRLs
represent an extensive class of multisubunit E3 ubiquitin ligases each
consisting
of a core module containing a member of the cullin family and the RING
domain
protein Rbx1p, which recruits the E2 ubiquitin conjugating enzymes
(UBCs)
to the ligase. This core is joined by one of several hundred adapter
proteins
each of which targets a distinct array of substrates for ubiquitylation
and
proteasomal degradation. The
COP9 signalosome
(CSN) complex removes the stimulatory
modification by the ubiquitin-related peptide NEDD8 from cullins.
CAND1 is a protein that associates with deneddylated
cullins. Our work has provided a model framework for how
CRL complex assembly with substrate adapters is
coordinated by the CSN and CAND1. We propose that CRL
core complexes toggle between two distinct CAND1 and CSN
cycles. In the CAND1 cycle, CRL core complexes undergo
continuous rapid exchange of their substrate adapters.
If this cycle is interrupted, CRL complexes with
abundant adapters accumulate at the expense of those
with rare adapters thus disturbing the disposition of
cellular CRL activity. Upon the availability of
substrate, specific CRL-adapter complexes are removed
from the CAND1 cycle by substrate driven neddylation.
This activates CRL-dependent substrate degradation. Upon
consumption of substrate, CRLs are toggled back into the
CAND1 cycle by CSN-mediated deneddylation. Our current
work focuses on biochemically testing several key
predictions of this model.
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1.2. Identification of CRL substrates
Although several
hundred putative CRLs were identified in the human genome, substrates
are known for only a handful of them. Whereas CRLs are relatively easy
to identify based on conserved motifs (e.g. RING, F-box, BTB domains), their
substrates seem to have little more in common than critical lysine
residues. Techniques to
systematically identify CRL substrates have been slow in coming. We are using biochemical and
quantitative proteomic techniques to identify substrates
of CRLs and E2 enzymes. Our approaches include activity-based affinity
capture assays as well as comprehensive proteomic
profiling by 2D-LC/MS-MS. Several
candidate substrates for fission yeast CRLs and human E2s are currently
being validated.
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2. Control of mRNA Translation
Like proteolysis, translational control has been
widely
implicated in the regulation of gene expression. Translation is
controlled
by the complex process of the stepwise assembly of translation
initiation
factors (eIFs) with mRNA and ribosomes. Our present activities in
this area include
the isolation and subunit characterization of fission yeast eIF
complexes. We have obtained evidence that fission yeast encodes two
distinct eIF3 complexes that are distinguished by a different set of
PCI domain proteins. Using polysome fractionation and deep sequencing, we are currently pursuing the hypothesis that
these complexes regulate the translation of distinct sets of mRNAs. We
are also analyzing other eIF complexes to determine whether they are
regulated by cellular stress or signal transduction pathways.
Using affinity
purification and tandem mass spectrometry, we have
recently identified the translasome, a supercomplex that
physically links the protein synthesis and degradation
machineries. The translasome contains eIF3, other eIFs,
translation elongation factors, the ribosome and
ribosome biogenesis factors, chaperones, and the
proteasome. These findings expand the repertoire of eIF3
functions and suggests its involvement in translation
initiation, elongation, and protein quality control.
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3. Studies on Prostate Cancer
The expression
of the CDK inhibitor and tumor suppressor p27 is downreglated in many cancers, primarily
through a post-transcriptional proteolytic mechanism. We
have performed cell-based screens of chemical libraries
to determine whether p27 levels in tumor cells can be
restored by small molecules. The screen revealed a panel
of drug-like compounds that upregulate p27, downregulate
CDK2 activity, induce cell cycle arrest and apoptosis of
prostate cancer cells. Our current efforts
concentrate on mechanism of action studies as well as on
the identification of the molecular targets of these
compounds.
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Publications
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Wu, S..; Zhou, W.; Nhan, T.; Toth J.I.; Petroski,
M.D.; Wolf, D.A. (2013) CAND1 controls in vivo
dynamics of the cullin 1-RING ubiquitin ligase
repertoire.
Nature Communications 4, 1642
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Rico-Bautista, E.; Wolf, D.A. (2012) Skipping
Cancer: Small Molecule Inhibitors of SKP2-Mediated
p27 Degradation.
Chemistry & Biology
19, 1497-1498 (preview invited)
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Bauer, F.;
Matsuyama, A.; Candiracci, J.; Dieu, M.; Scheliga,
S.; Wolf, D.A.; Yoshida, M.; Hermand, D. (2012)
Translational control of cell division by
elongator.
Cell Reports
1, 424-433
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Lackner, D.H.; Schmidt, M.W.; Wu, S.; Wolf, D.A.,
Bähler, J. (2012)
Regulation of transcriptome, translation, and
proteome in response to environmental stress in
fission yeast.
Genome Biology
13:R25
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| Keren-Kaplan,
T.; Attali, I., Motamedchaboki, K.; Davis, B.A.;
Tanner, N., Reshef, Y., Laudon, E.; Kolot, M.;
Levin-Kravets, O.; Kleifeld, O.; Glickman, M.;
Horazdovsky, B.F.; Wolf, D.A., Prag, G. (2012)
Synthetic biology approach to reconstituting the
ubiquitylation cascade in bacteria.
EMBO J., 31, 378 -
390
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Rico-Bautista, E.; Yang, C.-C.; Lu, L.; Roth, G.P.; Wolf, D.A. (2010)
Chemical genetics approach to restoring p27Kip1 reveals
novel compounds with antiproliferative activity in prostate
cancer cells.
BMC Biology
8:153
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Sha, Z., Brill, L.M., Cabrera, R., Kleifeld, O.,
Glickman, M.H., Chang, E.C., Wolf, D.A. (2009) The eIF3
interactome reveals a supercomplex linking protein
synthesis and degradation machineries.
Molecular Cell 36,
141-152
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Schmidt, M.W., McQuary, P.R., Wee, S., Hofmann, K.,
Wolf, D.A. (2009) F-box-directed CRL complex assembly
and regulation by the CSN and CAND1.
Molecular Cell
35,586-597
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Wu, S., Wolf, D.A. (2009)
RhoA destruction CULtivates actin.
Molecular Cell 35, 735-736 (Preview, invited)
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Jones, M.R., Quinton, L.J., Blahna, M.T., Neilson, J.R.,
Fu, S., Ivanov, A.R., Wolf, D.A., Mizgerd, J. (2009)
Zcchc11-dependent uridylation of microRNA directs
cytokine expression .
Nature
Cell Biology 11, 1157-1163
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Yang, C.-C., Wolf, D.A. (2009)
Inflamed Snail speed metastasis.
Cancer Cell 15, 355-357 (Preview, invited)
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Wolf, D.A., Petroski, M.D. (2009) Rfu1, stimulus for the
ubiquitin economy. Cell
137, 397-398 (Preview, invited)
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Brill, L.M.; Motamedchaboki, K.; Wu S.; Wolf, D.A. (2009)
Comprehensive proteomic analysis of Schizosaccharomyces
pombe by two-dimensional HPLC-tandem mass spectrometry.
Methods
48, 311-319
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Schmidt, M.W., Houseman, E.A.,
Ivanov, A.R, Wolf, D.A. (2007) Comparative proteomic and transcriptomic
profiling of the fission yeast Schizosaccharomyces pombe. Molecular Systems Biology, 3:79
[PDF File]
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Zhou,
C., Arslan, F., Wee, S., Krishnan, S., Ivanov, A.R., Oliva, A.,
Leatherwood,
J., Wolf, D.A. (2005) PCI proteins eIF3e and eIF3m define
distinct translation initiation factor 3 complexes.
BMC
Biology, 3:14
[PDF File]
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Wee,
S., Geyer, R., Toda, T., Wolf, D.A. (2005) CSN facilitates cullin-RING
ubiquitin ligase function by counteracting autocatalytic adapter
instability.
Nature
Cell Biology 7, 387-391
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Doud, M.K.,
Schmidt, M.W., Hines, D., Naumann, C., Kocourek, A., Kashani-Poor, N.,
Zeidler, R., Wolf,
D.A. (2004) Rapid prefractionation of complex protein lysates with
centrifugal membrane adsorber units improves the resolving power of
2D-PAGE-based proteome analysis. BMC
Genomics 5:25 [PDF File]
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Wolf, D.A.,
Wee, S., Zhou, C. (2003)
The COP9 signalosome: an assembly and maintenance
platform for cullin ubiquitin ligases? Nature
Cell Biology 5, 1029-1033
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Schmidt, M.,
Jain, A., Wolf. D.A. (2003) Multidimensional proteomic analysis of
proteolytic pathways involved in cell cycle control. In: Cell
Cycle Checkpoint Control Protocols. Lieberman H. B. ed. New
York: Humana Press (2003) Vol. 241: 235-245
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Geyer, R.,
Wee, S., Anderson, S., Yates J.R.III, Wolf. D.A. (2003) BTB/POZ domain
proteins are putative substrate adaptors for cullin 3 ubiquitin
ligases. Molecular Cell 12, 783-790
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Wolf, D.A., Geyer, R. (2003)
Dynamic release of Cdc34 from SCF: The hand that rocks the cradle. Cell 114, 532-533
(Preview, invited)
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Zhou, C.;
Wee, S.; Rhee, E.; Naumann, M.; Dubiel, W.; Wolf, D.A. (2003) Fission
yeast COP9/signalosome suppresses cullin activity through recruitment
of the deubiquitylating enzyme Ubp12p. Molecular
Cell 11, 927-938
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Wee, S., Hetfeld, B., Dubiel, W., Wolf, D.A.
(2002) Conservation of the COP9/signalosome in budding yeast. BMC
Genetics 3:15 [PDF
File]
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Lu, L., Schulz, H., Wolf, D.A. (2002)
The F-box protein SKP2 mediates androgen control of p27 stability in
LNCaP human prostate cancer cells. BMC Cell Biology
3:22 [PDF File]
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Seibert, V., Prohl, C., Schoultz, I., Rhee, E.,
Lopez, R., Abderazzaq, K., Zhou, C., Wolf, D.A. (2002) Combinatorial
diversity of fission yeast SCF ubiquitin ligases by homo- and
heterooligomeric assemblies of the F-box proteins Pop1p and Pop2p. BMC
Biochemistry 3:22 [PDF File]
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Zhou, C.; Seibert, V.; Geyer, R.; Rhee,
E.; Lyapina, S.; Cope, G.; Deshaies, R.J.; Wolf, D.A. (2001) The
fission yeast COP9/signalosome is involved in cullin modification by
ubiquitin-related Ned8p. BMC
Biochemistry 2:7 [PDF File]
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| Lyapina, S.;
Cope, G.; Shevchenko, A.; Serino, G.; Tsuge, T.; Zhou, C.; Wolf, D. A.;
Wei, N.; Shevchenko, A.; Deshaies, R. J. (2001). Promotion of
NEDD8-CUL1 conjugate cleavage by COP9 signalosome. Science 292, 1382-1385 |
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