Evolving Inteins An intein is a segment of a protein that can excise itself
Next, evolved intein variants inserted into the murine
and ligate the remaining portions to create an intact
transcription factors Gli1 and Gli3T were used to modulate
protein. The presence of an intein within a target protein
transcription-factor activity. Transcription-factor activity was
renders the macromolecule nonfunctional, but upon
dependent on the addition of 4-HT, and spliced Gli1 and
excision of the intein, the target can assume its native
Gli3T localized to their native subcellular compartments.
conformation. In yeast, directed evolution has been used
This finding provides additional evidence that spliced
to create inteins whose self-splicing is dependent on the
proteins behave similarly to their native counterparts. Finally,
small molecule 4-hydroxytamoxifen (4-HT). This method of
the researchers demonstrated that the evolved intein system is capable of producing protein levels
other approaches, such as the use of non-native promot-
sufficient to fulfill the role of the native
ers or heterologous control elements, because it requires
protein in the cell. Endogenous Gli1 can
minimal disruption of the cell’s regulatory networks. Now,
induce differentiation of mouse embry-
Yuen et al. ( J. Am. Chem. Soc. 2006, 128, 8939–8946)
onic cells into osteoblasts. Remarkably,
expand the application of evolved inteins to the manipula-
in the presence of 4-HT, mouse embry-
tion of protein function in mammalian cells. First, 4-HT-dependent intein splicing was characterized in mammalian cells transfected with a green fluorescent protein (GFP) intein variant. The researchers demonstrated that spliced, functional GFP was generated only in the presence of 4-HT in a dose- and time-dependent manner.
Manipulating Destruction Cells maintain precise mechanisms to control their protein levels, including regulating protein expression, function, and degradation. The ability to manipulate these processes helps us understand how they operate and enables the design of engineered protein circuits. While many methods are available to control protein expression, protein degradation has proven
Reprinted with permission from Journal of the American Chemical Society
controlling protein function has distinct advantages over
more difficult to manipulate. Now, McGinness et al. (Mol. Cell 2006, 22, 701–707) present a system to control destruction by exploiting properties of the adaptor protein SspB and the ssrA-tag, which mark proteins for degradation by the ClpXP protease. In Escherichia coli, proteins that contain the ssrA peptide sequence are targeted for destruction by the ClpXP protease, and
onic cells transfected with a Gli1-intein variant differentiated into osteoblasts. Further application of evolved, smallmolecule-dependent inteins harbors great potential for the exploration of protein function in cells. EG
this process is facilitated by SspB. To engineer a controllable system, the researchers cleverly designed a modified ssrA-tag, termed DAS+4, that has a similar affinity for SspB but a reduced affinity for the protease. It was first demonstrated in vitro that proteins carrying the modified tag are degraded poorly by ClpXP in the absence of SspB but are rapidly degraded when SspB is (continued on page 330)
Published online July 21, 2006 • 10.1021/cb600287r CCC: $33.50 © 2006 by American Chemical Society
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Introns Draw One from the DEK
TIPping the Target Identification Scale
The spliceosome is a cellular machine composed of RNA
Target identification is a key step and often a major hurdle in
and >100 proteins. In mammals, the complexity of the
chemical genetics experiments and drug-discovery efforts. One
spliceosome is dwarfed only by the essential task that it
method to identify targets of small molecules is photoaffinity
performs. It must recognize specific signals at the ends of noncoding regions known as introns and catalyze
labeling, in which UV irradiation is used to covalently cross-link
their removal. The majority of introns have ends marked
a photoactivatable derivative of the small molecule to its target.
by dinucleotides, GU at the 5′ end and AG at the 3′ end.
However, purification and identification of photoaffinity-labeled
Because these signals are often thousands of nucleotides
proteins are often complicated by the presence of contaminat-
apart, a puzzling question has long remained unanswered:
ing proteins. Now, Lamos et al. ( Angew. Chem., Int. Ed. 2006,
How does the spliceosome pick the correct dinucleotides among so many possible candidates? Now, Mendes-Soares
45, 4329–4333) present the synthesis and application of target
et al. (Science 2006, 312, 1961–1965) have uncovered a
identification probe (TIP) reagents that exploit the use of isoto-
new activity that helps explain the spliceosome’s selective
pic labels to facilitate target identification. TIP reagents are cleverly designed multifunctional molecules
nature. They monitored the binding of an early factor in 3′ splice site recognition known as the U2-auxillary factor
that contain a mixed isotope photoaffinity label and an affinity
(U2AF) to a segment of RNA that resembles an authentic
tag. The isotope label is composed of a benzophenone moiety
3′ splice site or a mutant. An isolated human U2AF complex was not highly selective for the authentic site, but an activity in nuclear extract
U2AF
made U2AF far more discrimiations followed this activity
DEK
U2AF
reagent is used as a 1:1 mixture of labeled to unlabeled U2AF
CG
etry identified the responsible protein,
difference compared with its unlabeled counterpart. The TIP
DEK AG
nating. Biochemical fractionuntil mass spectrom-
containing 11 deuterium atoms; this is a substantial mass
Image courtesy of J. Valcarcel
DEK
CG
molecules, and when incubated in a protein mixture and exposed to UV light, both labeled and unlabeled reagents are covalently attached to target proteins. Avidin affinity chromatography is used to isolate
DEK, a factor formerly associated with the chromatin coats
the protein–TIP conjugates from the mixture, and proteolysis
on DNA rather than RNA processing. In this study, DEK was
and subsequent mass spectrometry (MS) analysis of unlabeled
shown to interact with U2AF and act as a proctor during
peptides provide a list of candidate binding proteins. Of those
that factor’s choice of 3′ dinucleotide. DEK’s watchdog
peptide fragments, only those that also possess the unique MS
activity appears to be more complex than just enforcing the right binding event. Depletion of the protein from splicingcompetent extracts resulted in a loss of splicing catalysis
signature obtained from the isotopic label are derived from the target of interest. As proof of principle, a TIP reagent contain-
but no change in the assembly of the spliceosome onto
ing the immunosuppressive drug cyclosporine was synthesized
the intron. This implies that DEK may function during the
and successfully used to isolate and identify its binding protein
mysterious “catalytic activation” stage of the spliceosome
cyclophilin A from a mixture of four proteins.
cycle. This is especially interesting given its previous connection to chromatin regulation. Perhaps DEK acts as one of the nuclear coordinators to mitigate cross talk between transcriptional regulation and the splicing apparatus. Also
This approach paves the way for accelerated target identification for many applications. For example, TIP reagents can be used in live cells, and the large mass difference between labeled
of interest is that an overabundance or mutation of human
and unlabeled proteins enables resolution of intact proteins in
DEK has been linked with certain cancers. JU
top-down proteomics experiments. EG
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OH HO HO
Defeating Dementia
OH
OH OH
HO
OH
HO
1
OH 2
OH OH
HO
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3
Reprinted with permission from Nature Medicine
Alzheimer’s disease (AD) is the most common form of dementia, and the accumulation of amyloid β‑peptide (Aβ) aggregates in the brain is thought to be a critical element in the pathogenesis of the disease. It has been suggested that inhibiting
to evaluate two CHH stereoisomers, scyllo-CHH and epi-CHH, in mice expressing a mutant protein that causes AD in humans. In the first model, mice were treated prophylactically beginning at 6 weeks of age, before symptoms of AD surface, until either
Aβ aggregation could be an effective therapeutic strategy for AD, but the complexity of the disease and the challenge of creating appropriate disease models have hindered effective discovery and testing of inhibitors. McLaurin et al. (Nat. Med. 2006, 12, 801–808) now report the evaluation and efficacy of cyclohexanehexol (CHH) inhibitors of Aβ fibril assembly in a robust transgenic mouse model of AD. Phosphatidylinositol lipids have been previously shown to strongly facilitate Aβ oligomerization. It has been hypothesized that inhibition of Aβ fibril assembly by CHHs in vitro is due to their ability to directly compete with phosphatidylinositol binding to Aβ. Two different treatment models were used
4 or 6 months of age. Treatment with either inhibitor resulted in significant improvements in cognitive function and other AD-like phenotypes, including reductions in brain Aβ levels and mortality, although the scyllo-CHH stereoisomer was the more effective inhibitor. In the second treatment strategy, mice were given the inhibitors at 5 months of age, after the AD phenotype is established. Remarkably, mice treated with scyllo-CHH, but not epi-CHH, exhibited behavioral improvements and biochemical evidence for reduced Aβ plaques. The authors propose that CHH inhibitors directly prevent and possibly reverse Aβ oligomeric assembly in the brain: clearing of soluble Aβ through normal mechanisms and consequent prevention of disease may result. EG
Manipulating Destruction, continued present. The researchers Protein substrate next tested the system in vivo in sspB+, clpX+, ClpX sspB–, and clpX– strains SspB + L by tagging the protein titin ClpP A ssrA tag A with DAS+4 and placing Substrate Protease Delivery +adaptor complex it under the control of an inducible promoter. Upon induction, very low levels of titin-DAS+4 both SspB and ClpXP are neceswere found in sspB+ clpX+ strains, sary for efficient degradation of but substantially higher levels titin–DAS+4. Taking the system were present in the sspB– and one step further, the researchers clpX– strains, an indication that placed SspB under an inducible Reprinted with permission from Molecular Cell
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promoter in a ∆sspB strain expressing titin–DAS+4. Rapid degradation of titin–DAS+4 was observed Degradation only upon induction of SspB expression. The researchers further demonstrate that controlling degradation of proteins tagged with DAS+4 can be used to manipulate other cellular properties, including antibiotic resistance and enzyme production. EG
w w w. a c s c h e m i ca l biology.org
Asymmetry and Autoinhibition The epidermal growth factor receptor
Comparing the structures of the pro-
(EGFR) family of proteins plays critical
totypical tyrosine kinase Src and cyclin-
in an autoinhibited state. Further examination of previously described
roles in cell proliferation, differentiation,
dependent kinase (CDK) to the struc-
crystal structures of EGFR in activated
and motility and is activated in a variety
tures of EGFR, the authors hypothesized
conformations revealed a potentially
of human cancers, including malignan-
that EGFR is likely activated through
important structure in which the kinase
cies of the lung, head, and neck. EGFR
asymmetric interaction of two protein
domains of two molecules interacted in
is a transmembrane receptor tyrosine
molecules. They first showed that an
an asymmetric manner that is similar to
kinase that is activated upon dimeriza-
EGFR mutation frequently found in
the binding of cyclin to CDK. This cyclin
tion induced by ligand binding to the
binding to CDK leads to activation of
extracellular domain. Homodimerization
the CDK. Mutation of an EGFR residue
of the protein results in the activation
at the cyclin/CDK-like interface pro-
of the kinase and phosphorylation
duced crystals of EGFR that resembled
of tyrosine residues in the C-terminal
an inactive kinase. In addition, the
domain. These phosphotyrosines serve
authors showed that mutations at
as docking sites for other signaling
the cyclin/CDK-like interface prevent
proteins, leading to the activation of
EGFR activation. Taken together, these
numerous downstream pathways.
results suggest that EGFR is normally
Previous research has shown the structural basis for the dimerization of
autoinhibited and that ligand-induced Reprinted with permission from Cell
dimerization leads to an asymmetric
the EGFR extracellular domain. However,
human lung cancer activates EGFR. This
interaction of the kinase domains that
the mechanism by which EGFR kinase
mutation (mutating a neutral leucine to
aids in the activation of the molecule.
activity is induced upon ligand-binding
a charged arginine) interrupts several
Given the serious consequences of
has remained unclear. Now Zhang et al.
hydrophobic interactions necessary for
spurious activation of EGFR, this study
(Cell 2006, 125, 1137–1149) propose
maintaining the Src/CDK-like inactive
provides a glimpse at an elegant solu-
an allosteric mechanism for kinase
state. This result led the authors to
tion evolved to precisely regulate EGFR
activation.
hypothesize that EGFR naturally resides
signaling. ST
Upcoming Conferences Translational Control, CSHL September 6–10, 2006 Cold Spring Harbor, NY
2006 ACS Fall National Meeting September 10–14, 2006 San Francisco, CA
Dynamic Organization of Nuclear Function, CSHL September 27–October 1, 2006 Cold Spring Harbor, NY
RNA Chemistry Meets Biology September 29–30, 2006 Lund, Sweden
Neuroscience 2006 October 14–18, 2006 Atlanta, GA
HUPO World Congress October 28–November 1, 2006 Long Beach, CA
Spotlights written by Eva Gordon, Sarah Tegen, and Jason Underwood
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