Evolving Inteins Manipulating Destruction - American Chemical Society

Jul 21, 2006 - First, 4-HT-dependent intein splicing was characterized in mammalian .... Target identification is a key step and often a major hurdle ...
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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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ACS C H E M I C A L B I OLOGY • VOL.1 NO.6

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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

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HO

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1

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OH OH

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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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ACS C H E M I C A L B I OLOGY • VOL.1 NO.6

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

www.acschemicalbiolog y.o rg

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