Discovery and Characterization of a Small Molecule Inhibitor of the PDZ Domain of Dishevelled

Dishevelled (Dvl) is an essential protein in the Wnt signaling pathways; it uses its PDZ domain to transduce the Wnt signals from the membrane receptor Frizzled to downstream components. Here, we report identifying a drug-like small molecule compound through structure-based ligand screening and NMR spectroscopy and show the compound to interact at low micromolar affinity with the PDZ domain of Dvl. In a Xenopus testing system, the compound could permeate the cell membrane and block the Wnt signaling pathways. In addition, the compound inhibited Wnt signaling and reduced the levels of apoptosis in the hyaloid vessels of eye. Moreover, this compound also suppressed the growth of prostate cancer PC-3 cells. These biological effects suggest that by blocking the PDZ domain of Dvl, the compound identified in our studies effectively inhibits the Wnt signaling and thus provides a useful tool for studies dissecting the Wnt signaling pathways.The Wnt signaling pathways are regulated by a family of secreted Wnt glycoproteins. The canonical Wnt pathway, which is highly conserved, is best understood. In this pathway, Wnt molecules interact with the seven-transmembrane Frizzled (Fz)² proteins (1) by binding to an N-terminal cysteine-rich-domain (2). The signal is then transduced into the cell through an internal sequence of Fz, C-terminal to the seventh transmembrane domain, which binds directly to the PDZ (postsynaptic density-95/discs large/zonula occludens-1) domain of the cytoplasmic protein Dishevelled (Dvl) (3). Dvl then transduces the Wnt signals to downstream components (4). Three Dvl homologs (Dvl-1, -2, and -3) have been identified in humans; all are expressed in both embryonic and adult tissues, including brain, heart, lung, kidney, skeletal muscle, and others (4). Up-regulation and overexpression of Dvl proteins have been reported in many cancers, including those of breast, colon, prostate, mesothelium, and lung (non-small cell) (5–8).The Dvl protein is made up of three conserved domains: an N-terminal DIX domain, a central PDZ domain, and a C-terminal DEP domain (9). The central PDZ domain is of particular interest because of its interaction with Fz and other Wnt pathway proteins (3, 10). The direct interaction between the PDZ domain and Fz peptides is relatively weak, and other factors may play a role to ensure the communication between the two molecules (3). For example, several studies suggest that the DEP domain of Dvl has a membrane-targeting function that may facilitate PDZ-Fz interaction (11–14). However, the weak PDZ-Fz interaction provides an opportunity to block Wnt signaling at the Dvl level by using a small molecule inhibitor. An earlier study in our laboratories used an NMR-assisted virtual ligand screening approach to identify a peptide mimic that can bind to the Dvl PDZ domain (15). We have now used an improved algorithm to conduct an additional structure-based virtual screen of the PDZ domain of Dvl and have discovered a group of drug-like compounds that bind to the PDZ domain with moderate to low micromolar affinity. One of these compounds effectively blocked Wnt signaling in vivo and reduced the growth rate of a prostate cancer cell line.     

Heparan Sulfate Proteoglycan Modulation of Wnt5A Signal Transduction in Metastatic Melanoma Cells

Heparan sulfate proteoglycans (HSPGs) are important modulators for optimizing signal transduction of many pathways, including the Wnt pathways. We demonstrate that HSPG glycosaminoglycan levels increased with increasing metastatic potential of melanoma cells. Previous studies have demonstrated that Wnt5A increases the invasiveness of melanoma cells. We further demonstrate that HSPGs potentiate Wnt5A signaling, since enzymatic removal of the HSPG backbone resulted in a decrease in cellular Wnt5A levels, an increase in secreted Wnt5A in cell media, a decrease in downstream signaling, and ultimately, a decrease in invasiveness. Specifically, syndecan 1 and syndecan 4 expression correlated to Wnt5A expression and melanoma malignancy. Knockdown of syndecan 1 or 4 caused decreases in cell invasion, which could be restored by treating the cells with recombinant Wnt5A. These data indicate that syndecan 1 and 4 correlate to increased metastatic potential in melanoma patients and are an important component of the Wnt5A autocrine signaling loop, the activation of which leads to increased metastasis of melanoma.The American Cancer Society estimates that in 2009 there will be 68,720 new cases of melanoma in this country with ∼8,650 deaths. Recent studies have demonstrated that the non-canonical Wnt pathway, also known as the Wnt/Ca²⁺ pathway, plays an important role in increasing the metastatic potential of melanoma cells (1–5). Studies from our laboratory demonstrated that increasing Wnt5A, which mediates the non-canonical Wnt/Ca²⁺ signaling pathway, increased melanoma metastasis (1–3), and silencing Wnt5A levels via siRNA³ decreased invasion (2, 3). In addition, we have shown that Wnt5A acts via protein kinase C (PKC) to mediate the motility of melanoma cells via the inhibition of metastasis suppressors and an initiation of the epithelial to mesenchymal transition, characterized by the loss of E-cadherin and the up-regulation of Snail (2).Wnt signaling can be mediated by heparan sulfate proteoglycans (HSPGs) which are important signal transduction modulators. They mediate fibroblast growth factor, Hedgehog, epidermal growth factor, transforming growth factor-β, and WNT signaling pathways (6–11). HSPGs consist of two types, cell surface and basement membrane-associated HSPGs (12). Cell surface HSPGs are glycoproteins with covalently attached unbranched and modified sugar chains known as glycosaminoglycans (GAGs). There are two types of cell surface HSPGs, known as glypicans and syndecans (11, 13). Glypicans are attached to the cell surface via a glycosylphosphatidylinositol anchor, whereas syndecans are type 1 transmembrane proteins. HSPG GAG side chains are unbranched chains of modified repeating disaccharide units of N-acetylglucosamine and glucuronic acid. They are joined to the core protein via a tetrasaccharide linker attached to a serine residue. Following synthesis, these chains undergo modification with the addition of sulfates by N- and O-sulfation (14). The sulfation status determines to which specific portion of the GAG chains ligands, such as Wnt, will attach. The heparan sulfate endosulfatases Sulf1 and Sulf2 are cell surface enzymes that control growth factor signaling. The regulation of the 6-O-sulfation states by these endosulfatases changes the affinity of the GAG chains for ligand binding (15–17). Following sulfation modification, HSPGs can regulate signaling by dimerization (with other HSPGs or canonical signaling receptors), stabilization, or transport of the ligand to or away from the high affinity receptors (18–20). In addition, studies have suggested that the core proteins themselves may also play an important role in cell phenotype and function (21).HSPGs have been implicated in a number of pathological conditions, such as Simpson-Golabi-Behmel overgrowth syndrome (22), fibrodysplasia ossificans progressiva (23), and Alzheimer disease (24). In addition, HSPGs are overexpressed in many forms of cancer, including prostate cancer and melanoma (25, 26). Importantly, in cancer, proteoglycans can have both tumor-promoting and tumor-suppressing activities. This depends on the type of protein core, the GAGs attached, and the localization of the proteoglycan and the molecules they associate with. In addition, the tumor subtype, stages, and degree of tumor differentiation also affect the function of HSPGs (27). HSPGs are cleaved by heparanases or heparin lyases (heparinases), which have been shown to have differing effects on tumor cell activity. For example, treating cancer cells with heparanase-1, which cleaves heparin-like regions (specifically HLGAG sites with O-sulfated l-iduronic acid residues), results in an increase in both tumor growth and metastatic dissemination (28). However, treating tumor cells with heparinase III, which more specifically cleaves HSPGs (i.e. unsulfated d-glucorinic acid, heparan-sulfate-like regions) results in an inhibition of their metastatic capacity (29). Importantly, heparanase I cleaves only certain side chains, where heparinase III treatment cleaves the entire backbone of the HSPG. It is likely that cleavage of specific side chains facilitates cell motility by releasing cells from adhesion to neighboring cells, whereas cleavage of the entire molecule decreases the availability of secreted ligands to their receptors, especially those involved in autocrine signaling, such as Wnt5A.Historically, Wnt5A has been quite difficult to purify from cell culture media, despite the fact that it is a secreted protein. Further, in melanoma cells, Wnt5A appears to be signaling in an autocrine fashion (1, 2). These two observations, together with the fact that Wnt5A undergoes glycosylation (30), led us to hypothesize that HSPGs might be involved in increasing the availability of Wnt5A to its receptor, resulting in an increase in autocrine signaling and ultimately an increase in cellular invasion. In this study, we explore this hypothesis and investigate the role of HSPGs in the Wnt5A signaling cascade in metastatic melanoma cells.     

Ror2/Frizzled Complex Mediates Wnt5a-Induced AP-1 Activation by Regulating Dishevelled Polymerization

The receptor tyrosine kinase Ror2 acts as a receptor or coreceptor for Wnt5a to mediate Wnt5a-induced activation of the Wnt/JNK pathway and inhibition of the β-catenin-dependent canonical Wnt pathway. However, little is known about how Ror2 cooperates with another receptor component(s) to mediate Wnt5a signaling. We show here that Ror2 regulates Wnt5a-induced polymerization of Dishevelled (Dvl) and that this Ror2-mediated regulation of Dvl is independent of the cytoplasmic region of Ror2. Ror2 can associate with Frizzled7 (Fz7) via its extracellular cysteine-rich domain to form a receptor complex that is required for the regulation of Dvl and activation of the AP-1 promoter after Wnt5a stimulation. Suppressed expression of Fz7 indeed results in the inhibition of Wnt5a-induced polymerization of Dvl and AP-1 activation. Interestingly, both the DIX and the DEP domains of Dvl are indispensable for Dvl polymerization and subsequent AP-1 activation after Wnt5a stimulation. We further show that polymerized Dvl is colocalized with Rac1 and that suppressed expression of Rac1 inhibits Wnt5a-induced AP-1 activation. Collectively, our results indicate that Ror2/Fz receptor complex plays an important role in the Wnt5a/Rac1/AP-1 pathway by regulating the polymerization of Dvl.Wnt proteins can elicit β-catenin-dependent and -independent signaling pathways (2, 20, 46). Ror2 is a member of the Ror family of receptor tyrosine kinases and plays essential roles in developmental morphogenesis (21, 26, 31, 32, 44). Ror2 has been shown to act as a receptor or coreceptor for Wnt5a to activate the β-catenin-independent signaling pathway, involving JNK/c-Jun (AP-1), Src and Ca²⁺, which are essential for cell polarity, migration, and cancer cell invasion (8, 14, 28-31, 37). Wnt5a/Ror2 signaling also plays a crucial role in inhibiting the β-catenin-dependent signaling pathway (25). Structure-function analyses of Ror2 revealed that Ror2 mediates Wnt5a signaling through distinct mechanisms dependent on and independent of its kinase activity, i.e., Wnt5a-induced migration of fibroblast cells requires the cytoplasmic C-terminal portion of Ror2 but not its intrinsic kinase activity (28), whereas the intrinsic kinase activity of Ror2 is indispensable for extracellular matrix (ECM) degradation of osteosarcoma cells (8). In addition, inhibition of the β-catenin-dependent signaling pathway by Wnt5a also requires the intrinsic kinase activity of Ror2 (24). Importantly, the Caenorhabditis elegans ortholog of Ror2, CAM-1, also has the kinase activity-dependent and -independent functions (9, 12, 13). Furthermore, CAM-1 exhibits the cytoplasmic region-independent functions, including cell migration (17), synaptic transmission at the neuromuscular junction (10), and inhibition of the β-catenin-dependent signaling pathway (11), although their underlying molecular mechanisms remain to be determined. However, it is unknown whether or not Ror2 also exhibits the cytoplasmic region-independent functions in other organisms.Dishevelled (Dvl) is an essential mediator of both the β-catenin-dependent and -independent signaling pathways. We have previously reported that both Ror2 and Dvl are required for Wnt5a-induced cell migration (28). However, the relationship between Ror2 and Dvl in Wnt5a signaling remains unclear. It has been reported that Dvl has an ability to form dynamic polymers, which are crucial for activating the β-catenin-dependent signaling pathway probably by serving as a scaffold for Axin recruitment (39, 41). However, there is no direct evidence showing that Wnt stimulation indeed induces dynamic formation of Dvl polymers. In addition, it remains unclear whether or not the polymerization of Dvl is involved in the β-catenin-independent signaling pathway.In the present study we show that Wnt5a induces dynamic polymerization of Dvl2 via a receptor complex containing both Ror2 and Frizzled (Fz)7, even in the absence of the cytoplasmic region of Ror2. We further provide evidence indicating that Ror2/Fz7 receptor complex plays an important role in Wnt5a/Rac1/AP-1 pathway by regulating polymerization of Dvl2.     

Regulation of Phosphatidylinositol Kinases and Metabolism by Wnt3a and Dvl

Wnt signaling plays important roles in various physiological and pathophysiological processes. The pathway that leads to β-catenin stabilization is initiated by Wnt binding to its cell surface receptors, which induces the formation of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) via activation of phosphatidylinositol 4-phosphate 5-kinase (PIP5K) type I. Here, we show that Wnt also stimulated the production of phosphatidylinositol 4-phosphate (PtdIns(4)P), which depended on Frizzled (Fz), Dishevelled (Dvl), and phosphatidylinositol 4-kinase (PI4K) type IIα in HEK293T cells. Dvl directly interacted with and activated PI4KIIα by increasing its V_max for ATP and PtdIns. In addition, Dvl regulated PI4KIIα and PIP5KI via different domains. Moreover, Dvl, PI4KIIα, and PIP5KI appeared to form a ternary complex upon Wnt3a stimulation. This complex may allow efficient production of PtdIns(4,5)P₂ from PtdIns, which is far more abundant than PtdIns(4)P in cells. Therefore, this study provides new insights into the mechanism by which Wnt3a regulates the production of PtdIns(4,5)P₂.The Wnt family of secretory glycoproteins plays important roles in regulation of embryonic development and tumorigenesis. They also regulate many other physiological and pathophysiological processes, including bone development, neuronogenesis, adipogenesis, myogenesis, organogenesis, and lipid and glucose metabolism (1–5). Studies using Drosophila and Xenopus embryos as well as mammalian cells have established a canonical Wnt signaling pathway that leads to stabilization of β-catenin. In the absence of Wnt, a number of proteins, including Axin, adenomatous polyposis coli (APC), casein kinase 1 (CK1), glycogen synthase kinase-3β (GSK3β),³ form a complex that facilitates β-catenin phosphorylation by CK1 and GSK3β. This phosphorylation targets β-catenin for ubiquitination and proteasome-mediated proteolytic degradation (3, 6). Some of the Wnt proteins bind to two cell surface receptors Fz and low density lipoprotein receptor-related protein (LRP) 5/6 and initiate a signaling cascade that eventually leads to the suppression of β-catenin phosphorylation by GSK3β and stabilization of β-catenin.Because the finding that the canonical Wnt proteins transduce signals by inducing the interaction between LRP5/6 and Axin (7), more has been learned about the mechanisms by which this interaction is regulated by Wnt proteins. Studies have indicated that two phosphorylation events at the C-terminal intracellular domain of LRP5/6, the phosphorylation of Thr¹⁴⁷⁹ by CKIγ (8, 9) and of Ser¹⁴⁹⁰ by GSK3 (10, 11), were required for the interaction. We recently showed that Wnt3a stimulated the production of PtdIns (4,5)P₂, which in turn regulated the phosphorylation of LRP5/6 at Thr¹⁴⁷⁹ and Ser¹⁴⁹⁰ (12). We also showed that Wnt3a regulated phosphatidylinositol 4-phosphate 5-kinase type I (PIP5KI) activity by inducing the interaction between Dvl and PIP5KI (12). Moreover, Dvl could directly stimulate the lipid kinase activity of PIP5KI (12).PtdIns(4,5)P₂ plays important roles in various cellular functions, including membrane trafficking, cytoskeletal reorganization, migration, ion channel activation, and signal transduction (13). It, however, represents less than 1% of plasma membrane phospholipids and is primarily synthesized in most cells by sequential phosphorylation of PtdIns on the D4 and D5 positions of the inositol ring by two PtdIns kinases, PI4K and PIP5KI, respectively (14, 15). While PtdIns(4)P, the substrate for PIP5KI, is also accounted for around 1% of plasma membrane phospholipids, PtdIns, the substrate for PI4K, is very abundant. Thus, Wnt3a may have to stimulate PI4K activity to provide enough substrate for PIP5KI in PtdIns(4,5)P₂ production.Two types of PI4K (PI4KI and PI4KII) have been characterized in mammalian cells. There are two isoforms of PI4KII (PI4KIIα and PI4KIIβ) and two isoforms of PI4KI (PI4KIα and PI4KIβ) (16). In our previous study, we demonstrated the involvement of PI4KIIα in Wnt signaling. siRNA-mediated knockdown in mammalian cells and morpholino-mediated suppression in Xenopus embryos of PI4KIIα inhibited LRP6 phosphorylation and Wnt signaling. In this report, we examined whether Wnt3a regulates the lipid kinase activity of PI4KIIα and found that Wnt3a could induce an increase in the level of PtdIns(4)P in a Dvl- and Fz-dependent manner. In addition, the Dvl protein was found to directly interact with and activate PI4KIIα. Moreover, different domains of Dvl appeared to be involved in the regulation of PI4KIIα and PIP5KI, and Wnt3a induced the formation of a complex of Dvl, PI4KIIα, and PIP5KI possibly for more efficient production of PtdIns (4,5)P₂ in cells.     

High-definition De Novo Sequencing of Crustacean Hyperglycemic Hormone (CHH)-family Neuropeptides

A complete understanding of the biological functions of large signaling peptides (>4 kDa) requires comprehensive characterization of their amino acid sequences and post-translational modifications, which presents significant analytical challenges. In the past decade, there has been great success with mass spectrometry-based de novo sequencing of small neuropeptides. However, these approaches are less applicable to larger neuropeptides because of the inefficient fragmentation of peptides larger than 4 kDa and their lower endogenous abundance. The conventional proteomics approach focuses on large-scale determination of protein identities via database searching, lacking the ability for in-depth elucidation of individual amino acid residues. Here, we present a multifaceted MS approach for identification and characterization of large crustacean hyperglycemic hormone (CHH)-family neuropeptides, a class of peptide hormones that play central roles in the regulation of many important physiological processes of crustaceans. Six crustacean CHH-family neuropeptides (8–9.5 kDa), including two novel peptides with extensive disulfide linkages and PTMs, were fully sequenced without reference to genomic databases. High-definition de novo sequencing was achieved by a combination of bottom-up, off-line top-down, and on-line top-down tandem MS methods. Statistical evaluation indicated that these methods provided complementary information for sequence interpretation and increased the local identification confidence of each amino acid. Further investigations by MALDI imaging MS mapped the spatial distribution and colocalization patterns of various CHH-family neuropeptides in the neuroendocrine organs, revealing that two CHH-subfamilies are involved in distinct signaling pathways.Neuropeptides and hormones comprise a diverse class of signaling molecules involved in numerous essential physiological processes, including analgesia, reward, food intake, learning and memory (1). Disorders of the neurosecretory and neuroendocrine systems influence many pathological processes. For example, obesity results from failure of energy homeostasis in association with endocrine alterations (2, 3). Previous work from our lab used crustaceans as model organisms found that multiple neuropeptides were implicated in control of food intake, including RFamides, tachykinin related peptides, RYamides, and pyrokinins (4–6).Crustacean hyperglycemic hormone (CHH)¹ family neuropeptides play a central role in energy homeostasis of crustaceans (7–17). Hyperglycemic response of the CHHs was first reported after injection of crude eyestalk extract in crustaceans. Based on their preprohormone organization, the CHH family can be grouped into two sub-families: subfamily-I containing CHH, and subfamily-II containing molt-inhibiting hormone (MIH) and mandibular organ-inhibiting hormone (MOIH). The preprohormones of the subfamily-I have a CHH precursor related peptide (CPRP) that is cleaved off during processing; and preprohormones of the subfamily-II lack the CPRP (9). Uncovering their physiological functions will provide new insights into neuroendocrine regulation of energy homeostasis.Characterization of CHH-family neuropeptides is challenging. They are comprised of more than 70 amino acids and often contain multiple post-translational modifications (PTMs) and complex disulfide bridge connections (7). In addition, physiological concentrations of these peptide hormones are typically below picomolar level, and most crustacean species do not have available genome and proteome databases to assist MS-based sequencing.MS-based neuropeptidomics provides a powerful tool for rapid discovery and analysis of a large number of endogenous peptides from the brain and the central nervous system. Our group and others have greatly expanded the peptidomes of many model organisms (3, 18–33). For example, we have discovered more than 200 neuropeptides with several neuropeptide families consisting of as many as 20–40 members in a simple crustacean model system (5, 6, 25–31, 34). However, a majority of these neuropeptides are small peptides with 5–15 amino acid residues long, leaving a gap of identifying larger signaling peptides from organisms without sequenced genome. The observed lack of larger size peptide hormones can be attributed to the lack of effective de novo sequencing strategies for neuropeptides larger than 4 kDa, which are inherently more difficult to fragment using conventional techniques (34–37). Although classical proteomics studies examine larger proteins, these tools are limited to identification based on database searching with one or more peptides matching without complete amino acid sequence coverage (36, 38).Large populations of neuropeptides from 4–10 kDa exist in the nervous systems of both vertebrates and invertebrates (9, 39, 40). Understanding their functional roles requires sufficient molecular knowledge and a unique analytical approach. Therefore, developing effective and reliable methods for de novo sequencing of large neuropeptides at the individual amino acid residue level is an urgent gap to fill in neurobiology. In this study, we present a multifaceted MS strategy aimed at high-definition de novo sequencing and comprehensive characterization of the CHH-family neuropeptides in crustacean central nervous system. The high-definition de novo sequencing was achieved by a combination of three methods: (1) enzymatic digestion and LC-tandem mass spectrometry (MS/MS) bottom-up analysis to generate detailed sequences of proteolytic peptides; (2) off-line LC fractionation and subsequent top-down MS/MS to obtain high-quality fragmentation maps of intact peptides; and (3) on-line LC coupled to top-down MS/MS to allow rapid sequence analysis of low abundance peptides. Combining the three methods overcomes the limitations of each, and thus offers complementary and high-confidence determination of amino acid residues. We report the complete sequence analysis of six CHH-family neuropeptides including the discovery of two novel peptides. With the accurate molecular information, MALDI imaging and ion mobility MS were conducted for the first time to explore their anatomical distribution and biochemical properties.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

PR55��, a Regulatory Subunit of PP2A, Specifically Regulates PP2A-mediated ��-Catenin Dephosphorylation

A central question in Wnt signaling is the regulation of β-catenin phosphorylation and degradation. Multiple kinases, including CKIα and GSK3, are involved in β-catenin phosphorylation. Protein phosphatases such as PP2A and PP1 have been implicated in the regulation of β-catenin. However, which phosphatase dephosphorylates β-catenin in vivo and how the specificity of β-catenin dephosphorylation is regulated are not clear. In this study, we show that PP2A regulates β-catenin phosphorylation and degradation in vivo. We demonstrate that PP2A is required for Wnt/β-catenin signaling in Drosophila. Moreover, we have identified PR55α as the regulatory subunit of PP2A that controls β-catenin phosphorylation and degradation. PR55α, but not the catalytic subunit, PP2Ac, directly interacts with β-catenin. RNA interference knockdown of PR55α elevates β-catenin phosphorylation and decreases Wnt signaling, whereas overexpressing PR55α enhances Wnt signaling. Taken together, our results suggest that PR55α specifically regulates PP2A-mediated β-catenin dephosphorylation and plays an essential role in Wnt signaling.Wnt/β-catenin signaling plays essential roles in development and tumorigenesis (1–3). Our previous work found that β-catenin is sequentially phosphorylated by CKIα⁴ and GSK3 (4), which creates a binding site for β-Trcp (5), leading to degradation via the ubiquitination/proteasome machinery (3). Mutations in β-catenin or APC genes that prevent β-catenin phosphorylation or ubiquitination/degradation lead ultimately to cancer (1, 2).In addition to the involvement of kinases, protein phosphatases, such as PP1, PP2A, and PP2C, are also implicated in Wnt/β-catenin regulation. PP2C and PP1 may regulate dephosphorylation of Axin and play positive roles in Wnt signaling (6, 7). PP2A is a multisubunit enzyme (8–10); it has been reported to play either positive or negative roles in Wnt signaling likely by targeting different components (11–21). Toward the goal of understanding the mechanism of β-catenin phosphorylation, we carried out siRNA screening targeting several major phosphatases, in which we found that PP2A dephosphorylates β-catenin. This is consistent with a recent study where PP2A is shown to dephosphorylate β-catenin in a cell-free system (18).PP2A consists of a catalytic subunit (PP2Ac), a structure subunit (PR65/A), and variable regulatory B subunits (PR/B, PR/B′, PR/B″, or PR/B‴). The substrate specificity of PP2A is thought to be determined by its B subunit (9). By siRNA screening, we further identified that PR55α, a regulatory subunit of PP2A, specifically regulates β-catenin phosphorylation and degradation. Mechanistically, we found that PR55α directly interacts with β-catenin and regulates PP2A-mediated β-catenin dephosphorylation in Wnt signaling.     

Analysis of Proteins That Rapidly Change Upon Mechanistic/Mammalian Target of Rapamycin Complex 1 (mTORC1) Repression Identifies Parkinson Protein 7 (PARK7) as a Novel Protein Aberrantly Expressed in Tuberous Sclerosis Complex (TSC)

Many biological processes involve the mechanistic/mammalian target of rapamycin complex 1 (mTORC1). Thus, the challenge of deciphering mTORC1-mediated functions during normal and pathological states in the central nervous system is challenging. Because mTORC1 is at the core of translation, we have investigated mTORC1 function in global and regional protein expression. Activation of mTORC1 has been generally regarded to promote translation. Few but recent works have shown that suppression of mTORC1 can also promote local protein synthesis. Moreover, excessive mTORC1 activation during diseased states represses basal and activity-induced protein synthesis. To determine the role of mTORC1 activation in protein expression, we have used an unbiased, large-scale proteomic approach. We provide evidence that a brief repression of mTORC1 activity in vivo by rapamycin has little effect globally, yet leads to a significant remodeling of synaptic proteins, in particular those proteins that reside in the postsynaptic density. We have also found that curtailing the activity of mTORC1 bidirectionally alters the expression of proteins associated with epilepsy, Alzheimer''s disease, and autism spectrum disorder—neurological disorders that exhibit elevated mTORC1 activity. Through a protein–protein interaction network analysis, we have identified common proteins shared among these mTORC1-related diseases. One such protein is Parkinson protein 7, which has been implicated in Parkinson''s disease, yet not associated with epilepsy, Alzheimers disease, or autism spectrum disorder. To verify our finding, we provide evidence that the protein expression of Parkinson protein 7, including new protein synthesis, is sensitive to mTORC1 inhibition. Using a mouse model of tuberous sclerosis complex, a disease that displays both epilepsy and autism spectrum disorder phenotypes and has overactive mTORC1 signaling, we show that Parkinson protein 7 protein is elevated in the dendrites and colocalizes with the postsynaptic marker postsynaptic density-95. Our work offers a comprehensive view of mTORC1 and its role in regulating regional protein expression in normal and diseased states.The mechanistic/mammalian target of rapamycin complex 1 (mTORC1)¹ is a serine/threonine protein kinase that is highly expressed in many cell types (1). In the brain, mTORC1 tightly coordinates different synaptic plasticities — long-term potentiation (LTP) and long-term depression (LTD) — the molecular correlates of learning and memory (2–5). Because mTORC1 is at the core of many synaptic signaling pathways downstream of glutamate and neurotrophin receptors, many hypothesize that dysregulated mTORC1 signaling underlies cognitive deficits observed in several neurodegenerative diseases (3, 6–17). For example, mTORC1 and its downstream targets are hyperactive in human brains diagnosed with Alzheimer''s disease (AD) (18–20). Additionally in animal models of autism spectrum disorder (ASD), altered mTORC1 signaling contributes to the observed synaptic dysfunction and aberrant network connectivity (13, 15, 21–27). Furthermore, epilepsy, which is common in AD and ASD, has enhanced mTORC1 activity (28–32).Phosphorylation of mTORC1, considered the active form, is generally regarded to promote protein synthesis (33). Thus, many theorize that diseases with overactive mTORC1 arise from excessive protein synthesis (14). Emerging data, however, show that suppressing mTORC1 activation can trigger local translation in neurons (34, 35). Pharmacological antagonism of N-methyl-d-aspartate (NMDA) receptors, a subtype of glutamate receptors that lies upstream of mTOR activation, promotes the synthesis of the voltage-gated potassium channel, Kv1.1, in dendrites (34, 35). Consistent with these results, in models of temporal lobe epilepsy there is a reduction in the expression of voltage-gated ion channels including Kv1.1 (30, 31, 36). Interestingly in a model of focal neocortical epilepsy, overexpression of Kv1.1 blocked seizure activity (37). Because both active and inactive mTORC1 permit protein synthesis, we sought to determine the proteins whose expression is altered when mTORC1 phosphorylation is reduced in vivo.Rapamycin is an FDA-approved, immunosuppressive drug that inhibits mTORC1 activity (38). We capitalized on the ability of rapamycin to reduce mTORC1 activity in vivo and the unbiased approach of mass spectrometry to identify changes in protein expression. Herein, we provide evidence that mTORC1 activation bidirectionally regulates protein expression, especially in the PSD where roughly an equal distribution of proteins dynamically appear and disappear. Remarkably, using protein–protein interaction networks facilitated the novel discovery that PARK7, a protein thus far only implicated in Parkinson''s disease, (1) is up-regulated by increased mTORC1 activity, (2) resides in the PSD only when mTORC1 is active, and (3) is aberrantly expressed in a rodent model of TSC, an mTORC1-related disease that has symptoms of epilepsy and autism. Collectively, these data provide the first comprehensive list of proteins whose abundance or subcellular distributions are altered with acute changes in mTORC1 activity in vivo.     

Differential Regulation of Elastic Fiber Formation by Fibulin-4 and -5

Fibulin-4 and -5 are extracellular glycoproteins with essential non-compensatory roles in elastic fiber assembly. We have determined how they interact with tropoelastin, lysyl oxidase, and fibrillin-1, thereby revealing how they differentially regulate assembly. Strong binding between fibulin-4 and lysyl oxidase enhanced the interaction of fibulin-4 with tropoelastin, forming ternary complexes that may direct elastin cross-linking. In contrast, fibulin-5 did not bind lysyl oxidase strongly but bound tropoelastin in terminal and central regions and could concurrently bind fibulin-4. Both fibulins differentially bound N-terminal fibrillin-1, which strongly inhibited their binding to lysyl oxidase and tropoelastin. Knockdown experiments revealed that fibulin-5 controlled elastin deposition on microfibrils, although fibulin-4 can also bind fibrillin-1. These experiments provide a molecular account of the distinct roles of fibulin-4 and -5 in elastic fiber assembly and how they act in concert to chaperone cross-linked elastin onto microfibrils.Fibulins are a family of extracellular glycoproteins containing contiguous calcium-binding epidermal growth factor-like domains (cbEGFs)³ and a characteristic C-terminal fibulin (FC) domain (1–3). Recent studies have revealed that fibulin-4 and -5 are both essential for elastic fiber formation (4–7). Fibulin-4 is widely expressed from early embryogenesis and is necessary for normal vascular, lung, and skin development, since mice that lack fibulin-4 do not form elastic fibers and die perinatally (5). Furthermore, mice with reduced fibulin-4 expression develop aneurysms (8). Fibulin-5 is abundant in the aorta and large arteries during embryogenesis and following vascular injury (9, 10). Lack of fibulin-5 causes a less severe phenotype, with viable homozygous mice, but the elastic fibers in skin, lungs, and aorta are irregular and fragmented (6, 7), and there is altered vascular remodeling (11). These mice models also highlight that fibulin-4 and -5 have non-compensatory roles in elastic fiber formation. Mutations in both molecules can cause cutis laxa, a heritable disorder associated with elastic fiber degeneration leading to sagging skin, vascular tortuosity, and emphysematous lungs (12–15). A third isoform, fibulin-3, may play a minor role in elastic fiber formation, since its deficiency disrupts elastic fibers in Bruch''s membrane of the eye (16) and vaginal tissues (17).Elastic fiber formation is a complex multistep process (18–20). Initial pericellular microassembly of tropoelastin, which may involve the 67-kDa elastin-binding protein receptor, generates elastin globules that are stabilized by desmosine cross-links catalyzed mainly by lysyl oxidase (LOX) but also by LOXL1 (LOX-like 1). These globules are deposited on a fibrillin microfibril template, where they coalesce and undergo further cross-linking to form the elastin core of mature fibers. The ability of fibulin-4 and -5 to bind tropoelastin and fibrillin-1, the major structural component of microfibrils, supports a model in which these fibulins direct elastin deposition on microfibrils (4–7, 21–25). This model does not delineate the unique molecular contributions of fibulin-4 and -5 to elastic fiber formation, but some molecular differences have emerged. Tropoelastin was bound more strongly by fibulin-5 than by fibulin-4, whereas fibulin-5 was at the microfibril-elastin interface, but perichondrial fibulin-4 localized mainly to microfibrils (4).Fibulin-4 null mice offer tantalizing clues to how fibulin-4 contributes to elastic fiber formation (5). They had dramatically reduced (94%) desmosine cross-links despite no change in elastin or LOX expression levels, and electron-dense rodlike structures were prominent within elastin aggregates. Morphologically similar structures seen after chemically inhibiting LOX were previously identified as glycosaminoglycans, which can bind charged free ϵ-amino groups on lysines in tropoelastin (26). However, fibulin-4^+/− mice showed ∼20% increase in desmosine (5). LOX-null mice have phenotypic features similar to those of fibulin-4 null mice, dying perinatally with 60% reduced desmosine cross-links and major abnormalities in vascular and other elastic tissues (27, 28). In contrast, LOXL1-null mice are viable but have reduced desmosine (29), whereas fibulin-5 null mice have a 16% reduction in desmosine cross-links and survive well into adulthood (7). Detection of the LOXL1 pro-domain in fibulin-5 null mice skin but not wild-type skin implicates fibulin-5 in activation of LOXL1 (30).We and others have shown that fibrillin-1 and the microfibrillar protein MAGP-1 can both directly bind tropoelastin (31–34). However, the fibulin-null mice show that the fibrillin-1 interaction with tropoelastin is insufficient to support elastic fiber formation in vivo. Fibulin-5 has been reported to facilitate tropoelastin binding to the N-terminal half of fibrillin-1 (21). A study of elastin polypeptide self-assembly through coacervation and maturation phases showed that, although the N-terminal half of fibrillin-1 increased maturation velocity and droplet clustering, fibulin-4 and -5 both slowed maturation and limited globule growth (35). These studies imply that fibulins and fibrillin-1 act together to regulate elastin accretion on microfibrils.To gain further insights into the contributions of fibulin-4 and -5 to elastic fiber formation, we have delineated how they interact with tropoelastin, LOX, and fibrillin-1. Novel findings are that fibulin-4 directly binds LOX, and this interaction enhances fibulin-4 binding to tropoelastin, thus forming a ternary complex that may be critical for elastin cross-linking. Fibulin-5 can concurrently bind fibulin-4 and tropoelastin, but the interaction of both fibulins with fibrillin-1 strongly inhibits their binding to tropoelastin. These interactions indicate the molecular basis of how fibulins act as chaperones for deposition of elastin onto microfibrils. Our study thus provides a molecular account of the differential roles of fibulins-4 and -5 in elastic fiber formation.     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Fibulin 5 Forms a Compact Dimer in Physiological Solutions

Fibulin 5 is a 52-kDa calcium-binding epidermal growth factor (cbEGF)-rich extracellular matrix protein that is essential for the formation of elastic tissues. Missense mutations in fibulin 5 cause the elastin disorder cutis laxa and have been associated with age-related macular degeneration, a leading cause of blindness. We investigated the structure, hydrodynamics, and oligomerization of fibulin 5 using small angle x-ray scattering, EM, light scattering, circular dichroism, and sedimentation. Compact structures for the monomer were determined by small angle x-ray scattering and EM, and are supported by close agreement between the theoretical sedimentation of the structures and the experimental sedimentation of the monomer in solution. EM showed that monomers associate around a central cavity to form a dimer. Light scattering and equilibrium sedimentation demonstrated that the equilibrium between the monomer and the dimer is dependent upon NaCl and Ca²⁺ concentrations and that the dimer is dominant under physiological conditions. The dimerization of fragments containing just the cbEGF domains suggests that intermolecular interactions between cbEGFs cause dimerization of fibulin 5. It is possible that fibulin 5 functions as a dimer during elastinogenesis or that dimerization may provide a method for limiting interactions with binding partners such as tropoelastin.Fibulins are a family of seven extracellular matrix glycoproteins, some of which associate with elastic fibers and basement membranes (1, 2). They are involved in the assembly, organization, and stabilization of macromolecular complexes (3). Fibulins contain arrays of cbEGF²-like domains and a fibulin-type C-terminal (Fc) module (4). Fibulins 3–5 have a modified N-terminal cbEGF domain, followed by five cbEGF domains (4).Fibulin 5 (supplemental Fig. S1) is highly expressed in developing arteries with a low expression in adult vessels that is up-regulated following vascular injury and in atherosclerosis (5, 6). Expression has been detected in other elastin-rich tissues, including aorta, skin, uterus, lung, heart, ovary, and colon (5, 6). The extensibility of such tissues is provided by elastic fibers (7), and aging is associated with a loss of elasticity (8). Fibulin 5 is essential for elastinogenesis. The fibulin 5 knock-out mouse exhibits disorganized elastic fibers resulting in severe elastinopathies, with loose skin, vascular abnormalities, and emphysematous lungs. Similar changes are seen in an aged phenotype (9, 10). Mutations in fibulin 5 lead to the elastin disorder cutis laxa (11–13) and have been associated with age-related macular degeneration (14, 15).It has been shown that fibulin 5 binds elastic fibers (16) and interacts with tropoelastin (10), fibrillin 1 (17), lysyl oxidase-like protein 1 (18), -2, and -4 (19), latent transforming growth factor-β-binding protein 2 (19), emilin 1 (20), apolipoprotein (a) (21), and superoxide dismutase (22). Through an RGD motif fibulin 5 interacts with integrins (6, 9, 23).The assembly of elastic fibers is a complex hierarchical process. A model proposes that fibulin 5 associates with microfibrils via interactions with fibrillin 1; tropoelastin molecules bind fibulin 5 and coacervate, and lysyl oxidase-like protein 1 enzymes cross-link tropoelastin to form mature elastin (7, 16). Data that support this model indicate that fibulin 5 potentially increases the coacervation of tropoelastin, enhancing elastic fiber formation (24). However, other data suggest that fibulin 5 slows the maturation of elastin assemblies (25).Rotary-shadowing EM has suggested that fibulin 5 exists as a short rod with a globular domain at one end (26). We used size-exclusion column multiangle laser light scattering (SEC-MALLS), small angle x-ray scattering (SAXS), EM single particle analysis, analytical ultracentrifugation (AUC), CD, and isoelectric focusing to investigate the structures of fibulin 5 in monomeric and dimeric form, and the equilibrium between the two forms.