Cover letter - Sanford-Burnham Medical Research Institute

Sowmya Swaminathan
Chief Editor
Nature Cell Biology
Dieter A. Wolf, M.D.
Professor
Director, Proteomics
March 27, 2013
Dear Dr. Swaminathan,
We are submitting a manuscript entitled “CAND1 controls in vivo dynamics of the cullin-RING
ubiquitin ligase repertoire” for consideration for publication in Nature Cell Biology.
This study builds on our previous work on the regulation of multisubunit cullin-RING ubiquitin
ligases (CRLs) by neddylation and deneddylation (Wee et al. 2005, Nature Cell Biology 7, 387-
391; Schmidt et al. 2009, Molecular Cell 35, 586-597). CRLs are modular multisubunit enzymes
composed of a catalytic core for which vast numbers of substrate-specific receptor proteins
compete. This competitive layout presents special challenges to enzyme assembly by high
affinity protein interactions while simultaneously maintaining the flexibility to dynamically sample
the entire receptor repertoire.
In previous work published in Molecular Cell (Schmidt et al. 2009, 35, 586-597), we presented a
first model for the solution of this conundrum through a mechanism we referred to as the
“CAND1” cycle. CAND1 is a protein that binds to CUL1 only when the cullin is neither modified
with NEDD8 nor associated with substrate receptors (i.e. F-box proteins = FBPs). In fact, as
shown by the Hershko lab in 2006 (Bornstein et al. PNAS 103, 11515-11520), CAND1 prevents
the neddylation and activation of CUL1 in vitro. As such, CAND1 appears to be an inhibitor of
CRLs in vitro. However, as shown by us in the Schmidt et al. paper and by others in various
systems, CAND1 behaves as an activator of CRL function in vivo.
Reconciling this paradox, our model of the CAND1 cycle envisioned that CUL1 undergoes rapid
cycles of association and dissociation with CAND1. As a consequence, it would promote the
exchange of substrate receptors from unneddylated CUL1 thus maintaining the CRL repertoire.
In support of this model, we were first to demonstrate that, in fission yeast, only a very small
amount of CUL1 is in a stable complex with CAND1. This finding was subsequently reproduced
in human cells by Wade Harper’s and Steve Gygi’s groups (Bennett et al. 2010, Cell 143, 951–
965). However, this highly influential Cell paper also challenged our model (and the entire field
for that matter) by concluding that CAND1 has no role in CRL assembly and remodeling.
To definitively address this controversy, we have now developed cutting edge quantitative mass
spectrometry techniques to study the effect of CAND1 on the global CRL repertoire. We are not
only assessing its effect on the steady-state levels of CRL complexes, but unlike Harper’s
group, we can also measure CRL complex dynamics in vivo. These studies revealed a
pervasive role of CAND1 in maintaining the relative balance of the majority of cellular CRL
complexes. Lack of CAND1 leads to a dominance of some CUL1-FBP complexes over certain
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others, suggesting an impairment of FBPs in competing for CUL1 cores. This defect was
pinpointed to a requirement for CAND1 in the incorporation of newly synthesized FBPs into
preexisting CUL1 complexes. We further show that lack of CAND1 greatly impairs the efficiency
with which an ectopically expressed FBP can displace another FBP from a pre-existing CRL1
complex. Finally, purified CAND1 can displace substrate recpetors form CUL1 in vitro, an
activity we show to be required for substrate degradation. These results clearly demonstrate
that, contrary to the current believe in the field, CAND1 does play a major role in regulating the
dynamics of CRL complexes. Our data now prove our previous proposition of a “CAND1” cycle
and further suggest that this cycle functions to globally organize the CRL repertoire.
Not only do these findings solve long-standing confusions in the cullin field but they have
important ramifications beyond. For one, they enable the first coherent model of CRL assembly
and disassembly through biochemical coupling of CAND1 with the neddylation/deneddylation
cycle. At the same time, CAND1 appears to be the founding member of a potentially larger class
of protein exchange catalysts that regulate the assembly and disassembly of multi-protein
complexes with a similar degree of dynamicity as CRLs.
Due to their extreme plasticity, CRL enzymes permeate every aspect of biology in health and
disease. Not only are individual CRL family members frequently malfunctioning in cancer, but
defects at superior levels of control, such as studied in this work, can disrupt entire branches of
the CRL system for better or worse. On one hand, the resulting imbalances can give rise to
diseases; on the other hand, the underlying regulators are already being exploited as drug
targets in blood cancer. The mechanistic insights obtained in this work may contribute to the
development of even better anti-cancer drugs targeting the CRL system.
As reviewers we could suggest experts in the cullin and neddylation field which include in
alphabetical order: Gerhard Braus (University of Goettingen), Michele Pagano (NYU), Brenda
Schulman (St. Jude), Takashi Toda (London Research Institute), and Rick Vierstra (University
of Wisconsin-Madison), Ning Zheng (University of Wahsington).
For reasons of potential conflict, we ask that this manuscript not be reviewed by Wade Harper,
Steve Gygi or Matthias Peter.
Whereas our paper can stand entirely on its own, we are co-submitting it with a related paper
from Thimo Kurz’s lab that comes to the same conclusions using different approaches in
budding yeast. It may also be of interest to you that we are aware of similar work from Ray
Deshaies’ lab that is under consideration at Cell.
Sincerely,