Identification of Interactions in the NMD Complex Using Proximity-Dependent Biotinylation (BioID)

Proximity-dependent trans-biotinylation by the Escherichia coli biotin ligase BirA mutant R118G (BirA*) allows stringent streptavidin affinity purification of proximal proteins. This so-called BioID method provides an alternative to the widely used co-immunoprecipitation (co-IP) to identify protein-protein interactions. Here, we used BioID, on its own and combined with co-IP, to identify proteins involved in nonsense-mediated mRNA decay (NMD), a post-transcriptional mRNA turnover pathway that targets mRNAs that fail to terminate translation properly. In particular, we expressed BirA* fused to the well characterized NMD factors UPF1, UPF2 and SMG5 and detected by liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) the streptavidin-purified biotinylated proteins. While the identified already known interactors confirmed the usefulness of BioID, we also found new potentially important interactors that have escaped previous detection by co-IP, presumably because they associate only weakly and/or very transiently with the NMD machinery. Our results suggest that SMG5 only transiently contacts the UPF1-UPF2-UPF3 complex and that it provides a physical link to the decapping complex. In addition, BioID revealed among others CRKL and EIF4A2 as putative novel transient interactors with NMD factors, but whether or not they have a function in NMD remains to be elucidated.

Revealing Assembly of a Pore-Forming Complex Using Single-Cell Kinetic Analysis and Modeling

Many biological processes depend on the sequential assembly of protein complexes. However, studying the kinetics of such processes by direct methods is often not feasible. As an important class of such protein complexes, pore-forming toxins start their journey as soluble monomeric proteins, and oligomerize into transmembrane complexes to eventually form pores in the target cell membrane. Here, we monitored pore formation kinetics for the well-characterized bacterial pore-forming toxin aerolysin in single cells in real time to determine the lag times leading to the formation of the first functional pores per cell. Probabilistic modeling of these lag times revealed that one slow and seven equally fast rate-limiting reactions best explain the overall pore formation kinetics. The model predicted that monomer activation is the rate-limiting step for the entire pore formation process. We hypothesized that this could be through release of a propeptide and indeed found that peptide removal abolished these steps. This study illustrates how stochasticity in the kinetics of a complex process can be exploited to identify rate-limiting mechanisms underlying multistep biomolecular assembly pathways.

Antibodies against major histocompatibility complex class II antigens directly inhibit the growth of T cells infected with Theileria parva without affecting their state of activation

We have analyzed the effect of antibodies (Abs) directed against major histocompatibility complex (MHC) class II Abs on the proliferation of Theileria parva-infected (Tpi) T cells. Anti-MHC class II Abs exert a direct effect on Tpi T cells causing an acute block in their proliferation. The inhibition does not involve apoptosis and is also entirely reversible. The rapid arrest of DNA synthesis caused by anti-MHC class II Abs is not due to interference with the state of activation of the T cells since the transcriptional activator NF-kappa B remains activated in arrested cells. In addition, interleukin 2 (IL-2), IL-2R, and c-myc gene expression are also unaffected. By analyzing the cell-cycle phase distribution of inhibited cells, it could be shown that cells in all phases of the cell cycle are inhibited. The signal transduction pathway that results in inhibition was shown to be independent of protein kinase C and extracellular Ca2+. Tyrosine kinase inhibitors, however, partly reduced the level of inhibition and, conversely, phosphatase inhibitors enhanced it. The possible relevance of this phenomenon in other systems is discussed.

Identification of two Th1 cell epitopes on the Babesia bovis-encoded 77-kilodalton merozoite protein (Bb-1) by use of truncated recombinant fusion proteins

Previous studies have demonstrated the serologic and T-cell immunogenicity for cattle of a recombinant form of the apical complex-associated 77-kDa merozite protein of Babesia bovis, designated Bb-1. The present study characterizes the immunogenic epitopes of the Bb-1 protein. A series of recombinant truncated fusion proteins spanning the majority of the Bb-1 protein were expressed in Escherichia coli, and their reactivities with bovine peripheral blood mononuclear cells and T-cell clones derived from B. bovis-immune cattle and with rabbit antibodies were determined. Lymphocytes from two immune cattle were preferentially stimulated by the N-terminal half of the Bb-1 protein (amino acids 23 to 266, termed Bb-1A), localizing the T-cell epitopes to the Bb-1A portion of the molecule. CD4+ T-cell clones derived by stimulation with the intact Bb-1 fusion protein were used to identify two T-cell epitopes in the Bb-1A protein, consisting of amino acids SVVLLSAFSGN VWANEAEVSQVVK and FSDVDKTKSTEKT (residues 23 to 46 and 82 to 94). In contrast, rabbit antiserum raised against the intact fusion protein reacted only with the C-terminal half of the protein (amino acids 267 to 499, termed Bb-1B), which contained 28 tandem repeats of the tetrapeptide PAEK or PAET. Biological assays and Northern (RNA) blot analyses for cytokines revealed that following activation with concanavalin A, T-cell clones reactive against the two Bb-1A epitopes produced interleukin-2, gamma interferon, and tumor necrosis factors beta and alpha, but not interleukin-4, suggesting that the Bb-1 antigen preferentially stimulates the Th1 subset of CD4+ T cells in cattle. The studies described here report for the first time the characterization, by cytokine production, of the Th1 subset of bovine T cells and show that, as in mice, protozoal antigens can induce Th1 cells in ruminants. This first demonstration of B. bovis-encoded Th1 cell epitopes provides a rationale for incorporation of all or part of the Bb-1 protein into a recombinant vaccine.

Biochemical and genetic analysis of two protein kinases JNKK2 and MEKK3

Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryotic signaling modules consisting of a MAPK, a MAPKK and a MAP3K. MAPK cascades are involved in many cellular responses including proliferation, differentiation, apoptosis, stress and immune responses. ^ The first part of this thesis describes the cloning and biochemical analysis of JNKK2, a member of MAPKK gene family. Our results demonstrate that JNKK2 is a specific JNK activator and activates the JNK-dependent signal transduction pathway in vivo by inducing c-Jun and ATF2-mediated gene expression. We also found that JNKK2 is specifically activated by a MAP3K MEKK2 through formation of MEKK2-JNKK2-JNK1 triple complex module. JNKK2 is likely to mediate specific upstream signals to activate JNK cascade. ^ The second part of this thesis describes biochemical and gene disruption analysis of MEKK3, a member of MAP3K gene family. We showed that overexpression of MEKK3 strongly activates both JNK and p38 MAPKs but only weakly activates ERK. MEKK−/− embryos die at about embryonic day (E) 11. MEKK3−/− embryos displayed defects in blood vessel development in the yolk sacs, and in the myocardium and endocardium development at E9.5. The angiogenesis in the head, intersomitic region and placenta was also abnormal. These results demonstrate that MEKK3, a member of MAP3K MEKK/STE11 subgene family, is essential for early embryonic cardiovascular development. Furthermore, it was found that disruption of MEKK3 did not alter the expression of vascular endothelial growth factor-1 (VEGF-1), angiopoietin-1, -2 and their respective receptors Flt-1, Flk-1, Tie-1, Tie-2. Finally, MEKK3 was shown to activate myocyte-specific enhancer factor 2C (MEF2C), a crucial transcription factor for early embryonic cardiovascular development through the p38 MAPK cascade, suggesting that MEF2C is one of the key targets of the MEEKK3 signaling pathway during early embryonic cardiovascular development. ^

An interferon-inducible protein, p202, in cancer gene therapy

Interferons (IFNs) have been shown to exert antiviral, cell growth regulatory, and immunomodulatory effects on target cells. Both type I (α and β) and type II (γ) IFNs regulate cellular activities by specifically inducing the expression or activation of endogenous proteins that perform distinct biological functions. p202 is a 52 kDa nuclear phosphoprotein known to be induced by IFNs. p202 interacts with a variety of cellular transcription and growth regulatory factors and affects their functions. ^ In this report, we showed that the expression of p202 was associated with an anti-proliferative effect on human prostate cancer cells. Cells that expressed p202 showed reduced ability to grow in soft-agar, indicating a loss of transformation phenotype. More importantly, p202 expression reduced the tumorigenicity of human prostate cancer cells. p202-expressing cells exhibit an elevated level of hypophosphorylated form of pRb, and reduced level of cyclin B1 and p55CDC. ^ Our data suggest that p202 is a growth inhibitor gene in prostate cancer cells and its expression may also suppress transformation phenotype and tumorigenicity of prostate cancer cells. ^ In addition to inhibiting in vitro cell growth, suppressing the tumorigenicity of breast cancer cells in vivo, p202 expression could sensitize breast cancer cells to apoptosis induced by TNF-α treatment. One possible mechanism contributing to this sensitization is the inactivation of NF-κB by its interaction with p202. These results provide a scientific basis for a novel therapeutic strategy that combines p202 and TNF-α treatment against breast cancer. ^ It has been reported that NF-κB is constitutively active in human pancreatic cancer cells. Since p202 interacts with NF-κB and inhibits its activity, we examined a potential p202-mediated anti-tumor activity in pancreatic cancer. We used both ectopic and orthotopic xenograft models and demonstrated that p202 expression is associated with multiple anti-tumor activities that include inhibition of tumor growth, reduced tumorigenicity, prolonged survival, and remarkably, suppression of metastasis and angiogenesis. In vitro invasion assay also showed that p202-expressing pancreatic cancer cells are less invasive than those without p202 expression. That observation was supported by the findings that p202-expressing tumors showed reduced expression of angiogenic factors such as IL-8, and VEGF by inhibiting their transcription, and p202-expressing pancreatic cancer cells have reduced level of MAP-2 activity, a secreted protease activity important for metastasis. Together, our results strongly suggest that p202 expression mediates multiple anti-tumor activities against pancreatic cancer, and that may provide a scientific basis for developing a p202-based gene therapy in pancreatic cancer treatment. ^ Importantly, we demonstrated a treatment efficacy by using p202/SN2 liposome complex in a nude mice orthotopic breast cancer, and an ectopic pancreatic cancer xenograft model, through systemic and intra-tumor injection respectively. These results suggest a feasibility of using p202/SN2 liposome in future pre-clinical gene therapy experiments. ^

Loss of activator protein-2alpha results in overexpression of the thrombin receptor and correlates with the malignant phenotype of human melanoma

Increasing evidence demonstrates that the thrombin receptor (protease activated receptor-1, PAR-1) plays a major role in tumor invasion and contributes to the metastatic phenotype of human melanoma. We demonstrate that the metastatic potential of human melanoma cells correlates with overexpression of PAR-1. The promoter of the PAR-1 gene contains multiple putative AP-2 and Sp1 consensus elements. We provide evidence that an inverse correlation exists between the expression of AP-2 and the expression of PAR-1 in human melanoma cells. Re-expression of AP-2 in WM266-4 melanoma cells (AP-2 negative) resulted in decreased mRNA and protein expression of PAR-1 and significantly reduced the tumor potential in nude mice. ChIP analysis of the PAR-1 promoter regions bp −365 to −329 (complex 1) and bp −206 to −180 (complex 2) demonstrates that in metastatic cells Sp1 is predominantly binding to the PAR-1 promoter, while in nonmetastatic cells AP-2 is bound. In vitro analysis of complex 1 demonstrates that AP-2 and Sp1 bind to this region in a mutually exclusive manner. Transfection experiments with full-length and progressive deletions of the PAR-1 promoter luciferase constructs demonstrated that metastatic cells had increased promoter activity compared to low and nonmetastatic melanoma cells. Our data shows that exogenous AP-2 expression decreased promoter activity, while transient expression of Sp1 further activated expression of the reporter gene. Mutational analysis of complex 1 within PAR-1 luciferase constructs further demonstrates that the regulation of PAR-1 is mediated through interactions with AP-2 and Sp1. Moreover, loss of AP-2 in metastatic cells alters the AP-2 to Sp1 ratio and DNA-binding activity resulting in overexpression of PAR-1. In addition, we evaluated the expression of AP-2 and PAR-1 utilizing a tissue microarray of 93 melanocytic lesions spanning from benign nevi to melanoma metastasis. We report loss of AP-2 expression in malignant tumors compared to benign tissue while PAR-1 was expressed more often in metastatic melanoma cells than in benign melanocytes. We propose that loss of AP-2 results in increased expression of PAR-1, which in turn results in upregulation of gene products that contribute to the metastatic phenotype of melanoma. ^

CCTeta: A novel soluble guanylyl cyclase-interacting protein

Nitric oxide (NO) transduces most of its biological effects through activation of the heterodimeric enzyme, soluble guanylyl cyclase (sGC). Activation of sGC results in the production of 3′,5 ′-cyclic guanosine monophosphate (cGMP) from 5′ -guanosine triphosphate (GTP). In this thesis, we demonstrate a novel protein interaction between CCT (chaperonin containing t-complex polypeptide) subunit η and the α1β1 isoform of sGC. Using the yeast-two-hybrid system, CCTη was found to interact with the N-terminal portion of β1 subunit of sGC. This interaction was then confirmed in vitro with a co-immunoprecipitation from mouse brain. The interaction between these two proteins was further supported by a co-localization of the proteins within rat brain. Using the yeast-two-hybrid system, CCTη was found to bind to the N-terminal portion of sGC. In vitro assays with purified CCTη and Sf9 lysate expressing sGC resulted in a 33% inhibition of sodium nitroprusside (SNP)-stimulated sGC activity. The same assays were then performed using BAY41-2272, an NO-independent allosteric sGC activator, and CCTη had no effect on this activity. Furthermore, CCTη had no effect on the activity of αβCys105 sGC a constitutively active mutant that lacks a heme group. Of note is the fact that the full-length CCTη-expressing bacterial lysate inhibited the activity of sGC-expressing Sf9 lysate by 48% compared with GST alone. This indicates that the amino terminal 94 amino acids of CCTη are important to the inhibition of sGC activity. Lastly, a 45% inhibition of sGC activity by CCTη was seen in vivo in BE2 cells stably transfected with CCTη and treated with SNP. The fact that the inhibition of sGC was more pronounced with bacterial lysate expressing CCTη versus the purified CCTη implies that some factor in the bacterial lysate enhances the inhibitory effect of CCTη. Because the level of inhibition seen in bacterial lysate and in vivo experiments is similar, might imply that the factor that aids in CCTη effect on sGC is conserved. Together, these data suggest that CCTη is a novel type of sGC inhibitor that inhibits sGC by modifying the binding of NO to the heme group or the subsequent conformational changes induced by NO binding. ^

Identification and characterization of Xenopus Kazrin, a nucleocytoplasmic shuttling protein that interacts with ARVCF catenin

In common with other members of the p120-catenin subclass of catenins, ARVCF-catenin appears to have multiple cellular and developmental functions. In Xenopus, our lab recently demonstrated that xARVCF- and Xp120-catenins are each essential for early vertebrate embryogenesis, being functionally linked to Rho-family GTPases (RhoA, Rac) and cadherin metabolic stability. For the project described here, the yeast two-hybrid system was employed to screen a Xenopus laevis neurula library for proteins that interact with xARVCF, resulting in the identification of the Xenopus homolog of Kazrin (xKazrin). Kazrin is a variably-spliced protein of unknown function that has been shown to interact with periplakin and envoplakin, components of desmosomal junctions. Kazrin's primary sequence is highly conserved across vertebrate species and is composed of an amino-terminal nuclear export sequence (NES), a carboxy-terminal nuclear localization sequence (NLS) and a central predicted coiled-coil domain. In vitro and in vivo authenticity tests demonstrated that xARVCF-catenin interacts directly with xKazrin via xARVCF's Armadillo and carboxy-terminal regions and xKazrin's coiled-coil domain. The interaction of xARVCF-catenin with xKazrin is specific and does not extend to the related Xp120-catenin. xKazrin co-localized with E-cadherin at sites of cell-cell contact and could be co-immunoprecipitated with components of the cadherin complex. xKazrin was also present in the cytoplasm and nucleus. Suggestive of a nuclear role, mutation of xKazrin's predicted NLS resulted in nuclear exclusion, while deletion of the predicted NES resulted in loss of sensitivity to nuclear export inhibitors. Within Xenopus embryos, xKazrin was expressed across all developmental stages and appeared at varying levels in adult tissues. Morpholino depletion of xKazrin from Xenopus embryos resulted in axial elongation abnormalities and loss of tissue integrity after neurulation. Over-expression of xKazrin had no effect, while over-expression of a NLS mutant resulted in a mild phenotype similar to that seen in xKazrin depleted embryos. Interestingly, the axial phenotype resulting from reduced xKazrin levels was largely rescuable by xARVCF over-expression. In conjunction with xARVCF-catenin, xKazrin has properties consistent with its function at cell-cell contact sites and in the nucleus. ^

Signal relay between two integral membrane proteins: The sensory rhodopsin I/HtrI transducer molecular complex

The molecular complex of sensory rhodopsin I (SRI) and its transducer HtrI mediate color-sensitive phototaxis in the archaeon Halobacterium salinarum. Orange light causes an attractant response by a one-photon reaction and white light causes a repellent response by a two-photon reaction. Three aspects of this molecular complex were explored: (i) We determined the stoichiometry of SRI and HtrI to be 2:2 by gene fusion analysis. A SRI-HtrI fusion protein was expressed in H. salinarum and shown to mediate 1-photon and 2-photon phototaxis responses comparable to wild-type complex. Disulfide crosslinking demonstrated that the fusion protein is a homodimer in the membrane. Measurement of photochemical reaction kinetics and pH titration of absorption spectra established that both SRI domains are complexed to HtrI in the fusion protein, and therefore the stoichiometry is 2:2. (ii) Cytoplasmic channel closure of SRI by HtrI, an important aspect of their interaction, was investigated by incremental HtrI truncation. We found that binding of the membrane-embedded portion of HtrI is insufficient for channel closure, whereas cytoplasmic extension of the second HtrI transmembrane helix by 13 residues blocks proton conduction through the channel as well as full-length HtrI. The closure activity is localized to 5 specific residues, each of which incrementally contributes to reduction of proton conductivity. Moreover, these same residues in the dark incrementally and proportionally increase the pKa of the Asp76 counterion to the protonated Schiff base chromophore. We conclude that this critical region of HtrI alters the dark conformation of SRI as well as light-induced channel opening. (iii) We developed a procedure for reconstituting HtrI-free SRI and the SRI/HtrI complex into liposomes, which exhibit photocycles with opened and closed cytoplasmic channels, respectively, as in the membrane. This opens the way for study of the light-induced conformational change and the interaction in vitro by fluorescence and spin-labeling. Single-cysteine mutations were introduced into helix F of SRI, labeled with a nitroxide spin probe and a fluorescence probe, reconstituted into proteoliposomes, and light-induced conformational changes detected in the complex. The probe signals can now be used as the readout of signaling to analyze mutants and the kinetics of signal relay. ^

Alpha C protein of group B Streptococcus as a potential vaccine candidate

Group B Streptococcus (GBS) is a leading cause of life-threatening infection in neonates and young infants, pregnant women, and non-pregnant adults with underlying medical conditions. Immunization has theoretical potential to prevent significant morbidity and mortality from GBS disease. Alpha C protein (α C), found in 70% of non-type III capsule polysaccharide group B Streptococcus, elicits antibodies protective against α C-expressing strains in experimental animals and is an appealing carrier for a GBS conjugate vaccine. We determined whether natural exposure to α C elicits antibodies in women and if high maternal α C-specific serum antibody at delivery is associated with protection against neonatal disease. An ELISA was designed to measure α C-specific IgM and IgG in human sera. A case-control design (1:3 ratio) was used to match α C-expressing GBS colonized and non-colonized women by age and compare quantified serum α C-specific IgM and IgG. Sera also were analyzed from bacteremic neonates and their mothers and from women with invasive GBS disease. Antibody concentrations were compared using t-tests on log-transformed data. Geometric mean concentrations of α C-specific IgM and IgG were similar in sera from 58 α C strain colonized and 174 age-matched non-colonized women (IgG 245 and 313 ng/ml; IgM 257 and 229 ng/ml, respectively). Delivery sera from mothers of 42 neonates with GBS α C sepsis had similar concentrations of α C-specific IgM (245 ng/ml) and IgG (371 ng/ml), but acute sera from 13 women with invasive α C-expressing GBS infection had significantly higher concentrations (IgM 383 and IgG 476 ng/ml [p=0.036 and 0.038, respectively]). Convalescent sera from 5 of these women 16-49 days later had high α C-specific IgM and IgG concentrations (1355 and 4173 ng/ml, respectively). In vitro killing of α C-expressing GBS correlated with total α C-specific antibody concentration. Invasive disease but not colonization elicits α C-specific IgM and IgG in adults. Whether α C-specific IgG induced by vaccine would protect against disease in neonates merits further investigation. ^

Functional analysis of BLAP75, a BLM-associated protein

Bloom syndrome (BS) is an autosomal recessive disorder characterized by dwarfism, immunodeficiency, impaired fertility, and most importantly, early development of a broad range of cancers. The hallmark of BS cells is hyper-recombination, characterized by a drastically elevated frequency of sister chromatid exchange (SCE). BLM, the gene mutated in BS, encodes a DNA helicase of the RecQ protein family. BLM is thought to participate in several DNA transactions and to interact with many proteins involved in DNA replication, recombination, and repair. However, the precise function of BLM and the BLM-dependent anti-tumor mechanism remain obscure. ^ A novel protein, BLAP75 (BLM-associated polypeptide, 75KD), was identified to form an evolutionarily conserved complex with BLM and DNA topoisomerase IIIα (Topo IIIα). Our work demonstrates that loss of BLAP75 destabilized BLM and Topo IIIα proteins. BLAP75 colocalized with BLM in subnuclear foci in response to DNA damage and the recruitment of BLM to these foci was BLAP75-dependent. Moreover, depletion of BLAP75 by siRNA resulted in an elevated SCE rate similar to cells depleted of BLM by siRNA. In addition, RNAi-mediated silencing of BLAP75 greatly diminished cell viability. This cellular deficiency was rescued by expression of wild type BLAP75 but not BLAP75 with mutated conserved domain III, which abrogated the interaction between BLAP75, BLM and Topo IIIα, suggesting that the integrity of BLM-Topo IIIα-BLAP75 complex might be critical for cell survival. Finally, I found that BLAP75 was phosphorylated during mitosis and upon various DNA-damaging agents, implying that BLAP75 might also function in mitosis and DNA damage response. ^ Taken together, this study has defined BLAP75 as an integral component of the BLM complex to maintain genome stability. Our findings provide insights into the molecular mechanisms of the BLM helicase pathway and tumorigenesis process associated with these mechanisms. ^

Ligand-stimulated internalization of a Dictyostelium discoideum G protein-coupled cAMP receptor

The social amoeba, Dictyostelium discoideum, undergoes a remarkable starvation-induced program of development that transforms a population of unicellular amoebae into a fruiting body composed of resistant spores suspended on a stalk. During this development, secreted cAMP drives chemotaxis of the amoebae, leading to their aggregation, and subsequent differentiation and morphogenesis. Four sequentially expressed G protein-coupled receptors (GPCRs) for cAMP play critical roles in this process. The first of these, cAR1, is essential for aggregation as it mediates chemotaxis as well as the propagation of secreted cAMP waves throughout aggregating populations. Ligand-induced internalization has been shown to regulate a variety of GPCRs. However, little was known at the outset of this study about the role of internalization in the regulation of cAR1 function or, for that matter, in developmental systems in general. For this study, cAMP-induced cAR1 internalization was assessed by measuring (1) the reduction of cell surface binding sites for [ 3H]cAMP and (2) the redistribution of YFP-tagged receptors to the cell's interior, cAMP was found to induce little or no loss of ligand binding (LLB) in vegetative cells. However, the ability to induce LLB increased progressively over the initial 6 hrs of development, reaching ∼70% in cells undergoing aggregation. Despite these reductions in surface binding, detectable cAR1-YFP redistribution could be induced by cAMP only after the cells reached the mound stage (10 hrs) and was found to occur naturally by the ensuing slug stage (18 hrs). Site-directed substitution of a cluster of 5 serines in the receptor's cytoplasmic tail that was previously shown to be the principal site of cAMP-induced cAR1 phosphorylation impaired both LLB and receptor redistribution and furthermore resulted in mound-stage developmental arrest, suggesting that phosphorylation of cAR1 is a prerequisite for its internalization and that cAR1 internalization is required for post-aggregative development. To assess the involvement of clathrin mediated endocytosis, Dictyostelium cells lacking the clathrin light chain gene (clc-) or either of two dynamin genes were examined and found to be defective in LLB and, in the case of clc- cells, also cAR1 redistribution and turnover. Furthermore, cAR1 overexpression in clc- cells (like the serine mutant in wild-type cells) promoted developmental arrest in mounds. The mound-arrest phenotype was also recapitulated in a wild-type background by the specific expression of cAR1 in prestalk cells (but not prespore cells), suggesting that development depends critically on internalization and clearance of cAR1 from these cells. Persistent cAR1 expression following aggregation was found to be associated with aberrant expression of prestalk and prespore genes, which may adversely affect development in the prestalk cell lineage. The PI3 kinase-TORC2 signal transduction pathway, known to be important for Dictyostelium chemotaxis and internalization of yeast pheromone receptors, was examined using chemical inhibitors and null cells and found to be necessary for cAR1 internalization. In conclusion, cAR1 was shown to be similar to other GPCRs in that its internalization depends on phosphorylation of cytoplasmic domain serines, utilizes clathrin and dynamin, and involves the TORC2 complex. In addition, the findings presented here that cAR1 internalization is both developmentally regulated and required for normal development represent a novel regulatory paradigm that might pertain to other GPCRs known to play important roles in the development of humans and other metazoans. ^

The INO80 chromatin remodeling complex is involved in the nucleotide excision repair pathway

The genomic DNA of eukaryotic cells is well organized into chromatin structures. However, these repressed structures present barriers that block the access of regulatory factors to the genome during various nuclear events. To overcome the obstacle, two major cellular processes, post-modification of histone tails and ATP-dependent chromatin remodeling, are involved in reconfiguring chromatin structure and creating accessible DNA. Despite the current research progress, much remains to be explored concerning the relationship between chromatin remodeling and DNA repair. Recently, one member of the ATP-dependent chromatin remodeling complexes, INO80, has been found to play a crucial role in DNA damage repair. However, the functions of this complex in higher eukaryotes have yet to be determined. The goal of my study is to generate a human somatic INO80 conditional knockout model and investigate the functions of Ino80 in damage repair.^ By homologous targeting of the INO80 locus in human HCT116 colon epithelial cells, I established a human somatic INO80 conditional knockout model. I have demonstrated that the conditional INO80 cells exhibited a sufficiently viable period when the INO80 protein is removed. Moreover, I found that loss of INO80 resulted in deficient UV lesion repair in response to UV while the protein levels of the NER factors such as XPC, XPA, XPD were not affected. And in vitro repair synthesis assay showed that the NER incision and repair synthesis activities were intact in the absence of INO80. Examination on the damage recognition factor XPC showed its recruitment to damage sites was impaired in the INO80 mutant cells. Loss of INO80 also led to reduced enrichment of XPA at the site of UV lesions. Despite the reduced recruitment of XPC and XPA observed in INO80 mutants, no direct interaction was detected. Meanwhile, direct interaction between INO80 and DDB1, the initial UV lesion detector, was detected by coimmunoprecipitation. UV-induced chromosome relaxation was reduced in cells devoid of INO80. These results demonstrate the INO80 complex may participates in the NER by interacting with DDB1 and having a critical role of in creating DNA accessibility for the nucleotide excision pathway. ^

From protein microarray to biology: The discovery of epigenetic regulatory molecules

Chromatin, composed of repeating nucleosome units, is the genetic polymer of life. To aid in DNA compaction and organized storage, the double helix wraps around a core complex of histone proteins to form the nucleosome, and is therefore no longer freely accessible to cellular proteins for the processes of transcription, replication and DNA repair. Over the course of evolution, DNA-based applications have developed routes to access DNA bound up in chromatin, and further, have actually utilized the chromatin structure to create another level of complexity and information storage. The histone molecules that DNA surrounds have free-floating tails that extend out of the nucleosome. These tails are post-translationally modified to create docking sites for the proteins involved in transcription, replication and repair, thus providing one prominent way that specific genomic sequences are accessed and manipulated. Adding another degree of information storage, histone tail-modifications paint the genome in precise manners to influence a state of transcriptional activity or repression, to generate euchromatin, containing gene-dense regions, or heterochromatin, containing repeat sequences and low-density gene regions. The work presented here is the study of histone tail modifications, how they are written and how they are read, divided into two projects. Both begin with protein microarray experiments where we discover the protein domains that can bind modified histone tails, and how multiple tail modifications can influence this binding. Project one then looks deeper into the enzymes that lay down the tail modifications. Specifically, we studied histone-tail arginine methylation by PRMT6. We found that methylation of a specific histone residue by PRMT6, arginine 2 of H3, can antagonize the binding of protein domains to the H3 tail and therefore affect transcription of genes regulated by the H3-tail binding proteins. Project two focuses on a protein we identified to bind modified histone tails, PHF20, and was an endeavor to discover the biological role of this protein. Thus, in total, we are looking at a complete process: (1) histone tail modification by an enzyme (here, PRMT6), (2) how this and other modifications are bound by conserved protein domains, and (3) by using PHF20 as an example, the functional outcome of binding through investigating the biological role of a chromatin reader. ^