Fragment-Based Discovery of Novel Potent Sepiapterin Reductase Inhibitors.

Genome-wide-association studies in chronic low back pain patients identified sepiapterin reductase as a high interest target for developing new analgesics. Here we used 19F NMR fragment screening for the discovery of novel, ligand-efficient SPR inhibitors. We report the crystal structures of six chemically diverse inhibitors complexed with SPR, identifying relevant interactions and binding modes in the sepiapterin pocket. Exploration of our initial fragment screening hit led to double-digit nanomolar inhibitors of SPR with excellent ligand efficiency.

Identification of a multifunctional docking site on the catalytic unit of phosphodiesterase-4 (PDE4) that is utilised by multiple interaction partners.

Cyclic AMP (cAMP)-specific phosphodiesterase-4 (PDE4) enzymes underpin compartmentalised cAMP signalling by localising to distinct signalling complexes. PDE4 long isoforms can be phosphorylated by mitogen-activated protein kinase-activated protein kinase 2 (MK2), which attenuates activation of such enzymes through their phosphorylation by protein kinase A. Here we show that MK2 interacts directly with PDE4 long isoforms and define the sites of interaction. One is a unique site that locates within the regulatory upstream conserved region 1 (UCR1) domain and contains a core Phe141, Leu142 and Tyr143 (FLY) cluster (PDE4A5 numbering). Located with the second site is a critical core Phe693, Glu694, Phe695 (FQF) motif that is also employed in the sequestering of PDE4 long forms by an array of other signalling proteins, including the signalling scaffold ß-arrestin, the tyrosyl kinase Lyn, the SUMOylation E2 ligase UBC9, the dynein regulator Lis1 (PAFAH1B1) and the protein kinase Erk. We propose that the FQF motif lies at the heart of a multifunctional docking (MFD) site located within the PDE4 catalytic unit. It is clear from our data that, as well as aiding fidelity of interaction, the MFD site confers exclusivity of binding between PDE4 and a single specific partner protein from the cohort of signalling proteins whose interaction with PDE4 involves the FQF motif.

The Regulation of NF-κB Subunits by Phosphorylation.

The NF-κB transcription factor is the master regulator of the inflammatory response and is essential for the homeostasis of the immune system. NF-κB regulates the transcription of genes that control inflammation, immune cell development, cell cycle, proliferation, and cell death. The fundamental role that NF-κB plays in key physiological processes makes it an important factor in determining health and disease. The importance of NF-κB in tissue homeostasis and immunity has frustrated therapeutic approaches aimed at inhibiting NF-κB activation. However, significant research efforts have revealed the crucial contribution of NF-κB phosphorylation to controlling NF-κB directed transactivation. Importantly, NF-κB phosphorylation controls transcription in a gene-specific manner, offering new opportunities to selectively target NF-κB for therapeutic benefit. This review will focus on the phosphorylation of the NF-κB subunits and the impact on NF-κB function.

p75 Neurotrophin Receptor Regulates Energy Balance in Obesity.

Obesity and metabolic syndrome reflect the dysregulation of molecular pathways that control energy homeostasis. Here, we show that the p75 neurotrophin receptor (p75(NTR)) controls energy expenditure in obese mice on a high-fat diet (HFD). Despite no changes in food intake, p75(NTR)-null mice were protected from HFD-induced obesity and remained lean as a result of increased energy expenditure without developing insulin resistance or liver steatosis. p75(NTR) directly interacts with the catalytic subunit of protein kinase A (PKA) and regulates cAMP signaling in adipocytes, leading to decreased lipolysis and thermogenesis. Adipocyte-specific depletion of p75(NTR) or transplantation of p75(NTR)-null white adipose tissue (WAT) into wild-type mice fed a HFD protected against weight gain and insulin resistance. Our results reveal that signaling from p75(NTR) to cAMP/PKA regulates energy balance and suggest that non-CNS neurotrophin receptor signaling could be a target for treating obesity and the metabolic syndrome.

Nuclear pore complex remodeling by p75(NTR) cleavage controls TGF-ß signaling and astrocyte functions.

Astrocytes modulate neuronal activity and inhibit regeneration. We show that cleaved p75 neurotrophin receptor (p75(NTR)) is a component of the nuclear pore complex (NPC) required for glial scar formation and reduced gamma oscillations in mice via regulation of transforming growth factor (TGF)-ß signaling. Cleaved p75(NTR) interacts with nucleoporins to promote Smad2 nucleocytoplasmic shuttling. Thus, NPC remodeling by regulated intramembrane cleavage of p75(NTR) controls astrocyte-neuronal communication in response to profibrotic factors.

Screening for small molecule disruptors of AKAP-PKA interactions.

Protein-protein interactions (PPIs) are highly specific and diverse. Their selective inhibition with peptides, peptidomimetics, or small molecules allows determination of functions of individual PPIs. Moreover, inhibition of disease-associated PPIs may lead to new concepts for the treatment of diseases with an unmet medical need. Protein kinase A (PKA) is an ubiquitously expressed protein kinase that controls a plethora of cellular functions. A-kinase anchoring proteins (AKAPs) are multivalent scaffolding proteins that directly interact with PKA. AKAPs spatially and temporally restrict PKA activity to defined cellular compartments and thereby contribute to the specificity of PKA signaling. However, it is largely unknown which of the plethora of PKA-dependent signaling events involve interactions of PKA with AKAPs. Moreover, AKAP-PKA interactions appear to play a role in a variety of cardiovascular, neuronal, and inflammatory diseases, but it is unclear whether these interactions are suitable drug targets. Here we describe an enzyme-linked immunosorbent assay (ELISA) for the screening of small molecule libraries for inhibitors of AKAP-PKA interactions. In addition, we describe a homogenous time-resolved fluorescence (HTRF) assay for use in secondary validation screens. Small molecule inhibitors are invaluable molecular tools for elucidating the functions of AKAP-PKA interactions and may eventually lead to new concepts for the treatment of diseases where AKAP-PKA interactions represent potential drug targets.

PKA phosphorylation of p62/SQSTM1 regulates PB1 domain interaction partner binding.

p62, also known as SQSTM1, is a multi-domain signalling scaffold protein involved in numerous critical cellular functions such as autophagy, apoptosis and inflammation. Crucial interactions relevant to these functions are mediated by the N-terminal Phox and Bem1p (PB1) domain, which is divided into two interaction surfaces, one of predominantly acidic and one of basic character. Most known interaction partners, including atypical protein kinase C (aPKC), bind to the basic surface, and acidic-basic interactions at this interface also allow for p62 homopolymerisation. We identify here that the coupling of p62 to the cAMP signalling system is conferred by both the direct binding of cAMP degrading phosphodiesterase-4 (PDE4) to the acidic surface of the p62 PB1 domain and the phosphorylation of the basic surface of this domain by cAMP-dependent protein kinase (PKA). Such phosphorylation is a previously unknown means of regulating PB1 domain interaction partnerships by disrupting the interaction of p62 with basic surface binding partners, such as aPKCs, as well as p62 homopolymerisation. Thus, we uncover a new regulatory mechanism that connects cAMP signalling with the p62 multi-domain signalling scaffold and autophagy cargo receptor protein.

Phosphodiesterase-8A binds to and regulates Raf-1 kinase.

V-raf-1 murine leukemia viral oncogene homolog 1 (Raf-1) is a key activator of the ERK pathway and is a target for cross-regulation of this pathway by the cAMP signaling system. The cAMP-activated protein kinase, PKA, inhibits Raf-1 by phosphorylation on S259. Here, we show that the cAMP-degrading phosphodiesterase-8A (PDE8A) associates with Raf-1 to protect it from inhibitory phosphorylation by PKA, thereby enhancing Raf-1's ability to stimulate ERK signaling. PDE8A binds to Raf-1 with high (picomolar) affinity. Mapping of the interaction domain on PDE8A using peptide array technology identified amino acids 454-465 as the main binding site, which could be disrupted by mutation. A cell-permeable peptide corresponding to this region disrupted the PDE8A/Raf-1 interaction in cells, thereby reducing ERK activation and the cellular response to EGF. Overexpression of a catalytically inactive PDE8A in cells displayed a dominant negative phenotype on ERK activation. These effects were recapitulated at the organism level in genetically modified (PDE8A(-/-)) mice. Similarly, PDE8 deletion in Drosophila melanogaster reduced basal ERK activation and sensitized flies to stress-induced death. We propose that PDE8A is a physiological regulator of Raf-1 signaling in some cells.

Comparative analysis of vaginal versus robotic-assisted hysterectomy for benign indications.

We aimed to compare perioperative outcomes of robotic-assisted hysterectomy versus vaginal hysterectomy in patients with benign gynecologic conditions, using a retrospective chart review of 240 consecutive benign hysterectomies from May 2008 to April of 2010 performed by a single surgical team at the Eisenhower Medical Center. The analysis included an equal number of cases in each group: 120 robotic-assisted total laparoscopic hysterectomies and 120 total vaginal hysterectomies. Consecutive cases met the inclusion criteria of benign disease. There were no statistically significant differences related to age, body mass index, history of prior abdominal surgery, or uterine weight. Operative times in the robotic group were significantly longer by an average of 59 min (p < 0.001). Patients with robotic-assisted hysterectomy had clinically equivalent estimated blood loss (55.5 ml vs. 84.7 ml, p < 0.001) and the intraoperative complication rates were 1.7% vaginal versus 0% robotic (p = 0.156). There was one conversion in the vaginal group due to pelvic adhesions and no conversions in the robotic group. Length of hospital stay was 1 day for both groups. The perioperative complication rates were equivalent between groups (6.7 vs. 11.7%, p = 0.180), but there were more major complications in the vaginal group (0 vs. 3.3%, p = 0.044). We conclude that, in a comparable group of patients, robotic-assisted hysterectomy takes longer to complete but results in fewer major complications.

cAMP: novel concepts in compartmentalised signalling.

Cyclic adenosine 3,'5'-monophosphate (cAMP) is the archetypal second messenger produced at the membrane by adenylyl cyclase following activation of many different G protein-coupled receptor (GPCR) types. Although discovered over fifty years ago, the notion that cAMP responses were compartmentalised was born in the 1980s. Since then, modern molecular techniques have facilitated visualisation of cellular cAMP dynamics in real time and helped us to understand how a single, ubiquitous second messenger can direct receptor-specific functions in cells. The aim of this review is to highlight emerging ideas in the cAMP field that are currently developing the concept of compartmentalised cAMP signalling systems.

Oxygen-dependent cleavage of the p75 neurotrophin receptor triggers stabilization of HIF-1α.

Homeostatic control of oxygen availability allows cells to survive oxygen deprivation. Although the transcription factor hypoxia-inducible factor 1α (HIF-1α) is the main regulator of the hypoxic response, the upstream mechanisms required for its stabilization remain elusive. Here, we show that p75 neurotrophin receptor (p75(NTR)) undergoes hypoxia-induced γ-secretase-dependent cleavage to provide a positive feed-forward mechanism required for oxygen-dependent HIF-1α stabilization. The intracellular domain of p75(NTR) directly interacts with the evolutionarily conserved zinc finger domains of the E3 RING ubiquitin ligase Siah2 (seven in absentia homolog 2), which regulates HIF-1α degradation. p75(NTR) stabilizes Siah2 by decreasing its auto-ubiquitination. Genetic loss of p75(NTR) dramatically decreases Siah2 abundance, HIF-1α stabilization, and induction of HIF-1α target genes in hypoxia. p75(NTR-/-) mice show reduced HIF-1α stabilization, vascular endothelial growth factor (VEGF) expression, and neoangiogenesis after retinal hypoxia. Thus, hypoxia-induced intramembrane proteolysis of p75(NTR) constitutes an apical oxygen-dependent mechanism to control the magnitude of the hypoxic response.

Elucidation of a structural basis for the inhibitor-driven, p62 (SQSTM1)-dependent intracellular redistribution of cAMP phosphodiesterase-4A4 (PDE4A4).

A survey of PDE4 inhibitors reveals that some compounds trigger intracellular aggregation of PDE4A4 into accretion foci through association with the ubiquitin-binding scaffold protein p62 (SQSTM1). We show that this effect is driven by inhibitor occupancy of the catalytic pocket and stabilization of a "capped state" in which a sequence within the enzyme's upstream conserved region 2 (UCR2) module folds across the catalytic pocket. Only certain inhibitors cause PDE4A4 foci formation, and the structural features responsible for driving the process are defined. Switching to the UCR2-capped state induces conformational transition in the enzyme's regulatory N-terminal portion, facilitating protein association events responsible for reversible aggregate assembly. PDE4-selective inhibitors able to trigger relocalization of PDE4A4 into foci can therefore be expected to exert actions on cells that extend beyond simple inhibition of PDE4 catalytic activity and that may arise from reconfiguring the enzyme's protein association partnerships.

Small molecule AKAP-protein kinase A (PKA) interaction disruptors that activate PKA interfere with compartmentalized cAMP signaling in cardiac myocytes.

A-kinase anchoring proteins (AKAPs) tether protein kinase A (PKA) and other signaling proteins to defined intracellular sites, thereby establishing compartmentalized cAMP signaling. AKAP-PKA interactions play key roles in various cellular processes, including the regulation of cardiac myocyte contractility. We discovered small molecules, 3,3'-diamino-4,4'-dihydroxydiphenylmethane (FMP-API-1) and its derivatives, which inhibit AKAP-PKA interactions in vitro and in cultured cardiac myocytes. The molecules bind to an allosteric site of regulatory subunits of PKA identifying a hitherto unrecognized region that controls AKAP-PKA interactions. FMP-API-1 also activates PKA. The net effect of FMP-API-1 is a selective interference with compartmentalized cAMP signaling. In cardiac myocytes, FMP-API-1 reveals a novel mechanism involved in terminating ß-adrenoreceptor-induced cAMP synthesis. In addition, FMP-API-1 leads to an increase in contractility of cultured rat cardiac myocytes and intact hearts. Thus, FMP-API-1 represents not only a novel means to study compartmentalized cAMP/PKA signaling but, due to its effects on cardiac myocytes and intact hearts, provides the basis for a new concept in the treatment of chronic heart failure.

p62 (SQSTM1) forms part of a novel, reversible aggregate containing a specific conformer of the cAMP degrading phosphodiesterase, PDE4A4.

Cells employ macroautophagy to deliver aggregates of misfolded and/or ubiquitinated proteins for lysosomal degradation or supply of essential macromolecules under conditions of nutrient deprivation. The former seems complementary to the proteasome system, which is likely to degrade only soluble proteins. While bulk degradation via the autophagy pathway during starvation is rather nonspecific, the recognition of proteins that are either misfolded or adopt a particular conformation and/or become ubiquitinated, requires some form of specificity. This is brought about, at least in part, by the scaffold and ubiquitin-binding protein, p62 (SQSTM1). p62 is a multidomain scaffold that sequesters other proteins and polymerizes through its Phox and Bem1p domains and binds K63-ubiquitinated proteins through its UBA domain. p62 interaction with LC3 appears critical for membrane encapsulation seen in autophagosomes. However, there is a growing body of evidence indicating that p62 is not exclusively involved in autophagy regulation, and that there are nonmembrane encapsulated, soluble subpopulations of p62 in cells. The role of these subpopulations has yet to be resolved, although one function appears to be to regulate signaling, as indicated through p62's ability to regulate NFκB activation. Signaling through the ubiquitous cyclic AMP (cAMP) system is compartmentalized, with tethered subpopulations of cAMP-degrading phosphodiesterases sculpting cAMP gradients around specific signaling complexes so as to regulate them at a spatial level. We have recently demonstrated that p62 colocalizes with a specific conformer of the cAMP degrading phosphodiesterase, PDE4A4, so as to form reversible, membrane-free cytosolic aggregates lacking LC3. This results in PDE4A4 becoming sequestered away from signaling proteins that normally sequester it, providing a means of reprogramming compartmentalized cAMP signaling in cells.

p62 (SQSTM1) and cyclic AMP phosphodiesterase-4A4 (PDE4A4) locate to a novel, reversible protein aggregate with links to autophagy and proteasome degradation pathways.

Chronic challenge of cyclic AMP phosphodiesterase-4A4 (PDE4A4) with certain PDE4 selective inhibitors causes it to reversibly form intracellular aggregates that are not membrane-encapsulated. These aggregates are neither stress granules (SGs) nor processing bodies (PBs) as they contain neither PABP-1 nor Dcp1a, respectively. However, the PDE4 inhibitor rolipram decreases arsenite-induced SGs and increases the amount of PBs, while arsenite challenge ablates rolipram-induced PDE4A4 aggregates. PDE4A4 aggregates are neither autophagic vesicles (autophagosomes) nor aggresomes, although microtubule disruptors ablate PDE4A4 aggregate formation. PDE4A4 constitutively co-immunoprecipitates with p62 protein (sequestosome1, SQSTM1), which locates to both PDE4A4 aggregates and autophagosomes in cells constitutively challenged with rolipram. The mTor inhibitor, rapamycin, activates autophagy, prevents PDE4A4 from forming intracellular aggregates and triggers the loss of bound p62 from PDE4A4. siRNA-mediated knockdown of p62 attenuates PDE4A4 aggregate formation. The p62-binding protein, light chain 3 (LC3), is not found in PDE4A4 aggregates. Blockade of proteasome activity and activation of autophagy with MG132 both increases the level of ubiquitinated proteins found associated with PDE4A4 and inhibits PDE4A4 aggregate formation. Activation of autophagy with either thapsigargin or ionomycin inhibits PDE4A4 aggregate formation. Inhibition of autophagy with either wortmannin or LY294002 activates PDE4A4 aggregate formation. The protein kinase C inhibitors, RO 320432 and GO 6983, and the ERK inhibitors UO 126 and PD 98059 all activated PDE4A4 aggregate formation, whilst roscovitine, thalidomide and the tyrosine kinase inhibitors, genistein and AG17, all inhibited this process. We suggest that the fate of p62-containing protein aggregates need not necessarily be terminal, through delivery to autophagic vesicles and aggresomes. Instead, we propose a novel regulatory mechanism where a sub-population of p62-containing protein aggregates would form in a rapid, reversible manner so as to sequester specific cargo away from their normal, functionally important site(s) within the cell. Thus an appropriate conformational change in the target protein would confer reversible recruitment into a sub-population of p62-containing protein aggregates and so provide a regulatory function by removing these cargo proteins from their functionally important site(s) in a cell.

Glycogen synthase kinase 3beta interaction protein functions as an A-kinase anchoring protein.

A-kinase anchoring proteins (AKAPs) include a family of scaffolding proteins that target protein kinase A (PKA) and other signaling proteins to cellular compartments and thereby confine the activities of the associated proteins to distinct regions within cells. AKAPs bind PKA directly. The interaction is mediated by the dimerization and docking domain of regulatory subunits of PKA and the PKA-binding domain of AKAPs. Analysis of the interactions between the dimerization and docking domain and various PKA-binding domains yielded a generalized motif allowing the identification of AKAPs. Our bioinformatics and peptide array screening approaches based on this signature motif identified GSKIP (glycogen synthase kinase 3beta interaction protein) as an AKAP. GSKIP directly interacts with PKA and GSK3beta (glycogen synthase kinase 3beta). It is widely expressed and facilitates phosphorylation and thus inactivation of GSK3beta by PKA. GSKIP contains the evolutionarily conserved domain of unknown function 727. We show here that this domain of GSKIP and its vertebrate orthologues binds both PKA and GSK3beta and thereby provides a mechanism for the integration of PKA and GSK3beta signaling pathways.

Compartmentalized cAMP signalling in regulated exocytic processes in non-neuronal cells.

Cyclic adenosine monophosphate (cAMP) is a central second messenger controlling a plethora of vital functions. Studies of cAMP dynamics in living cells have revealed markedly inhomogeneous concentrations of the second messenger in different compartments. Moreover, cAMP effectors such as cAMP-dependent protein kinase (PKA) and cAMP-activated GTP-exchange factors (Epacs) are tethered to specific cellular sites. Both the tailoring of cAMP concentrations, and the activities of cAMP-dependent signalling systems at specific cellular locations are prerequisites for most, if not all, cAMP-dependent processes. This review focuses on the role of compartmentalized cAMP signalling in exocytic processes in non-neuronal cells. Particularly, the insertion of aquaporin-2 into the plasma membrane of renal principal cells as an example for a cAMP-dependent exocytic process in a non-secretory cell type, renin secretion from juxtaglomerular cells as a cAMP-triggered exocytosis from an endocrine cell, insulin release from pancreatic beta-cells as a Ca2+-mediated and cAMP-potentiated exocytic processes in an endocrine cell, and cAMP- or Ca2+ -triggered H+ secretion from gastric parietal cells as an exocytic process in an exocrine cell are discussed. The selected examples of cAMP-regulated exocytic pathways are reviewed with regard to key proteins involved: adenylyl cyclases, phosphodiesterases, PKA, A kinase anchoring proteins (AKAPs) and Epacs.

High-affinity AKAP7delta-protein kinase A interaction yields novel protein kinase A-anchoring disruptor peptides.

PKA (protein kinase A) is tethered to subcellular compartments by direct interaction of its regulatory subunits (RI or RII) with AKAPs (A kinase-anchoring proteins). AKAPs preferentially bind RII subunits via their RII-binding domains. RII-binding domains form structurally conserved amphipathic helices with unrelated sequences. Their binding affinities for RII subunits differ greatly within the AKAP family. Amongst the AKAPs that bind RIIalpha subunits with high affinity is AKAP7delta [AKAP18delta; K(d) (equilibrium dissociation constant) value of 31 nM]. An N-terminally truncated AKAP7delta mutant binds RIIalpha subunits with higher affinity than the full-length protein presumably due to loss of an inhibitory region [Henn, Edemir, Stefan, Wiesner, Lorenz, Theilig, Schmidtt, Vossebein, Tamma, Beyermann et al. (2004) J. Biol. Chem. 279, 26654-26665]. In the present study, we demonstrate that peptides (25 amino acid residues) derived from the RII-binding domain of AKAP7delta bind RIIalpha subunits with higher affinity (K(d)=0.4+/-0.3 nM) than either full-length or N-terminally truncated AKAP7delta, or peptides derived from other RII binding domains. The AKAP7delta-derived peptides and stearate-coupled membrane-permeable mutants effectively disrupt AKAP-RII subunit interactions in vitro and in cell-based assays. Thus they are valuable novel tools for studying anchored PKA signalling. Molecular modelling indicated that the high affinity binding of the amphipathic helix, which forms the RII-binding domain of AKAP7delta, with RII subunits involves both the hydrophobic and the hydrophilic faces of the helix. Alanine scanning (25 amino acid peptides, SPOT technology, combined with RII overlay assays) of the RII binding domain revealed that hydrophobic amino acid residues form the backbone of the interaction and that hydrogen bond- and salt-bridge-forming amino acid residues increase the affinity of the interaction.