Systematic analysis of the Frazzled receptor interactome establishes previously unreported regulators of axon guidance.

The Netrin receptor Dcc and its Drosophila homolog Frazzled play crucial roles in diverse developmental process, including axon guidance. In Drosophila, Fra regulates midline axon guidance through a Netrin-dependent and a Netrin-independent pathway. However, what molecules regulate these distinct signaling pathways remain unclear. To identify Fra-interacting proteins, we performed affinity purification mass spectrometry to establish a neuronal-specific Fra interactome. In addition to known interactors of Fra and Dcc, including Netrin and Robo1, our screen identified 85 candidate proteins, the majority of which are conserved in humans. Many of these proteins are expressed in the ventral nerve cord, and gene ontology, pathway analysis and biochemical validation identified several previously unreported pathways, including the receptor tyrosine phosphatase Lar, subunits of the COP9 signalosome and Rho-5, a regulator of the metalloprotease Tace. Finally, genetic analysis demonstrates that these genes regulate axon guidance and may define as yet unknown signaling mechanisms for Fra and its vertebrate homolog Dcc. Thus, the Fra interactome represents a resource to guide future functional studies.

PPFIA4 Promotes Proliferation, Migration and Glycolysis of Esophageal Cancer Cells.

PPFIA4 has been reported to be associated with cancer glycolysis, but its role in esophageal cancer (EC) is unclear. OBJECTIVE: To investigate the role of PPFIA4 in EC. METHODS: qRT-PCR and WB were used to detect the expression of PPFIA4 in EC cells and normal cells. PPFIA4 was inhibited to detect changes in the invasion and migration ability of EC cells. WB detected the expression of cell invasion and migration marker proteins MMP-2 and MMP-9, and the kit detected changes in ATP and lactate levels in EC cells. RESULTS: PPFIA4 was highly expressed in EC cells. Inhibition of PPFIA4 inhibited the invasion and migration ability as well as the expression of MMP-2 and MMP-9 in EC cells, and decreased the levels of ATP and lactate in EC cells. CONCLUSION: Inhibition of PPFIA4 inhibited invasion, migration ability and glycolysis of EC cells.

The RNA-binding protein Musashi controls axon compartment-specific synaptic connectivity through ptp69D mRNA poly(A)-tailing.

Synaptic targeting with subcellular specificity is essential for neural circuit assembly. Developing neurons use mechanisms to curb promiscuous synaptic connections and to direct synapse formation to defined subcellular compartments. How this selectivity is achieved molecularly remains enigmatic. Here, we discover a link between mRNA poly(A)-tailing and axon collateral branch-specific synaptic connectivity within the CNS. We reveal that the RNA-binding protein Musashi binds to the mRNA encoding the receptor protein tyrosine phosphatase Ptp69D, thereby increasing poly(A) tail length and Ptp69D protein levels. This regulation specifically promotes synaptic connectivity in one axon collateral characterized by a high degree of arborization and strong synaptogenic potential. In a different compartment of the same axon, Musashi prevents ectopic synaptogenesis, revealing antagonistic, compartment-specific functions. Moreover, Musashi-dependent Ptp69D regulation controls synaptic connectivity in the olfactory circuit. Thus, Musashi differentially shapes synaptic connectivity at the level of individual subcellular compartments and within different developmental and neuron type-specific contexts.

Type IIa RPTPs and Glycans: Roles in Axon Regeneration and Synaptogenesis.

Type IIa receptor tyrosine phosphatases (RPTPs) play pivotal roles in neuronal network formation. It is emerging that the interactions of RPTPs with glycans, i.e., chondroitin sulfate (CS) and heparan sulfate (HS), are critical for their functions. We highlight here the significance of these interactions in axon regeneration and synaptogenesis. For example, PTPσ, a member of type IIa RPTPs, on axon terminals is monomerized and activated by the extracellular CS deposited in neural injuries, dephosphorylates cortactin, disrupts autophagy flux, and consequently inhibits axon regeneration. In contrast, HS induces PTPσ oligomerization, suppresses PTPσ phosphatase activity, and promotes axon regeneration. PTPσ also serves as an organizer of excitatory synapses. PTPσ and neurexin bind one another on presynapses and further bind to postsynaptic leucine-rich repeat transmembrane protein 4 (LRRTM4). Neurexin is now known as a heparan sulfate proteoglycan (HSPG), and its HS is essential for the binding between these three molecules. Another HSPG, glypican 4, binds to presynaptic PTPσ and postsynaptic LRRTM4 in an HS-dependent manner. Type IIa RPTPs are also involved in the formation of excitatory and inhibitory synapses by heterophilic binding to a variety of postsynaptic partners. We also discuss the important issue of possible mechanisms coordinating axon extension and synapse formation.

Understanding the causes of errors in eukaryotic protein-coding gene prediction: a case study of primate proteomes.

BACKGROUND: Recent advances in sequencing technologies have led to an explosion in the number of genomes available, but accurate genome annotation remains a major challenge. The prediction of protein-coding genes in eukaryotic genomes is especially problematic, due to their complex exon-intron structures. Even the best eukaryotic gene prediction algorithms can make serious errors that will significantly affect subsequent analyses. RESULTS: We first investigated the prevalence of gene prediction errors in a large set of 176,478 proteins from ten primate proteomes available in public databases. Using the well-studied human proteins as a reference, a total of 82,305 potential errors were detected, including 44,001 deletions, 27,289 insertions and 11,015 mismatched segments where part of the correct protein sequence is replaced with an alternative erroneous sequence. We then focused on the mismatched sequence errors that cause particular problems for downstream applications. A detailed characterization allowed us to identify the potential causes for the gene misprediction in approximately half (5446) of these cases. As a proof-of-concept, we also developed a simple method which allowed us to propose improved sequences for 603 primate proteins. CONCLUSIONS: Gene prediction errors in primate proteomes affect up to 50% of the sequences. Major causes of errors include undetermined genome regions, genome sequencing or assembly issues, and limitations in the models used to represent gene exon-intron structures. Nevertheless, existing genome sequences can still be exploited to improve protein sequence quality. Perspectives of the work include the characterization of other types of gene prediction errors, as well as the development of a more comprehensive algorithm for protein sequence error correction.

Drosophila enabled promotes synapse morphogenesis and regulates active zone form and function.

BACKGROUND: Recent studies of synapse form and function highlight the importance of the actin cytoskeleton in regulating multiple aspects of morphogenesis, neurotransmission, and neural plasticity. The conserved actin-associated protein Enabled (Ena) is known to regulate development of the Drosophila larval neuromuscular junction through a postsynaptic mechanism. However, the functions and regulation of Ena within the presynaptic terminal has not been determined. METHODS: Here, we use a conditional genetic approach to address a presynaptic role for Ena on presynaptic morphology and ultrastructure, and also examine the pathway in which Ena functions through epistasis experiments. RESULTS: We find that Ena is required to promote the morphogenesis of presynaptic boutons and branches, in contrast to its inhibitory role in muscle. Moreover, while postsynaptic Ena is regulated by microRNA-mediated mechanisms, presynaptic Ena relays the output of the highly conserved receptor protein tyrosine phosphatase Dlar and associated proteins including the heparan sulfate proteoglycan Syndecan, and the non-receptor Abelson tyrosine kinase to regulate addition of presynaptic varicosities. Interestingly, Ena also influences active zones, where it restricts active zone size, regulates the recruitment of synaptic vesicles, and controls the amplitude and frequency of spontaneous glutamate release. CONCLUSION: We thus show that Ena, under control of the Dlar pathway, is required for presynaptic terminal morphogenesis and bouton addition and that Ena has active zone and neurotransmission phenotypes. Notably, in contrast to Dlar, Ena appears to integrate multiple pathways that regulate synapse form and function.

Lar maintains the homeostasis of the hematopoietic organ in Drosophila by regulating insulin signaling in the niche.

Stem cell compartments in metazoa get regulated by systemic factors as well as local stem cell niche-derived factors. However, the mechanisms by which systemic signals integrate with local factors in maintaining tissue homeostasis remain unclear. Employing the Drosophila lymph gland, which harbors differentiated blood cells, and stem-like progenitor cells and their niche, we demonstrate how a systemic signal interacts and harmonizes with local factor/s to achieve cell type-specific tissue homeostasis. Our genetic analyses uncovered a novel function of Lar, a receptor protein tyrosine phosphatase. Niche-specific loss of Lar leads to upregulated insulin signaling, causing increased niche cell proliferation and ectopic progenitor differentiation. Insulin signaling assayed by PI3K activation is downregulated after the second instar larval stage, a time point that coincides with the appearance of Lar in the hematopoietic niche. We further demonstrate that Lar physically associates with InR and serves as a negative regulator for insulin signaling in the Drosophila larval hematopoietic niche. Whether Lar serves as a localized invariable negative regulator of systemic signals such as insulin in other stem cell niches remains to be explored.

Chemical activation of divergent protein tyrosine phosphatase domains with cyanine-based biarsenicals.

Strategies for the direct chemical activation of specific signaling proteins could provide powerful tools for interrogating cellular signal transduction. However, targeted protein activation is chemically challenging, and few broadly applicable activation strategies for signaling enzymes have been developed. Here we report that classical protein tyrosine phosphatase (PTP) domains from multiple subfamilies can be systematically sensitized to target-specific activation by the cyanine-based biarsenical compounds AsCy3 and AsCy5. Engineering of the activatable PTPs (actPTPs) is achieved by the introduction of three cysteine residues within a conserved loop of the PTP domain, and the positions of the sensitizing mutations are readily identifiable from primary sequence alignments. In the current study we have generated and characterized actPTP domains from three different subfamilies of both receptor and non-receptor PTPs. Biarsenical-induced stimulation of the actPTPs is rapid and dose-dependent, and is operative with both purified enzymes and complex proteomic mixtures. Our results suggest that a substantial fraction of the classical PTP family will be compatible with the act-engineering approach, which provides a novel chemical-biological tool for the control of PTP activity and the study of PTP function.

First Genome-wide Association Analysis for Growth Traits in the Largest Coral Reef-Dwelling Bony Fishes, the Giant Grouper (Epinephelus lanceolatus).

The giant grouper, Epinephelus lanceolatus, is the largest coral reef-dwelling bony fish species. However, despite extremely fast growth performance and the considerable economic importance in this species, its genetic regulation of growth remains unknown. Here, we performed the first genome-wide association study (GWAS) for five growth traits in 289 giant groupers using 42,323 single nucleotide polymorphisms (SNPs) obtained by genotyping-by-sequencing (GBS). We identified a total of 36 growth-related SNPs, of which 11 SNPs reached a genome-wide significance level. The phenotypic variance explained by these SNPs varied from 7.09% for body height to 18.42% for body length. Moreover, 22 quantitative trait loci (QTLs) for growth traits, including nine significant QTLs and 13 suggestive QTLs, were found on multiple chromosomes. Interestingly, the QTL (LG17: 6934451) was shared between body weight and body height, while two significant QTLs (LG7: 22596399 and LG15: 11877836) for body length were consistent with the associated regions of total length at the genome-wide suggestive level. Eight potential candidate genes close to the associated SNPs were selected for expression analysis, of which four genes (phosphatidylinositol transfer protein cytoplasmic 1, protein tyrosine phosphatase receptor type E, alpha/beta hydrolase domain-containing protein 17C, and vascular endothelial growth factor A-A) were differentially expressed and involved in metabolism, development, response stress, etc. This study improves our understanding of the complex genetic architecture of growth in the giant grouper. The results contribute to the selective breeding of grouper species and the conservation of coral reef fishes.

Demystifying the extracellular matrix and its proteolytic remodeling in the brain: structural and functional insights.

The extracellular matrix (ECM) plays diverse roles in several physiological and pathological conditions. In the brain, the ECM is unique both in its composition and in functions. Furthermore, almost all the cells in the central nervous system contribute to different aspects of this intricate structure. Brain ECM, enriched with proteoglycans and other small proteins, aggregate into distinct structures around neurons and oligodendrocytes. These special structures have cardinal functions in the normal functioning of the brain, such as learning, memory, and synapse regulation. In this review, we have compiled the current knowledge about the structure and function of important ECM molecules in the brain and their proteolytic remodeling by matrix metalloproteinases and other enzymes, highlighting the special structures they form. In particular, the proteoglycans in brain ECM, which are essential for several vital functions, are emphasized in detail.

Planar-Polarized Semaphorin-5c and Plexin A Promote the Collective Migration of Epithelial Cells in Drosophila.

Collective migration of epithelial cells is essential for morphogenesis, wound repair, and the spread of many cancers, yet how individual cells signal to one another to coordinate their movements is largely unknown. Here, we introduce a tissue-autonomous paradigm for semaphorin-based regulation of collective cell migration. Semaphorins typically regulate the motility of neuronal growth cones and other migrating cell types by acting as repulsive cues within the migratory environment. Studying the follicular epithelial cells of Drosophila, we discovered that the transmembrane semaphorin, Sema-5c, promotes collective cell migration by acting within the migrating cells themselves, not the surrounding environment. Sema-5c is planar polarized at the basal epithelial surface such that it is enriched at the leading edge of each cell. This location places it in a prime position to send a repulsive signal to the trailing edge of the cell ahead to communicate directional information between neighboring cells. Our data show that Sema-5c can signal across cell-cell boundaries to suppress protrusions in neighboring cells and that Plexin A is the receptor that transduces this signal. Finally, we present evidence that Sema-5c antagonizes the activity of Lar, another transmembrane guidance cue that operates along leading-trailing cell-cell interfaces in this tissue, via a mechanism that appears to be independent of Plexin A. Together, our results suggest that multiple transmembrane guidance cues can be deployed in a planar-polarized manner across an epithelium and work in concert to coordinate individual cell movements for collective migration.

Liprin-α4 as a New Therapeutic Target for SCLC as an Upstream Mediator of HIF1α.

BACKGROUND/AIM: Small-cell lung cancer (SCLC) remains one of deadliest types of cancers. Cis-diamminedichloroplatinum (CDDP) is a key chemotherapeutic agent for SCLC, however, its therapeutic effect is limited. Recently, hypoxia in the cancer microenvironment has been suggested to influence the effect of cancer therapy. MATERIALS AND METHODS: Using small interfering RNA inhibition of leukocyte common antigen-related interacting protein alpha 4 (liprin-α4), and of hypoxia-inducible factor (HIF)-1α, proliferation, invasion, migration and chemosensitivity were investigated in SBC-5 SCLC cells, under normoxia and hypoxia. RESULTS: Liprin-α4 was found to contribute to proliferation, but not migration and invasion of SBC-5 cells both under normoxia and hypoxia. Inhibition of liprin-α4 increased chemosensitivity of SBC-5 cells under hypoxia. Liprin-α4 signaling occurs through mitogen-activated protein kinase pathways via activation of HIF1α expression. Inhibition of HIF1α reduced proliferation and increased chemosensitivity of SBC-5 cells under hypoxia. CONCLUSION: Liprin-α4 inhibition may enhance the effect of CDDP and liprin-α4 might be a novel therapeutic target in SCLC.

The roles of protein tyrosine phosphatases in bone-resorbing osteoclasts.

Maintaining the proper balance between osteoblast-mediated production of bone and its degradation by osteoclasts is essential for health. Osteoclasts are giant phagocytic cells that are formed by fusion of monocyte-macrophage precursor cells; mature osteoclasts adhere to bone tightly and secrete protons and proteases that degrade its matrix. Phosphorylation of tyrosine residues in proteins, which is regulated by the biochemically-antagonistic activities of protein tyrosine kinases and protein tyrosine phosphatases (PTPs), is central in regulating the production of osteoclasts and their bone-resorbing activity. Here we review the roles of individual PTPs of the classical and dual-specificity sub-families that are known to support these processes (SHP2, cyt-PTPe, PTPRO, PTP-PEST, CD45) or to inhibit them (SHP1, PTEN, MKP1). Characterizing the functions of PTPs in osteoclasts is essential for complete molecular level understanding of bone resorption and for designing novel therapeutic approaches for treating bone disease.

The structure of SALM5 suggests a dimeric assembly for the presynaptic RPTP ligand recognition.

Synaptic adhesion molecules play a crucial role in the regulation of synapse development and maintenance. Recently, several families of leucine-rich repeat (LRR) domain-containing neuronal adhesion molecules have been characterised, including netrin-G ligands, LRRTMs and the synaptic adhesion-like molecule (SALM) family proteins. Most of these are expressed at the excitatory glutamatergic synapses, and dysfunctions of these genes are genetically linked with cognitive disorders, such as autism spectrum disorders and schizophrenia. The SALM family proteins SALM3 and SALM5, similar to SLITRKs, have been shown to bind to the presynaptic receptor protein tyrosine phosphatase (RPTP) family ligands. Here, we present the 3.1 Å crystal structure of the SALM5 LRR-Ig-domain construct and biophysical studies that verify the crystallographic results. We show that SALM1, SALM3 and SALM5 form similar dimeric structures, in which the LRR domains form the dimer interface. Both SALM3 and SALM5 bind to RPTP immunoglobulin domains with micromolar affinity. SALM3 shows a clear preference for the RPTP ligands with the meB splice insert. Our structural studies and sequence conservation analysis suggests a ligand-binding site and mechanism for RPTP binding via the dimeric LRR domain region.

The receptor protein tyrosine phosphatase CLR-1 is required for synaptic partner recognition.

During neural circuit formation, most axons are guided to complex environments, coming into contact with multiple potential synaptic partners. However, it is critical that they recognize specific neurons with which to form synapses. Here, we utilize the split GFP-based marker Neuroligin-1 GFP Reconstitution Across Synaptic Partners (NLG-1 GRASP) to visualize specific synapses in live animals, and a circuit-specific behavioral assay to probe circuit function. We demonstrate that the receptor protein tyrosine phosphatase (RPTP) clr-1 is necessary for synaptic partner recognition (SPR) between the PHB sensory neurons and the AVA interneurons in C. elegans. Mutations in clr-1/RPTP result in reduced NLG-1 GRASP fluorescence and impaired behavioral output of the PHB circuit. Temperature-shift experiments demonstrate that clr-1/RPTP acts early in development, consistent with a role in SPR. Expression and cell-specific rescue experiments indicate that clr-1/RPTP functions in postsynaptic AVA neurons, and overexpression of clr-1/RPTP in AVA neurons is sufficient to direct additional PHB-AVA synaptogenesis. Genetic analysis reveals that clr-1/RPTP acts in the same pathway as the unc-6/Netrin ligand and the unc-40/DCC receptor, which act in AVA and PHB neurons, respectively. This study defines a new mechanism by which SPR is governed, and demonstrates that these three conserved families of molecules, with roles in neurological disorders and cancer, can act together to regulate communication between cells.

Targeting Receptor-Type Protein Tyrosine Phosphatases with Biotherapeutics: Is Outside-in Better than Inside-Out?

Protein tyrosine phosphatases (PTPs), of the receptor and non-receptor classes, are key signaling molecules that play critical roles in cellular regulation underlying diverse physiological events. Aberrant signaling as a result of genetic mutation or altered expression levels has been associated with several diseases and treatment via pharmacological intervention at the level of PTPs has been widely explored; however, the challenges associated with development of small molecule phosphatase inhibitors targeting the intracellular phosphatase domain (the "inside-out" approach) have been well documented and as yet there are no clinically approved drugs targeting these enzymes. The alternative approach of targeting receptor PTPs with biotherapeutic agents (such as monoclonal antibodies or engineered fusion proteins; the "outside-in" approach) that interact with the extracellular ectodomain offers many advantages, and there have been a number of exciting recent developments in this field. Here we provide a brief overview of the receptor PTP family and an update on the emerging area of receptor PTP-targeted biotherapeutics for CD148, vascular endothelial-protein tyrosine phosphatase (VE-PTP), receptor-type PTPs σ, γ, ζ (RPTPσ, RPTPγ, RPTPζ) and CD45, and discussion of future potential in this area.

Hepatic protein tyrosine phosphatase receptor gamma links obesity-induced inflammation to insulin resistance.

Obesity-induced inflammation engenders insulin resistance and type 2 diabetes mellitus (T2DM) but the inflammatory effectors linking obesity to insulin resistance are incompletely understood. Here, we show that hepatic expression of Protein Tyrosine Phosphatase Receptor Gamma (PTPR-γ) is stimulated by inflammation in obese/T2DM mice and positively correlates with indices of inflammation and insulin resistance in humans. NF-κB binds to the promoter of Ptprg and is required for inflammation-induced PTPR-γ expression. PTPR-γ loss-of-function lowers glycemia and insulinemia by enhancing insulin-stimulated suppression of endogenous glucose production. These phenotypes are rescued by re-expression of Ptprg only in liver of mice lacking Ptprg globally. Hepatic PTPR-γ overexpression that mimics levels found in obesity is sufficient to cause severe hepatic and systemic insulin resistance. We propose hepatic PTPR-γ as a link between obesity-induced inflammation and insulin resistance and as potential target for treatment of T2DM.

Two receptor tyrosine phosphatases dictate the depth of axonal stabilizing layer in the visual system.

Formation of a functional neuronal network requires not only precise target recognition, but also stabilization of axonal contacts within their appropriate synaptic layers. Little is known about the molecular mechanisms underlying the stabilization of axonal connections after reaching their specifically targeted layers. Here, we show that two receptor protein tyrosine phosphatases (RPTPs), LAR and Ptp69D, act redundantly in photoreceptor afferents to stabilize axonal connections to the specific layers of the Drosophila visual system. Surprisingly, by combining loss-of-function and genetic rescue experiments, we found that the depth of the final layer of stable termination relied primarily on the cumulative amount of LAR and Ptp69D cytoplasmic activity, while specific features of their ectodomains contribute to the choice between two synaptic layers, M3 and M6, in the medulla. These data demonstrate how the combination of overlapping downstream but diversified upstream properties of two RPTPs can shape layer-specific wiring.

Dendritic space-filling requires a neuronal type-specific extracellular permissive signal in Drosophila.

Neurons sometimes completely fill available space in their receptive fields with evenly spaced dendrites to uniformly sample sensory or synaptic information. The mechanisms that enable neurons to sense and innervate all space in their target tissues are poorly understood. Using Drosophila somatosensory neurons as a model, we show that heparan sulfate proteoglycans (HSPGs) Dally and Syndecan on the surface of epidermal cells act as local permissive signals for the dendritic growth and maintenance of space-filling nociceptive C4da neurons, allowing them to innervate the entire skin. Using long-term time-lapse imaging with intact Drosophila larvae, we found that dendrites grow into HSPG-deficient areas but fail to stay there. HSPGs are necessary to stabilize microtubules in newly formed high-order dendrites. In contrast to C4da neurons, non-space-filling sensory neurons that develop in the same microenvironment do not rely on HSPGs for their dendritic growth. Furthermore, HSPGs do not act by transporting extracellular diffusible ligands or require leukocyte antigen-related (Lar), a receptor protein tyrosine phosphatase (RPTP) and the only known Drosophila HSPG receptor, for promoting dendritic growth of space-filling neurons. Interestingly, another RPTP, Ptp69D, promotes dendritic growth of C4da neurons in parallel to HSPGs. Together, our data reveal an HSPG-dependent pathway that specifically allows dendrites of space-filling neurons to innervate all target tissues in Drosophila.

The secreted MSP domain of C. elegans VAPB homolog VPR-1 patterns the adult striated muscle mitochondrial reticulum via SMN-1.

The major sperm protein domain (MSPd) has an extracellular signaling function implicated in amyotrophic lateral sclerosis. Secreted MSPds derived from the C. elegans VAPB homolog VPR-1 promote mitochondrial localization to actin-rich I-bands in body wall muscle. Here we show that the nervous system and germ line are key MSPd secretion tissues. MSPd signals are transduced through the CLR-1 Lar-like tyrosine phosphatase receptor. We show that CLR-1 is expressed throughout the muscle plasma membrane, where it is accessible to MSPd within the pseudocoelomic fluid. MSPd signaling is sufficient to remodel the muscle mitochondrial reticulum during adulthood. An RNAi suppressor screen identified survival of motor neuron 1 (SMN-1) as a downstream effector. SMN-1 acts in muscle, where it colocalizes at myofilaments with ARX-2, a component of the Arp2/3 actin-nucleation complex. Genetic studies suggest that SMN-1 promotes Arp2/3 activity important for localizing mitochondria to I-bands. Our results support the model that VAPB homologs are circulating hormones that pattern the striated muscle mitochondrial reticulum. This function is crucial in adults and requires SMN-1 in muscle, likely independent of its role in pre-mRNA splicing.