Spotlight

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Chromosome Congress During each round of cell division, chromosomes are replicated and remain paired as sister chromatids until later in cell division. The mitotic spindle, a bipolar apparatus in the dividing cell, is responsible for capturing the chromosomes, separating the chromatids, and distributing them into the daughter cells. In preparation for segregation, the chromatids connect to the spindle microtubule bundles (green signal), called kinetochore fibers (K-fibers) that emanate from each spindle at either end of the dividing cell. The captured chromosomes (red signal) move (congress) to the metaphase plate prior to the separation of sister chromatids. If one chromosome is attached through one chormatid to a single K-fiber at one end of the cell, how does the sister chromatid find its K-fiber emanating from the opposite pole? Now Kapoor et al. (Science 2006, 311, 388–391) use a combination of microscopy, chemical biology, and RNA interference (RNAi) to examine chromosome congression.

They find that chromosomes attached to a K-fiber at one spindle pole slide along neighboring fibers toward the center of the cell. This process brings the unattached sister chromatid in range with a K-fiber

from the opposite pole. Using chemical inhibitors that slow mitotic progression, the authors show that this sliding process occurs in ~85% of choromosomes. Combining chemical biology with RNAi methods, the authors determined that CENP-E, a kinesin-7 family microtubule motor, is likely responsible for movement of the unattached chormatid along K-fibers. These data explain why chromosome congression is a cooperative process. As more chromosomes become attached at both spindle

poles, additional K-fiber tracks are generated for the movement of other chromosomes. These results also show that a combination of techniques is required to mechanistically dissect complex processes such as mitosis. EJ

Cannabinoids… Legally Cannabinoids, such as those found in

the Cannabis plant, are known to affect cellular function. Endocannabinoids and their

receptors are found in neurons, the messenger cells of the central nervous system (CNS), and in microglial cells, the immune cells of the CNS. In microglia, endocannabinoids are believed to protect neurons from inflammatory damage, but the mechanism is not well understood. Elijaschewitsch et al. (Neuron 2006, 49, 67–79) now elucidate a defined pathway through which the endocannabinoid anandamide (AEA) limits neuronal damage after CNS injury.

As part of their role in immune surveillance, microglia use the mitogen-activated protein kinase (MAPK) pathway to produce inflammatory signals. In healthy brain tissue, AEA release results in induction of the MAPK pathway. In contrast, after primary injury, accumulation of AEA in brain tissue results in protection of neurons from inflammatory damage. To investigate the mechanism of protection, the effects of AEA release on the MAPK pathway in activated microglia were examined. It was discovered that AEA release turns off the MAPK pathway, as evidenced by a decrease in nitric oxide production and a reduction in phosphorylation of extracellular signal-regulated kinase-1/2 (ERK-1/2) and ERK kinase. Furthermore, the authors see an induction of the dephosphorylating enzymes mitogen-activated protein kinase-phosphatases-1 and -2 (MKP-1 and MKP-2) as a direct result of phosphorylation of Histone H3 on the mkp-1 gene, providing further insight into the mechanism of the protective effect of AEA.

The results suggest that AEA acts as a gatekeeper for signal transduction in microglial cells, imposing a negative feedback loop to control the inflammatory response and neurodegenerative immune reactions after primary brain damage. As the clinical use of cannabinoids or related compounds (including those found in the Cannabis plant) for treatment of CNS inflammation and multiple sclerosis are under consideration, these findings represent compelling evidence that the cannabinoid system is a valid target for therapeutic intervention in neuroinflammatory and neurodegenerative disorders. EG

Published online February 17, 2006 • 10.1021/cb0600055

SpotlightThe β2 integrins are cell surface proteins that mediate leukocyte recruitment during the inflammatory response. β2 integrins are potential drug targets for inflammatory and immune disorders, but their structural and functional complexity has hindered the discovery of effective small molecule inhibitors. Björklund et al. (Biochemistry 2006, published online 09 February 2006; 10.1021/bi052238b) have developed a novel high throughput screen for the β2 integrins and have discovered a new class of small molecule inhibitors.

Exploiting the sensitivity and efficiency of phage display technology, the researchers developed a competition assay to identify small molecules that displace phage displaying a known 18-mer peptide, abbreviated DDGW, from the binding domain, or I domain, of the αmβ2 integrin. The assay design overcomes the limitations of comparable assays that may lack sensitivity, require labeling reagents that can affect peptide activity, or have undesired avidity effects. The screen yielded several compounds containing a 2-thioxothiazolidin-4-one substructure as specific inhibitors of the interaction between the

DDGW-phage and the I domain. IMB-10 was identified as the most active compound, and additional experiments revealed that this compound stabilized binding of the I domain to its endogenous ligands, proMMP-9

and fibrinogen. Moreover, molecular modeling and other studies using mutant I domains indicated that IMB-10 shifts the equilibrium of the I domain structure toward the active conformation. Remarkably, leukemia cell migration in vitro and leukocyte recruitment in vivo were both potently and selectively inhibited by IMB-10, suggesting that stabilization of integrin ligand binding is a viable approach for development of anti-inflammatory agents.

The novel mechanism through which IMB-10 asserts its effects could have advantages over integrin inhibi-tors that prevent ligand binding. Targeting activated integrins could confer selectivity toward cells already triggered by an inflammatory signal, and upregulation of other integrins to compensate for loss of function is not a concern. These inhibitors could lead to exciting new drugs for immune and inflammatory diseases. EG

An abundance of ATP-dependent enzymes catalyze DNA and RNA rearrangements in the cell. These motors and remodelers play critical roles at all stages of gene replication and expression. The mechanisms coupling ATP hydrolysis with nucleic acid acrobatics have been of interest for decades, yet appropriate assays to view these enzymes in action have remained a barrier. Now, two such enzymes, a bacterial DNA gyrase and a viral RNA helicase, have been tracked at the single molecule level in unique high

resolution assays. In both cases, the experimental design allows direct observation of a nucleic acid substrate during the enzyme’s catalytic cycle.

DNA gyrase introduces negative supercoils into DNA. These supercoils are essential because they compact the bacterial chromosome and promote any reaction that involves an untwisting of DNA. There are two basic movements DNA is capable of, bending and twisting. Bending has been studied for a long time, but twisting is more challeng-

ing to examine. To observe this activity, Gore et al. (Nature 2006, 439, 100–104) engi-neered molecular tweezers to pull a DNA substrate containing a magnetic bead taut by a magnet. The center of the DNA helix contained a nick to facili-tate rotation and a fluorescent bead to visualize rotation. When DNA wraps around the enzyme to form a complex poised for catalysis, approxi-mately one rotation of the DNA is observed. With ATP present, a catalytic cycle causes two rotations of the DNA. This setup

allowed the measurement of a variable rarely accessible to enzymologists, tension. As the DNA tension was increased in tiny increments, both the gyrase initiation rate and the ability to catalyze multiple rotations without dissociation dropped markedly. Interest-ingly, as DNA tension was increased, the velocity of gyrase catalysis was unchanged. Using this assay, the authors detect two pauses correspond-ing to two kinetic steps in the gyrase cycle and show that the

New Diagnostics for Tiny Motors

Neutralizing Neutrophils

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Untangling Webs Efforts to investigate and treat viral infections have increased our understanding of interactions between viral and host proteins. However, interactions among intraviral proteins, including those in the herpesvirus family, have not yet been well-characterized. Uetz et al. (Science 2006, 311, 239–242) have now generated genome-wide intraviral protein interaction maps for Kaposi’s sarcoma-associated herpesvirus (KSHV), linked to Kaposi’s sarcoma and B-cell lymphomas, and varicella-zoster virus (VSV), a herpesvirus associated with chickenpox and shingles. These data provide insights into the properties of herpesviruses and their interaction with the human protein interactome.

Using yeast two-hybrid technology, the researchers identified 123 and 173 intraviral protein pairs in KSHV and VSV, respectively. They observed that, in contrast to cellular networks which typically exhibit the properties of scale-free networks, where most proteins have few interaction partners but a few have many, viral protein networks emerge as single, highly coupled modules. Further examination of the KSHV interactome coupled with data available from orthologous proteins in other herpesviruses allowed the researchers to predict 114

orthologous intraviral protein interactions in four different herpesviruses.

The researchers connected the KSHV interactome with a prototypical human protein interaction network using 20 predicted interactions between 8 KSHV and 20 human proteins. Notably, when the viral and human interactomes were docked together, the topology of the KSHV network changed from a highly coupled module to a scale-free network of interacting submodules, indicating that the combined virus–host network takes on host network properties. The researchers hypothesize that although the viral and human interactomes have distinct network topologies in their isolated states infection may result in the emergence of new system properties that embody specific features of viral pathogenesis. Further insight into these interactions will enhance understanding of viral mechanisms and may lead to new strategies for treatment of viral infections. EG

rate-limiting step of the super-coiling reaction is at the end of the cycle.

Optical tweezers were used to watch a helicase unwind a duplex RNA at high resolu-tion. Dumont et al. (Nature 2006, 439, 105–108) mounted a hairpin of double-stranded RNA between beads such that unwinding of the RNA increases the bead-to-bead distance. In this case, the test subject was NS3, an ATP-dependent 3 ́to 5 ́helicase critical for hepatitis C viral replication.

Upon addition of NS3 and saturating ATP, the RNA duplex was unwound in bursts and pauses like an inchworm rather than a steady train along RNA tracks. An average of 11 base pairs were unwound by NS3 before a pause. By varying ATP, the authors show that exit from a pause is a two-step kinetic mechanism and, as with gyrase, only one step requires ATP binding. At low ATP concentra-tions, “substeps” separated by subpauses appeared, and these indicated that unwinding

of 11 base pairs occurs in three distinct events. The authors propose a dual-function model for NS3 helicase. In the model, a “translocator” activity moves in 11 base pair steps and monitors double-stranded RNA directly ahead of the unwinding activity. The second activity or “helix opener” moves in 3–5 base pair steps to disrupt the helix. The data indicate that ATP is critical for coordinating both of these inchworming functions.

With both gyrase and NS3, changing the force on the

nucleic acid substrate alters some aspects of enzyme activity. In a cell, the force on a DNA or RNA is influenced by proteins which bind, compact or copy the strands. These regu-latory contributions will be an interesting direction for future research now that the study of such ATP-dependent molecular machines has such a high resolution. These assays also enable careful mutant studies and dissection of the mysteri-ous role of ATP in such cellular processes. JU

New Diagnostics for Tiny Motors, continued

Spotlight

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Spotlight

HUPO-USAHuman Proteome OrganizationMarch 12–15, 2006Boston, MA

ACS Spring National MeetingAmerican Chemical SocietyMarch 26–30, 2006 Atlanta, GA

ASBMB Annual Meeting and Centennial CelebrationAmer. Soc. Biochem.

Mol. BiologyApril 1–5, 2006San Francisco, CA

97th Annual MeetingAmerican Association for

Cancer ResearchApril 1–5, 2006Washington, DC

Looking at RNA InteractionsRNA binding proteins (RBPs)

are essential components

of many cellular machines,

including those that control

pre-messenger RNA (mRNA)

splicing, mRNA editing,

mRNA transport, translation

regulation, and RNA

degradation. Understanding how and when these

proteins interact with their target RNA is crucial

to studying gene regulation. Few methodologies

exist for identifying all of the RBPs that bind to

one mRNA of interest. Zielinski et al. (PNAS 2006,

103, 1557–­1562) have now developed a peptide–­

nucleic acid (PNA)-assisted technique to identify

proteins that interact with a specific RNA in vivo.

The authors coupled a PNA, a nucleic acid

analog in which the sugar-phosphate backbone

is replaced with a polyamide backbone, to a

cell-penetrating peptide called transportin 10

(TP10), and a photoactivatable amino acid adduct

p-benzoylphenylalanine (Bpa). The authors chose

to apply their new PNA-assisted identification

tool to find RBPs that bind to

ankylosis RNA, a dendrically

localized mRNA coding for a

pyrophosphate transporter.

After transport into the

neuronal cells, the covalent

bond between TP10 and the

PNA was reduced and the

PNA released to hybridize

with the target mRNA (red

signal). UV irradiation of the cells activated Bpa

and generated a free phenylalanine radical

able to crosslink the nearest protein. These

PNA-mRNA-protein complexes were isolated and

the RBPs identified using mass spectrometry.

The authors also examined the effect of external

stimuli on these complexes and found that RNA-

protein complexes (yellow signal) are remodeled

in response to a physiological change, a result

consistent with the current thought that RNA-

protein interactions are dynamic. This new in vivo

methodology allows scientists to quantify these

changes in RNA-protein interactions which in turn

will expand our understanding of RBP-mediated

gene regulation. EJ

UPCOMING CONfERENCES

Spotlights written by Eva Gordon, Evelyn Jabri, and Jason Underwood.

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