NSAID gels and COX-2 selectivity in topical pain killers

Research from Bing Xu’s lab, published in November in Journal of the American Chemical Society, has recently been featured in C&E News. The Xu lab researchers, including Chemistry grad students Jiayang Li, Yi Kuang, Yuan Gao, Xuewen Du and Junfeng Shi, synthesized hydrogels by synthetically coupling small D-amino acid peptides to naproxen (a non-steroidal antiinflammatory drug – NSAID). This was done with the idea of forming gels that can be used for topical pain treatment.

Studies on the compounds formed showed that not only are gels formed, but the D-peptide conjugates of naproxen showed better selectivity towards COX-2 (the therapeutic target) compared to COX-1 (a source of side effects) than naproxen alone or an L-peptide conjugate. Clinical applications are still far away, but this finding opens exciting new avenues for research.

Li J, Kuang Y, Gao Y, Du X, Shi J, Xu B. d-Amino Acids Boost the Selectivity and Confer Supramolecular Hydrogels of a Nonsteroidal Anti-Inflammatory Drug (NSAID). J Am Chem Soc. 2012.

Damaged DNA and self-eating (autophagy) in budding yeast.

Chromosome double-strand breaks (DSBs) threaten the integrity of the genome. Cells respond to DSBs by activating the DNA damage checkpoint that blocks cells prior to mitosis, allowing more time for the repair of damaged DNA. When the DSB can be repaired, the cell cycle checkpoint is turned off so that cells can resume cell cycle progression, a process termed recovery. If the DSB remains unrepaired, G2/M arrest persists for a long time but eventually cells adapt and – despite the persistent DNA damage – complete mitosis and divide. Much of our understanding of the DNA damage response has come from the study of the budding yeast Saccharomyces cerevisiae, where it is possible to create DSB damage synchronously in all cells of the population. This can be accomplished either by uncapping telomeres, exposing their normally protected ends or by creating a single, defined DSB by inducing the site-specific HO endonuclease. From such studies, it was possible to identify a highly evolutionarily conserved DNA damage sensing and signaling cascade that is initiated by Mec1, the yeast homolog of mammalian ATR protein kinase (reviewed in Ref. (1)). Yeast genetic approaches revealed a number of adaptation-defective mutants, a subset of which also are recovery-defective. Previous studies also demonstrated that triggering the DNA damage checkpoint affects not only mitosis and the efficiency of DNA repair within the nucleus; it also affects cytoplasmic responses (2, 3). In a new paper from the Haber lab published in PNAS, we uncovered mutations in the Golgi-Associated Retrograde Protein (GARP) complex that are adaptation-defective. We show that the defect in these mutants can be mimicked by activating the cytoplasm-to-vacuole (CVT) pathway of autophagy that prevents the nuclear accumulation of separase, Esp1, in the nucleus, thus preventing the cells both adapting and recovering from DSB damage.

In budding yeast, a single unrepaired double-strand break (DSB) triggers the Mec1-dependent cell cycle arrest prior to anaphase for 12-15 before they adapt. Adaptation is accompanied by the loss of hyperphosphorylation of Rad53, yeast’s Chk2 homolog.  Rad53 remains phosphorylated in a number of adaptation-defective mutations, including deletion of the two PP2C phosphatases, ptc2ptc3D, that normally dephosphorylate Rad53.  Adaptation is also blocked by ablating a number of proteins with diverse roles in DSB repair, including srs2D, rdh54D as well as by a mutation in yeast’s polo kinase cdc5-ad.

In our paper, we find that hyperactivation of the cytoplasm-to-vacuole (CVT) autophagy pathway causes the permanent G2/M arrest of cells with a single DSB that is reflected in the nuclear exclusion of both separase, Esp1, and its chaperone/inhibitor, securin, Pds1(See figure).  Autophagy in response to DNA damage can be induced in three different ways: (1) by deleting members of the Golgi-Associated Retrograde Protein complex (GARP) such as vps51D; (2) by adding rapamycin; or (3) by overexpressing a dominant-negative ATG13-8SA mutation.  The permanent checkpoint-mediated arrest in any of these three conditions can be overcome in three ways: (1) by blocking autophagy with mutations such as atg1D, atg5D or atg11D; (2) by deleting the vacuolar protease Prb1 or its activator, Pep4; or (3) by driving Esp1 into the nucleus with a SV40 nuclear localization signal.  In contrast, these same alterations fail to suppress the adaptation defects of ptc2ptc3D or cdc5-ad.  Transient accumulation of Pds1 in the vaucole is also seen in wild type cells lacking PEP4 after induction of a DSB.  Unlike other adaptation-defective mutations, G2/M arrest persists even as the DNA damage-dependent phosphorylation of Rad53 diminishes, suggesting that cells have become unable to activate separase to initiate anaphase after DNA damage.  In addition, we have found that cells fail to recover when VPS51 is deleted or when ATG13-8SA is overexpressed.

Increased autophagy causes the delocalization of both Pds1 (securin) and Esp1 (separase) from the nucleus in checkpoint-arrested budding yeast cells. A. GFP-tagged Pds1 and Esp1 localize to the nucleus at the neck of G2/M-arrested wild type (WT) cells that have suffered a single unrepaired chromosome double-strand break (DSB). Both rdh54Δ and vps51Δ prevent cells from adapting and resuming cell cycle progression, but only ablating Vps51 – part of the Golgi-associated retrograde protein (GARP) complex – causes the mislocalization of Pds1 and Esp1 and the partial degradation of Pds1 by vacuolar proteases. Preventing degradation of Pds1 (and possibly other mitotic regulators) results in the suppression of permanent arrest and the relocalization of sufficient Esp1 into the nucleus to release cells from their pre-anaphase arrest. A similar suppression of arrest in vps51Δ cells is obtained by disabling autophagy (not shown). B. Induction of autophagy by overexpression of ATG13-8SA (6) prevents adaptation in wild type cells. Expression of ATG13-SA was induced at the same time that a single, unrepairable DSB was created. Whereas normal cells adapt by 24 h, increased autophagy prevents cells from progressing beyond the G2/M stage of the cell cycle. Deletion of the PEP4 gene that activates vacuolar proteases or ATG1 that is required for autophagy suppresses the arrest and allows cells to divide and resume cell cycle progression.

Taken together with other recent results (4, 5), these observations emphasize that the DNA damage response can trigger the mislocalisation and cytoplasmic proteolysis of important nuclear proteins that regulate DNA repair and cell cycle progression. These results broaden our perspective on the ways in which cells respond to DNA damage and delay cell cycle progression while such damage persists.

Ex MCB grad Farokh Dotiwala, current MCB grad Vinay Eapen and ex-postdoc Jake Harrison were the co-first authors on this paper. Assistant professor Satoshi Yoshida also contributed significantly to this project.

Dotiwala F(*), Eapen VV(*), Harrison JC(*), Arbel-Eden A, Ranade V, Yoshida S & Haber JE (2012) DNA damage checkpoint triggers autophagy to regulate the initiation of anaphase, PNAS (Published online before print November 19, 2012, doi: 10.1073/pnas.1218065109)

1.         Harrison JC & Haber JE (2006) Surviving the breakup: the DNA damage checkpoint. Annu Rev Genet 40:209-235.
2.         Dotiwala F, Haase J, Arbel-Eden A, Bloom K, & Haber JE (2007) The yeast DNA damage checkpoint proteins control a cytoplasmic response to DNA damage. Proc Natl Acad Sci U S A 104(27):11358-11363.
3.         Smolka MB, et al. (2006) An FHA domain-mediated protein interaction network of Rad53 reveals its role in polarized cell growth. J Cell Biol 175(5):743-753.
4.         Robert T, et al. (2011) HDACs link the DNA damage response, processing of double-strand breaks and autophagy. Nature 471(7336):74-79.
5.         Dyavaiah M, Rooney JP, Chittur SV, Lin Q, & Begley TJ (2011) Autophagy-dependent regulation of the DNA damage response protein ribonucleotide reductase 1. Mol Cancer Res 9(4):462-475.
6.         Kamada Y (2010) Prime-numbered Atg proteins act at the primary step in autophagy: unphosphorylatable Atg13 can induce autophagy without TOR inactivation. Autophagy 6(3):415-416.

Materials Science poster session

The NSF funded Materials Research Science & Engineering Center (MRSEC) received its 5 year review on Oct 11-12, 2012 when a panel of 5 scientists and 2 NSF officials visited Brandeis and kicked the tires of our Center. The highlight of the review was lunch between the panelists and 20 MRSEC graduates students and postdocs and the poster session, shown here, in which 30 posters describing research in the Center was presented to the panel. The four MRSEC thrusts were represented in the poster session: Active Matter, Chiral Self-Assembly, Oscillating Chemical Dynamics, and Confined Polymers, plus posters on our Seeds and Facilities. Join us again in Spring for our on-campus retreat.
 
Cincopa WordPress plugin

Math graduate student training grant renewed

Mathematics Ph.D. students and faculty at Brandeis should be happy to learn that the department’s training grant from the US Dept. of Education’s Graduate Assistance in Areas of National Need (GAANN) program is being renewed for another three years. Training grants are a vital piece of the puzzle for supporting graduate education in the sciences, allowing Ph.D. students to focus on research.

 

Writing on the ceiling

BrandeisNOW answers the question Who’s been writing on the Physics ceiling?

How does a hard-wired simple circuit generate multiple behaviors?

In a paper appearing in last week’s issue of Neuron, members of the Sengupta Lab and their collaborators from the Bargmann Lab describe how a fixed neural circuit produces multiple behaviors in a context-dependent manner.  The study was led by former Brandeis post-doctoral fellow Kyuhyung Kim in the Sengupta Lab (currently Assistant Professor at DGIST, Korea) and Rockefeller student Heeun Jang in the Bargmann Lab. Also involved in the study were current Brandeis MCB students Scott Neal and Danna Zeiger, and Dongshin Kim, the head of the Brandeis Microfluidics Facility.

For this study the researchers used the nematode Caenorhabditis elegans. The nervous system of C. elegans consists of only 302 neurons (in the adult hermaphrodite) whose anatomical connectivities are well-mapped. Despite its relatively small nervous system, C. elegans exhibits a wide range of behaviors in response to environmental stimuli. For instance, C. elegans exhibits varied responses to pheromones – small chemical substances used for intra-specific communication. Some pheromones are repulsive to adult hermaphrodite C. elegans but neutral to male C. elegans. However, reducing the function of the neuropeptide Y-like receptor NPR-1 results in hermaphrodites now exhibiting neutral pheromone responses and males becoming strongly attracted. The researchers asked how the sex and neuromodulatory state of the animal allows it to interpret the pheromone stimulus differently to generate distinct behavioral responses.

To answer this question, the researchers used behavioral assays, genetic manipulations of neuronal output, and in vivo measurements of pheromone-induced neuronal activity (using genetically encoded calcium sensors and customized microfluidics devices designed by the Brandeis Microfluidics Facility). They found that flexible output of a neuronal ‘hub-and-spoke’ circuit motif was responsible for generating these distinct pheromone responses under different conditions.

In this circuit, pheromone-sensing neurons ASK and ADL are connected to the central RMG motor/interneuron by gap junctions (see Figure). Jang et al. showed that in hermaphrodites with high levels of NPR-1 activity, the ADL sensory neurons respond strongly to a specific pheromone component and drive avoidance behavior via their chemical synapses to command interneurons for locomotion. However, sexual dimorphism in the circuit results in males having reduced ADL pheromone responses.  Moreover, Jang et al. showed that ADL synaptic output in males is further decreased via RMG and ASK-mediated antagonism (see Figure). As a result, males are indifferent to this pheromone.

The next issue the authors addressed is the role of NPR-1 activity in regulating pheromone responses. The Bargmann Lab had previously shown that high NPR-1 activity inhibits RMG, and under these conditions, pheromone responses of the ASK sensory neurons are low. Conversely, when NPR-1 activity is reduced or absent, ASK pheromone responses are enhanced. Jang et al. found that in the absence of NPR-1 activity, ADL chemical synaptic output in response to pheromones is antagonized by the RMG-ASK gap junction circuit. In other words, avoidance mediated by ADL chemical synaptic output is balanced by attraction mediated by the RMG-ASK gap junction circuit, resulting in hermaphrodites being neither attracted to nor avoiding this pheromone. In males with reduced NPR-1 activity the same effects are observed, however, since the ADL pheromone response is already lower in males, the RMG-ASK attraction-mediating arm “wins” resulting in attraction to pheromones.  The authors refer to these as overlapping ‘push-pull’ circuits in analogy with electronic circuits.

These results begin to explain how a small fixed circuit can generate a remarkable range of behaviors via alteration of sensory response properties as well as choice of specific synaptic output pathway as a function of neuromodulatory state and sex. The general theme of a circuit functioning differently under different neuromodulatory conditions has been extensively studied in the Marder Lab in the crustacean nervous system, and is an important principle to be kept in mind when interpreting functionality from structurally described connectomes.

Jang H(*), Kim K(*), Neal SJ, Macosko E, Kim D, Butcher RA, Zeiger DM, Bargmann CI, Sengupta P. Neuromodulatory State and Sex Specify Alternative Behaviors through Antagonistic Synaptic Pathways in C. elegans. Neuron. 2012;75(4):585-92.

Protected by Akismet
Blog with WordPress

Welcome Guest | Login (Brandeis Members Only)