Naproxen ((S)-6-methoxy-alpha-methyl-2-naphthaleneacetic acid) is a powerful non-selective non-steroidal anti-inflammatory drug that is extensively used as a prescription and over-the-counter medication. Naproxen exhibits gastrointestinal toxicity but its cardiovascular toxicity may be reduced compared to other drugs in its class. Despite the fact that naproxen has been marketed for many years, the molecular basis of its interaction with cyclooxygenase (COX) enzymes is unknown. We performed a detailed study of naproxen-COX-2 interactions using site-directed mutagenesis, structure-activity analysis and X-ray crystallography. The results indicate that each of the pendant groups of the naphthyl scaffold are essential for COX inhibition and only minimal substitutions are tolerated. Mutation of Trp-387 to Phe significantly reduced inhibition by naproxen, a result that appears unique to this inhibitor. Substitution of S or CH2 for the O atom of the p-methoxy group yielded analogs that were not affected by the W387F substitution and that exhibited increased COX-2 selectivity relative to naproxen. Crystallization and X-ray analysis yielded structures of COX-2 complexed to naproxen and its methylthio analog at 1.7 and 2.3 angstrom resolution, respectively. The combination of mutagenesis, structure-analysis, and X-ray crystallography provided comprehensive information on the unique interactions responsible for naproxen binding to COX-2.

Most mammalian cell types depend on multiple Src-family kinases (SFKs) to regulate diverse signaling pathways. Strict control of SFK activity is essential to normal cellular function and loss of kinase regulation contributes to several forms of cancer and other diseases. Previous X-ray crystal structures of the SFKs c-Src and Hck revealed that intramolecular association of their SH3 domains and SH2-kinase linker regions has a key role in downregulation of kinase activity. However, the amino acid sequence of the Hck linker represents a suboptimal ligand for the isolated SH3 domain, suggesting that it may form the PPII helical conformation required for SH3 docking only in the context of the intact structure. To test this hypothesis directly, we determined the crystal structure of a truncated Hck protein consisting of the SH2 and SH3 domains plus the linker. Despite the absence of the kinase domain, the structures and relative orientations of the SH2 and SH3 domains in this shorter protein were very similar to those observed in near-full-length, downregulated Hck. However, the SH2-kinase linker adopted a modified topology and failed to engage the SH3 domain. This new structure supports the idea that these non-catalytic regions work together as a ''conformational switch'' that modulates kinase activity in a manner unique to the SH3 domain and linker topologies present in the intact Hck protein. Our results also provide fresh structural insight into the facile induction of Hck activity by HIV-1 Nef and other Hck SH3-domain binding proteins, and implicate the existence of innate conformational states unique to individual Src-family members that ''fine-tune'' their sensitivities to activation by SH3-based ligands.

Epigenetic silencing of Ras-Association Domain Family (RASSF) genes, RASSF1 and RASSF5 (also called NORE1), by CpG hypermethylation is found frequently in many cancers. While the physiological roles of RASSF1 have been studied in some detail, the exact functions of RASSF5 are not well understood. Here, we show that RASSF5 plays an important role in mediating apoptosis in response to death receptor ligands, Tumor Necrosis Factor-alpha (TNF-alpha) and TNF-related Apoptosis-inducing Ligand (TRAIL). Depletion of RASSF5 by siRNA significantly reduced TNF-alpha mediated apoptosis, likely through its interaction with proapoptotic kinase MST1, a mammalian homolog of Hippo. Consistent with this, siRNA knockdown of MST1 also resulted in resistance to TNF-alpha induced apoptosis. To further study the role of Rassf5 in vivo, we generated Rassf5-deficient mouse. Inactivation of Rassf5 in mouse embryonic fibroblasts (MEFs) resulted in resistance to TNF-alpha- and TRAIL-mediated apoptosis. Importantly, Rassf5-null mice were significantly more resistant to the TNF-alpha induced apoptosis and failed to activate Mst1. Loss of Rassf5 also resulted in spontaneous immortalization of MEFs at earlier passages than the control MEFs, and Rassf5-null immortalized MEFs, but not the immortalized wildtype MEFs, were fully transformed by K-RasG12V. Together, our results demonstrate a direct role for RASSF5 in death receptor ligand-mediated apoptosis and provide further evidence for RASSF5 as a tumor suppressor.

Many organisms use sophisticated systems to acquire growth-limiting iron. Iron-limitation is especially apparent in bacterial pathogens of mammalian hosts where free-iron concentrations are physiologically negligible. A common strategy is to secrete low molecular weight iron-chelators, termed siderophores, and express high affinity receptors for the siderophore-iron complex. Staphylococcus aureus, a widespread pathogen, produces two siderophores, staphyloferrin A (SA) and staphyloferrin B (SB). We have determined the crystal structure of the staphyloferrin B receptor, SirA, at high resolution in both the apo- and Fe(III)-SB (FeSB)-bound forms. SirA, a member of the class III binding protein family of metal receptors, has N- and C-terminal domains, each comprised of mainly a beta-stranded core and alpha-helical periphery. The domains are bridged by a single alpha-helix and together form the FeSB binding site. SB coordinates Fe(III) through five oxygen atoms and one nitrogen atom in distorted octahedral geometry. SirA undergoes conformational change upon siderophore binding, largely securing two loops from the C-terminal domain to enclose FeSB with a low nM dissociation constant. The staphyloferrin A receptor, HtsA, homologous to SirA, also encloses its cognate siderophore (FeSA); however, the largest conformational rearrangements involve a different region of the C-terminal domain. FeSB is uniquely situated in the binding pocket of SirA with few of the contacting residues being conserved with those of HtsA interacting with FeSA. Though both SirA and HtsA bind siderophores from the same alpha-hydroxycarboxylate class, the unique structural features of each receptor provides an explanation for their distinct specificity.

Ubiquitin pathway E3 ligases are an important component conferring specificity and regulation in ubiquitin attachment to substrate proteins. The Arabidopsis thaliana RING (Really Interesting New Gene) domain-containing proteins BRIZ1 and BRIZ2 are essential for normal seed germination and post-germination growth. Loss of either BRIZ1 (At2g42160) or BRIZ2 (At2g26000) results in a severe phenotype. Heterozygous parents produce progeny that segregate 3:1 for wild-type: growth-arrested seedlings. Homozygous T-DNA insertion lines are recovered for BRIZ1 and BRIZ2 after introduction of a transgene containing the respective coding sequence, demonstrating that disruption of BRIZ1 or BRIZ2 in the T-DNA insertion lines is responsible for the observed phenotype. Both proteins have multiple predicted domains in addition to the RING domain; a BRAP2 (BRCA1 associated protein 2), a ZnF UBP (Zinc finger ubiquitin binding protein), and a coiled-coil domain. In vitro, both BRIZ1 and BRIZ2 are active as E3 ligases, but only BRIZ2 binds ubiquitin. In vitro synthesized and purified recombinant BRIZ1 and BRIZ2 preferentially form hetero-oligomers rather than homo-oligomers, and the coiled-coil domain is necessary and sufficient for this interaction. BRIZ1 and BRIZ2 co-purify after expression in tobacco leaves, which also requires the coiled-coil domain. BRIZ1 and BRIZ2 coding regions with substitutions in the RING domain are inactive in vitro and after introduction, fail to complement their respective mutant lines. Our current model is that BRIZ1 and BRIZ2 together are required for formation of a functional ubiquitin E3 ligase in vivo, and this complex is required for germination and early seedling growth.

The Notch signaling pathway is important for cell-fate decisions in embryonic development and adult life. Defining the functional importance of the Notch pathway in these contexts requires the elucidation of essential signal transduction components that have not been fully characterized. Here, we show that Rabconnectin-3B is required for the Notch pathway in mammalian cells. siRNA-mediated silencing of Rabconnectin-3B in mammalian cells attenuated Notch signaling and disrupted the activation and nuclear accumulation of the Notch target Hes1. Rabconnectin-3B knockdown also disrupted V-ATPase activity in mammalian cells, consistent with previous observations in Drosophila. Pharmacological inhibition of the V-ATPase complex significantly reduced Notch signaling in mammalian cells. Finally, Rabconnectin-3B knockdown phenocopied functional disruption of Notch signaling during osteoclast differentiation. Collectively, these findings define an important role for Rabconnectin-3 and V-ATPase activity in the Notch signaling pathway in mammalian cells.

TRIM5alpha is a member of the tripartite motif (TRIM) family of proteins and affects both early and late phases of the retroviral life cycle. Although TRIM5alpha multimerizes to form cytoplasmic bodies, which are thought to play an important role in viral restriction, the identity of TRIM5alpha-containing cytoplasmic bodies remains elusive. To better understand TRIM5alpha cytoplasmic body constituents and the cellular proteins which could be involved in the TRIM5alpha-mediated antiviral activities, we sought TRIM5alpha-binding factors. We identified a lipid microdomain protein flotillin-1/reggie-2 as an interacting partner of TRIM5alpha via co-immunoprecipitation. Immuno-histochemistry studies confirmed the co-localization of rhesus monkey TRIM5alpha (TRIM5alpharh) cytoplasmic bodies with flotillin-1/reggie-2. Caveolin-1, another lipid microdomain-associated protein, also co-localized with TRIM5alpha cytoplasmic bodies. Intriguingly, disruption of cellular cholesterol by cyclodextrin perturbed TRIM5alpha cytoplasmic body formation. Furthermore, lipid starvation partially relieved the endogenous post-entry restriction of HIV-1 infection, which could be subsequently restored by lipid repletion. These observations indicate the involvement of cellular lipids in TRIM5alpha-mediated antiviral activities. Given that many viruses utilize cellular lipid microdomains for viral entry and assembly, it is plausible that lipid-enriched domains provide microenvironments where TRIM5alpha recognizes retroviral components.

Stbd1 is a protein of previously unknown function that is most prevalent in liver and muscle, the major sites for storage of the energy reserve glycogen. The protein is predicted to contain a hydrophobic N-terminus and a C-terminal CBM20 glycan binding domain. Here we show that Stbd1 binds to glycogen in vitro and that endogenous Stbd1 locates to perinuclear compartments in cultured mouse FL83B or Rat1 cells. When over-expressed in COSM9 cells, Stbd1 concentrated at enlarged perinuclear structures, co-localized with glycogen, the late endosomal/lysosomal marker LAMP1 and the autophagy protein GABARAPL1. Mutant Stbd1 lacking the N-terminal hydrophobic segment had a diffuse distribution throughout the cell. Point mutations in the CBM20 domain did not change the perinuclear localization of Stbd1 but glycogen was no longer concentrated in this compartment. Stable over-expression of glycogen synthase in Rat1WT4 cells resulted in accumulation of glycogen as massive perinuclear deposits, where a large fraction of the detectable Stbd1 co-localized. Starvation of Rat1WT4 cells for glucose resulted in dissipation of the massive glycogen stores into numerous and much smaller glycogen deposits that retained Stbd1. In vitro, in cells and in animal models, Stbd1 consistently tracked with glycogen. We conclude that Stbd1 is involved in glycogen metabolism, by binding to glycogen and anchoring it to membranes, thereby affecting its cellular localization and its intra-cellular trafficking to lysosomes.

The SH2 domain-containing inositol 5-phosphatase 1 (SHIP1) dephosphorylates PtdIns-3,4,5-P3 to PtdIns-3,4-P2 in hematopoietic cells to regulate multiple cell signaling pathways. SHIP1 can be phosphorylated by the cyclic AMP-dependent protein kinase (PKA) resulting in an increase in SHIP1 activity (J. Biol. Chem., 284, 20070-8, 2009). Using a combination of approaches, we identified the serine residue regulating SHIP1 activity. After mass spectrometric identification of seventeen serine and threonine residues on SHIP1 as being phosphorylated by PKA in vitro, studies with truncation mutants of SHIP1 narrowed the phosphorylation site to the catalytic region between residues 400 - 866. Of the two candidate phosphorylation sites located in this region (Ser440 and Ser774), only mutation of Ser440 to Ala abolished the ability of PKA to phosphorylate the purified, catalytic domain of SHIP1 (resides 401-866). Mutation of Ser440 to Ala in full length SHIP1 abrogated the ability of PKA to increase the activity of SHIP1 in mammalian cells. Using flow cytometry we found that the PKA activator, Sp-adenosine 3,5-cyclic monophosphorothioate (Sp-cAMPS) blunted the phosphorylation of Akt downstream of B cell antigen receptor engagement in SHIP1-null DT40 B lymphocytes expressing native mouse SHIP1. The inhibitory effect of Sp-cAMPS was absent in cells expressing the S440A mutant of SHIP1. These results suggest that activation of SHIP1 by PKA via phosphorylation on Ser440 is an important regulatory event in hematopoietic cells.

Genomic integrity is maintained by the coordinated interaction of many DNA damage response pathways, including checkpoints, DNA repair processes, and cell cycle restart. In Saccharomyces cerevisiae,, the BRCT domain-containing protein Rtt107/Esc4 is required for restart of DNA replication after successful repair of DNA damage, and for cellular resistance to DNA damaging agents. Rtt107 and its interaction partner Slx4 are phosphorylated during the initial phase of DNA damage response by the checkpoint kinases Mec1 and Tel1. Since the natural chromatin template plays an important role during the DNA damage response, we tested whether chromatin modifications affected the requirement for Rtt107 and Slx4 during DNA damage repair. Here, we report that the sensitivity to DNA damaging agents of rtt107Delta and slx4Delta mutants was rescued by inactivation of the chromatin regulatory pathway leading to H3 K79 trimethylation. Further analysis revealed that lack of Dot1, the H3 K79 methyltransferase, led to activation of the translesion synthesis pathway, thereby allowing the survival in the presence of DNA damage. The DNA damage-induced phosphorylation of Rtt107 and Slx4, which was mutually dependent, were not restored in the absence of Dot1. The antagonistic relationship between Rtt107 and Dot1 was specific for DNA damage-induced phenotypes, whereas the genomic instability caused by loss of Rtt107 was not rescued. These data revealed a multifaceted functional relationship between Rtt107 and Dot1 in the DNA damage response and maintenance of genome integrity.

A meta-cleavage pathway for the aerobic degradation of aromatic hydrocarbons is catalyzed by extradiol dioxygenases via a two-step mechanism: catechol substrate binding and dioxygen incorporation. The binding of substrate triggers the release of water, thereby opening a coordination site for molecular oxygen. The crystal structures of AkbC, a type I extradiol dioxygenase, and the enzyme-substrate (3-methylcatechol) complex revealed the substrate-binding process of extradiol dioxygenase. AkbC is composed of an N-domain and an active C-domain, which contains iron coordinated by a 2-His-1-carboxylate facial triad motif. The C-domain includes a beta-hairpin structure and a C-terminal tail. In substrate-bound AkbC, 3-methylcatechol interacts with the iron via a single hydroxyl group, which represents an intermediate stage in the substrate-binding process. Structure-based mutagenesis revealed that the C-terminal tail and beta-hairpin form part of the substrate-binding pocket that is responsible for substrate specificity by blocking substrate entry. Once a substrate enters the active site, these structural elements also play a role in the correct positioning of the substrate. Based on the results presented here, a putative substrate-binding mechanism is proposed.

Several mammalian forkhead transcription factors have been shown to impact on cell cycle regulation and are themselves linked to cell cycle control systems. Here we have investigated the little studied mammalian forkhead transcription factor FOXK2 and demonstrate that it is subject to control by cell cycle-regulated protein kinases. FOXK2 exhibits a periodic rise in its phosphorylation levels during the cell cycle, with hyperphosphorylation occurring in mitotic cells. Hyperphosphorylation occurs in a CDK-cyclin-dependent manner with CDK1/CLNB as the major kinase complex, although CDK2 and CLNA also appear to be important. We have mapped two CDK phosphorylation sites, serines 368 and 423, which play a role in defining FOXK2 function through regulating its stability and its activity as a transcriptional repressor protein. These two CDK sites appear vital for FOXK2 function as expression of a mutant lacking these sites cannot be tolerated and causes apoptosis.

The SLIT-ROBO GTPase-activating proteins (srGAPs) are critical for neuronal migration through inactivation of Rho GTPases Cdc42, Rac1 and RhoA. Here we report that srGAP2 physically interacts with protein arginine methyltransferase 5 (PRMT5). srGAP2 localizes to the cytoplasm and plasma membrane protrusion. srGAP2 knockdown reduces cell spreading and increases cell migration, but has no effects on cell proliferation. PRMT5 binds to the N-terminal of srGAP2 (225-538 aa) and methylates its C-terminal arginine residue R927. The methylation mutant srGAP2-R927A fails to rescue cell spreading defect, is unable to localize to plasma membrane leading edge, and perturbs the srGAP2 homo-dimer formation mediated by the F-BAR domain. These results suggest that srGAP2 arginine methylation plays important roles on cell spreading and cell migration through influencing membrane protrusion.

Cells in mechanically active tissues undergo constant plasma membrane damage that must be repaired to allow survival. To identify wound-associated proteins, a cell impermeant, thiol-reactive biotinylation reagent was used to label and subsequently isolate intracellular proteins that become exposed on the surface of cultured cells after plasma membrane damage induced by scraping from substratum, or crushing with glass beads. Scrape-damaged cells survived injury and were capable of forming viable colonies. Proteins that were exposed to the cell surface were degraded or internalized a few seconds to several minutes after damage, except for vimentin, which was detectable on the cell surface for at least an hour after injury. Seven major biotinylated protein bands were identified on SDS-PAGE gels. Mass spectrometric studies identified cytoskeletal proteins (caldesmon-1, vimentin), endoplasmic reticulum proteins (ERp57, ERp5, HSP47), and nuclear proteins (lamin C, hnRNPF, nucleophosmin-1) as major proteins exposed after injury. While caldesmon was a major wound-associated protein in calpain small subunit knockout fibroblasts, it was rapidly degraded in wild-type cells, probably by calpains. Lamin C exposure after wounding was most likely the consequence of nuclear envelope damage. These studies document major intracellular proteins associated with the cell surface of reversibly damaged somatic cells. The studies also show that externalization of some proteins reported to have physiologic or pathologic roles on the cell surface can occur in cells undergoing plasma membrane damage and subsequent repair.

The evolutionary loss of hepatic urate oxidase (uricase) has resulted in humans with elevated serum uric acid (urate). Uricase loss may have been beneficial to early primate survival. However, elevated serum urate has predisposed man to hyperuricemia; a metabolic disturbance leading to gout, hypertension, and various cardiovascular diseases. Human serum urate levels are largely determined by urate reabsorption and secretion in the kidney. Renal urate reabsorption is controlled via two proximal tubular urate transporters: apical URAT1 (SLC22A12) and basolateral URATv1/GLUT9 (SLC2A9). In contrast, molecular mechanism(s) for renal urate secretion remain unknown. In this report, we demonstrate that an orphan transporter hNPT4 (human sodium-phosphate transporter 4; SLC17A3) was a multispecific organic anion efflux transporter expressed in the kidneys and liver. hNPT4 was localized at the apical side of renal tubules and functioned as a voltage-driven urate transporter. Furthermore, loop diuretics, such as furosemide and bumetanide, substantially interacted with hNPT4. Thus, this protein is likely to act as a common secretion route for both drugs and may play an important role in diuretics-induced hyperuricemia. In vivo role of hNPT4 was suggested by two hyperuricemia patients with missense mutations in SLC17A3. These mutated hNPT4 exhibited reduced urate efflux when they were expressed in Xenopus oocytes. Our findings will complete a model of urate secretion in the renal tubular cell, where intracellular urate taken up via OAT1 and/or OAT3 from the blood, exits from the cell into the lumen via hNPT4.

The recently discovered heliobacteria are the only gram-positive photosynthetic bacteria that have been cultured. One of the unique features of heliobacteria is that they have properties of both the photosynthetic green sulfur bacteria (containing the type I reaction center) and Clostridia (forming heat-resistant endospores). Most of the previous studies of heliobacteria, which are strict anaerobes and have the simplest known photosynthetic apparatus, have focused on energy and electron transfer processes. It has been assumed that like green sulfur bacteria, the major carbon flow in heliobacteria is through the (incomplete) reductive (reverse) tricarboxylic acid (RTCA) cycle, whereas the lack of CO2-enhanced growth has not been understood. Here, we report studies to fill the knowledge gap of heliobacterial carbon metabolism. We confirm that the CO2-anaplerotic pathway is active during phototrophic growth, and that isoleucine is mainly synthesized from the citramalate pathway. Further, to our surprise, our results suggest that the oxidative (forward) TCA (OTCA) cycle is operative and more active than the previously reported RTCA cycle. Both isotopomer analysis and activity assays suggest that citrate is produced by a putative (Re)-citrate synthase and then enters the OTCA cycle. Moreover, in contrast to (Si)-citrate synthase, (Re)-citrate synthase produces a different isomer of 2-fluorocitrate that is not expected to inhibit the activity of aconitase.

We identified that activation of the Gq-linked dopamine D1-D2 receptor heterooligomer generates a PLC-dependent intracellular calcium signal. Confocal FRET between endogenous dopamine D1 and D2 receptors in striatal neurons confirmed a physical interaction between them. Pre-treatment with SKF 83959, which selectively activates the D1-D2 receptor heteromer, or SKF 83822 which only activates the D1 receptor homooligomer, led to rapid desensitization of the D1-D2 receptor heteromer mediated calcium signal in both heterologous cells and striatal neurons. This desensitization response was mediated through selective occupancy of the D1 receptor binding pocket. Although SKF 83822 was unable to activate the D1-D2 receptor heteromer, it still permitted desensitization of the calcium signal. This suggested that occupancy of the D1 receptor binding pocket by SKF 83822 resulted in conformational changes sufficient for desensitization without heteromer activation. BRET and co-immunoprecipitation studies indicated an agonist induced physical association between the D1-D2 receptor heteromeric complex and GRK2. Increased expression of GRK2 led to a decrease in the calcium signal with or without prior exposure to either SKF 83959 or SKF 83822. GRK2 knockdown by siRNA led to an increase in the signal after pre-treatment with either agonist. Expression of the catalytically inactive and RGS mutated GRK2 constructs each led to a partial recovery of the GRK2-attenuated calcium signal. These results indicated that desensitization of the dopamine D1-D2 receptor heteromer mediated signal can occur by agonist occupancy even without activation and is dually regulated by both the catalytic and RGS domains of GRK2.

Endothelin converting enzyme-2 (ECE-2) is a membrane bound zinc-dependent metalloprotease which shares a high degree of sequence homology with endothelin converting enzyme-1 (ECE-1), but displays an acidic pH optimum characteristic of maturing enzymes acting late in the secretory pathway. Although ECE-2, like ECE-1, can cleave the big-endothelin intermediate to produce the vasoconstrictive endothelin peptide, its true physiological function remains to be elucidated, a task which is hampered by the lack of specific tools to study and discriminate ECE-2 from ECE-1, i.e. specific substrates and/or specific inhibitors. To fill this gap, we searched for novel ECE-specific peptide substrates. To this end, peptides derived from the big-endothelin intermediate were tested using ECE-1 and ECE-2, leading to the identification of an ECE-1 specific substrate. Moreover, screening of our proprietary fluorigenic peptide Fluofast libraries using ECE-1 and ECE-2 allowed the identification of AcSKGPya-F-W-NopGGKNH2 (PL405), as a specific and high affinity ECE-2 substrate. Indeed, ECE-2 cleaved PL405 at the Pya-F amide bond with a specificity constant (kcat/Km) of 8.1 103 M-1s-1. Using this novel substrate, we also characterized the first potent (Ki = 7.7 nM) and relatively selective ECE-2 inhibitor and developed a quantitative fluorigenic ECE-2 assay. The assay was used to study the ex vivo ECE-2 activity in wild type and ECE-2 knock out tissues and was found to truly reflect ECE-2 expression patterns. The PL405 assay is thus the first tool to study ECE-2 inhibition using high throughput screening or for ex vivo ECE-2 quantification.

Amyloid-beta (Abeta) peptide is thought to have a significant role in the progressive memory loss observed in patients with Alzheimer's disease (AD) and inhibits synaptic plasticity in animal models of learning. We previously demonstrated that brain-derived neurotrophic factor (BDNF) is critical for synaptic AMPA receptor delivery in an in vitro model of eyeblink classical conditioning. Here, we report that acquisition of conditioned responses (CRs) was significantly attenuated by bath application of oligomeric (200 nM), but not fibrillar, Abeta peptide. Western blotting revealed that BDNF protein expression during conditioning is significantly reduced by treatment with oligomeric Abeta, as was phosphorylation levels of CREB, CaMKII, CaMKIV and ERK. However, levels of PKA and PKCzeta/lambda were unaffected, as was PDK-1. Protein localization studies using confocal imaging indicate that oligomeric Abeta, but not fibrillar or scrambled forms, suppress colocalization of GluR1 and GluR4 AMPAR subunits with synaptophysin, indicating that trafficking of these subunits to synapses during the conditioning procedure is blocked. In contrast, coapplication of BDNF with oligomeric Abeta significantly reversed these findings. Interestingly, a tolloid-like metalloproteinase in turtle, tTLLs, which normally processes the precursor proBDNF into mature BDNF, was found to degrade oligomeric Abeta into small fragments. These data suggest that an Abeta-induced reduction in BDNF, perhaps due to interference in the proteolytic conversion of proBDNF to BDNF, results in inhibition of synaptic AMPAR delivery and suppression of the acquisition of conditioning.

The lipase activity of most phospholipase Cs (PLCs) is basally repressed by a highly degenerate and mostly disordered X/Y-linker inserted within the catalytic domain. Release of this auto-inhibition is driven by electrostatic repulsion between the plasma membrane and the electronegative X/Y-linker. In contrast, PLC-gamma isozymes (-gamma1, -gamma2) are structurally distinct from other PLCs since multiple domains are present in their X/Y-linker. Moreover, although many tyrosine kinases directly phosphorylate PLC-gamma isozymes to enhance their lipase activity, the underlying molecular mechanism of this activation remains unclear. Here we define the mechanism for the unique regulation of PLC-gamma isozymes by their X/Y-linker. Specifically, we identify the C-terminal SH2 domain within the X/Y-linker as the critical determinant for auto-inhibition. Tyrosine phosphorylation of the X/Y-linker mediates high-affinity intramolecular interaction with the C-terminal SH2 domain that is coupled to a large conformational rearrangement and release of auto-inhibition. Consequently, the PLC-gamma isozymes link phosphorylation to phospholipase activation by elaborating upon primordial regulatory mechanisms found in other PLCs.