Stem Cells Go Global The clinical promise of human embryonic stem cell research (hESCR) is tantalizing scientists throughout the world, but controversial ethical issues continually jeopardize progress in the field. 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 scientists, ethicists, journal editors, lawyers, and policy makers from 14 countries recently convened in Hinxton, Cambridge, U.K., to create guiding principles 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 prohibit their scientists from participating in hESCR collaborations in countries in which it is legal? Currently, at least one Image courtesy of Getty Images country appears to assert extraterritorial jurisdiction over their scientists, while others do not. It is not reasonable to expect that all countries will eventually adopt similar 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 scientific and ethical integrity in hESCR. For example, it was suggested that scientists should submit new stem cell lines to depositories that subscribe 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
Networking Mycobacteria Astonishingly, 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 networks 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 effective method for exploring Mtb protein–protein interactions in mycobacteria. The method is based on the functional reconstitution of murine dihydrofolate reductase (mDHFR) upon the interaction of two mycobacterial proteins, which are independently fused to two mDHFR domains. Active mDHFR confers mycobacterial resistance against trimethoprim (TRIM), and thus mycobacterial growth in the presence Rv3873
Rv3875 (Esat-6) Rv0686
of TRIM is
Rv3870 (Snm1)
indicative of a
Rv3871 (Snm2) Rv2151c (FtsQ)
protein–protein interaction. Several
Rv2240c Rv3596c (ClpC1)
Rv3800c (Pks13)
identified
Rv2460c (ClpP2) Rv3874 (Cfp-10)
Reprinted with permission from Proceedings of the National Academy of Sciences
protein pairs, including the
Mtb secreted immunogenic antigens Esat-6 and Cfp-10, were used to initially validate the system. In addition, a modified assay was developed to quantify the strength of specific protein–protein interactions, significantly (continued on page 475)
Published online September 15, 2006 • 10.1021/cb600392e CCC: $33.50 © 2006 by American Chemical Society
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A Link to Zinc zinc ion sensitive fluorescent 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′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) caused an increase in cell surface expression of MHC class II and induced CD4+ T cell activation. By contrast, increasing intracellular DC zinc levels led to inhibition of TPENmediated 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 surface. Intracellular zinc levels are modulated by zinc importer and exporter proteins, and examination of transporter levels upon LPS treatment revealed a TLR-dependent net increase in zinc export. The connection between LPS exposure and free zinc levels was verified 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 concentrations, 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 indicate that the level of intracellular free Untreated 4
LPS 8.2
45
23
60
21
10
MHCII
Zinc has received substantial attention for its purported ability to prevent or alleviate symptoms of the common cold. Regardless of the efficacy 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) maturation. An integral part of the immune response depends on the maturation of DCs, which is concomitant with expression of class II major histocompatibility 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
27
Zn indicator
Reprinted with permission from Nature Immunology
zinc changes in response to extracellular 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 finding in the field of signaling pathways. EG
Networking Mycobacteria, continued expanding the versatility of the method. To dem-
potential functions of these proteins revealed sugges-
onstrate the capacity of M-PFC to identify unknown
tive linkages to the secretory system, including spe-
protein interactions, a mycobacterial genomic library
cific involvement in membrane targeting and translo-
was screened for proteins that interact with Cfp-10.
cation. This powerful method enabled the elucidation
Six proteins were uncovered in the screen: one was
of several components of the protein interaction
Esat-6, and the other five were previously unknown to
network of Cfp-10, paving the way for delineating the
interact with Cfp-10. Intriguingly, examination of the
elusive secretory mechanisms of Mtb. EG
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B. p s
eu ma do
llei 844
Making Sense of Quorum Sensing lei, which is responsible for the life-threatening u disease melioidosis and has potential uses as a bioweapon. It was initially demonstrated that genetic disruption of PQS synthesis in P. aeruginosa can be Reprinted with permission from Chemistry & Biology restored by complementation 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 synthesize 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 production. The authors propose that the ability of certain bacteria to generate distinct AHQs may be a critical component of the intricate mechanisms by which quorum sensing is regulated. EG A
m
t tan
844 hhq
Quorum 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 biofilm development. The opportunistic human pathogen 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 communication 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 combined 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-
Cannabinoid Crossing The molecular details behind the enticing
degradation of fatty acid amides, including
model promotes simple diffusion, aided
therapeutic and psychological effects of
anandamide, but the mechanism by which
by the lipophilic nature of fatty acid
cannabinoids like those found in marijuana
anandamide crosses into the cell for deliv-
amides. A second hypothesis argues for
have been the subject of investigation for
ery to this enzyme remains elusive. Two
the existence of a plasma-membrane-
decades. The active components of mari-
recent studies (Dickason-Chesterfield et al.,
associated transporter that facilitates
juana, such as Δ9-tetrahydrocannabinol, as
Cell. Mol. Neurobiol., published online
anandamide uptake. Still another
well as endogenous cannabinoids (called
May 31, 2006, DOI: 10.1007/s10571-006-
paradigm invokes an endocytic process
endocannabinoids), such as anandamide,
9072-6, and Alexander and Cravatt, JACS
for uptake and transport of fatty acid
elicit their biological effects through
2006, 128, 9699–9704) employ inhibitors
amides to FAAH. Though structurally
interactions with cannabinoid receptors
of endocannabinoid transport to provide
unrelated to anandamide, the potent,
in the brain and select peripheral tissues.
insight into fatty acid amide metabolism.
small-molecule inhibitor of anandamide
However, the uptake and catabolism of
Several hypotheses are circulating about
uptake LY2183240 enabled researchers
these compounds are less well understood.
how fatty acid amides are delivered from
to refine existing models of endocan-
The serine hydrolase fatty acid amide
outside the cell to the intracellular mem-
nabinoid transport and define the utility
hydrolase (FAAH) has been implicated in the
brane compartments that house FAAH. One
of compounds of this class. (continued on page 477)
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Cannabinoid Crossing, continued (activity-based protein profiling multidimensional
Dickason-Chesterfield and colleagues compared several reported inhibitors of anandamide transport, including
protein identification technology) was used to identify
LY2183240, for their ability to prevent cellular uptake of
the other serine hydrolases inhibited by LY2183240,
anandamide and to block FAAH hydrolytic activity in vitro.
which included α/β-hydrolase 6 and monoacylglycerol
Cellular uptake was assessed in rat basophilic leukemia
lipase. The authors suggest that the promiscuity of this
cells, which actively express FAAH, and in HeLa cervical
inhibitor is likely due to the reactivity of its heterocyclic
cancer cells, which do not express FAAH. All of the com-
urea group, precluding its incorporation into potential
pounds tested prevented cellular uptake and inhibited
pharmaceutical agents. However, use of LY2183240 as
FAAH activity, but the potency of each inhibitor was dra-
a tool to probe endocannabinoid transport suggested
matically right-shifted in functional anandamide uptake in
the intimate involvement of FAAH in anandamide
the HeLa cells. In addition, in cell membranes from both
metabolism. The authors propose that hydrolysis of
rat basophilic leukemia and HeLa cell lines, 3H-LY2183240
anandamide by FAAH results in a concentration gradient
identified a high-affinity plasma membrane associated
that drives uptake of the molecule. The question remains: does a specific anandamide
binding site independent of FAAH. Notably, the rank order and Ki values for displacing 3H-LY2183240 matched
transport protein exist? Evidence presented in these
the functional anandamide uptake inhibitory constants,
papers provides compelling circumstantial evidence for
lending support for a specific reuptake N
anandamide transport protein. The authors propose that taken together,
δ– Ser241–O
the data suggest the existence of a distinct transport protein for anandamide that can adopt high and low binding affinity states, depending on the presence or absence of FAAH.
O N
Carbamylation
N N N
H
Ser241
Arg243
O
H Ser217–O
Ser213
O LY2183240
N
N NH N N
+
δ+ Lys142–NH2 FAAH
Inactivated FAAH
Reprinted with permission from the Journal of the American Chemical Society
In a separate study, Alexander and Cravatt scrutinize
the presence of a transport protein, but it also suggests
the inhibitory properties of LY2183240 using func-
that inhibition of anandamide uptake by LY2183240
tional proteomics. Brain proteomes were treated with
involves direct interaction of LY2183240 with FAAH.
LY2183240, and use of the activity-based serine hydrolase
However, the inability of the radiolabeled structural
probe fluorophosphonate-biotin led to the identification
analogue of LY2183240 to cross the plasma membrane
of several enzymes, including FAAH, that were inhibited by
complicates reconciliation of all the data. Clearly, more
this compound. Tryptic digestion and mass spectrometry
studies are needed to elucidate the puzzling process
analysis of purified FAAH treated with LY2183240 revealed
by which anandamide enters into the cell. Whether
that the compound covalently inhibits FAAH. Furthermore,
anandamide uptake occurs through simple diffusion
studies in mice confirmed that LY2183240 covalently
or is carried in by a transporter is uncertain, but if the
inhibits FAAH and several other serine hydrolases in vivo
putative transporter does exist, selective small-
at pharmacologically efficacious doses. An advanced
molecule probes would be welcome tools to help clarify
functional proteomic platform termed ABPP-MudPIT
the mechanism. EG
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Flipping the Lid Protein degradation in eukaryotic
insight, specific parent ion peaks
Trypanosomes Synthesize to a Different Drummer
Trypanosoma brucei, the parasite that causes sleeping sickness, exploits two hosts during its life essential process. The proteasome increased pressure chamber known cycle, insects and mammals. In the mammalian is the large ATP-dependent trash as a collision cell. Under these conbloodstream, T. brucei cleverly eludes immune barrel for proteins that have been ditions, subunits that are on the detection by changing its surface coating of variant surface glycoprotein (VSG) molecules. Fatty acids tagged for disposal. Proteins are periphery or are less stably associ(FAs) are a critical component of the glycosylphosmarked for degradation by covaated are thought to dissociate, phatidylinositol anchor that tethers the VSGs to the lent attachment of a 76 amino and their identity can be assessed plasma membrane. Interestingly, the insect form acid polypeptide known as ubiqui- using mass spectrometry. Using and the mammalian form of T. brucei have distinct tin. Ubiquitin status is recognized different collision cell conditions, FA needs. Lee et al. (Cell 2006, 126, 691–699) have discovered that although most organisms, both by the top of the trash barrel, the the authors showed that a number prokaryotic and eukaryotic, use type I or II synthases 19S regulatory lid of the proof subunits can be released from to synthesize FAs, T. brucei uses microsomoal elonteasome. After the lid checks for the lid. The topology of the lid was gases to generate its FAs. ubiquitin and removes it, the trash also investigated using a chemiSeveral pieces of circumstantial evidence sugbarrel portion, the 20S proteolytic cal cross-linking reagent coupled gested that T. brucei did not use the typical pathway for FA synthesis. This prompted investigation of particle, is unmasked, and the tar- with MS to identify multimers the potential role of elongases (ELOs), which are geted protein is degraded. A high- of the various protein subunits. known to extend FAs to longer-chain FAs in other resolution look at the proteasome Several of the associations are in organisms. A cell-free system containing T. brucei has long proved difficult because comforting agreement with genetic membranes was used to evaluate FA synthesis, and of its large size, dozens of protein interaction assays. This study, it was observed that RNAi silencing of ELO1 caused a dramatic reduction in FA synthesis. In addition, subunits, and a complex in vivo along with other recent studies on knockout strains for each of the four ELO genes in assembly pathway. A recent study large machines like the ribosome, T. brucei were generated, and thin-layer chromatogfrom Sharon et al. (PLoS Biol. highlights biochemical applicaraphy and phosphorimaging analysis revealed that 2006, 4, 1314–1324), however, tions for MS not only for protein T. brucei uses a sequential pathway for FA synthesis. has taken a new approach to identification but also for spatial ELO1 is responsible for extending a 4 carbon chain (C4) to a 10 carbon chain (C10), ELO2 extends proteasome study by harnessing and temporal clues into complex C10 to C14, and ELO3 extends C14 to C18. ELO4 the power of mass spectrometry assemblies. JU does not appear to be involved in FA synthesis; (MS) and chemical cross-linkIntact lid rather, it elongates the unsaturated long-chain FA (Catalytic subunit) 47+ ing. The authors developed a 100 46+ arachidonate. It was further demonstrated that I robust method for purifying the Base subunit the ELO pathway is responsible for FA synthesis in 41+ 45+ cultured T. brucei. Notably, culturing T. brucei in a intact regulatory subunit, the lid, 48+ * low-lipid environment induced FA synthesis, revealdirectly from yeast cell extracts. II 42+* Hinge region ing that mechanisms are in place for regulation of % MS confirmed the presence of all 49+ 44+ this pathway. The authors propose that T. brucei 43+* known protein subunits as one Sub-complex has purposely evolved a unique pathway for FA 34+ 33+ 43+ major complex and also displayed synthesis that is readily adaptable to the vastly 32+ 35+ different environments in which some putative intermediates and 0 m/z 5500 6000 6500 7000 7500 8000 8500 9000 9500 trypanosomes must thrive. EG subcomplexes. To gain further
cells is a carefully regulated and
were accelerated through an
Image courtesy of Carol Robinson
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Sc ie nc e m fro n pe rm is si o wi th Re pr in te d
p53 Partners with Collagen Antiangiogenic therapy is a promising strategy for treating cancer, essentially depriving a tumor of its
angiogenesis regulation. It was demonstrated that p53 dependent expression of α(II)PH ultimately results
lifeline by cutting off its blood supply. Understanding the under-
in an increase in the generation of the antiangiogenic collagen
lying mechanisms of angiogenesis, along with the discovery of
fragments, presumably because of increased collagen synthesis
novel antiangiogenic agents, will greatly contribute to this field.
and subsequent processing. Inhibition of α(II)PH with the small-
Several types of collagen proteins possess C-terminal fragments,
molecule inhibitor ethyl-3,4-dihydroxy benzoate or an antisense
such as endostatin and tumstatin, that have promising antian-
oligonucleotide against α(II)PH resulted in decreased endostatin
giogenic properties. The seemingly unrelated tumor suppressor
levels, indicating that α(II)PH is a necessary component of endo-
protein p53 has also recently been implicated in the regulation
statin production. Moreover, expression of α(II)PH in the absence
of angiogenesis. Teodoro et al. (Science 2006, 313, 968–971)
of p53 was sufficient to stimulate the emergence of endostatin and
now report an intriguing link between p53 and α(II) collagen
tumstatin in conditioned media. Implications of these findings on
prolyl-4-hydroxylase (α(II)PH), an essential enzyme in collagen
angiogenesis were demonstrated when conditioned media contain-
biosynthesis, that provides insight into the impact of angiogen-
ing antiangiogenic collagen fragments selectively triggered apop-
esis regulation on cancer.
tosis in human umbilical vein endothelial cells. Furthermore, when
Various techniques, including polymerase chain reaction
cells expressing α(II)PH were xenografted into nude mice, tumor
based subtractive hybridization and chromatin immunoprecipi-
growth was dramatically suppressed. The authors propose that p53
tation, were used to establish that the α(II)PH gene is a direct
induction of α(II)PH expression initiates a pathway for
target of p53 transcriptional activation. Several human cancer
increased collagen synthesis and processing, yet
cells lines expressing variants of p53, collagen, or α(II)PH
another mechanism to add to p53’s repertoire
were then created to investigate the roles of these proteins in
of tumor suppressor activities. EG
Reprinted with permission from Journal of Proteome Research
NH2
H2N
Histone-eomics
H2N
In the eukaryotic nucleus, DNA is com-
2380–2388), a short consensus peptide
in DNA damage condi-
pacted, in part, by the histone family of
that resembles all of the histone protein
tions, but also 44 new ones
proteins. This locked-down configuration
tails was synthesized and used as bait in
appeared to bind in response to damage. The
of DNA, known as chromatin, must rapidly
a nuclear fishing expedition. The peptide
study goes further and identifies the phos-
respond to regulatory cues in the cell and
was immobilized on a resin, and nuclear
phorylation state of many of the bound pro-
open up specific gene regions to allow
extracts from human immune cells were
teins. These modifications may play key roles
processes like transcription or DNA repair
applied to this matrix. Factors that bound
in modulating factor binding to the histone
to occur. The histone proteins themselves
to the histone-like peptide were identi-
tails. This study demonstrates that a relatively
play a key role in such transformations.
fied in a comprehensive manner via mass
simple experiment combined with a sensitive
Each histone carries a charged amino-
spectrometry of eluted material. Next, the
detection method can yield a host of interest-
terminal peptide tail that protrudes from
authors employed the same proteomics
ing candidates and new directions for more
the compact chromatin structure. These
but with nuclear extracts from cells that
careful inquiry. Also, because DNA damage is
peptides can recruit or repel trans-acting
were treated with bleomycin, an agent that
just one of the cellular phenomena to which
factors, which might alter the compact
induces double-stranded breaks in the
chromatin must adapt, this methodology
state of the DNA. In a new study by
DNA. Interestingly, they found that 40 dif-
may prove useful to those who look closely at
Dirksen et al. ( J. Proteome Res. 2006, 5,
ferent proteins were no longer recovered
gene expression and cell signaling. JU
NH2 NH2
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