Molecular and Cellular Pharmacology Student Seminars 2006-2007

Ka Young Chung
Walker Lab
Monday, September 18, 2006

Endothelin-1 (ET-1) regulates contractility and growth of the mammalian heart by binding ETA and ETB G-protein coupled receptors (GPCRs). 

To establish which growth signaling pathways are associated with ET-1 receptors in adult myocardium, a proteomic analysis was performed.  Classical signaling proteins thought to function downstream of ETA such asGaq, PLC-b1, PKC-e and PKC-d were identified in co-immunoprecipitates of ETA by mass spectrometry.  Also prominent were the growth factor receptor tyrosine kinases erbB2 and erbB4, as well as their downstream growth signaling effectors PI3kinase, Akt, MEK and Erk.  Another group of proteins in ETA immunoprecipitates was scaffolding proteins such as Shank3 and SAP97.

Western blot analysis confirmed co-immunoprecipitation of erbB2/4, PI3kinase, and Akt with ETA, and confocal microscopy revealed co-localization of each with ETA in cardiac transverse tubules (T-tubules).  Treatment of isolated ventricular myocytes with the erbB4 receptor ligand neuregulin 1b (NRG 1b) promoted erbB2/4 tryosine phosphorylation and Akt serine phosphorylation, whereas treatment with ET-1 did not.  This observation argues against ET-1 growth signaling occurring via erbB2/4 transactivation in adult myocardium.  ETA did however undergo cross-talk with this pathway as ET-1 pretreatment greatly reduced tyrosine phosphorylation of erbB2/4, serine phosphorylation of Akt, and negative inotropy mediated by NRG 1b.  This inhibitory cross-communication between different receptor classes in adult myocardium is distinct from the transactivation phenomenon observed in neonatal myocytes and cell lines.  The results suggest a new conceptual framework for understanding human cardiomyopathies associated with i) prolonged exposure to ET-1 and ii) therapies targeted at erbB2 inhibition.

Cardiac T-tubules are invaginations of surface membrane occurring in the vicinity of Z-lines, which facilitates the propagation of external signals into the inner cross-section of myocytes.  In pathological conditions such as hypertrophic human heart, the structure of T-tubules appears to be changed.  However, despite the importance of T-tubules in myocyte function, mechanisms of T-tubule development or maintenance and mechanisms underlying how proteins localize in T-tubules are not completely understood.  In the proteomic study of ETA immunoprecipitates, Shank3, a scaffolding protein was identified.  To investigate the role of the scaffolding protein on T-tubule formation, we will characterize Shank3 in cardiac myocytes.  To investigate, the importance and the mechanism of T-tubular localization of ETA, we will express wild type ETA-CFP and PDZ-binding domain mutant ETA-CFP in cardiac myocytes.

Monica Gavala
Bertics Lab
Monday, September 25th, 2006

Association of P2X7 Purinoreceptor (P2X7R) Signaling with Macrophage Activation Status

Inflammation is a localized response to injury or infection and is mediated by factors released from leukocytes present in the inflammatory microenvironment. Usually accompanying infection or inflammation is the release of a high concentration of extracellular nucleotides, supplying a source of ligands for purinergic receptors that are present on the cell surface of many immune cells. One particular nucleotide receptor that is mainly expressed on leukocytes is the P2X7 receptor (P2X7R). Previous work with P2X7R has linked this receptor to the modulation of macrophage and monocyte (macrophage precursor cell) responsiveness to inflammatory stimuli, such as the production of pro-inflammatory cytokines, reactive oxygen species, and prostaglandins. Interestingly, the promoter for many P2X7R-modulated inflammatory genes (i.e. TNF-a, iNOS, COX2) contains consensus sites for cyclic-AMP response element binding protein (CREB) binding. Previous studies, along with my preliminary data, suggest that stimulation of mitogen-activated protein kinase (MAPK) cascades, and the control of transcription via CREB-linked pathways, are potentially involved in the nucleotide regulation of macrophage-induced inflammation.

Although the activated macrophage used to be defined as a cell that secretes pro-inflammatory mediators and kills intracellular pathogens, recent research supports the idea that macrophage activation is a heterogeneous process wherein, depending on the stimuli, different classes of activated macrophages are generated that exhibit diverse immunological functions and physiologies. In this regard, macrophage activation has been proposed to be regulated by select products from activated Type 1 (TH1) or Type 2 (TH2) activated T helper lymphocytes at sites of inflammation to produce M1- or M2-activated macrophages, respectively. M1 macrophage activation involves priming of naïve monocytic cells with interferon-gamma (IFN-_) that is released from TH1 cells in response to invasive bacterial, protozoal and viral infections. Conversely, when the host is invaded by extracellular parasites or helminths, or if the host is asthmatic or atopic, TH2 cells readily release cytokines such as interleukin (IL)-4 which primes macrophages toward M2-activation.

The activity of the P2X7R has been demonstrated to enhance macrophage responsiveness to pro-inflammatory stimuli, which is consistent with M1 macrophage activation. Intriguingly, when the P2X7R activity is attenuated, either through genetic polymorphisms, pharmacological inhibitors, or generated knockouts, hallmarks of M1 macrophage activation are also attenuated (e.g.,  decrease in TNF-_ production) and a more M2 macrophage phenotype develops (e.g.,   enhanced IL-10 release). Thus, it is of interest to know how P2X7R signaling is affected in monocytic cells by cytokines that would be present in different disease states (a.k.a. TH1-mediated versus TH2-mediated microenvironments). Accordingly, I am examining the effect of an M1 activator (i.e., IFN-_) versus an M2 activator (i.e., IL-4) on the activation of select proteins found to be downstream of P2X7R activation using pharmacological and molecular approaches on various macrophage model systems. These studies will provide a better understanding of the signaling capacity of differentially-primed macrophages as well as a better understanding of what impact the P2X7R has in different disease states.

Haichuan Duan
Jefcoate Lab
Monday, October 2, 2006

The steroidogenic acute regulatory protein (StAR) is a key mediator of cholesterol transfer from the outer to the inner mitochondrial membrane, a rate limiting step in the rapid stimulation of steroidogenesis. Two StAR transcripts are expressed in rodent steroidogenic cells, 1.6kb and 3.5kb. They differ only in their 3' untranslated regions (3'UTR). An AU-rich element (AURE), which is the most common mRNA stability regulatory element, is found only in the 3.5kb mRNA. Regulation of mRNA stability provides a means to rapidly changing gene expression levels and is typically seen in genes involved in acute responses. We tested whether StAR expression can be rapidly regulated through the 3.5 kb mRNA. We examined the effects of StAR 3'UTR on transcript stability by using both StAR cDNA vectors and luciferase-StAR 3'UTR chimeric constructs. A direct mRNA transfection method was also employed to measure mRNA half-life. We found that the extended StAR 3'UTR causes decreased expression levels through both enhanced cytoplasmic instability and nuclear degradation. The basal destabilization effect was narrowed down to a 700 base basal instability region (BIR). Prolonged cAMP stimulation decreases the half-life of 3.5 kb message 2 fold. The AURE binding proteins HuR, TIS11b and AUF1 are all abundantly expressed in steroidogenic cells. TIS11b is also rapidly stimulated by cAMP. Cotransfection studies showed that TIS11b selectively destabilizes through AURE of StAR extended 3'UTR. We are now assessing the effect of knocking down HuR and TIS11b on StAR transcript expression. Recently we also found that histone deacetylase (HDAC) inhibitors have opposite effects on cAMP and PMA induction of StAR, which cannot be fully accounted for by promoter constructs. This raises the question of whether post-transcriptional mechanisms are also involved.

Gennifer Mager
Svaren Lab
Monday, October 9, 2006

Active Gene Repression by the Egr2/NAB complex during Peripheral Nerve Myelination

Myelin formation in the peripheral nervous system by Schwann cells requires interaction of the Egr2/Krox20 transcriptional regulator with the NAB family of corepressors. NAB proteins could limit EGR2 activation to maintain myelin target genes at appropriate levels, or they could be involved in active repression of specific genes during myelination. To define the molecular role of NAB corepression, we screened for NAB-regulated genes that decline during the course of peripheral nerve myelination. We identified several genes-including Id2 and Rad,-that are induced in peripheral nerve of Egr2/Krox20 knockout mice. In vivo chromatin immunoprecipitation analysis of myelinating sciatic nerve was used to show developmental association of both Egr2 and NAB2 on the Id2 and Rad promoters as they are being repressed during the myelination process. In addition, recent results have indicated that NAB2 represses transcription by interaction with the chromodomain helicase DNA-binding protein 4 (CHD4) subunit of the NuRD chromatin remodeling complex, and we demonstrate that CHD4 occupies NAB-repressed promoters in a developmentally regulated manner. Furthermore, elevated expression of Id2 decreases endogenous Mpz expression, as well as Egr2-stimulated Mpz expression.  These results illustrate a novel aspect of genetic regulation of peripheral nerve myelination, by showing that Egr2 actively represses genes during myelination in conjunction with NAB corepressors.

Angela Mabb
Miyamoto Lab
Monday, October 16, 2006

The Role of Proteins in NEMO Sumoylation:  Players in the Infield

Nuclear Factor-kappaB (NF-kB) is a transcription factor that regulates a diverse subset of genes such as those involved in immune function, growth control, and regulation of apoptosis.  NF-kB can be activated by a wide variety of stimuli such as tumor necrosis factor alpha (TNFa), interleukin-1 (IL-1), lipopolysacharride (LPS), and DNA damaging agents (ionizing radiation and chemotherapeutic drugs such as camptothecin and etoposide).  Given the evidence of NF-kB's role in chemoresistance, it is critical to define the pathways that contribute to NF-kB activation in hopes of finding novel molecular targets to inhibit NF-kB upon treatment with chemotherapeutics.  The orchestration of events responsible for activation of NF-kB in response to DNA damaging agents requires sequential posttranslational modifications of a regulatory subunit essential for NF-kB activation, known as NEMO.  We are interested in the first known posttranslational modification of NEMO in this pathway, sumoylation.  Here, we demonstrate that the protein PIASy serves as a SUMO ligase for NEMO both in vitro and in vivo.  Furthermore, we investigate another protein that has been implicated in DNA damage induced NEMO sumoylation, RIP, and are currently defining its role in mediating NEMO sumoylation. 

Eosinophils are terminally differentiated innate immune cells that have both advantageous and detrimental functions within the host. It has been reported that eosinophils kill parasites and alert the immune system to presence of a foreign invaders, but eosinophils are also thought to contribute to disordered airway function that is characteristic of asthma. Interleukin-5 (IL-5) strongly affects many aspects of eosinophil biology. Expression of the IL-5 receptor has been detected on eosinophils and basophils only, giving IL-5 functions that are specific to only a few cell types. Interleukin-5-family cytokines have been shown to mediate survival of peripheral blood eosinophils /in vitro/, suggesting that they potentiate the lifespan of airway eosinophils. The potentiation of lifespan thereby contributes to the pathology of the disease through enabling the persistence of eosinophils in the tissues, exacerbating the inflammation already found in the airways. However, the mechanisms by which IL-5-family cytokines mediate eosinophil survival have not been elucidated.

Expression of the survival promoting gene Pim-1 has been observed following IL-5, GM-CSF (Granulocyte Macrophage - Colony Stimulating Factor), and IL-3 stimulation. Our lab has demonstrated that STAT5 phosphorylation and activation occurs following IL-5, GM-CSF, or IL-3 stimulation of peripheral blood eosinophils, and expression of Pim-1 is thought to be STAT5-dependent. We have also demonstrated that Pim-1 is expressed in airway eosinophils following segmental allergen challenge (SBP-Ag). We have been able to attenuate Pim-1 expression in human peripheral blood eosinophils following IL-5 stimulation. Accordingly, the overall hypothesis of these studies is that the eosinophil survival induced by IL-5-family cytokines is mediated through STAT5-dependent expression of Pim-1 and other anti-apoptotic proteins.

Bone morphogenetic protein 1 (BMP1) is an astacin-like metalloproteinase, known to process several extracellular matrix components as well as some proteins involved in TGF-beta-like BMP signaling. Chordin is an extracellular antagonist of TGF- beta-like BMPs.  The antagonism is achieved by the binding of chordin to the BMP, preventing receptor binding.  This antagonism is relieved when either bone morphogenetic 1 (BMP1) or mammalian tolloid-like 1 (TLL1) processes chordin and releases the BMP from its latent complex, allowing the BMP to continue signaling.  Chordin-like 1 and chordin-like 2 are homologous to chordin and have been shown to bind several TGF-beta-like BMPs and function via a similar mechanism to chordin.  These findings led our lab to pursue these proteins as possible substrates for BMP1/TLD-like metalloproteinases.  This question will be investigated using biochemical studies, cellular assays, and determining the effects of these proteins In zebrafish development.  Thus far, no homologue of the chordin-like proteins has been discovered in zebrafish.  It is one of our goals to clone zebrafish chl1 and chl2 using database mining and PCR techniques and characterize their potential roles in zebrafish development.

Dominique Fontanilla
Ruoho Lab
Monday, November 20, 2006

Pharmacological and Functional Characterization of the Sigma-1 Receptor

The sigma receptor represents a ubiquitously expressed unique binding site in the CNS and is a member of the orphan receptor class for which no endogenous ligand is known. It is known, however, that the sigma receptor binds with high affinity to several classes of chemically unrelated ligands such as neurosteroids, neuroleptics, dextrobenzomorphans, and several psychostimulants (e.g. cocaine, methamphetamine, MDMA, methacathinone). Consequently, it is thought that the sigma receptor may mediate the immunosuppressant, antipsychotic, and neuroprotective effects of drugs. Functionally, sigma-1 receptor ligands have been shown to modulate voltage-gated K+ channels independently of G-proteins or kinases (1). They have also been shown to mediate calcium release from intracellular stores (2), regulate compartmentalization of lipids on ER (3), and have antitumour activity in vitro and in vivo (4).The sigma1 receptor has been shown to have three hydrophobic regions, two of which, have highly conserved residues between the sigma receptor and the yeast C8-C7 sterol isomerase (ERG2) (5). These regions have thus been termed steroid binding domains 1 and 2 (SBD1 and SBD2). Previous photoaffinity labeling work in our lab has implicated Asp 188 in SBD2 as a ligand-binding target using a 3-iodo-4-azidococaine photoprobe. Currently, we aim to probe the SBD1 region by synthesizing photoprobes that have their photoreactive group on moieties that we hypothesize are interacting with this domain. Therefore, N-alkyl-N'-aralkyl derivatives, trace amine derivatives, and methcathinone-like compounds, all of which mimic the pharmacophore found in many sigma1 ligands have been synthesized in addition to an N-substituted cocaine derivative.  The binding affinities of these compounds have been characterized by competitive displacement against [3H] (+) pentazocine, which is selective for sigma1. These compounds have also been characterized functionally at the cellular level through whole cell voltage clamp methods.  At the whole animal level, preliminary data indicates that (+)SKF-10047 induces hypermobility and compound C52 is a sigma-1 antagonist.  Biochemically, preliminary data for an N-alkyl-N'-aralkyl derivative photoprobe and an N-substituted cocaine photoprobe show sigma1-specific binding that can be protected by haloperidol, a neuroleptic with high affinity for sigma1 receptors.

References:
1) Wilke, R.A. et al. (1999). J. PHysiol. 517, 391-406.
2) Hayashi, T. and Su. T.P. (2001). PNAS. USA. 98, 491-496.
3) Hayashi, T. and Su, T.P.(2003). JPET. 306, 718-725.
4) Berthois, Y. et al. (2003). B.J. Cancer. 88, 438-446.
5) Jbilo, O. et al. (1997) JBC. 272(43):27107

Characterization of sphingolipid binding at the sigma-1 receptor

The sigma-1 receptor once considered an opioid receptor is a transmembrane mammalian protein distinguished by its affinity for a wide range of pharmacological agents. The sigma-1 receptor has affinity for antipsychotics such as haloperidol, steroids such as progesterone, drugs of abuse such as cocaine and methamphetamine. Despite its promiscuous binding ability the endogenous ligand for the sigma-1 receptor is not known. The sigma-1 receptor has been implicated in anxiety, depression, schizophrenia, analgesia and drug dependence. Although its signaling mechanisms are not completely understood, various functions have been proposed for sigma-1 receptor such as regulation of intracellular Ca2+ release (1), inhibition of voltage gated K+ channels (2) and antitumour effects both in vitro and in vivo (3). The sigma-1 receptor was found to be present in ER lipid domains enriched in complex sphingolipids and have roles in lipid transport (4). We have previously expressed the guinea pig sigma-1 receptor in E. coli as a maltose binding protein (MBP) fusion protein and developed a purification scheme to obtain pure sigma-1 receptor (5). Studies aimed at characterizing the pharmacological properties of the purified protein showed that members of the class of endogenous sphingolipids (sphingosine, N, N dimethyl sphingosine, sphinganine) bind to the sigma-1 receptor with considerable affinity both to the purified protein as well as in membrane preparations. It was also seen that while sphingosine binds to the sigma-1 receptor its phosphorylated counterpart sphingosine-1 phosphate and its acylated derivatives such as ceramide and ceramide-1 phosphate do not bind. The observation that long chain aliphatic amines bind to the sigma-1 receptor prompted us to explore the structural features of the sigma-1 receptor pharmacophore by testing a series of aliphatic amines and their N substituted phenylpropyl derivatives. It was found that addition of the electron withdrawing phenylpropyl or p-nitro-phenylpropyl to alkyl amines with low affinity increases the affinity of these compounds for the sigma-1 receptor binding site by 2-3 orders of magnitude. We are currently involved in the synthesis of high affinity aliphatic amine photoprobes for the sigma-1 receptor that will be employed to identify the location of the alkyl amine binding site on the sigma-1 receptor.

References:

1.  Hayashi, T., and Su, T. P. (2001) Proc Natl Acad Sci U S A 98, 491-496
2.  Wilke, R. A., Lupardus, P. J., Grandy, D. K., Rubinstein, M., Low, M. J., and Jackson, M. B. (1999) J Physiol 517 ( Pt 2), 391-406
3. Berthois, Y., Bourrie, B., Galiegue, S., Vidal, H., Carayon, P., Martin, P. M., and Casellas, P. (2003) Br J Cancer 88, 438-446
4.  Hayashi, T., and Su, T. P. (2004) Proc Natl Acad Sci U S A 101, 14949-14954
5. Ramachandran, S., Lu, H., Prabhu, U., and Ruoho, A. E. (2006) Protein Expr Purif

Matt Marengo
David Wassarman Lab
Monday, December 4, 2006

Mechanisms of Signal-Dependent Alternative Splicing

Alternative pre-mRNA splicing is a major mechanism utilized by eukaryotic organisms to
expand their protein-coding capacity. We are using the Drosophila melanogaster TAF1 gene
as a model system for understanding how signaling pathways can regulate alternative
splicing. TAF1 encodes a subunit of TFIID, which is broadly required for RNA polymerase
II transcription.  Previously, our laboratory demonstrated that TAF1 alternative
splicing generates four mRNAs and that an alternative exon encodes a domain that changes
the DNA binding properties of TAF1 in vitro. In the Drosophila cell line S2, TAF1
alternative splicing is regulated by the ATR DNA damage signaling pathway in response to
treatment with camptothecin (CPT). A TAF1 minigene assay was developed to identify the
pre-mRNA elements necessaryfor CPT-induced alternative splicing.  This assay identified
two intronic elements that are necessary for the CPT-induced upregulation of the two
alternative exons.  Disrupting one element did not prevent the CPT-induced inclusion of
the other alternative exon and vice versa.  These data suggest that the two CPT-induced
splicing events can be uncoupled and signaled independently. Furthermore, inclusion of
one of the alternative exons was rendered consitituitive by changing a weak 5' splice
site to the consensus sequence for U1 snRNP binding.  We hypothesize that CPT signals
inclusion of the alternative exon through regulation of a splicing factor that enhances
U1 snRNP binding.  These results are a step towards understanding how DNA damage
signaling impinges directly on the splicing machinery.

Nick Schill
R.A. Anderson Lab
Monday, December 11, 2006

A role for PIPKIg splice variants in cellular trafficking pathways

The spatial and temporal metabolism of phosphoinositides regulates many cell signaling pathways. Phosphatidylinositol 4,5-bisphosphate (PI4,5P2) is a lipid signaling molecule which plays a role in the regulation of endocytosis, actin assembly, cell migration, and the maintenance of cellular adhesion structures. Phosphatidylinositol 4-phosphate 5-kinase type I gamma (PIPKIg) synthesizes PI4,5P2 at specific subcellular sites via interactions with distinct protein binding partners. Variation of the C-terminal region of PIPKIg is the mechanism by which the kinase interacts with these different protein subsets. Four PIPKIg splice variants have been identified in human cells: PIPKIg640, 668, 700, and 707. These splice variants all exhibit kinase activity and distinctive subcellular localization patterns. Importantly, each splice variant seems to interact with a set of specific targeting proteins. PIPKIg668 interacts with both E-cadherin, an adhesion molecule present at cell-cell junctions, and adaptor complexes (AP), which mediate aspects of intracellular trafficking. With PIPKIg668 acting as a bridge between E-cadherin and AP1B, the efficient delivery of E-cadherin to the basolateral membrane is facilitated. Interestingly, we have observed that endogenousPIPKIg668 and PIPKIg707 can be co-immunoprecipitated with E-cadherin, although all four splice variants associate with E-cadherin when overexpressed. Consistent with a role for PIPKIg in the trafficking of cellular proteins, PIPKIg707 also interacts with proteins of the sorting nexin family (SNX) via its unique C-terminal sequence. Members of the SNX family are known to alter the turnover of receptors on the plasma membrane. Given the observed role of PIPKIg668 in E-cadherin trafficking, PIPKIg707 may also regulate aspects of E-cadherin transport via its interaction with the SNX proteins.

Bryan T. Glaser
Burgess Lab
Monday, December 18, 2006

Characterizing Inhibitors of the Sigma70-RNA Polymerase Interaction as Potential Antibiotics

Antibiotic resistance is an increasing problem that could pose serious health risks in the future.  In order to address this situation, antibiotic candidates utilizing novel mechanisms of action need to be developed.  The Escherichia coli RNA polymerase holoenzyme is a desirable target because its role in transcription is critical to cell survival.  The E. coli holoenzyme is comprised of core RNA polymerase (a2bb'w) along with one of seven sigma factors.  Each sigma factor is responsible for driving transcription of a different set of genes with sigma70 regulating the "house keeping" genes.  If the sigma70-core interaction can be prevented, the bacteria can no longer transcribe and grow.  We designed a high throughput screen using luminescence resonance energy transfer (LRET) to monitor this protein-protein interaction.  Using this assay we screened a library of 16,000 small molecules and identified four lead structures.  These four compounds have been further characterized using in vitro assays such as LRET, electrophoretic mobility shift assays (EMSA), and in vitro transcription in both prokaryotic and eukaryotic systems.   In vivo assays such as disc diffusion inhibition, growth curve analysis, and time-kill assays were performed in multiple bacterial strains.  Two of the compounds show activity against both gram positive and gram negative strains of bacteria.  Finally, structure-activity relationship (SAR) studies are under way to determine ways to improve the activity of the compounds to enable future drug development.

Ashley Doan
Anna Huttenlocher Lab
Monday, January 22, 2007

Receptor for Activated C Kinase, RACK1, is an adaptor protein that binds to and regulates signaling via Src and PKC-dependent pathways, and has been implicated in cell migration.  In this study we demonstrate novel functions for RACK1 in regulating adhesion dynamics during cell migration.  We report that cells lacking RACK1 are less motile and show reduced dynamics of paxillin and talin at focal complexes. To investigate the role of the RACK1/Src interactions in paxillin dynamics, we used RACK1 in which the Src binding site has been mutated (RACK1 Y246F).  Expression of wild type RACK1, but not RACK Y246F, was able to rescue the adhesion and migration defects observed in the RACK1-deficient cells.  Furthermore, our findings indicate that RACK1 functions to regulate paxillin phosphorylation and that its effects on paxillin dynamics requires the Src-mediated phosphorylation of tyrosine 31/118 on paxillin.  Taken together, these findings support a role for RACK1 as a key regulator of cell migration and adhesion dynamics through its association with Src, and the modulation of paxillin phosphorylation at early adhesions.

Jennifer Lamberski
Richard Burgess Lab
Monday, January 29, 2007

GRIP1-ERRÉø interaction as a possible new target for anti-breast cancer therapeutic

Breast cancer is the second leading cause of cancer-related death in women.  The steroid nuclear receptor estrogen receptor alpha (ERÉø) is the primary target of current breast cancer therapies, which block ERÉø activation or estrogen synthesis.  However, cancers that lack ERÉø (ERÉø-negative) are more aggressive and function through unknown mechanisms.  Estrogen-related receptor alpha (ERRÉø), a protein with high sequence similarity to ERÉø, has functional similarity to ERÉø in certain breast cancer cell types; though unlike ERÉø, ERRÉø acts independently of steroid ligand.  Instead, we hypothesize that its activity may be due in part to its interaction with co-activator proteins.  In cell types in which ERRÉø is present but ERÉø is either absent or inactive, we believe ERRÉø may be functionally substituting for ERÉø and playing an important role in the progression of a subset of breast cancers.  We found that co activator recruitment is indeed important for full ERRÉø activation of transcription.  We also determined that of the three members of the p160 family of transcriptional co-activators, GRIP1 (SRC-2, TIF-2) was cell type- and promoter- specific in its co-activation of transcription with a constitutive active form of ERRÉø.  We used this information to develop a cell-based, high-throughput screen for small molecules that inhibit the transcription of an estrogen-response element (ERE)-containing luciferase reporter gene in the presence of overexpressed GRIP1 and "activated" ERRÉø.  This screen, performed in MCF-7 cells treated with ICI-182,780 (a compound that inhibits ERÉø; also known as Faslodex; AstraZeneca), used a 4,160 "known bioactives" compound library.  We are currently validating "hits" from the cell-based screen by performing counter screens to rule out compounds that are toxic to cells or that generally inhibit transcription or translation.  We then plan to screen the effects of remaining "hits" by inhibition of the GRIP1-ERRÉø interaction in vitro. Finally, we will characterize the in vivo and in vitro "hits" by dose response curves and by the activity of closely related compounds.  We believe that the GRIP1-ERRÉø interaction is a promising target for new breast cancer therapies.

Neil Daily
Tom Martin Lab
Monday, March 19, 2007

CAPS interacts with syntaxin: possible clue for CAPS function in regulated membrane fusion

CAPS (Ca2+ -dependent activator protein for secretion) is a novel 1289 amino acid protein that was discovered by its activity in reconstituting Ca2+-dependent dense-core vesicle exocytosis in permeable neuroendocrine cells.  CAPS is expressed in neural and peptide hormone-secreting endocrine cells and has an essential role in pre-fusion events by conferring competence on docked dense-core vesicles for Ca2+ -triggered fusion.  Vesicle fusion with the plasma membrane is mediated through SNARE protein interactions.  Syntaxin is an essential SNARE protein resident on the plasma membrane that is part of the core machinery (which includes VAMP2 and SNAP25) needed for membrane fusion in exocytosis. Recent studies have shown that CAPS promotes fusion of liposomes containing VAMP2 with those containing syntaxin and SNAP25 in a reconstituted liposome fusion assay.  Flotation of liposomes from fusion assays on Accudenz gradients showed that CAPS binds liposomes.  Subsequent flotation studies showed that CAPS binds to liposomes containing syntaxin/SNAP25 or syntaxin but not to liposomes containing VAMP2 or lacking protein.  Systematic binding studies with syntaxin-containing liposomes showed that CAPS exhibits a KD ~ 200 nM and a Bmax equivalent to 1 mol CAPS bound per mol syntaxin.  Preliminary mutational studies indicated that CAPS binds to a truncated transmembrane syntaxin that lacks its amino terminal Habc domain but preserves the membrane-proximal H3 SNARE motif.  These studies indicate that CAPS exhibits high affinity stoichiometric interactions with syntaxin, an essential core component of the membrane fusion machinery.  This interaction may play a key role in CAPS function in dense-core vesicle exocytosis by promoting localization and assembly of SNARE complexes required for fusion. 

Uyen Chu
Dr. Arnold Ruoho's Lab
Monday, March 26, 2007

Sigma receptors were first identified by Martin et al. (1) as a subtype of opioid receptors. However, they were later reclassified as a unique binding site in the central nervous system due to their poor affinity for opioid receptors antagonists such as naloxone and naltrexone. Because of their high affinity for most common neuroleptics, sigma receptors received much attention as a potential alternative targets for the development of antipsychotic drugs. While the endogenous ligand of the sigma-1 receptor has yet to be identified and its exact function in the cell is unclear, the sigma-1 receptor has been implicated in diverse cellular processes. Among sigma-1 receptors many functions are potentiating the calcium release from intracellular stores through its interaction with ankyrin B and IP3R (2), modulating the voltage gated K+ channel independent of G-protein or kinases (3), and regulating cellular proliferation (4). Our lab recently demonstrated that sphingosine binds to the sigma-1 receptor while sphingosine-1 phosphate does not, which potentially identifies long N-alkyl chain compounds as regulator of sigma-1 receptor function. Using chemical synthesis, competitive displacement binding assays against [3H]-(+)-pentazocine, and photoaffinity labeling, my work focuses on studying biochemical properties of the sigma-1 receptor interaction with compounds structurally similar to sphingosine, including phenylpropyl- and nitrophenylpropyl-N-alkylamines. In addition, very preliminary data on sigma-1 receptor intrinsic tryptophan fluorescence in the presence of the sigma ligand, haloperidol will be discussed.

Brian Hoffmann
Dr. Deane Mosher Lab
Monday, April 2, 2007

Dissecting the Plasma Fibronectin and Fibrin Interaction Through Combining Transglutaminase-catalyzed Cross-linking, Immuno-isolation, and Mass Spectrometry

Complex formation between plasma fibronectin (pFN) and fibrin plays an important role in the promotion of platelet and cell attachment at sites of injury via cell surface receptors.  The pFN-fibrin interaction is stabilized by thrombin-activated Factor XIII (FXIII), a transglutaminase that catalyzes formation of ε-amino(γ-glutamyl)-lysine isopeptide bonds during blood coagulation.  Glutamine residues in the most amino-terminal region of pFN are thought to form the isopeptide bonds with lysine residue(s) in carboxy-terminal 450 residues of the 609-residue fibrin α-chain, perhaps within residues residues 221-391 that mediate binding of pFN to the α-chain.  Thrombin also converts fibrinogen to fibrin during coagulation through cleavage of its central domain, presumably allowing this portion of the fibrin α-chain to become readily available for interaction with pFN.  In order to understand the involvement of the α-chain, I have undertaken studies to identify the exact site(s) of covalent cross-linking between pFN and fibrin. Monoclonal antibody to the amino-terminal region of pFN was used to immuno-purify cross-linked pFN-fibrin complexes after limited trypsinization.  The purified cross-linked sample from limited trypsinization yielded a ~37-kDa fragment containing the pFN amino-terminal ~27-kDa region and a smaller portion of the fibrin α-chain.  Immuno-purified materials were analyzed by MALDI MS and MALDI MS/MS mass spectrometry.  The amino-terminal 2834-Da tryptic peptide of pFN was missing from digests of cross-linked samples when compared to the non-cross-linked control samples.  Tryptic peptides representing residues 226 to 238 and 259 to 308 of the fibrin α-chain were identified in digests of the ~37-kDa complex.  The missing sequence between 239 and 258 contains lysines at residues 243 and 249.  The MS and MS/MS experiments indicate that at least some of the 2834-Da tryptic peptide from pFN is cross-linked to such a “double” peptide representing residues 239 to 249 of the fibrin α-chain, implicating K243 as a site of cross-linking.  K249, which is found in the sequence EWKAL conserved in fibrin(ogen) α-chain of diverse mammalian species, is also highly suspected in the cross-linking interaction from these studies.  Identification of the cross-linking site(s) will give insight into the pFN-fibrin interaction involvement in wound and tissue repair.

Jessica Townsend
Dr. Jeff Johnson Lab
Monday, April 9, 2007

The Role of Glutathione Depletion in Neurodegenerative Disease

Many neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's Diseases are believed to be partially induced by decreased levels of the antioxidant glutathione in given areas of the brain leading to intraneuronal accumulation of reactive oxygen species. Nuclear factor E2-related factor 2 (Nrf2) is a transcription factor associated with the induction of many genes essential for detoxification and maintenance of cellular reducing potential including those involved in glutathione synthesis. Nrf2 achieves this through the binding and activation of the cis-acting regulatory element known as the antioxidant response element (ARE). In previous work it was shown that overexpression of Nrf2 significantly increases total intracellular glutathione levels through activation of the ARE. It has also been shown that pretreatment with a chemical inhibitor of glutathione biosynthesis, butathione sulfoxamine, abolishes Nrf2-mediated neuronal protection from oxidative stress. This data suggests glutathione as the essential antioxidant involved in protective effects seen by Nrf2 upregulation. However, in preliminary experiments from our lab, using a genetic mouse model for glutathione depletion, we show that activation of ARE-mediated transcription is still able to protect against hydrogen peroxide induced oxidative stress in primary cortical cultures. Results from these experiments suggest that glutathione is not in fact essential to protection from oxidative stress mediated through the Nrf2-ARE pathway.

Kelly Christopherson
Lab of Dr. Cynthia Czajkowski
Monday, April 16, 2007

Stoichiometry and Alcohol Sensitivity of alpha4-beta2-delta GABA(A) Receptors

The gamma-aminobutyric acid type A, GABA (A), receptor mediates the majority of fast synaptic inhibition in the brain and is a target of many depressants, such as the benzodiazipines, barbiturates, and alcohol. The most prevalent receptor subtype contains alpha, beta, and gamma subunits. However, GABA (A) receptors containing the delta subunit, instead of gamma, display a unique sensitivity to ethanol and may play a key role in mediating the behavioral effects of alcohol at physiologically relevant doses. Evidence also indicates that chronic use of alcohol may cause a rearrangment of receptor subtype levels in the brain, which may ultimately result in increased substance dependence and altered receptor pharmacology.

Alcoholism and alcohol abuse are serious problems that have severe repercussions on physical and mental health as well as family and lifestyle. 13.8 million American adults have problems with drinking, 8.1 million of which are alcoholic. Chronic alcohol abuse causes major damage to the brain as well as other symptoms requiring extensive medical care that in total costs society billions of dollars each year.

Alpha-beta-delta receptors have several important and unique characteristics, such as extrasynaptic positioning and modulation by neuroactive steroids and alcohol, making them interesting proteins to study. Furthermore, recent research supports the hypothesis that these receptors may, in fact, be responsible for mediating the physiological effects of moderately intoxicating doses of alcohol in the brain. However, alpha-beta-delta receptors are not as well studied as their alpha-beta-gamma counterparts, and fundamental properties, such as the stoichiometry of these receptors is unknown.

My research projects aim to characterize delta-containing receptors by determining their subunit stoichiometry, using alpha4-beta2-delta receptors as a model. In addition, the responsiveness of the receptors to low doses of alcohol will be established and the putative alcohol binding site on the receptors will be mapped. The drug Ro15-4513, which is currently used clinically to antagonize the behavioral effects of excessive alcohol consumption, has been reported to bind with high affinity to delta-containing GABA(A) receptors and is thought to compete for the same binding site as alcohol. Ro15-4513 contains a photo-activatable azido group that will be used to photolabel delta-containing receptors. The specific subunit into which the drug photoincorporates will then be identified and the photolabeled region of the subunit will be narrowed down using a series of proteolytic digestions. The success of this project will provide a better understanding of how GABA(A) receptors mediate the effects of alcohol in the brain and will provide insight that will aid in the development of novel pharmacotherapeutics for the treatment of many symptoms of chronic alcohol abuse.

Jessica Heck
Richard Anderson Laboratory
Monday, April 16, 2007

Characterization of a Novel Interaction between Type I Phosphatidylinositol-Phosphate Kinases and _-catenin

Adherens junctions are specialized adhesive complexes between cells that contribute to cell polarity, tissue integrity, and coordinated cell movements. In epithelial cells, E-cadherin is the transmembrane core of adherens junctions and functions via an extracellular interaction with cadherin molecules on adjacent cells and intracellular interactions with a scaffolding and signaling protein complex which includes _-catenin. Besides linking E-cadherin to the actin cytoskeleton, _-catenin also functions as a transcriptional co-activator downstream of Wnt signaling to regulate the transcription of a number of genes including E-cadherin. Phosphatidylinositol-phosphate kinase type I_ isoform (PIPKI_) has been shown to bind E-cadherin independent of _-catenin and regulate E-cadherin trafficking to the basolateral membrane for assembly of cell-cell contacts. Here we show that a direct interaction between multiple type I isoforms and _-catenin has also been identified. Considering the dual role of _-catenin within the cell, confirmation of this interaction leads to intriguing possibilities for the regulation of _-catenin function by the PIP kinases or modulation of PIP kinase function by _-catenin.

Jon Wang
Rapraeger Lab
Monday, April 23, 2007

Syndecan-1 core protein regulates proliferationin cultured adherent cells

Many growth factors, including FGFs, heparin-binding members of the EGF family, HGF, Wnts, hedgehogs and some members of the TGF superfamily, depend on heparan sulfate proteoglycans (HSPGs) as co-factors in receptor binding and/or signaling. Overexpression of many of these growth factors and their receptors plays a pivotal role in tumor progression. However, the role of HSPGs in this process has not been fully appreciated. We find that syndecan-1 (Sdc1), a member of the syndecan family of HSPGs and the predominant PG expressed in mammary epithelium, is required for the proliferation of MDA-MB-231 and MCF-7 human mammary carcinoma cells. Treatment of these cells with rabbit polyclonal antibody directed against the Sdc1 ectodomain dramatically inhibits their growth (~ 50% reduction) in both a 2D and 3D culture environment. Moreover, this growth inhibition is substratum independent, occurring in cells cultured in or on reconstituted basement membrane (Matrigel), type I collagen or tissue culture plastic. However, this effect is Sdc1 specific because treatment with rabbit IgG alone or rabbit polyclonal syndecan-4 ectodomain antibody has no effect. Transfection of human-specific siRNA to silence Sdc1 expression arrests MDA-MB-231 cells in the G0/G1 phase of the cell cycle. Stable expression of full length mouse Sdc1 (mSdc1) or GPI-linked mouse Sdc1 ectodomain (GPI-mS1ED), which is unaffected by the human-specific siRNA, rescues cell proliferation confirming that the Sdc1 ectodomain alone retains growth-promoting activity. Because the syndecan transmembrane and cytoplasmic domains are not required, these results suggest that Sdc1 must signal in cooperation with a cell surface partner(s). Surprisingly, expression of a Sdc1 ectodomain mutant (TDM) that lacks its heparan sulfate chains, which are absolutely required for ligand binding (e.g., growth factors and extracellular matrix), also rescues proliferation. Together, these findings highlight a potentially novel role for the Sdc1 core protein ectodomain in the regulation of mammary carcinoma cell proliferation.

Shin-Il Kim
Bresnick Lab
Monday, April 23, 2007

Dissecting Molecular Steps in Chromatin Domain Activation During Hematopoietic Differentiation

GATA factors orchestrate hematopoiesis via multistep transcriptional mechanisms, but the interrelationships and importance of individual steps are poorly understood. Using complementation analysis in GATA-1-null cells and mice containing a hypomorphic allele of the chromatin remodeler BRG1, we dissected the pathway from GATA-1 binding to cofactor recruitment, chromatin loop formation, and transcriptional activation. Analysis of GATA-1-mediated activation of the b-globin locus, in which GATA-1 assembles dispersed complexes at the promoters and the distal LCR, revealed molecular intermediates, including GATA-1-independent and GATA-1-containing LCR subcomplexes, both defective in promoting loop formation. An additional intermediate consisted of an apparently normal LCR complex and a promoter complex with reduced levels of total RNA Polymerase II (Pol II) and Pol II phosphorylated at serine 5 of the Carboxy-Terminal Domain. Reduced BRG1 activity solely compromised Pol II and serine 5-phosphorylated Pol II occupancy at the promoter, phenocopying the LCR-deleted mouse. These studies defined a hierarchical order of GATA-1-triggered events at a complex locus and establish a novel mechanism of long-range gene regulation.

Yiming Zhu
Bertics lab
Monday, April 30, 2007

Eosinophil, a type of leukocyte, is thought to contribute to the pathology of allergic disorders including asthma. Interleukin-5 (IL-5) is a primary eosinophil-active cytokine and it regulates many aspects of eosinophil biology. In vivo and in vitro studies have shown that IL-5 can potentiate cell responsiveness to subsequent chemokine stimulation in human eosinophils, a process known as “priming”. Eosinophils isolated from asthmatic patients show increased release of cytotoxic granule proteins and enhanced production of proinflammatory mediators in response to chemokine stimulation. The increased cellular responses contribute to the chronic inflammation in asthma. Although priming has been known for years, the intracellular mechanism is not yet clear. My studies suggest that IL-5 not only potentiates chemokine receptor mediated pathways but also pathways of other G Protein-Coupled Receptors (GPCRs) in human eosinophils. Preliminary data also support the hypothesis that Src kinase family members and b-arrestin are important intracellular players for priming process.

Lindsay M. Hill
Bertics Lab
Monday, April 30, 2007

Extracellular nucleotides are present in high concentrations at sites of inflammation, cell lysis and platelet degranulation. This extracellular ATP can act as a ligand for nucleotide receptor P2X7 found on leukocytes such as monocytes and macrophages. Activation of P2X7 is involved in LPS-mediated macrophage action. Recently, our lab found that stimulation of P2X7 in LPS-primed macrophages results in enhanced reactive oxygen species (ROS) production, greater than that from either stimulus alone. ROS production is an essential component of macrophage immune function, maintaining roles in bacterial cytotoxicity and macrophage signaling. In my studies I have tested the hypothesis that the capacity of P2X7 to synergize with LPS entails the regulation of the ROS producing enzyme NADPH oxidase. Enhanced ROS production was found in LPS-primed murine macrophages and human primary blood monocytes after P2X7 stimulation but not in P2X7-deficient cells. This enhanced ROS production was attenuated by a NADPH oxidase inhibitor. Additionally, these treatment conditions resulted in enhanced phosphorylation of a NADPH oxidase component. These data support a model wherein P2X7 signaling is critical for the modulation of the macrophage immune response.

Felix Yeh
Chapman Lab
Monday, May 7, 2007

Two Distinct Receptors Mediate Tetanus Neurotoxin Entry

The mechanism in which tetanus neurotoxin enters neurons is not well established. Literature suggests two mutually exclusive entry pathways: through recycling synaptic vesicles (Matteoli et al., 1996) and through another vesicular organelle (Deinhardt et al., 2006a; Roux et al., 2005). To understand the extreme toxicity of tetanus neurotoxin and discover clues about the receptors for tetanus toxin, it is important to determine the methods in which tetanus toxin travels through victims. My hypothesis is that tetanus toxin is able to travel from the periphery and arrive at its final destination, the spinal cord, through two unique protein receptors that direct its transport in the motorneuron and its entry in central nervous system. My current studies are directed towards elucidating the entry pathways of tetanus neurotoxin and also identifying the two receptors that mediate tetanus neurotoxin's entry into motorneurons and inhibitory interneurons, the two targets of tetanus toxin. This research will help us develop new methods to deliver drugs through tetanus toxin's utilization of a pathway to shuttle molecules from the periphery into the central nervous system. Tetanus neurotoxin can also be engineered into a nontoxic carrier of DNA to help in debilitating diseases associated with motorneurons and inhibitory interneurons, such as amyotrophic lateral sclerosis (Lou Gehrig's disease) or Parkinson's disease. For third world countries, novel methods to prevent the disease can also be developed, that may be save millions of lives a year.

Zhen Zhang
Meyer Jackson Lab
Monday, May 7, 2007

Regulation of exocytosis by synaptotagmin isoforms and divalent cation

Synaptotagmin (syt) I acts as a Ca2+ sensor in regulated exocytosis. Kinetic analysis of syt isoforms and mutants has shown that syt controls a step leading to fusion pore opening and a step involving open fusion pores. Members of the syt family bind to the divalent cations Sr 2+ and Ba2+, as well as to Ca2+, and the affinities of these interactions vary with Syt isoform. Here we overexpressed syt I, syt VII or syt IX in PC12 cells and used amperometry to study how they regulate secretion triggered by Ca 2+, Sr2+, and Ba2+. In exocytosis triggered by Ca2+, syt VII shortened the first-spike latency and accelerated the initial secretion rate, while syt IX had the opposite effect and syt I left these parameters unchanged. In exocytosis triggered by Sr 2+, syt VII shortened the first-spike latency and accelerated the initial secretion rate, while syt I and syt IX left these parameters unchanged. For all three isoforms, Sr2+ prolonged the first-spike latency and slowed the initial secretion rate compared to Ca 2+. Ba2+ triggered exocytosis even without depolarization by a high concentration of KCl. These results suggest that syt VII promotes and syt IX impedes the assembly of the fusion apparatus, compared to syt I. Sr 2+ also slows fusion apparatus assembly. In exocytosis triggered by Ca2+, syt I, syt VII and syt IX all prolonged prespike foot (PSF) duration indicating a stabilization of the fusion pore. In exocytosis triggered by Sr 2+, only syt VII, which has a higher affinity for Sr2+ than syt I and syt IX, prolonged PSF duration. In exocytosis triggered by Ba2+, again syt VII, which is unique in its higher affinity for Ba 2+, prolonged PSF duration. Sr2+ and Ba2+ prolonged PSF duration to similar degrees (~80% and 100% increase respectively) compared to Ca2+. These results suggest that syt I, syt VII and syt IX all stabilize the open fusion pore. Sr 2+ and Ba2+ also stabilize the fusion pore compared to Ca2+. Syt I, syt VII and syt IX regulate exocytosis at multiple steps, and comparisons between Ca2+, Sr2+, and Ba 2+ may help in resolving how synaptotagmin isoforms regulate different steps of exocytosis. (Supported by NS44057)

Brian Torres
Dr. Arnold Ruoho Lab
Monday, May 14, 2007

N-terminus Regulation of vesicular monoamine transporter 2 Function

Essential for the normal function of the monoaminergic system is the sequestration of monoamines into protective vesicular compartments for storage and subsequent exocytotic release. The vesicular monoamine tranporter 2 (VMAT2) sequesters monoamines into vesicular compartments in cell populations throughout the human body. The VMAT2 is targeted by anti-hypertensive drugs and has been implicated in exerting a protective effect against Parkinson's Disease like states. The cytosolic VMAT2 N-terminus, containing five (+) arginine and lysine residues, was examined as a potential intra- or intermolecular enabler and regulator of monoamine transport. VMAT2 containing vesicles isolated from human VMAT2 transfected cos-7 cells rapidly accumulate 3H serotonin in a reserpine repressible manner. Removal of VMAT2 N-terminus residues or treatment of human VMAT2 transfected cos-7 vesicles with an N-terminus peptide inhibited 3H serotonin accumulation. However, a peptide comprised of N-terminal residues in randomized order, or an N-terminal peptide phosphorylated serine residues 15 and 18 failed to inhibit 3H serotonin accumulation. GST constructs of the N-terminus undergo in vitro phosphorylation by PKC but not PKA at serines 15 and 18. Finally, assessment of transport in cos-7 vesicles transfected with constructs in which N-terminus serine residues 15 and 18 were mutated to 1. alanine or 2. aspartate residues showed enhanced and diminished transport, respectively, in comparison to WT. Taken together these data indicate sequence specific and phosphorylation specific regulation of VMAT2 monoamine transport by targeting serine residues at the N-terminus. The seminar will close with a brief introduction on the leucine rich repeat kinase 2 (LRRK2), a kinase in which gain of function mutations have been correlated to Parkinson's Disease in up to 40% of particular familial Parkinson's Disease patients.

James Kinter
Dr. Dovat Lab
Monday, May 14, 2007

The Use of Differentiation Therapy in the Treatment of Malignant Disease

Cellular Differentiation is the process by which immature cells become more specialized cell types, through intricately regulated alterations in gene expression patterns. Differentiation therapy is based on the concept that malignant cells have a block in differentiation, which can be overcome by hyperphysiological concentrations of differentiating agents. The goal of differentiation therapy is to push malignant cells towards a terminally differentiated state associated with replicative senescence. To investigate alterations in gene expression responsible for replicative senescence associated with terminal differentiation, we induced HL-60 cells towards terminal differentiation via three distinct differentiation pathways followed by MicroArray analysis. We discovered that upon onset of terminal differentiation the expression of ~125 gene transcripts involved in DNA repair were down-regulated by greater then two fold in all three pathways. Interestingly, only ~3 gene transcripts involved in DNA repair were commonly up-regulated under the same criteria. We hypothesize that the combination of differentiation agents with DNA damaging Chemotherapeutics reduces HL-60 viable cell number while increasing cytotoxic effectat doses significantly lower than achievable chemotherapeutic plasma concentrations. Although differentiating agents, such as All-Trans Retinoic Acid, are already used with success in combination with DNA Damaging Chemotherapeutics in the treatment of acute promyelocytic leukemia, our data provides support for a new pharmacogenomic based targeted combined chemotherapy, in which, DNA Damaging agents can be used with greater effect by coupling their administration with differentiation agents, increasing cytotoxicity while potentially decreasing side effects in the treatment of malignant disease.

Derek Pavelec
Kennedy Lab
140 Bardeen
Monday, May 21, 2007

Identification of Novel Proteins Required for Endogenous RNAi

I am interested in understanding the endogenous biological functions of the RNAi machinery. In order to further our understanding of RNAi we are conducting genetic screens in the model organism C. elegans to identify components of the endogenous RNAi machinery. Previous genetic screens in the laboratory have identified a series of factors termed ERI-1, RRF-3, ERI-3, and ERI-5 that exist in a protein complex with the catalytic engine of RNAi, the RNase III like enzyme Dicer (DCR-1). This complex has been termed the ERI/DCR complex. Further genetic screening has yielded additional components of the endogenous RNAi machinery. Animals carrying Eri mutations fail to accumulate a subset of cellular small RNAs. These endogenous small RNAs are also missing in the new Eri genes that I have identified. The loss of these small RNAs correlates a miss-regulation of the target mRNA. I hypothesize that the ERI/DCR complex and the additional RNAi genes that I have identified are essential for regulating hte levels of endogenous mRNAs in C. elegans.