N-terminal Interaction Research Articles

Effects of Myosin Binding Protein-C Isoform, Phosphorylation, and Domains on the Rotational Dynamics of Actin Filaments

We have determined the effects of Myosin binding protein-C (MyBP-C) and its domains on the microsecond time-scale rotational dynamics of actin, using time-resolved phosphorescence anisotropy (TPA). MyBP-C is a multi-domain thick filament-associated modulator of striated muscle contraction, spanning the interfilament spacing to contact both myosin and actin. Cardiac (c-) and slow skeletal (ss-) MyBP-C are known substrates for Protein kinase-A (PKA), and phosphorylation of cMyBP-C alters contractile properties and myofilament structure. To determine the effects of MyBP-C on actin's microsecond structural dynamics, we labeled actin at C374 with erythrosine iodoacetamide and performed TPA experiments. The interaction of all three MyBP-C isoforms with actin increased the final anisotropy (r∞) of the TPA decay in a concentration-dependent manner, indicating restriction of the rotational amplitude of actin dynamics. While PKA phosphorylation had little effect on fast skeletal (fs-) MyBP-C, phosphorylation of cMyBP-C and ssMyBP-C nearly eliminated the effects of these proteins to restrict actin dynamics, despite no change in binding affinity with phosphorylation. Skeletal MyBP-C (C1-C10) affected actin anisotropy at lower concentrations than cMyBP-C (C0-C10), suggesting that skeletal and cardiac N-terminal MyBP-C interactions with actin have distinct properties. The effects of truncated cMyBP-C on actin anisotropy determined that C-terminal domains are important for restricting rotational dynamics, whereas N-terminal domains are important for regulating this effect. These MyBP-C-induced changes in actin dynamics may play a role in the known effects of MyBP-C on the functional actin-myosin interaction. This work was funded by grants from NIH (F32 HL107039-01 to BAC and T32 AR007612 to DDT).

The N-Terminal Domain of the Escherichia coli PriA Helicase Contains Both the DNA- and Nucleotide-Binding Sites. Energetics of Domain–DNA Interactions and Allosteric Effect of the Nucleotide Cofactors

Functional interactions of the Escherichia coli PriA helicase 181N-terminal domain with the DNA and nucleotide cofactors have been quantitatively examined. The isolated 181N-terminal domain forms a stable dimer in solution, most probably reflecting the involvement of the domain in specific cooperative interactions of the intact PriA protein--double-stranded DNA (dsDNA) complex. Only one monomer of the domain dimer binds the DNA; i.e., the dimer has one effective DNA-binding site. Although the total site size of the dimer--single-stranded DNA (ssDNA) complex is ~13 nucleotides, the DNA-binding subsite engages in direct interactions with approximately five nucleotides. A small number of interacting nucleotides indicates that the DNA-binding subsites of the PriA helicase, i.e., the strong subsite on the helicase domain and the weak subsite on the N-terminal domain, are spatially separated in the intact enzyme. Contrary to current views, the subsite has an only slight preference for the 3'-end OH group of the ssDNA and lacks any significant base specificity, although it has a significant dsDNA affinity. Unlike the intact helicase, the DNA-binding subsite of the isolated domain is in an open conformation, indicating the presence of the direct helicase domain--N-terminal domain interactions. The discovery that the 181N-terminal domain possesses a nucleotide-binding site places the allosteric, weak nucleotide-binding site of the intact PriA on the N-terminal domain. The specific effect of ADP on the domain DNA-binding subsite indicates that in the intact helicase, the bound ADP not only opens the DNA-binding subsite but also increases its intrinsic DNA affinity.

Amelogenin Supramolecular Assembly in Nanospheres Defined by a Complex Helix-Coil-PPII Helix 3D-Structure

Tooth enamel, the hardest material in the human body, is formed within a self-assembled matrix consisting mostly of amelogenin proteins. Here we have determined the complete mouse amelogenin structure under physiological conditions and defined interactions between individual domains. NMR spectroscopy revealed four major amelogenin structural motifs, including an N-terminal assembly of four α-helical segments (S9-V19, T21-P33, Y39-W45, V53-Q56), an elongated random coil region interrupted by two 310 helices (∼P60-Q117), an extended proline-rich PPII-helical region (P118-L165), and a charged hydrophilic C-terminus (L165-D180). HSQC experiments demonstrated ipsilateral interactions between terminal domains of individual amelogenin molecules, i.e. N-terminal interactions with corresponding N-termini and C-terminal interactions with corresponding C-termini, while the central random coil domain did not engage in interactions. Our HSQC spectra of the full-length amelogenin central domain region completely overlapped with spectra of the monomeric Amel-M fragment, suggesting that the central amelogenin coil region did not involve in assembly, even in assembled nanospheres. This finding was confirmed by analytical ultracentrifugation experiments. We conclude that under conditions resembling those found in the developing enamel protein matrix, amelogenin molecules form complex 3D-structures with N-terminal α-helix-like segments and C-terminal PPII-helices, which self-assemble through ipsilateral interactions at the N-terminus of the molecule.

P2.17 Fukutin-related protein resides in the Golgi cisternae of human skeletal muscle fibres and forms disulfide-linked homodimers via an N-terminal interaction interface

P2.17 Fukutin-related protein resides in the Golgi cisternae of human skeletal muscle fibres and forms disulfide-linked homodimers via an N-terminal interaction interface

Fukutin-Related Protein Resides in the Golgi Cisternae of Skeletal Muscle Fibres and Forms Disulfide-Linked Homodimers via an N-Terminal Interaction

Limb-Girdle Muscular Dystrophy type 2I (LGMD2I) is an inheritable autosomal, recessive disorder caused by mutations in the FuKutin-Related Protein (FKRP) gene (FKRP) located on chromosome 19 (19q13.3). Mutations in FKRP are also associated with Congenital Muscular Dystrophy (MDC1C), Walker-Warburg Syndrome (WWS) and Muscle Eye Brain disease (MEB). These four disorders share in common an incomplete/aberrant O-glycosylation of the membrane/extracellular matrix (ECM) protein α-dystroglycan. However, further knowledge on the FKRP structure and biological function is lacking, and its intracellular location is controversial. Based on immunogold electron microscopy of human skeletal muscle sections we demonstrate that FKRP co-localises with the middle-to-trans-Golgi marker MG160, between the myofibrils in human rectus femoris muscle fibres. Chemical cross-linking experiments followed by pairwise yeast 2-hybrid experiments, and co-immune precipitation, demonstrate that FKRP can exist as homodimers as well as in large multimeric protein complexes when expressed in cell culture. The FKRP homodimer is kept together by a disulfide bridge provided by the most N-terminal cysteine, Cys6. FKRP contains N-glycan of high mannose and/or hybrid type; however, FKRP N-glycosylation is not required for FKRP homodimer or multimer formation. We propose a model for FKRP which is consistent with that of a Golgi resident type II transmembrane protein.

The N Terminus of the Adhesion G Protein-coupled Receptor GPR56 Controls Receptor Signaling Activity

GPR56 is an adhesion G protein-coupled receptor that plays a key role in cortical development. Mutations to GPR56 in humans cause malformations of the cerebral cortex, but little is known about the normal function of the receptor. We found that the large N terminus (NT) of GPR56 is cleaved from the rest of the receptor during processing but remains non-covalently associated with the seven-transmembrane region of the receptor, as indicated by coimmunoprecipitation of the two GPR56 fragments from both transfected cells and native tissue. We also found that truncation of the GPR56 NT results in constitutive activation of receptor signaling, as revealed by increased GPR56-stimulated signaling upon transfection of HEK-293 cells with truncated GPR56, greatly enhanced binding of β-arrestins by truncated GPR56 relative to the full-length receptor, extensive ubiquitination of truncated GPR56, and cytotoxicity induced by truncated GPR56 that could be rescued by cotransfection of cells with β-arrestin 2. Furthermore, we found that the GPR56 NT is capable of homophilic trans-trans interactions that enhance receptor signaling activity. On the basis of these findings, we suggest a model of receptor activation in which the large N terminus of GPR56 constrains receptor activity but N-terminal interactions (GPR56 NT with an extracellular ligand and/or GPR56 NT homophilic trans-trans associations) can remove this inhibitory influence of the N terminus to activate receptor signaling.

Calpain regulates N-terminal interaction of GSK-3β with 14-3-3ζ, p53 and PKB but not with axin

Calpain produces a truncation of GSK3β that removes the N-terminal inhibitory domain. Here we analyze the effect of that truncation on protein–protein interaction. We pulled down GST-tagged proteins in the presence of full length GSK-3β and calpain-cleaved GSK-3β. Commercial GSK-3β was first incubated with calpain for 2.5min in vitro, and then with GST-tagged proteins in the presence of calpeptin, a synthetic calpain inhibitor. Western blot analyses were performed to determine if there is an interaction between these GST-tagged proteins and truncated GSK-3β. Using axin GST-tagged, we pulled down the protein in the presence of full length GSK-3β and calpain-cleaved GSK-3β. Western blot analyses showed full length GSK-3β in the pellet as well GSK-3β cleaved by calpain. Thus axin was able to bind GSK-3β without the N-terminal end. When the same experiment was carried out with GST-tagged 14-3-3ζ, p53 and PKB, full length GSK-3β was observed in the pellet, but GSK-3β truncated by calpain was not pulled down demonstrating that GSK-3β N-terminal end is necessary to interact with these three proteins. Our data demonstrate that N-terminal end is necessary for 14-3-3ζ, p53 and PKB interaction. However, the interaction of GSK3β with axin is not altered by calpain. These data support a physiological role for GSK3β truncation mediated by calpain.

Aquaporins at a glance

With a concentration of 55,000 mM, water is by far the most prevalent molecule in biological systems. For many years it was assumed that biological membranes are freely water permeable, so that the existence of a family of water channels would not have been predicted. However, biophysical studies in

Abstract LB-128: The N-terminus of the ASPP2 tumor suppressor binds Ras and enhances Ras/Raf/MEK activation to promote oncogene-induced cellular senescence

Abstract The ASPP2 tumor suppressor is a pro-apoptotic member of a family of p53 binding proteins. It functions in part by selectively enhancing p53 transcriptional activation of pro-apoptotic target genes. Although the ASPP2 C-terminal domain binds the p53 core domain, little is known about the physical and functional interactions of other proteins to the highly structured ASPP2 N-terminus. Moreover, mounting evidence from our lab and others demonstrate that ASPP2 also mediates non-apoptotic p53-independent functions. However, the molecular mechanisms remain poorly understood. Previous reports have found that ASPP2 can localize to the cytoplasm and have identified a potential Ras-association domain in the ASPP2 N-terminus. Therefore, we wondered if novel ASPP2 functions could be mediated by ASPP2 N-terminal interaction with cytoplasmic Ras. Using co-immunoprecipitation and immunofluorescence microscopy, we identified ASPP2 in a complex with active Ras on the plasma membrane. Moreover, we also found that this interaction required the ASPP2 N-terminus since BBP, a naturally occurring alternatively spliced ASPP2 isoform lacking the N-terminus, showed markedly reduced ability to bind Ras. To determine if ASPP2 could modulate Ras function, we ectopically expressed ASPP2 in cells and found enhancement of Ras activation and subsequent stimulation of ERK phosphorylation. Conversely, ASPP2 knockdown by siRNA drastically reduced ERK phosphorylation and activation by EGF. Although it is oncogenic in tumor cells, constitutive Ras activation in normal cells leads to premature senescence in a Raf-MEK-ERK activation dependent manner. To explore if ASPP2 enhanced senescence, we knocked down ASPP2 expression with siRNA and found that senescence induced by RasV12 lentiviral infection in normal human fibroblasts was significantly attenuated. This was similar to the attenuation of senescence achieved by the MEK inhibitor PD98059. Together, our results demonstrate a novel function of the ASPP2 N-terminus in promoting cellular senescence via Ras-interaction and stimulation of Raf-MEK-ERK activity. These findings suggest a novel mechanism by which ASPP2-induced senescence may mediate its tumor suppression ability, and opens new avenues for investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-128. doi:10.1158/1538-7445.AM2011-LB-128

Inter- and intra-molecular interactions of Arabidopsis thaliana DELLA protein RGL1

The phytohormone gibberellin and the DELLA proteins act together to control key aspects of plant development. Gibberellin induces degradation of DELLA proteins by recruitment of an F-box protein using a molecular switch: a gibberellin-bound nuclear receptor interacts with the N-terminal domain of DELLA proteins, and this event primes the DELLA C-terminal domain for interaction with the F-box protein. However, the mechanism of signalling between the N- and C-terminal domains of DELLA proteins is unresolved. In the present study, we used in vivo and in vitro approaches to characterize di- and tri-partite interactions of the DELLA protein RGL1 (REPRESSOR OF GA1-3-LIKE 1) of Arabidopsis thaliana with the gibberellin receptor GID1A (GIBBERELLIC ACID-INSENSITIVE DWARF-1A) and the F-box protein SLY1 (SLEEPY1). Deuterium-exchange MS unequivocally showed that the entire N-terminal domain of RGL1 is disordered prior to interaction with the GID1A; furthermore, association/dissociation kinetics, determined by surface plasmon resonance, predicts a two-state conformational change of the RGL1 N-terminal domain upon interaction with GID1A. Additionally, competition assays with monoclonal antibodies revealed that contacts mediated by the short helix Asp-Glu-Leu-Leu of the hallmark DELLA motif are not essential for the GID1A–RGL1 N-terminal domain interaction. Finally, yeast two- and three-hybrid experiments determined that unabated communication between N- and C-terminal domains of RGL1 is required for recruitment of the F-box protein SLY1.

N-terminal Half of the cGMP Phosphodiesterase γ-Subunit Contributes to Stabilization of the GTPase-accelerating Protein Complex

In the visual signal terminating transition state, the cyclic GMP phosphodiesterase (PDE6) inhibitory γ-subunit (PDEγ) stimulates GTPase activity of the α-subunit of transducin (αt) by enhancing the interaction between αt and its regulator of G protein signaling (RGS9), which is constitutively bound to the type 5 G protein β-subunit (β5). Although it is known from a crystal structure of partial molecules that the PDEγ C terminus contacts with both αt and RGS9, contributions from the intrinsically disordered PDEγ N-terminal half remain unclear. In this study, we were able to investigate this issue using a photolabel transfer strategy that allows for mapping the interface of full-length proteins. We observed label transfer from PDEγ N-terminal positions 50, 30, and 16 to RGS9·β5 in the GTPase-accelerating protein (GAP) complex composed of PDEγ·αt·RGS9·β5. In support of a direct PDEγ N-terminal interaction with RGS9·β5, the PDEγ N-terminal peptide PDEγ(1-61) abolished label transfer to RGS9·β5, and another N-terminal peptide, PDEγ(10-30), disassembled the GAP complex in label transfer and pulldown experiments. Furthermore, we determined that the PDEγ C-terminal interaction with αt was enhanced whereas the N-terminal interaction was weakened upon changing the αt conformation from the signaling state to the transition state. This "rearrangement" of PDEγ domain interactions with αt appears to facilitate the interaction of the PDEγ N-terminal half with RGS9·β5 and hence its contribution to optimal stabilization of the GAP complex.

Small molecule inhibitor of the RPA70 N-terminal protein interaction domain discovered using in silico and in vitro methods

The pharmacological suppression of the DNA damage response and DNA repair can increase the therapeutic indices of conventional chemotherapeutics. Replication Protein A (RPA), the major single-stranded DNA binding protein in eukaryotes, is required for DNA replication, DNA repair, DNA recombination, and DNA damage response signaling. Through the use of high-throughput screening of 1500 compounds, we have identified a small molecule inhibitor, 15-carboxy-13-isopropylatis-13-ene-17,18-dioic acid (NSC15520), that inhibited both the binding of Rad9–GST and p53–GST fusion proteins to the RPA N-terminal DNA binding domain (DBD), interactions that are essential for robust DNA damage signaling. NSC15520 competitively inhibited the binding of p53-GST peptide with an IC 50 of 10 μM. NSC15520 also inhibited helix destabilization of a duplex DNA (dsDNA) oligonucleotide, an activity dependent on the N-terminal domain of RPA70. NSC15520 did not inhibit RPA from binding single-stranded oligonucleotides, suggesting that the action of this inhibitor is specific for the N-terminal DBD of RPA, and does not bind to DBDs essential for single-strand DNA binding. Computer modeling implicates direct competition between NSC15520 and Rad9 for the same binding surface on RPA. Inhibitors of protein–protein interactions within the N-terminus of RPA are predicted to act synergistically with DNA damaging agents and inhibitors of DNA repair. Novel compounds such as NSC15520 have the potential to serve as chemosensitizing agents.

Mitotic Inhibition of GRASP65 Organelle Tethering Involves Polo-like Kinase 1 (PLK1) Phosphorylation Proximate to an Internal PDZ Ligand

GRASP65 links cis-Golgi cisternae via a homotypic, N-terminal PDZ interaction, and its mitotic phosphorylation disrupts this activity. Neither the identity of the PDZ ligand involved in the GRASP65 self-interaction nor the mechanism by which phosphorylation inhibits its interaction is known. Phospho-mimetic mutation of known cyclin-dependent kinase 1/cyclin B sites, all of which are in the C-terminal "regulatory domain" of the molecule, failed to block organelle tethering. However, we identified a site phosphorylated by Polo-like kinase 1 (PLK1) in the GRASP65 N-terminal domain for which mutation to aspartic acid blocked tethering and alanine substitution prevented mitotic Golgi unlinking. Further, using interaction assays, we discovered an internal PDZ ligand adjacent to the PLK phosphorylation site that was required for tethering. These results reveal the mechanism of phosphoinhibition as direct inhibition by PLK1 of the PDZ ligand underlying the GRASP65 self-interaction.

Novel Interactions of CAPS (Ca2+-dependent Activator Protein for Secretion) with the Three Neuronal SNARE Proteins Required for Vesicle Fusion

CAPS (aka CADPS) is required for optimal vesicle exocytosis in neurons and endocrine cells where it functions to prime the exocytic machinery for Ca(2+)-triggered fusion. Fusion is mediated by trans complexes of the SNARE proteins VAMP-2, syntaxin-1, and SNAP-25 that bridge vesicle and plasma membrane. CAPS promotes SNARE complex formation on liposomes, but the SNARE binding properties of CAPS are unknown. The current work revealed that CAPS exhibits high affinity binding to syntaxin-1 and SNAP-25 and moderate affinity binding to VAMP-2. CAPS binding is specific for a subset of exocytic SNARE protein isoforms and requires membrane integration of the SNARE proteins. SNARE protein binding by CAPS is novel and mediated by interactions with the SNARE motifs in the three proteins. The C-terminal site for CAPS binding on syntaxin-1 does not overlap the Munc18-1 binding site and both proteins can co-reside on membrane-integrated syntaxin-1. As expected for a C-terminal binding site on syntaxin-1, CAPS stimulates SNARE-dependent liposome fusion with N-terminal truncated syntaxin-1 but exhibits impaired activity with C-terminal syntaxin-1 mutants. Overall the results suggest that SNARE complex formation promoted by CAPS may be mediated by direct interactions of CAPS with each of the three SNARE proteins required for vesicle exocytosis.

Experimental evidence that the membrane-spanning helix of PufX adopts a bent conformation that facilitates dimerisation of the Rhodobacter sphaeroides RC–LH1 complex through N-terminal interactions

The PufX polypeptide is an integral component of some photosynthetic bacterial reaction center-light harvesting 1 (RC–LH1) core complexes. Many aspects of the structure of PufX are unresolved, including the conformation of its long membrane-spanning helix and whether C-terminal processing occurs. In the present report, NMR data recorded on the Rhodobacter sphaeroides PufX in a detergent micelle confirmed previous conclusions derived from equivalent data obtained in organic solvent, that the α-helix of PufX adopts a bent conformation that would allow the entire helix to reside in the membrane interior or at its surface. In support of this, it was found through the use of site-directed mutagenesis that increasing the size of a conserved glycine on the inside of the bend in the helix was not tolerated. Possible consequences of this bent helical structure were explored using a series of N-terminal deletions. The N-terminal sequence ADKTIFNDHLN on the cytoplasmic face of the membrane was found to be critical for the formation of dimers of the RC–LH1 complex. It was further shown that the C-terminus of PufX is processed at an early stage in the development of the photosynthetic membrane. A model in which two bent PufX polypeptides stabilise a dimeric RC–LH1 complex is presented, and it is proposed that the N-terminus of PufX from one half of the dimer engages in electrostatic interactions with charged residues on the cytoplasmic surface of the LH1α and β polypeptides on the other half of the dimer.

N‐terminal fusion of a hyperthermophilic chitin‐binding domain to xylose isomerase from Thermotoga neapolitana enhances kinetics and thermostability of both free and immobilized enzymes

Immobilization of a thermostable D-xylose isomerase (EC 5.3.1.5) from Thermotoga neapolitana 5068 (TNXI) on chitin beads was accomplished via a N-terminal fusion with a chitin-binding domain (CBD) from a hyperthermophilic chitinase produced by Pyrococcus furiosus (PF1233) to create a fusion protein (CBD-TNXI). The turnover numbers for glucose to fructose conversion for both unbound and immobilized CBD-TNXI were greater than the wild-type enzyme: k(cat) (min(-1)) was approximately 1,000, 3,800, and 5,800 at 80 degrees C compared to 1,140, 10,350, and 7,000 at 90 degrees C, for the wild-type, unbound, and immobilized enzymes, respectively. These k(cat) values for the glucose to fructose isomerization measured are the highest reported to date for any XI at any temperature. Enzyme kinetic inactivation at 100 degrees C, as determined from a bi-phasic inactivation model, showed that the CBD-TNXI bound to chitin had a half-life approximately three times longer than the soluble wild-type TNXI (19.9 hours vs. 6.8 hours, respectively). Surprisingly, the unbound soluble CBD-TNXI had a significantly longer half-life (56.5 hours) than the immobilized enzyme. Molecular modeling results suggest that the N-terminal fusion impacted subunit interactions, thereby contributing to the enhanced thermostability of both the unbound and immobilized CBD-TNXI. These interactions likely also played a role in modifying active site structure, thereby diminishing substrate-binding affinities and generating higher turnover rates in the unbound fusion protein.

Removal of the Cardiac Troponin I N-terminal Extension Improves Cardiac Function in Aged Mice

The cardiac troponin I (cTnI) isoform contains a unique N-terminal extension that functions to modulate activation of cardiac myofilaments. During cardiac remodeling restricted proteolysis of cTnI removes this cardiac specific N-terminal modulatory extension to alter myofilament regulation. We have demonstrated expression of the N-terminal-deleted cTnI (cTnI-ND) in the heart decreased the development of the cardiomyopathy like phenotype in a beta-adrenergic-deficient transgenic mouse model. To investigate the potential beneficial effects of cTnI-ND on the development of naturally occurring cardiac dysfunction, we measured the hemodynamic and biochemical effects of cTnI-ND transgenic expression in the aged heart. Echocardiographic measurements demonstrate cTnI-ND transgenic mice exhibit increased systolic and diastolic functions at 16 months of age compared with age-matched controls. This improvement likely results from decreased Ca(2+) sensitivity and increased cross-bridge kinetics as observed in skinned papillary bundles from young transgenic mice prior to the effects of aging. Hearts of cTnI-ND transgenic mice further exhibited decreased beta myosin heavy chain expression compared to age matched non-transgenic mice as well as altered cTnI phosphorylation. Finally, we demonstrated cTnI-ND expressed in the heart is not phosphorylated indicating the cTnI N-terminal is necessary for the higher level phosphorylation of cTnI. Taken together, our data suggest the regulated proteolysis of cTnI during cardiac stress to remove the unique cardiac N-terminal extension functions to improve cardiac contractility at the myofilament level and improve overall cardiac function.

The Fission Yeast Inhibitor of Growth (ING) Protein Png1p Functions in Response to DNA Damage

In budding yeast and human cells, ING (inhibitor of growth) tumor suppressor proteins play important roles in response to DNA damage by modulating chromatin structure through collaborating with histone acetyltransferase or histone deacetylase complexes. However, the biological functions of ING family proteins in fission yeast are poorly defined. Here, we report that Png1p, a fission yeast ING homolog protein, is required for cell growth under normal and DNA-damaged conditions. Png1p was further confirmed to regulate histone H4 acetylation through collaboration with the MYST family histone acetyltransferase 1 (Mst1). Additionally, both fission yeast PNG1 and MST1 can functionally complement their budding yeast correspondence homologs YNG2 and ESA1, respectively. These results suggest that ING proteins in fission yeast might also conserve function, similar to ING proteins in budding yeast and human cells. We also showed that decreased acetylation in Deltapng1 cells resulted in genome-wide down-regulation of 756 open reading frames, including the central DNA repair gene RAD22. Overexpression of RAD22 partially rescued the png1 mutant phenotype under both normal and DNA-damaged conditions. Furthermore, decreased expression of RAD22 in Deltapng1 cells was confirmed to be caused by decreased H4 acetylation at its promoter. Altogether, these results indicate that Png1p is required for histone H4 acetylation and functions upstream of RAD22 in the DNA damage response pathway.

Abstract 3780: Plumbagin a non-toxic natural agent, induces apoptosis and inhibits the growth of prostate cancer LNCaP and C4-2 cells via blocking of both androgen and non-androgen activation of androgen receptor signaling

Abstract Prostate cancer (PCa) is one of the most commonly diagnosed cancers and leading cause of cancer related death in men in Western countries including the Unites States. Androgen receptor (AR)-mediated signaling plays a critical role in the development and progression of PCa. Most androgen independent (AI) prostate tumors continue to express AR as well as its target gene PSA, which indicates that these tumor cells maintain functional AR signaling. Evidence suggests that AR signaling is activated through androgen and androgen bypass mechanism. We believe that identification of non-toxic natural agents capable of blocking both androgen and non-androgen activation of AR signaling could be effective for the treatment of PCa. We have previously shown that a novel plant derived napthoquinone plumbagin (PL) induces apoptosis and inhibits growth of PCa cells in in vitro as well as in in vivo model systems (Cancer Research 2008, PMID: 18974148). We present here that PL inhibits both androgen and non-androgen activation of AR signaling in PCa cells. We observed that PL inhibits synthetic androgen methyltrienolone (R1881) induced growth of androgen-dependent (AD) LNCaP and AI C4-2 cells. PL treatment was found to inhibit protein levels of AR and AR-target gene PSA and accelerates AR degradation. To analyze the effect of PL on decreased AR transactivation, transient transfections using androgen responsive reporters PSA-luc were carried out. Treatment of LNCaP and C4-2 cells with R1881 led to induction of PSA reporter in these cells. Co-treatment of LNCaP and C4-2 cells with PL led to a dose-dependent decrease in AR transactivation as reflected by a decrease in the PSA reporter activity. These results suggest that PL interferes with androgen-induced transactivation function of AR. It has been reported that IL-6 signaling acts as a ligand for the activation of AR signaling. IL-6 induced phosphorylated Stat3 interacts with the N-terminal domain of AR and activates its target genes. We have previously shown that PL treatment inhibits Stat3 phosphorylation in PCa DU-145 cells xenograft tumor tissues (Cancer Research 2008, PMID: 18974148). Therefore, we hypothesized that PL treatment will inhibit Stat3 and AR N-terminal domain interaction in PCa cells. Reciprocal immunoprecipitation/blotting experiments revealed that PL treatment inhibited IL-6 induced interaction of Stat3 with AR N-terminal domain in both the PCa LNCaP and C4-2 cells. PL treatment also inhibited IL-6 induced Tyr 705 and Ser727 phosphorylation of Stat3. These results establish that PL has the potential to block the androgen and non-androgen activation of AR signaling in PCa and provide further evidence that PL could be used as a chemopreventive natural agent in ongoing anti-hormone therapy of PCa. (Support: NIH grants, CA35368). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3780.

Aquaporin-4 (AQP4) Associations and Array Dynamics Probed by Photobleaching and Single-molecule Analysis of Green Fluorescent Protein-AQP4 Chimeras

The plasma membrane assembly of aquaporin-4 (AQP4) water channels into orthogonal arrays of particles (OAPs) involves interactions of AQP4 N-terminal domains. To study in live cells the site of OAP assembly, the size and dynamics of plasma membrane OAPs, and the heterotetrameric associations of AQP4, we constructed green fluorescent protein (GFP)-labeled AQP4 "long" (M1) and "short" (M23) isoforms in which GFP was inserted at the cytoplasm-facing N or C terminus or between Val-141 and Val-142 in the second extracellular loop of AQP4. The C-terminal and extracellular loop GFP insertions did not interfere with the rapid unrestricted membrane diffusion of GFP-labeled M1 or the restricted diffusion and OAP assembly of GFP-labeled M23. Photobleaching of brefeldin A-treated cells showed comparable and minimally restricted diffusion of M1 and M23, indicating that OAP assembly occurs post-endoplasmic reticulum. Single-molecule step photobleaching and intensity analysis of GFP-labeled M1 in the absence versus presence of excess unlabeled M1 or M23 with an OAP-disrupting mutation indicated heterotetrameric AQP4 association. Time-lapse total internal reflection fluorescence imaging of M23 in live cells at 37 degrees C indicated that OAPs diffuse slowly (D approximately 10(-12) cm(2)/s) and rearrange over tens of minutes. Our biophysical measurements in live cells thus reveal extensive AQP4 monomer-monomer and tetramer-tetramer interactions.