17Å
Carbohydrate Channeling Many bacteria produce extracellular polysaccharides (EPSs) that play pivotal roles in their structural integrity and pathogenicity. Certain EPSs form a discrete structural layer called the capsule, and group 1 capsule assembly is characterized by the synthesis of individual lipid-linked polysaccharide repeats that are exported to the periplasm for assembly of the fulllength polymer. Once assembled, the polymer is transported across the outer membrane by the lipoprotein Wza, a member of the outer membrane auxiliary protein family. Dong et al. (Nature 2006, 444, 226-229) now present the crystal structure of Wza, providing insight 105Å into the regulation and mechanism Both images reprinted by permission from Macmillan Publishers Ltd: Nature, Dong, C., et al., 444, 226-229, copyright 2006. by which capsular polysaccharide is exported through the bacterial membrane. The structure revealed that Wza is an octamer containing a large internal cavity open to the extracellular environment but closed to the periplasm. Each mono-
mer of Wza has four domains, with domains 2 and 3 being duplicates. The eight copies of domain 1 form a ring structure with a concave surface at the base of the protein. Domain 4 is an amphipathic helix, and 105Å the eight monomers create an a-helical barrel at the top of the structure. The central cavity and the exterior surface of the protein are polar, with the exception of the markedly hydrophobic a-helical barrel that is predicted to be the transmembrane region. Notably, all other known integral outer membrane proteins possess transmembrane b-barrels; Wza is the first example of a transmembrane a-helical barrel. On the basis of this structure, the authors propose that the carbohydrate moves from the periplasm to the central cavity of Wza. The polar environment enables water molecules to effectively lubricate the cavity for translocation of the polysaccharides to the membrane, where they exit through the a-helical barrel. They also hypothesize that the opening of the cavity is regulated via a conformational change at the concave base of the protein, possibly through interaction with the inner membrane tyrosine autokinase Wzc. Eva Gordon
Pluripotency Aplenty Embryonic stem (ES) cells are plu-
understanding of basic developmen-
erocycles against an mES cell line
ripotent, which means they have the
tal biology and facilitate potential
that, in the absence of self-renewal
extraordinary ability to differentiate
ES cell-based therapies. Chen et al.
conditions such as feeder cells or
into all cell types and thus harbor
(Proc. Natl. Acad. Sci. U.S.A. 2006,
exogenous factors, has limited self-
tremendous therapeutic promise.
103, 17,266-17,271) now report the
propagation ability. Remarkably,
Under self-renewal conditions,
discovery of a synthetic small mol-
SC-1-treated cells could self-renew
however, ES cells can propagate in
ecule, SC-1 (also named pluripotin),
for >10 passages, were phenotypi-
the pluripotent state indefinitely.
that on its own sustains the self-
cally indistinguishable from ES cells
Molecular tools that help define
renewal of murine ES (mES) cells.
grown under self-renewal condi-
the mechanisms governing ES cell self-renewal will contribute to our
SC-1 was discovered from a highthroughput screen of 50,000 het-
tions, and, upon removal of SC-1, could differentiate into various cell (continued on page 673)
Published online December 15, 2006 • 10.1021/cb600478c CCC: $33.50 © 2006 by American Chemical Society
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continued from page 672 types in culture and contribute to the germ line in mice in vivo. After initial studies failed to link SC-1 to known self-renewal pathways, target identification experiments using an SC-1-linked affinity matrix led to the isolation of two proteins, extracellular signal-regulated protein kinase-1 (Erk1) and Ras GTPase-activating protein (RasGAP). In cells, SC-1 was shown to inhibit Erk1 phosphorylation and to activate the Ras pathway by inhibiting RasGAP. The authors subsequently demonstrated that overexpression of Erk1 or RasGAP promotes ES cell differentiation, either of which can be partially rescued by exposure to SC-1. Notably, the dual inhibition of Erk1 and RasGAP was necessary for maintaining the cells in an undifferentiated state; blocking either pathway alone by other methods was not sufficient to sustain self-renewal. The authors propose that SC-1 functions by simultaneously blocking Erk1 pathways, which are known to contribute to cell differentiation, and by inhibiting RasGAP pathways, which may activate self-renewal pathways through Ras signaling. Eva Gordon
www.acschemicalbiolog y.o rg
Flexibly Folded N-Linked glycosylation of proteins serves important structural and functional roles, including involvement in protein folding, stability, cell adhesion, and the immune response. In eukaryotes, it is known that N-linked
containing the glycosylation consensus sequence of AcrA was inserted into a loop in GFP and the GFP-derivative was exposed to PglB, the protein was glycosylated and retained full fluorescence, a further demonstration that PglB is capable of glycosylating folded proteins in vitro. Finally, four folding variants of bovine ribonuclease A (RNaseA), which included a folded, enzymatically active species, a partially folded species, and two random coils, were examined for their ability to act as PglB substrates. PglB glycosylated all four forms of RNaseA, but notably, the nonstructured species were better PglB substrates than the folded proteins. The authors propose that in bacteria, glycosylation sites likely adopt specific conformations in locally flexible areas of folded proteins, providing insight into the different mechanisms that have evolved for prokaryotic and eukaryotic glycosylation systems. Eva Gordon
glycosylation is coupled to protein translocation and that the enzyme responsible, oligosaccharyltransferase (OTase), recognizes its substrate in an unfolded state. However, the homologous glycosylation pathway in bacteria is less well characterized. Now, Kowarik et al. (Science 2006, 314, 1148-1150) report that bacterial N-glycosylation is not linked to the protein translocation machinery. Using pulse chase experiments, the authors first observed that the bacterial protein AcrA was glycosylated by the bacterial oligosaccharyltransferase (PglB) post-translationally, a sign of a translocation-independent reaction. When they explored this further, they found that an AcrA fusion that was directed to the periplasm as a folded protein was Reduced Native (inactive) (active) also glycosylated, Oxidative DTT, ∆T an indication that Inclusion refolding bodies GnCl PglB can glycosylate folded, exported 1. Oxidation proteins. They next Alkylation 2. Alkylation S-peptide demonstrated that removal PglB is capable of glycosylating purified AcrA in vitro, in sharp Reduced-alkylated “Scrambled” S-protein “Nicked” contrast to eukaryotic (inactive) (inactive) (inactive) (active) OTases, which do not possess this ability. -CH2CONH2 SS-bond Cys-residue When a peptide From Kowarik, M., et al., Science, Nov 17, 2006, DOI: 10.1126/science.1134351. Reprinted with permission from AAAS. pr Limi o (su teo ted bti lys lis isin)
Pluripotency Aplenty,
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Chaperoning Chromatin Not unlike teenagers at the prom, the biomolecules in the nuclei of cells that make up chromatin need chaperones to ensure that they behave properly. Chromatin consists of DNA wound around proteins called histones; whereas the interaction between histones and DNA has been extensively characterized, the interactions of histones with each other and with chaperones in the absence of DNA are not well understood. Now, English et al. (Cell 2006, 127, 495-508) present the crystal structure of the histone-chaperone protein antisilencing function 1 (Asf1) bound to the H3/H4 histone heterodimer, providing novel insights into the interactions between histones and their chaperones. The Asf1-H3/H4 structure revealed that Asf1 interacts extensively with both H3 and H4, an unexpected finding because previous studies had suggested that Asf1 directly interacts only with H3. Notably, the last 10 residues of H4 add an additional b-strand to an antiparallel b-sheet within Asf1. Moreover, the carboxyl terminus of H4 undergoes a dramatic conformational change in order to form this b-sheet with Asf1. Interest-
Reprinted from Cell, 127, English, C. M., et al., Structural basis for the histone chaperone activity of asf1, 495-508, Copyright 2006, with permission from Elsevier.
ingly, it is these same residues that form a b-sheet with histone 2A in the nucleosome, stabilizing nucleosome structure. To explore the biological relevance of the key Asf1-H3/H4 contacts revealed in the crystal structure, the authors mutated various residues within the binding interface between Asf1, H3, and H4. Yeast expressing these mutant proteins exhibited growth defects, reduced chromatin-disassembly activity, and diminished Asf1H3/H4 interactions; this confirms the importance of the Asf1-H3/H4 interaction in chromatin regulation. On the basis of structural and biological data from these studies and the established role of the H4 carboxyl terminus in stabilizing histone structure, the authors propose that once this region of H4 is exposed, the interprotein bsheet formed between Asf1 and H4 captures the H3/H4 complex and facilitates further chromatin disassembly, putting forth a potential mechanism by which histone chaperones mediate nucleosome assembly and disassembly. Eva Gordon
Channeling Autoimmunity Certain autoimmune diseases such as multiple sclerosis (MS), rheumatoid arthritis (RA), or type-1 diabetes mellitus (T1DM) are caused in part by a specific type of autoreactive white blood cell called an effector memory T (TEM) cell. TEM cells from patients with MS have distinct phenotypic characteristics, including elevated expression levels of Kv1.3 potassium channels. Beeton et al. (Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 17,414-17,419) now demonstrate that TEM cells from patients with RA or T1DM also exhibit high levels of Kv1.3 and that Kv1.3 blockers selectively target these TEM cells and can diminish RA progression and T1DM incidence in rats. Using patch-clamp technology, immunostaining, and flow cytometry, the authors first showed that disease-associated autoreactive TEM cells from T1DM and RA patients express high levels of Kv1.3. Next, they evaluated immunological synapse formation, Ca2+ signaling, cytokine production, and [3H]thymidine incorporation and determined that selective Kv1.3 blockers preferentially block TEM cell function without affecting the activity of other T cells, an important distinction to ensure that other aspects of immune function are not compromised. The therapeutic potential of Kv1.3 blockers was next assessed in RA and T1DM disease models in rats. Treatment with Kv1.3 blockers significantly alleviated RA symptoms and reduced the incidence of experimental autoimmune diabetes in the animals. Notably, toxicity studies using repeated doses of the blockers revealed that they did not appear to cause adverse effects. These exciting findings indicate that Kv1.3 channels may be a promising therapeutic target for any autoimmune disease in which levels of Kv1.3 on TEM cells are elevated. Thus, discovery of new small-molecule Kv1.3 blockers could lead to effective drugs for a variety of autoimmune diseases. Eva Gordon
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Shigella’s Path of Destruction Shigella, the pathogenic bacteria responsible for
ing down MTs that were in their path. Suspi-
causing dysentery, motor through the cytoplasm
ciously, VirA just happened to be secreted on
of host cells by inducing actin polymerization and
the bacterial surface via the type III secretion
hitching a ride on the resulting propulsive force.
system in the exact vicinity of the MT fragmen-
This motility helps firmly establish infection and
tation. Perusal of the secondary structure of
facilitates access to neighboring host cells, but
VirA revealed a strong resemblance to a family
the mechanisms that guide the movement are
of cysteine proteases; thus, VirA was examined
not clearly defined. Now, Yoshida et al. (Science
for cysteine-protease-like activity. Indeed, VirA
2006, 314, 985-989) report that the Shigella
was capable of degrading a-tubulin, and this
effector protein VirA degrades host cell microtu-
activity was inhibited by cysteine protease
bules (MTs) via a tubulin-specific cysteine-pro-
inhibitors. Importantly, mice infected with vari-
tease-like activity, effectively clearing a path for
ous Shigella mutants with compromised VirA
movement through the cytoplasm.
activity had increased survival rates compared
In a comparison of wild-type (WT) and virA
with the WT bacteria. This study illuminates
mutant (virA-) bacterial movement through mam-
how VirA contributes to the remarkable strat-
malian cells, immunofluorescence microscopy
egy by which Shigella sabotages its intracel-
revealed that virA- mutants did not move, an indi- lular obstacles. cation of the critical role that VirA plays in Shigella Other cytoplasmic motility. The authors also demonstrated that MT
invading bacterial
structure acts as a barrier to movement; WT bac-
pathogens such as
teria exhibited smoother movement with reduced
Listeria monocyto-
variability in cells treated with the MT-destabiliz-
genes, Mycobac-
ing drug nocodazole compared with untreated
terium marinum,
cells. Further probing with immunofluorescence
Rickettsia prowaze-
and freeze-fracture electron microscopy revealed
kii, and Burkholderia pseudomallei also move
that as the bacteria move, they create tunnel-like
through the host cell cytoplasm by inducing
areas through MT networks in the precise area
actin polymerization at one pole of the bac-
where the actin tail had formed. Moreover, the
terium; thus, similar methods to deforest the
MT networks were frequently fragmented in the
MT jungle may be used by these pathogens as
tunnel zone, a sign that the bacteria were break-
well. Eva Gordon
www.acschemicalbiolog y.o rg
From Yoshida, S., et al., Science, Nov 10, 2006, DOI: 10.1126/science.1133174. Reprinted with permission from AAAS.
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Driving Cancer Cells to Suicide Cancer cells have the notorious ability to sidestep the
apoptosis if Bak and/or Bax are present. Investiga-
innate pathways that direct a cell to commit suicide,
tion of several putative small-molecule BH3 mimics
an important and natural part of a normal cell’s life
using Bax and Bak-deficient cells revealed that only
cycle. Moreover, many cancer cells develop resistance
ABT-737 actually induced apoptosis via the same
to the very drugs designed to kill them by induc-
mechanism that BH3-containing proteins do. This is
ing cell suicide. The Bcl-2 protein family consists of
an important consideration in the design of effective
several proteins that play important roles in guiding
anticancer agents to ensure selectivity and to reduce
cells toward survival, such as the prosurvival proteins
the likelihood of adverse side effects. ABT-737 was
Bcl-2, Bcl-xL, or Mcl-1, or death, including the prodeath
shown to bind selectively to Bcl-2 and Bcl-xL and
proteins Noxa,
to disrupt the association of Bcl-2 with Bax in the
Bim, Bad,
leukemia cell line HL-60. Notably, however, ABT-737
Bax, and Bak.
did not bind to the prosurvival protein Mcl-1.
Control
Interaction of
20 mg/kg
30 mg/kg
Day 1
Day 19
Reprinted from Cancer Cell, 10, Konopleva, M., et al., Mechanisms of apoptosis sensitivity and resistance to the BH3 mimetic ABT-737 in acute myeloid leukemia, 375-388, Copyright 2006, with permission from Elsevier.
The human leukemia cell line HL-60, which does
a conserved
not express high levels of Mcl-1, underwent apop-
dimerization
tosis upon exposure to ABT-737, whereas other cell
motif, the BH3
lines that express high levels of Mcl-1 were resistant
domain on cer-
to ABT-737-induced apoptosis. Both research groups
tain prodeath
hypothesized that treatment of resistant cell lines
members, with
with ABT-737 in combination with agents that reduce
a hydrophobic
Mcl-1 expression might lead to enhanced cytotox-
groove on
icity. Several strategies known to diminish Mcl-1
prosurvival
levels, including treatment with short hairpin RNA
members can
against Mcl-1, exposure to extracellular signal-regu-
initiate cell suicide, otherwise known as apoptosis.
lated protein kinase (Erk) phosphorylation inhibitors,
This suggests that small molecules that mimic the
overexpression of Noxa, or IL-3 deprivation, each
BH3 domain could be effective anticancer agents. Two
resulted in greatly enhanced sensitivity of otherwise
recent reports (Konopleva et al., Cancer Cell 2006,
resistant cells to ABT-737. Additionally, treatment
10, 375-388, and van Delft et al., Cancer Cell 2006,
of resistant cell lines with other agents, such as the
10, 389-399) dissect the molecular mechanism of
chemotherapeutic drugs doxorubicin, cytosine ara-
ABT-737, a small-molecule BH3 mimetic, and provide
binoside, or etoposide; the cyclin-dependent kinase
enticing evidence for the consideration of ABT-737 as a
inhibitor R-roscovitine; or the protein synthesis
therapeutic agent for a variety of cancers.
inhibitor cycloheximide, also resulted in enhanced
It is important to note that interaction of BH3 domains with prosurvival members only initiates
sensitivity to ABT-737, likely in part by down-regulating Mcl-1 expression. (continued on page 677)
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FRET No More Over O-GlcNAc
Post-translational modification of proteins with b-O-N-acetyl-D-glucosamine (O-GlcNAc) is a dynamic event that, like phosphorylation, contributes to cell signaling in nuclear and cytosolic proteins and has been linked to certain disease states. Unfortunately, little is known about the spatial and temporal regulation of O-GlcNAc during cell signaling because of a lack of methods to study this modification in live cells. Now, Carrillo et al. ( J. Am. Chem. Soc. 2006, 128, 14,768-14,769) present a FRET-based sensor for detecting the dynamics of O-GlcNAc modifications in cells. The sensor was designed so that the addition of an O-GlcNAc to an appropriate substrate results in a binding event, which in turn brings two fluorophores together to cause an increase in FRET. A fusion protein is composed of an enhanced cyan fluorescent protein, the bacterial lectin GafD (which binds terminal b-O-GlcNAc), the casein kinase II-derived peptide substrate for O-GlcNAc transferase, and a yellow fluorescent protein variant made up the components of the sensor. To establish that the sensor could detect the addition and the removal of O-GlcNAc from proteins, the authors used bacterially expressed sensors to conduct an in vitro assay. As expected, a significant increase in fluorescence emission at 528 nm was observed when the sensor was incubated with recombinant O-GlcNAc transferase and www.acschemicalbiolog y.o rg
Driving Cancer Cells to Suicide, continued from page 676 Both studies also revealed the
Exploration of ABT-737’s mecha-
efficacy of ABT-737 as an anticancer nism of action enabled both research agent in animal models. Konopleva
groups to determine why certain
et al. showed that ABT-737 signifi-
cancer cells become suicidal upon
cantly reduced leukemia burden
exposure to ABT-737 while others
and extended survival of mice in
resist being pushed down that
leukemia models, with no adverse
path. On the basis of these studies,
effects on normal tissue. In addi-
ABT-737 has exciting prospects as a
tion, van Delft et al. demonstrated
single agent in tumors with low Mcl-1
that ABT-737 prolonged survival of
expression or in combination with
mice transplanted with either con-
other anticancer agents in high Mcl-
trol or Bcl-2 expressing lymphoma
1-expressing tumors. Moreover, these
cells. Notably, mice transplanted
discoveries have revealed that Mcl-1
with Mcl-1 expressing lymphoma
expression levels could be a valuable
cells were resistant to ABT-737
prognostic marker for response to
treatment.
ABT-737. Eva Gordon
t=0h
t = 1.5 h
O-GlcNAc sensor
1.4
1.0
30 µm Reprinted with permission from Carrillo, L. D., et al., J. Am. Chem. Soc., 128, 14,768-14,769. Copyright 2006 American Chemical Society.
the requisite sugar donor UDP-GlcNAc. Likewise, a significant loss of signal was observed when O-GlcNAcase, the enzyme that removes O-GlcNAc from proteins, was added to a sensor that contained O-GlcNAc. The sensor was next tested for its ability to detect O-GlcNAc dynamics in live cells. Indeed, treatment of a human cancer cell line transiently transfected with the sensor that had reagents that promoted a dynamic increase in O-GlcNAc resulted in a significant increase in FRET. This sensor represents the first method for exploring O-GlcNAc dynamics in a cellular setting, and its modular design will enable examination of the interplay between O-GlcNAc and phosphorylation networks. Eva Gordon
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