Spotlight Clockwork Lithium
Lithium, a common treatment for bipolar disorder, carries a number of side effects. Sleep disturbance is among the most common. Sleep patterns are controlled by circadian rhythm, the 24-hour internal clock in organisms. These rhythms, which are also displayed by cells, are maintained at the mole cular level through interconnected transcriptional feedback loops of clock genes. Post-translational modification also plays a role in regulation of the cellular clocks. Now, Yin et al. (Science 2006, 311, 1002–1005) suggest a molecular link between lithium, a kinase, an orphan nuclear receptor, and the clock genes. The orphan nuclear receptor Rev-erbα is responsible for modulating the transcription and rhythmic expression of Bmal1, one of many clock proteins. Previous studies showed that Rev-erbα turns down its own transcription and that of Bmal1 during circadian night. Rev‑erbα contains several potential phosphorylation sites for glycogen synthase kinase 3β (GSK3β). Interestingly, this kinase is selectively inhibited by lithium ions and a mutation found in the fly homologue of GSK3β affects circadian timing. To test if this
kinase modulates the activity of Rev-erbα, the authors depleted GSK3β from tissue culture cells using RNA interference. To their surprise, depletion of the kinase resulted in the disappearance of Rev-erbα as well. Since a number of timing events in the circadian circuit are regulated by positive and negative feedback on transcription, the mRNA levels of both Rev-erbα and master clock regulator Bmal1 were tested. When GSK3β is depleted, the mRNA levels of both regulators increased. These data suggested that GSK3β activity was modulating the amount of Rev-erbα through post-translational modification. To test this hypothesis, the authors inhibited GSK3β kinase activity with lithium at concentrations that mimic patient serum levels. Steve Campbell/Getty Images They found very little Rev-erbα protein suggesting that it was being degraded. The authors show that ubiquitin-mediated proteasomal degradation of Rev-erbα is modulated by two key serine residues which are GSK3β phosphorylation sites. These and other data indicate that the kinase activity of GSK3β is critical for stabilizing Rev-erbα and explain how lithium inhibition of a kinase alters the circadian clock. JU
Trigger Points Many age-related degenerative diseases, including Alzheimer’s, and diseases connected with medical therapy, such as dialysis-related amyloidosis (DRA), are associated with the oligomerization of protein into amyloid fibrils. The constituents of these amyloids vary widely, but the fibrils share common structural properties. In vitro studies indicate that fibril formation begins with a nucleation event that is followed by a rapid and cooperative
association. As the amyloid product is a heterogeneous aggregate, the molecular basis for these changes are, to date, poorly understood. Now, Eakin et al. (Nat. Struct. Mol. Biol. 2006; 13, 202–208) determine the chemical basis and resultant structural changes required for oligomerization of β-2‑microglobulin (β2m) in DRA. β2m is a subunit necessary for the cell surface expression of class I-like complexes such as the major histocompatibility
complex (MHC). As part of MHC turnover, β2m is released and subsequently broken down by the kidney. The concentration of β2m is higher in patients with kidney disease, but this increased level of the protein by itself does not result in aggregation. Under physiological conditions, the transient presence of stoichiometric Cu2+ is required for β2m fibrillogenesis. The authors show that Cu2+ catalyzes the formation of (continued on page 59)
Published online March 17, 2006 • 10.1021/cb600107t CCC $33.50 © 2006 by American Chemical Society
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Spotlight Designer Collagen Collagen, which is Greek for “glue producer”, is the most abundant protein in humans. True to its name, collagen literally holds the human body together as the major component of connective tissue. It also contributes to skin strength and elasticity and has a greater tensile strength than steel. Collagen’s many unique properties have stimulated attempts to develop materials for medicinal and nanotechnological applications. However, natural collagen is difficult to modify and can cause pathological side effects when transplanted into humans. Furthermore, generation of synthetic collagen has proved difficult to control. Now Kotch and Raines (Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 3028–3033) have used synthetic chemistry to create unique fragments that self-assemble into collagen. Natural collagen is comprised of three polypeptide strands that fold into a triple
helix, with each strand containing a repeating tripeptide sequence. The researchers generated synthetic peptides containing these tripeptide sequences along with strategically placed cysteines to enable covalent linkage of the strands through disulfide bonds. The peptides were designed so that the locations of the cysteines resulted in “sticky ends” that enabled self-assembly of collagenlike triple helices. The authors used a variety of techniques to characterize the synthetic collagen triple helices. Circular dichroism spectra confirmed a triple helical structure, and thermal stability experiments demonstrated cooperative denaturation,
characteristic of triple helices. Dynamic light scattering, atomic force microscopy, and transmission electron microscopy provided information about the hydrodynamic radius and length of the fibrils, which had a width near 1 nm and a length approaching 1 µm, depending on amino acid composition, temperature, f yo and solvent. The length of em d ca lA na natural collagen is approximately io .A. t Na U.S 06 s, 20 ce 300 nm; thus this “sticky-ends” © ien Sc method for producing synthetic collagen is capable of producing strands longer than those of natural collagen. This unique approach presents intriguing opportunities for creating collagen-based materials for diverse applications, ranging from tissue engineering to providing nanowires for electronic devices. EG
Trigger Points, continued
Rep ri
nted
with p
erm
issio n fro mN SMB
M*, an alternative nativelike conformation of the protein. This intermediate assembles into dimeric building blocks that are stabilized by Cu2+. The authors hypothesized that divalent cation– mediated backbone isomerization of one proline residue was responsible for M* formation. To test this hypothesis, Eakin et al. mutated the proline to alanine trapping this residue
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in a trans conform ation. This mutant bound Cu2+ 10 000 fold more tightly than WT β2m. The crystal structure showed that, like WT β2m, the mutant adopts a β‑sandwich structure but its hydrophobic core is repacked. These and other structural rearrangements result in the formation of an amphipathic dimer. The dimeric structure reveals a basis for its role as
an intermediate building block for the formation of higher oligomeric states. Metal ions are implicated in other amyloid diseases. The proteins in these systems, like β2m, show cation-triggered conformational transitions possibly involving backbone rearrangements. Understanding the relationships between these metal-triggered transitions and fibril formation will be valuable in understanding amyloid diseases. EJ
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Spotlight Assisted Suicide E6-mediated drug resistance, the researchers used an E6‑expressing colon carcinoma cell line called RKO-E6 that is two to four times more resistant to doxorubicin-mediated apoptosis than RKO cells alone. Approximately 27 000 small molecules were screened for their ability to increase the toxicity of doxorubicin, and 88 compounds were identified that selectively increase doxorubicin’s lethality by at least 20%. The compounds were grouped into five structural classes, including quaternary ammonium compounds, protein synthesis inhibitors, 11-deoxyprostaglandin E1 analogues, 1,3‑bis(4‑morpholinylmethyl)-2-imidazolidinethione analogues, and a collection that the authors named indoxins. Examination of the mechanisms by which the compounds exerted their effects revealed that doxorubicin resistance is tied to regulation of its
target, topoisomer ase IIα. Many of the compounds were found either to increase cellular topoisomerase IIα levels or to induce S phase arrest. The latter could affect topoisomerase IIα transcription. The indoxins were especially interesting because they upregulated topoisomerase IIα and caused S phase arrest, increasing the sensitivity of resistant cells to these compounds. Photoaffinity pull-down experiments revealed that the molecular target of the indoxins is a nuclear actin-related protein complex involving myosin 1c and ARP2, proteins that are known to be involved in transcriptional regulation. The mechanisms of action of the compounds identified in this screen shed light on pathways of doxorubicin resistance in cancer, and this knowledge could lead to improved treatment strategies for drug-resistant cancers. EG Image courtesy of B. Stockwell
Some anticancer agents work by inducing cancer cells to undergo apoptosis or commit suicide. Cancer cells unfortunately are notorious for acquiring resistance to apoptosis-inducing drugs, including the DNA topoisomer ase II-based DNA-damaging agent doxorubicin. Smukste et al. (Cancer Cell 2006, 9, 133–146) have developed an assay to identify small molecules that boost the toxicity of doxorubicin in resistant cancer cells. Human papillomavirus (HPV), the major causative factor in cervical cancer, produces an oncogenic protein called E6. This protein increases the resistance of cancerous cells to anticancer drugs through various mechanisms. E6 forms a complex with E6 Associated Protein, an E3 ligase, and induces p53 ubiquitination and degradation. This allows tumor cells to become resistant to apoptosis. E6 also causes the degradation of a proapoptotic protein BAK and activates telomerase, which extends the life of cancer cells. To identify small molecules that overcome
Reforming Bubbles How do cells know where to end transcription of a gene, and what do they do when the transcription process stalls? Transcription termination is the answer, and E. coli have evolved a number of different mechanisms to accomplish this task. Termination can be accomplished by an intrinsic mechanism relying on the formation of a hairpin structure within the RNA molecule. Alternatively termination can rely on an enzymatic process involving either the Rho terminator factor or the Mfd protein, which is important for removing stalled polymerases. Parks and Roberts (Proc. Natl. Acad. Sci U.S.A.; doi:10.1073/pnas.0600145103) have explored the similarities between these termination mechanisms and have found
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there are common elements in each of them, all involving the transcription bubble. The transcription bubble is the region of unwound DNA in which RNA polymerase elongates the new RNA molecule. Termination and release of the RNA involve the rewinding of the DNA duplex within this region and the unwinding of the DNA/RNA hybrid. Intrinsic RNA termination requires the formation of a hairpin structure that disrupts the RNA/DNA hybrid. This disruption allows the DNA transcription bubble to be partially reformed, favoring dissociation of the RNA/DNA hybrid. Parks and Roberts examined whether Rho-mediated or Mfd‑mediated
termination also involved the rewinding of the transcription bubble. They show that transcription bubbles prevented from reannealing by incorporation of mismatches within the bubble region are less efficient at releasing transcripts in both Rho-dependent and Mfd-dependent termination. They further show that both Rho and Mfd translocate the transcription bubble downstream, effectively moving the transcription bubble and further favoring dissociation of the RNA from the DNA template. These results indicate that all known mechanisms of termination involves the remodeling of the transcription bubble, and yield a clearer picture of the mechanism of prokaryotic transcription termination. ST w w w. a c s c h e m i ca l biology.org
Spotlight Capturing the Capsid Many viruses, including hepatitis B virus (HBV), West Nile
ated a fluorescent capsid protein, termed C150BO. This
virus, and HIV, have macromolecular protein cores, or capsids,
modified protein was highly fluorescent prior to assembly,
that contain the viral genome. Assembly of the core is an
but fluorescence dropped markedly upon capsid assembly
integral part of the viral life cycle, has no counterpart in the infected cell, and may be particularly sensitive to small changes in local conformation, making it an attractive target for antiviral therapy. However, capsid assembly has not been extensively pursued as a drug target, in part due to difficulty in adapting relevant assays for high throughput screening.
due to fluorescence quenching. Two small molecules known to disrupt capsid assembly, urea and a heteroaryldihydro pyrimidine (HAP-1), were used to determine whether perturbation of capsid assembly could be detected using C150BO. When HAP‑1, which is known to accelerate capsid assembly
Stray et al. (Nat. Biotechnol. 2006, 24, 358–362) has devel-
and nucleate the formation of irregular particles, was added
oped an in vitro assay for HBV capsid assembly, facilitating
to the C150BO assembly reactions, a decrease in fluorescence
the search for small molecule inhibitors. This assay can be
was observed. This result suggests that HAP-1 increased
readily adapted to other virus assembly systems (such as
the rate and extent of C150BO assembly as it does with
avian influenza).
wild-type capsid protein. Unlike HAP-1, urea inhibits capsid
In HBV, the icosahedral core consists of 240 copies of capsid protein that enclose the viral DNA and reverse transcriptase. In vitro, assembly of the capsid protein into a corelike particle is dependent on many factors, such as protein concentration, pH, and ionic conditions, and is nucleated by a trimer of protein dimers. This assembly process results
assembly. When it was added to C150BO assembly reactions, an increase in fluorescence was observed, confirming the decrease in assembly. These results indicate the C150BO assay can identify small molecule inhibitors of virus assembly. Furthermore, this assay is amenable to a high throughput
in close proximity of the C termini of the capsid proteins,
format, providing a novel method for discovering small
suggesting that fluorescence quenching could be employed
molecule antiviral agents that misdirect capsid formation to
to monitor assembly progression. The researchers gener-
yield aberrant and noninfectious virus particles. EG
Image courtesy of A. Zlotnick
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Spotlight Misfortunes of Misfolding to explore the effects
such as Huntington’s, Alzheimer’s, Parkin-
of the presence of an
son’s, and amyotrophic lateral sclerosis
aggregation-prone pro-
(Lou Gehrig’s disease), are associated
tein on cell homeostasis.
with the misfolding of proteins. In some
Temperature-sensitive
of these disorders, protein misfolding
mutant proteins are dependent on the
and aggregation are caused by expan-
folding environment, so the extent of
that normally the presence of misfolded
sion of polyglutamine (polyQ) residues
aggregation versus folded protein in the
protein activates a stress response
in specific proteins, but the link between
ts mutant worms serves as an indicator of
that increases protein refolding. The
aggregation and disease pathogenesis
the state of the protein-folding quality-
researchers hypothesize that the chronic
is not clearly defined. Gidalevitz et al.
control system. C. elegans strains contain-
presence of aggregation-prone proteins,
(Science, 2006, 311, 1471–1474.) have
ing either nonaggregating or aggrega-
while insufficient to activate the stress
uncovered a connection between polyQ
tion-prone polyQ proteins were crossed
response, initiates a positive feedback
Reprinted with permission from AAAS.
Several neurodegenerative disorders,
that expression of the aggregation-prone polyQ protein generally interferes with the protein-folding capacity of the cell.
The results are surprising considering
with several structurally
mechanism that enhances disruption of
malfunction that helps
and functionally unrelated
protein folding. Over time, the gradual
explain the role of
ts mutants. The authors
accumulation of misfolded and damaged
observed that the presence
proteins compromises cellular function
of an aggregation-prone
and results in a disease state. This work
polyQ protein, but not the
puts forward a general mechanism for the
nonaggregating protein,
catastrophic effects of protein misfolding,
Reprinted with permission from AAAS.
expansion and cellular
aberrant protein folding in neurodegenerative conditions. The researchers employed genetic methods using
was sufficient for the worms to exhibit
and further discernment of this process
Caenorhabditis elegans (C. elegans)
the mutant phenotype at the permissive
could lead to new therapeutic strategies
temperature-sensitive (ts) mutations
condition. These observations indicate
to treat neurodegenerative disorders. EG
Integrating Your RNA Is data coming at you faster than you can catalogue it? You’re not alone but don’t despair. A group of RNA scientists, the RNA Ontology Consortium (ROC) is developing an RNA Ontology (RO) (RNA 2006, published online February 16, 2006, 10.1261/ rna.2343206). According to Wikipedia, ontology has one basic question: “What actually exists?” For the RNA scientists what exist are the kinds, structures, and sequences of RNA, and the events and processes in which they are involved. RNA Ontology is an initiative to create a taxonomy of all this information along with 62
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the methodologies used to examine and interpret it. RO also aims to create software to bring computational tools to the bench scientists and to make possible precise searches for all RNA information. With this aggregate of taxonomies, tools, and common vocabulary, scientists with different backgrounds, experiences, and interests can discuss their favorite RNA using a meaningful, mutually intelligible dialect. The ROC invites those interested in RNA to contribute to the on-going discussion of RNA ontology through their Community Discussion Board (http://roc.bgsu.edu) GM w w w. a c s c h e m i ca l biology.org
Spotlight Distinguishing Cathepsins Cathepsins are cysteine proteases whose expression levels and activity are increased in human and mouse cancer cells. In some tumors, cathepsins are also mislocalized to the cell surface and can be secreted into the extracellular space. There are 11 cathepsin family members but their individual functions in cancer and normal cells are not clear. In a recent study, Gocheva et al. (Genes Dev. 2006, 20, 543–556) used a genetic approach to define the roles of four cathepsin genes in cancer cells. The author generated knockouts of four different cathepsin genes in cancer bearing mice and examined the phenotypes of the mutants. Mutations in cathepsins B and S impaired the formation of tumors and angiogenesis. Removing cathepsin B, L or S resulted in reduced tumor growth. Absence of any one of these three cathepsins impaired tumor invasion. In contrast, removing cathepsin C had no significant effect on the tumor parameters examined. Further analysis showed that cathepsin B, L, and S cleaved E-cadherin, a protein involved in cell adhesion, in vitro, but cathepsin C did not. In mice lacking cathepsin B, L, and S, the levels of E‑cadherin levels are maintained. The authors propose that the cathepsin B, L, and S-mediated tumor invasion is likely due to loss of cell adhesion resulting from the cleavage of E-cadherin. The molecular mechanism by which each cathepsin mediates other aspects of tumorigensis requires additional studies. However, the data from Gocheva et al. may be useful in designing inhibitors that target specific cathepsins and thereby specific stages of cancer progression. EJ
A Life or Death Decision Cellular response to the proinflammatory cytokine tumor necrosis factor (TNF) can result in cell survival or cell death, depending on the circumstances. TNF activates the c‑Jun NH2-terminal kinase (JNK) signaling pathway, but understanding how JNK determines the cell’s destiny requires deciphering its apparent dual role in mediating both life and death. TNF-induced activation of Image courtesy of R. Davis JNK is characterized by an early, robust but transient phase followed by a late, low-level but sustained period. Ventura et al. (Mol. Cell 2006, 21, 701–710) have used chemical genetics to investigate how the time course of JNK activity affects the fate of the cell. The redundancy of kinases in the cell has hampered the ability to find specific small molecule inhibitors to probe kinase function, and JNK is no exception. To circumvent this problem, the researchers created mutant JNK (m-JNK) containing an enlarged ATP binding pocket and an inhibitor, 1‑naphthylmethyl-4-amino-1-tert-butyl-3-(p-methyl phenyl)pyrazolo[3,4‑d]pyrimidine (1NM‑PP1), that binds specifically to this pocket but not to wild-type JNK or other kinases. The researchers also created murine embryonic fibroblasts (MEFs) that express wild-type JNK, no JNK, or m-JNK to facilitate interpretation of the specific function of JNK in the cell. The temporal role of JNK in apoptosis and survival pathways was investigated using these unique biological tools. The authors found that JNK activity during the late phase of JNK signaling mediates proapoptotic signaling. In contrast, it was observed that the early phase of TNF-induced JNK activation is critical for signaling survival. These data explain the apparent paradoxical roles of JNK in the cell and indicate that a cell’s fate is critically dependent on the temporal regulation of this kinase. The details of how JNK controls each signaling process will require additional mechanistic studies. EG
Upcoming Conferences Channels, Receptors and Synapses Cold Spring Harbor Meeting April 18–21, 2006 Cold Spring Harbor Laboratory, NY
First Annual Meeting Japanese Society for Chemical Biology May 8–9, 2006 Tokyo, Japan
71st symposium : Regulatory RNAs May 31–June 5, 2006 Cold Spring Harbor Laboratory, NY
Molecular Chaperones and the Heat Shock Response May 3–7, 2006 Cold Spring Harbor Laboratory, NY
Yale Chemical Biology Symposium May 12, 2006 New Haven, CT
RNA 2006 Annual Meeting of the RNA Society June 20–25, 2006 Seattle, WA
Spotlights written by Eva Gordon, Evelyn Jabri, Grace Miller, Sarah Tegen, and Jason Underwood.
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