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474 ACS CHEMICAL BIOLOGY • VOL.1 NO.8 www.acschemicalbiology.org

Published online September 15, 2006 • 10.1021/cb600392e CCC: $33.50

© 2006 by American Chemical Society

Stem Cells Go GlobalThe clinical promise of human embryonic stem cell research (hESCR) is tantalizing scientists throughout the world, but contro-versial ethical issues continually jeopardize progress in the fi eld. Countries around the globe have distinct regulations guarding hESCR, but borders can become blurred when scientists from different countries attempt to work together. More than 50 scien-

tists, ethicists, journal editors, lawyers, and policy makers from 14 countries recently convened in Hinxton, Cam-bridge, U.K., to create guiding prin-ciples for international collaborations in hESCR (Mathews et al., Science 2006, 313, 921–922).

One hotly contested topic centered on extraterritorial jurisdiction over hESCR. Should countries that have banned hESCR have the power to pro-hibit their scientists from participating in hESCR collaborations in countries in which it is legal? Currently, at least one country appears to assert extraterrito-

rial jurisdiction over their scientists, while others do not. It is not reasonable to expect that all countries will eventually adopt simi-lar policies with respect to hESCR. However, the Hinxton Group urged lawmakers, research institutions, and journal editors to take appropriate measures to ensure that, provided the research is conducted in a legal and ethical manner and that participation in hESCR is not expressly prohibited, scientists feel comfortable pursuing international collaborations without fear of prosecution, restriction, or discrimination.

The Hinxton Group also discussed the responsibilities that researchers and journal editors bear to ensure scientifi c and ethical integrity in hESCR. For example, it was suggested that sci-entists should submit new stem cell lines to depositories that sub-scribe to internationally accepted standards of quality. In addition, editors and authors should work together to make all pertinent information regarding hESCR research readily available, including details about the cell lines used and ethical considerations taken.

In the fall of 2006, the Hinxton Group will make available a public database for the deposition of documents relevant to the policies and ethics of hESCR. The group stressed that the rapid evolution of hESCR will require continual development of ethical practices that consider academic, professional, and public opinion. EG

(continued on page 475)

Networking MycobacteriaAstonishingly, nearly one-third of the world’s population

is infected with Mycobacterium tuberculosis (Mtb), but

the molecular details of its pathogenicity are not well

understood. Deciphering the protein interaction networks

utilized by Mtb would help unravel some of the mysteries

of Mtb virulence and facilitate drug development against

tuberculosis. Yeast two-hybrid (Y2H) technology has been

an invaluable tool for unveiling protein interaction net-

works in many organisms, but the use of yeast as a host

can pose various limitations. Singh et al. (PNAS 2006,

103, 11346–11351) now report the development of a

mycobacteria-based cousin of Y2H, termed mycobacterial

protein fragment complementation (M-PFC), as an effec-

tive method for exploring Mtb protein–protein interac-

tions in mycobacteria.

The method is based on the functional reconstitution

of murine dihydrofolate reductase (mDHFR) upon the

interaction of two mycobacterial proteins, which are inde-

pendently fused to two mDHFR domains. Active mDHFR

confers mycobacterial resistance against trimethoprim

(TRIM), and thus mycobacterial growth in the presence

of TRIM is

indicative of a

protein–pro-

tein interac-

tion. Several

identifi ed

protein pairs,

including the

Mtb secreted immunogenic antigens Esat-6 and Cfp-10,

were used to initially validate the system. In addition, a

modifi ed assay was developed to quantify the strength

of specifi c protein–protein interactions, signifi cantly

Image courtesy of Getty Images

Rv3874 (Cfp-10)

Rv3800c(Pks13)

Rv2240c

Rv0686

Rv3875(Esat-6)

Rv3871(Snm2)

Rv2151c(FtsQ)

Rv3596c(ClpC1)

Rv2460c(ClpP2)

Rv3873Rv3870(Snm1)

Reprinted with permission from Proceedings of the National Academy of Sciences

cb600392e.indd 474cb600392e.indd 474 9/13/2006 6:05:50 PM9/13/2006 6:05:50 PM

475www.acschemicalbiology.org VOL.1 NO.8 • ACS CHEMICAL BIOLOGY

A Link to ZincZinc has received substantial attention for its purported ability to prevent or alleviate symptoms of the common cold. Regardless of the effi cacy of this metal as a cold remedy, zinc is an essential element that plays a role in a wide variety of cellular processes, including a well-established but not well-characterized effect on the immune response. Kitamura et al. (Nature Immunology 2006, 7, 971–977) explore the relationship between zinc homeostasis and immune cell function by examining the effects of manipulating intracellular free zinc levels on dendritic cell (DC) matura-tion.

An integral part of the immune response depends on the maturation of DCs, which is concomitant with expression of class II major histo-compatibility complex (MHC class II) proteins through which antigens are presented to T cells. It is known that the endotoxin lipopolysaccharide (LPS) induces DC maturation through Toll-like receptor (TLR) stimulation. A

expanding the versatility of the method. To dem-

onstrate the capacity of M-PFC to identify unknown

protein interactions, a mycobacterial genomic library

was screened for proteins that interact with Cfp-10.

Six proteins were uncovered in the screen: one was

Esat-6, and the other fi ve were previously unknown to

interact with Cfp-10. Intriguingly, examination of the

zinc ion sensitive fl uorescent probe was used to observe that LPS-induced DC stimulation resulted in decreased intracellular zinc concentrations. In addition, treatment of DCs with the zinc chelating reagent N,N,N′,N′-tetra-kis(2-pyridylmethyl)ethylenediamine (TPEN) caused an increase in cell sur-face expression of MHC class II and induced CD4+ T cell activation. By contrast, increasing intracellular DC zinc levels led to inhibition of TPEN-mediated increases in surface expression of MHC class II and LPS-induced movement of MHC class II positive vesicles from the perinuclear area to the cell sur-face. Intracellular zinc levels are modulated by zinc importer and exporter proteins, and examination of transporter levels upon LPS treat-ment revealed a TLR-dependent net increase in zinc export. The connection between LPS exposure and free zinc levels was verifi ed in vivo when mice injected with LPS exhibited decreased intracellular zinc concentrations and

altered zinc transporter expression. Taken together, these results provide a biochemical connection between intracellular free zinc concentra-tions, zinc transporter levels, and TLR signaling, illuminating one pathway by which zinc homeostasis modulates the immune response. Furthermore, the implications could go well beyond the immune system; these data indi-cate that the level of intracellular free

zinc changes in response to extracel-lular stimuli, suggesting that zinc acts as a signaling molecule like calcium. If this process can be generalized to other cell types, this would be an exciting fi nding in the fi eld of signal-ing pathways. EG

potential functions of these proteins revealed sugges-

tive linkages to the secretory system, including spe-

cifi c involvement in membrane targeting and translo-

cation. This powerful method enabled the elucidation

of several components of the protein interaction

network of Cfp-10, paving the way for delineating the

elusive secretory mechanisms of Mtb. EG

Networking Mycobacteria, continued

Untreated LPS

4 8.2

27 60

45

21 10

23

Zn indicator

MH

CII

Reprinted with permission from Nature Immunology



Cannabinoid CrossingThe molecular details behind the enticing

therapeutic and psychological effects of

cannabinoids like those found in marijuana

have been the subject of investigation for

decades. The active components of mari-

juana, such as Δ9-tetrahydrocannabinol, as

well as endogenous cannabinoids (called

endocannabinoids), such as anandamide,

elicit their biological effects through

interactions with cannabinoid receptors

in the brain and select peripheral tissues.

However, the uptake and catabolism of

these compounds are less well understood.

The serine hydrolase fatty acid amide

hydrolase (FAAH) has been implicated in the

Making Sense of Quorum SensingQuorum sensing refers to the ability of bacteria to use

signaling molecules to communicate with one another. A

variety of structurally diverse small molecules produced

by bacteria regulate critical aspects of their function,

including pathogenicity, secondary metabolism, and

biofi lm development. The opportunistic human patho-

gen Pseudomonas aeruginosa synthesizes dozens of

2-alkyl-4(1H )-quinolones (AHQs), including a molecule

termed pseudomonas quinolone signal (PQS) that is

known to regulate virulence gene expression. However,

the roles that other AHQs play in cellular communica-

tion mechanisms in P. aeruginosa and other bacteria are

not well characterized. Diggle et al. (Chem. Biol. 2006,

13, 701–710) now report that AHQs are synthesized by

several species of bacteria and are likely to be an integral

part of their quorum-sensing network.

A combination of bioinformatics, bacterial genetics,

bioreporters, and analytical chemistry were cleverly com-

bined to determine the existence and potential function

of AHQs in several bacterial species. Genomic analysis

revealed that in addition to P. aeruginosa, other strains

of Pseudomonas and Burkholderia produced AHQs. Of

special interest was the human pathogen B. pseudomal-

lei, which is responsible

for the life-threatening

disease melioidosis and

has potential uses as a

bioweapon. It was initially

demonstrated that genetic

disruption of PQS synthe-

sis in P. aeruginosa can be

restored by complementa-

tion with the corresponding gene in B. pseudomallei,

verifying that the genes have a similar function in both

species. Use of an innovative combination of thin-layer

chromatography and an AHQ bioreporter revealed that,

of 20 bacterial strains tested, 9 had the ability to synthe-

size AHQs, although notably only P. aeruginosa strains

were capable of producing PQS. A critical role for AHQ

signaling was demonstrated in B. pseudomallei, when

genetic disruption of AHQ synthesis resulted in a striking,

wrinkled phenotype and an increase in elastase produc-

tion. The authors propose that the ability of certain bacte-

ria to generate distinct AHQs may be a critical component

of the intricate mechanisms by which quorum sensing is

regulated. EG

B. pseudomallei 844

844

hhqA

mutant

Reprinted with permission from Chemistry & Biology

degradation of fatty acid amides, including

anandamide, but the mechanism by which

anandamide crosses into the cell for deliv-

ery to this enzyme remains elusive. Two

recent studies (Dickason-Chesterfi eld et al.,

Cell. Mol. Neurobiol., published online

May 31, 2006, DOI: 10.1007/s10571-006-

9072-6, and Alexander and Cravatt, JACS

2006, 128, 9699–9704) employ inhibitors

of endocannabinoid transport to provide

insight into fatty acid amide metabolism.

Several hypotheses are circulating about

how fatty acid amides are delivered from

outside the cell to the intracellular mem-

brane compartments that house FAAH. One

model promotes simple diffusion, aided

by the lipophilic nature of fatty acid

amides. A second hypothesis argues for

the existence of a plasma-membrane-

associated transporter that facilitates

anandamide uptake. Still another

paradigm invokes an endocytic process

for uptake and transport of fatty acid

amides to FAAH. Though structurally

unrelated to anandamide, the potent,

small-molecule inhibitor of anandamide

uptake LY2183240 enabled researchers

to refi ne existing models of endocan-

nabinoid transport and defi ne the utility

of compounds of this class.(continued on page 477)



Cannabinoid Crossing, continued

Dickason-Chesterfi eld and colleagues compared several

reported inhibitors of anandamide transport, including

LY2183240, for their ability to prevent cellular uptake of

anandamide and to block FAAH hydrolytic activity in vitro.

Cellular uptake was assessed in rat basophilic leukemia

cells, which actively express FAAH, and in HeLa cervical

cancer cells, which do not express FAAH. All of the com-

pounds tested prevented cellular uptake and inhibited

FAAH activity, but the potency of each inhibitor was dra-

matically right-shifted in functional anandamide uptake in

the HeLa cells. In addition, in cell membranes from both

rat basophilic leukemia and HeLa cell lines, 3H-LY2183240

identifi ed a high-affi nity plasma membrane associated

binding site independent of FAAH. Notably, the rank order

and Ki values for displacing 3H-LY2183240 matched

the functional anandamide uptake inhibitory constants,

lending support for a specifi c reuptake

anandamide transport protein. The

authors propose that taken together,

the data suggest the existence of a dis-

tinct transport protein for anandamide

that can adopt high and low binding

affi nity states, depending on the pres-

ence or absence of FAAH.

In a separate study, Alexander and Cravatt scrutinize

the inhibitory properties of LY2183240 using func-

tional proteomics. Brain proteomes were treated with

LY2183240, and use of the activity-based serine hydrolase

probe fl uorophosphonate-biotin led to the identifi cation

of several enzymes, including FAAH, that were inhibited by

this compound. Tryptic digestion and mass spectrometry

analysis of purifi ed FAAH treated with LY2183240 revealed

that the compound covalently inhibits FAAH. Furthermore,

studies in mice confi rmed that LY2183240 covalently

inhibits FAAH and several other serine hydrolases in vivo

at pharmacologically effi cacious doses. An advanced

functional proteomic platform termed ABPP-MudPIT

(activity-based protein profi ling multidimensional

protein identifi cation technology) was used to identify

the other serine hydrolases inhibited by LY2183240,

which included α/β-hydrolase 6 and monoacylglycerol

lipase. The authors suggest that the promiscuity of this

inhibitor is likely due to the reactivity of its heterocyclic

urea group, precluding its incorporation into potential

pharmaceutical agents. However, use of LY2183240 as

a tool to probe endocannabinoid transport suggested

the intimate involvement of FAAH in anandamide

metabolism. The authors propose that hydrolysis of

anandamide by FAAH results in a concentration gradient

that drives uptake of the molecule.

The question remains: does a specifi c anandamide

transport protein exist? Evidence presented in these

papers provides compelling circumstantial evidence for

the presence of a transport protein, but it also suggests

that inhibition of anandamide uptake by LY2183240

involves direct interaction of LY2183240 with FAAH.

However, the inability of the radiolabeled structural

analogue of LY2183240 to cross the plasma membrane

complicates reconciliation of all the data. Clearly, more

studies are needed to elucidate the puzzling process

by which anandamide enters into the cell. Whether

anandamide uptake occurs through simple diffusion

or is carried in by a transporter is uncertain, but if the

putative transporter does exist, selective small-

molecule probes would be welcome tools to help clarify

the mechanism. EG

N N

NN

ON

NN

NHN

LY2183240

Carbamylation

Inactivated FAAH

Ser213 Ser241 Arg243

N

OO +

FAAH

Ser241–O

Ser217–O

Lys142–NH2

H

H

δ+

δ–

Reprinted with permission from the Journal of the American Chemical Society



Flipping the LidProtein degradation in eukaryotic

cells is a carefully regulated and

essential process. The proteasome

is the large ATP-dependent trash

barrel for proteins that have been

tagged for disposal. Proteins are

marked for degradation by cova-

lent attachment of a 76 amino

acid polypeptide known as ubiqui-

tin. Ubiquitin status is recognized

by the top of the trash barrel, the

19S regulatory lid of the pro-

teasome. After the lid checks for

ubiquitin and removes it, the trash

barrel portion, the 20S proteolytic

particle, is unmasked, and the tar-

geted protein is degraded. A high-

resolution look at the proteasome

has long proved diffi cult because

of its large size, dozens of protein

subunits, and a complex in vivo

assembly pathway. A recent study

from Sharon et al. (PLoS Biol.

2006, 4, 1314–1324), however,

has taken a new approach to

proteasome study by harnessing

the power of mass spectrometry

(MS) and chemical cross-link-

ing. The authors developed a

robust method for purifying the

intact regulatory subunit, the lid,

directly from yeast cell extracts.

MS confi rmed the presence of all

known protein subunits as one

major complex and also displayed

some putative intermediates and

subcomplexes. To gain further

insight, specifi c parent ion peaks

were accelerated through an

increased pressure chamber known

as a collision cell. Under these con-

ditions, subunits that are on the

periphery or are less stably associ-

ated are thought to dissociate,

and their identity can be assessed

using mass spectrometry. Using

different collision cell conditions,

the authors showed that a number

of subunits can be released from

the lid. The topology of the lid was

also investigated using a chemi-

cal cross-linking reagent coupled

with MS to identify multimers

of the various protein subunits.

Several of the associations are in

comforting agreement with genetic

interaction assays. This study,

along with other recent studies on

large machines like the ribosome,

highlights biochemical applica-

tions for MS not only for protein

identifi cation but also for spatial

and temporal clues into complex

assemblies. JU

Trypanosoma brucei, the parasite that causes sleeping sickness, exploits two hosts during its life cycle, insects and mammals. In the mammalian bloodstream, T. brucei cleverly eludes immune detection by changing its surface coating of variant surface glycoprotein (VSG) molecules. Fatty acids (FAs) are a critical component of the glycosylphos-phatidylinositol anchor that tethers the VSGs to the plasma membrane. Interestingly, the insect form and the mammalian form of T. brucei have distinct FA needs. Lee et al. (Cell 2006, 126, 691–699) have discovered that although most organisms, both prokaryotic and eukaryotic, use type I or II synthases to synthesize FAs, T. brucei uses microsomoal elon-gases to generate its FAs.

Several pieces of circumstantial evidence sug-gested that T. brucei did not use the typical pathway for FA synthesis. This prompted investigation of the potential role of elongases (ELOs), which are known to extend FAs to longer-chain FAs in other organisms. A cell-free system containing T. brucei membranes was used to evaluate FA synthesis, and it was observed that RNAi silencing of ELO1 caused a dramatic reduction in FA synthesis. In addition, knockout strains for each of the four ELO genes in T. brucei were generated, and thin-layer chromatog-raphy and phosphorimaging analysis revealed that T. brucei uses a sequential pathway for FA synthesis. ELO1 is responsible for extending a 4 carbon chain (C4) to a 10 carbon chain (C10), ELO2 extends C10 to C14, and ELO3 extends C14 to C18. ELO4

does not appear to be involved in FA synthesis; rather, it elongates the unsaturated long-chain FA arachidonate. It was further demonstrated that the ELO pathway is responsible for FA synthesis in cultured T. brucei. Notably, culturing T. brucei in a low-lipid environment induced FA synthesis, reveal-ing that mechanisms are in place for regulation of this pathway. The authors propose that T. brucei has purposely evolved a unique pathway for FA

synthesis that is readily adaptable to the vastly different environments in which trypanosomes must thrive. EG

Trypanosomes Synthesize to a Different Drummer

5500 6000 6500 7000 7500 8000 8500 9000 9500m/z0

100

%

Sub-complex

Intact lid

47+

34+

46+

45+

44+

43+

48+

49+

33+32+35+

*

*

*

42+

41+

43+*

Hinge region

Base subunit

II

I(Catalytic subunit)

Image courtesy of Carol Robinson



Reprinted w

ith perm

ission fr

om Science

Spotlights written by Eva Gordon and Jason Underwood

Histone-eomicsIn the eukaryotic nucleus, DNA is com-

pacted, in part, by the histone family of

proteins. This locked-down confi guration

of DNA, known as chromatin, must rapidly

respond to regulatory cues in the cell and

open up specifi c gene regions to allow

processes like transcription or DNA repair

to occur. The histone proteins themselves

play a key role in such transformations.

Each histone carries a charged amino-

terminal peptide tail that protrudes from

the compact chromatin structure. These

peptides can recruit or repel trans-acting

factors, which might alter the compact

state of the DNA. In a new study by

Dirksen et al. ( J. Proteome Res. 2006, 5,

p53 Partners with CollagenAntiangiogenic therapy is a promising strategy for

treating cancer, essentially depriving a tumor of its

lifeline by cutting off its blood supply. Understanding the under-

lying mechanisms of angiogenesis, along with the discovery of

novel antiangiogenic agents, will greatly contribute to this fi eld.

Several types of collagen proteins possess C-terminal fragments,

such as endostatin and tumstatin, that have promising antian-

giogenic properties. The seemingly unrelated tumor suppressor

protein p53 has also recently been implicated in the regulation

of angiogenesis. Teodoro et al. (Science 2006, 313, 968–971)

now report an intriguing link between p53 and α(II) collagen

prolyl-4-hydroxylase (α(II)PH), an essential enzyme in collagen

biosynthesis, that provides insight into the impact of angiogen-

esis regulation on cancer.

Various techniques, including polymerase chain reaction

based subtractive hybridization and chromatin immunoprecipi-

tation, were used to establish that the α(II)PH gene is a direct

target of p53 transcriptional activation. Several human cancer

cells lines expressing variants of p53, collagen, or α(II)PH

were then created to investigate the roles of these proteins in

angiogenesis regulation. It was demonstrated that

p53 dependent expression of α(II)PH ultimately results

in an increase in the generation of the antiangiogenic collagen

fragments, presumably because of increased collagen synthesis

and subsequent processing. Inhibition of α(II)PH with the small-

molecule inhibitor ethyl-3,4-dihydroxy benzoate or an antisense

oligonucleotide against α(II)PH resulted in decreased endostatin

levels, indicating that α(II)PH is a necessary component of endo-

statin production. Moreover, expression of α(II)PH in the absence

of p53 was suffi cient to stimulate the emergence of endostatin and

tumstatin in conditioned media. Implications of these fi ndings on

angiogenesis were demonstrated when conditioned media contain-

ing antiangiogenic collagen fragments selectively triggered apop-

tosis in human umbilical vein endothelial cells. Furthermore, when

cells expressing α(II)PH were xenografted into nude mice, tumor

growth was dramatically suppressed. The authors propose that p53

induction of α(II)PH expression initiates a pathway for

increased collagen synthesis and processing, yet

another mechanism to add to p53’s repertoire

of tumor suppressor activities. EG

2380–2388), a short consensus peptide

that resembles all of the histone protein

tails was synthesized and used as bait in

a nuclear fi shing expedition. The peptide

was immobilized on a resin, and nuclear

extracts from human immune cells were

applied to this matrix. Factors that bound

to the histone-like peptide were identi-

fi ed in a comprehensive manner via mass

spectrometry of eluted material. Next, the

authors employed the same proteomics

but with nuclear extracts from cells that

were treated with bleomycin, an agent that

induces double-stranded breaks in the

DNA. Interestingly, they found that 40 dif-

ferent proteins were no longer recovered

in DNA damage condi-

tions, but also 44 new ones

appeared to bind in response to damage. The

study goes further and identifi es the phos-

phorylation state of many of the bound pro-

teins. These modifi cations may play key roles

in modulating factor binding to the histone

tails. This study demonstrates that a relatively

simple experiment combined with a sensitive

detection method can yield a host of interest-

ing candidates and new directions for more

careful inquiry. Also, because DNA damage is

just one of the cellular phenomena to which

chromatin must adapt, this methodology

may prove useful to those who look closely at

gene expression and cell signaling. JU

NH2

NH2

NH2

H2N

H2N

Reprinted with permission from Journal of Proteome Research


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