Glypican Is a Modulator of Netrin-Mediated Axon Guidance

Netrin is a key axon guidance cue that orients axon growth during neural circuit formation. However, the mechanisms regulating netrin and its receptors in the extracellular milieu are largely unknown. Here we demonstrate that in Caenorhabditis elegans, LON-2/glypican, a heparan sulfate proteoglycan, modulates UNC-6/netrin signaling and may do this through interactions with the UNC-40/DCC receptor. We show that developing axons misorient in the absence of LON-2/glypican when the SLT-1/slit guidance pathway is compromised and that LON-2/glypican functions in both the attractive and repulsive UNC-6/netrin pathways. We find that the core LON-2/glypican protein, lacking its heparan sulfate chains, and secreted forms of LON-2/glypican are functional in axon guidance. We also find that LON-2/glypican functions from the epidermal substrate cells to guide axons, and we provide evidence that LON-2/glypican associates with UNC-40/DCC receptor–expressing cells. We propose that LON-2/glypican acts as a modulator of UNC-40/DCC-mediated guidance to fine-tune axonal responses to UNC-6/netrin signals during migration.     

Glypican LON-2 is a conserved negative regulator of BMP-like signaling in Caenorhabditis elegans

Bone morphogenetic protein (BMP) pathways are required for a wide variety of developmental and homeostatic decisions, and mutations in signaling components are associated with several diseases. An important aspect of BMP control is the extracellular regulation of these pathways. We show that LON-2 negatively regulates a BMP-like signaling pathway that controls body length in C. elegans. lon-2 acts genetically upstream of the BMP-like gene dbl-1, and loss of lon-2 function results in animals that are longer than normal. LON-2 is a conserved member of the glypican family of heparan sulfate proteoglycans, a family with several members known to regulate growth-factor signaling in many organisms. LON-2 is functionally conserved because the Drosophila glypican gene dally rescues the lon-2(lf) body-size defect. We show that the LON-2 protein binds BMP2 in vitro, and a mutant variation of LON-2 found in lon-2(e2140) animals diminishes this interaction. We propose that LON-2 binding to DBL-1 negatively regulates this pathway in C. elegans by attenuating ligand-receptor interactions. This is the first report of a glypican directly interacting with a growth-factor pathway in C. elegans and provides a mechanistic model for glypican regulation of growth-factor pathways.     

crm-1 facilitates BMP signaling to control body size in Caenorhabditis elegans

We have identified in Caenorhabditis elegans a homologue of the vertebrate Crim1, crm-1, which encodes a putative transmembrane protein with multiple cysteine-rich (CR) domains known to have bone morphogenetic proteins (BMPs) binding activity. Using the body morphology of C. elegans as an indicator, we showed that attenuation of crm-1 activity leads to a small body phenotype reminiscent of that of BMP pathway mutants. We showed that the crm-1 loss-of-function phenotype can be rescued by constitutive supply of sma-4 activity. crm-1 can enhance BMP signaling and this activity is dependent on the presence of the DBL-1 ligand and its receptors. crm-1 is expressed in neurons at the ventral nerve cord, where the DBL-1 ligand is produced. However, ectopic expression experiments reveal that crm-1 gene products act outside the DBL-1 producing cells and function non-autonomously to facilitate dbl/sma pathway signaling to control body size.     

Protein arginine methyltransferase 7 has a novel homodimer-like structure formed by tandem repeats

Protein arginine methyltransferase 7 (PRMT7) is a member of a family of enzymes that catalyze the transfer of methyl groups from S-adenosyl-l-methionine to nitrogen atoms on arginine residues. Here, we describe the crystal structure of Caenorhabditis elegans PRMT7 in complex with its reaction product S-adenosyl-l-homocysteine. The structural data indicated that PRMT7 harbors two tandem repeated PRMT core domains that form a novel homodimer-like structure. S-adenosyl-l-homocysteine bound to the N-terminal catalytic site only; the C-terminal catalytic site is occupied by a loop that inhibits cofactor binding. Mutagenesis demonstrated that only the N-terminal catalytic site of PRMT7 is responsible for cofactor binding.     

HLA-B-associated transcript 3 (Bat3/Scythe) negatively regulates Smad phosphorylation in BMP signaling

Members of the transforming growth factor-β (TGF-β) superfamily participate in numerous biological phenomena in multiple tissues, including in cell proliferation, differentiation, and migration. TGF-β superfamily proteins therefore have prominent roles in wound healing, fibrosis, bone formation, and carcinogenesis. However, the molecular mechanisms regulating these signaling pathways are not fully understood. Here, we describe the regulation of bone morphogenic protein (BMP) signaling by Bat3 (also known as Scythe or BAG6). Bat3 overexpression in murine cell lines suppresses the activity of the Id1 promoter normally induced by BMP signaling. Conversely, Bat3 inactivation enhances the induction of direct BMP target genes, such as Id1, Smad6, and Smad7. Consequently, Bat3 deficiency accelerates the differentiation of primary osteoblasts into bone, with a concomitant increase in the bone differentiation markers Runx2, Osterix, and alkaline phosphatase. Using biochemical and cell biological analyses, we show that Bat3 inactivation sustains the C-terminal phosphorylation and nuclear localization of Smad1, 5, and 8 (Smad1/5/8), thereby enhancing biological responses to BMP treatment. At the mechanistic level, we show that Bat3 interacts with the nuclear phosphatase small C-terminal domain phosphatase (SCP) 2, which terminates BMP signaling by dephosphorylating Smad1/5/8. Notably, Bat3 enhances SCP2–Smad1 interaction only when the BMP signaling pathway is activated. Our results demonstrate that Bat3 is an important regulator of BMP signaling that functions by modulating SCP2–Smad interaction.     

HSPG modulation of BMP signaling in fibrodysplasia ossificans progressiva cells

Cell surface heparan sulfate proteoglycans (HSPGs) play important roles in morphogen gradient formation and cell signaling. Bone morphogenetic protein (BMP) signaling is dysregulated in fibrodysplasia ossificans progressiva (FOP), a disabling disorder of progressive heterotopic bone formation. Here, we investigated the role of HSPG glycosaminoglycan (GAG) side chains on BMP signaling and found increased total and HSPG-specific GAG chain levels and dysregulation in HSPG modulation of BMP signaling in FOP lymphoblastoid cells (LCLs). Specifically, HSPG profiling demonstrated abundant mRNA and protein levels of glypican 1 and syndecan 4 on control and FOP LCLs, with elevated core protein levels on FOP cells. Targeted downregulation of glypican 1 core protein synthesis by siRNA enhanced BMP signaling in control and FOP cells, while reduction of syndecan 4-core protein synthesis decreased BMP signaling in control, but not FOP cells. These results suggest that FOP cells are resistant to the stimulatory effects of cell surface HSPG GAG chains, but are susceptible to the inhibitory effects, as shown by downregulation of glypican 1. These data support that HSPG modulation of BMP signaling is altered in cells from patients with FOP and that altered HSPG-related BMP signaling may play a role in the pathogenesis of the disease.     

Kekkon5 is an extracellular regulator of BMP signaling

Precise spatial and temporal control of Drosophila Bone Morphogenetic Protein (BMP) signaling is achieved by a host of extracellular factors that modulate ligand distribution and activity. Here we describe Kekkon5 (Kek5), a transmembrane protein containing leucine-rich repeats (LRRs), as a novel regulator of BMP signaling in Drosophila. We find that loss or gain of kek5 disrupts crossvein development and alters the early profile of phosphorylated Mad and dSRF in presumptive crossvein cells. kek5 phenotypic effects closely mimic those observed with Short gastrulation (Sog), but do not completely recapitulate the effects of dominant negative BMP receptors. We further demonstrate that Kek5 is able to antagonize the BMP ligand Glass bottom boat (Gbb) and that the Kek5 LRRs are required for BMP inhibitory activity, while the Ig domain is dispensable in this context. Our identification of Kek5 as a modulator of BMP signaling supports the emerging notion that LIG proteins function as diverse regulators of cellular communication.     

Smurf-mediated differential proteolysis generates dynamic BMP signaling in germline stem cells during Drosophila testis development

Germline stem cells (GSCs) produce gametes throughout the reproductive life of many animals, and intensive studies have revealed critical roles of BMP signaling to maintain GSC self-renewal in Drospophila adult gonads. Here, we show that BMP signaling is downregulated as testes develop and this regulation controls testis growth, stem cell number, and the number of spermatogonia divisions. Phosphorylated Mad (pMad), the activated Drosophila Smad in germ cells, was restricted from anterior germ cells to GSCs and hub-proximal cells during early larval development. pMad levels in GSCs were then dramatically downregulated from early third larval instar (L3) to late L3, and maintained at low levels in pupal and adult GSCs. The spatial restriction and temporal down-regulation of pMad, reflecting the germ cell response to BMP signaling activity, required action in germ cells of E3 ligase activity of HECT domain protein Smurf. Analyses of Smurf mutant testes and dosage-dependent genetic interaction between Smurf and mad indicated that pMad downregulation was required for both the normal decrease in stem cell number during testis maturation in the pupal stage, and for normal limit of four rounds of spermatogonia cell division for control of germ cell numbers and testis size. Smurf protein was expressed at a constant low level in GSCs and spermatogonia during development. Rescue experiments showed that expression of exogenous Smurf protein in early germ cells promoted pMad downregulation in GSCs in a stage-dependent but concentration-independent manner, suggesting that the competence of Smurf to attenuate response to BMP signaling may be regulated during development. Taken together, our work reveals a critical role for differential attenuation of the response to BMP signaling in GSCs and early germ cells for control of germ cell number and gonad growth during development.     

hunchback and Ikaros-like zinc finger genes control reproductive system development in Caenorhabditis elegans

Here we provide evidence for a C2H2 zinc finger gene family with similarity to Ikaros and hunchback. The founding member of this family is Caenorhabditis elegans ehn-3, which has important and poorly understood functions in somatic gonad development. We examined the expression and function of four additional hunchback/Ikaros-like (HIL) genes in C. elegans reproductive system development. Two genes, ehn-3 and R08E3.4, are expressed in somatic gonadal precursors (SGPs) and have overlapping functions in their development. In ehn-3; R08E3.4 double mutants, we find defects in the generation of distal tip cells, anchor cells, and spermatheca; three of the five tissues derived from the SGPs. We provide in vivo evidence that C. elegans HIL proteins have functionally distinct zinc finger domains, with specificity residing in the N-terminal set of four zinc fingers and a likely protein-protein interaction domain provided by the C-terminal pair of zinc fingers. In addition, we find that a chimeric human Ikaros protein containing the N-terminal zinc fingers of EHN-3 functions in C. elegans. Together, these results lend support to the idea that the C. elegans HIL genes and Ikaros have similar functional domains. We propose that hunchback, Ikaros, and the HIL genes arose from a common ancestor that was present prior to the divergence of protostomes and deuterostomes.     

The Drosophila LEM-domain protein MAN1 antagonizes BMP signaling at the neuromuscular junction and the wing crossveins

BMP signaling responses are refined by distinct secreted and intracellular antagonists in different cellular and temporal contexts. Here, we show that the nuclear LEM-domain protein MAN1 is a tissue-specific antagonist of BMP signaling in Drosophila. MAN1 contains two potential Mad-binding sites. We generated MAN1^ΔC mutants, harbouring a MAN1 protein that lacks part of the C-terminus including the RNA recognition motif, a putative Mad-binding domain. MAN1^ΔC mutants show wing crossvein (CV) patterning defects but no detectable alterations in nuclear morphology. MAN1^ΔC pupal wings display expanded phospho-Mad (pMad) accumulation and ectopic expression of the BMP-responsive gene crossveinless-2 (cv-2) indicating that MAN1 restricts BMP signaling. Conversely, MAN1 overexpression in wing imaginal discs inhibited crossvein development and BMP signaling responses. MAN1 is expressed at high levels in pupal wing veins and can be activated in intervein regions by ectopic BMP signaling. The specific upregulation of MAN1 in pupal wing veins may thus represent a negative feedback circuit that limits BMP signaling during CV formation. MAN1^ΔC flies also show reduced locomotor activity, and electrophysiology recordings in MAN1^ΔC larvae uncover a new presynaptic role of MAN1 at the neuromuscular junction (NMJ). Genetic interaction experiments suggest that MAN1 is a BMP signaling antagonist both at the NMJ and during CV formation.     

Activity of Lymphostatin,A Lymphocyte Inhibitory Virulence Factor of Pathogenic Escherichia coli,is Dependent on a Cysteine Protease Motif

Lymphostatin (LifA) is a 366 kDa protein expressed by attaching & effacing Escherichia coli. It plays an important role in intestinal colonisation and inhibits the mitogen- and antigen-stimulated proliferation of lymphocytes and the synthesis of proinflammatory cytokines. LifA exhibits N-terminal homology with the glycosyltransferase domain of large clostridial toxins (LCTs). A DTD motif within this region is required for lymphostatin activity and binding of the sugar donor uridine diphosphate N-acetylglucosamine. As with LCTs, LifA also contains a cysteine protease motif (C1480, H1581, D1596) that is widely conserved within the YopT-like superfamily of cysteine proteases. By analogy with LCTs, we hypothesised that the CHD motif may be required for intracellular processing of the protein to release the catalytic N-terminal domain after uptake and low pH-stimulated membrane insertion of LifA within endosomes. Here, we created and validated a C1480A substitution mutant in LifA from enteropathogenic E. coli strain E2348/69. The purified protein was structurally near-identical to the wild-type protein. In bovine T lymphocytes treated with wild-type LifA, a putative cleavage product of approximately 140 kDa was detected. Appearance of the putative cleavage product was inhibited in a concentration-dependent manner by bafilomycin A1 and chloroquine, which inhibit endosome acidification. The cleavage product was not observed in cells treated with the C1480A mutant of LifA. Lymphocyte inhibitory activity of the purified C1480A protein was significantly impaired. The data indicate that an intact cysteine protease motif is required for cleavage of lymphostatin and its activity against T cells.     

93-kDa twin-domain serine protease inhibitor (Serpin) has a regulatory function on the beetle Toll proteolytic signaling cascade

Serpins are protease inhibitors that play essential roles in the down-regulation of extracellular proteolytic cascades. The core serpin domain is highly conserved, and typical serpins are encoded with a molecular size of 35–50 kDa. Here, we describe a novel 93-kDa protein that contains two complete, tandemly arrayed serpin domains. This twin serpin, SPN93, was isolated from the larval hemolymph of the large beetle Tenebrio molitor. The N-terminal serpin domain of SPN93 forms a covalent complex with the Spätzle-processing enzyme, a terminal serine protease of the Toll signaling cascade, whereas the C-terminal serpin domain of SPN93 forms complexes with a modular serine protease and the Spätzle-processing enzyme-activating enzyme, which are two different enzymes of the cascade. Consequently, SPN93 inhibited β-1,3-glucan-mediated Toll proteolytic cascade activation in an in vitro system. Site-specific proteolysis of SPN93 at the N-terminal serpin domain was observed after activation of the Toll proteolytic cascade in vivo, and down-regulation of SPN93 by RNAi sensitized β-1,3-glucan-mediated larval death. Therefore, SPN93 is the first serpin that contains twin tandemly arrayed and functionally active serpin domains that have a regulatory role in the larval Toll proteolytic signaling cascade.     

The Rubella Virus Nonstructural Protease Requires Divalent Cations for Activity and Functions in trans

The rubella virus (RUB) nonstructural (NS) protease is a papain-like cysteine protease (PCP) located in the NS-protein open reading frame (NSP-ORF) that cleaves the NSP-ORF translation product at a single site to produce two products, P150 (the N-terminal product) and P90 (the C-terminal product). The RUB NS protease was found not to function following translation in vitro in a standard rabbit reticulocyte lysate system, although all of the other viral PCPs do so. However, in the presence of divalent cations such as Zn²⁺, Cd²⁺, and Co²⁺, the RUB NS protease functioned efficiently, indicating that these cations are required either as direct cofactors in catalytic activity or for correct acquisition of three-dimensional conformation of the protease. Since other viral and cell PCPs do not require cations for activity and the RUB NS protease contains a putative zinc binding motif, the latter possibility is more likely. Previous in vivo expression studies of the RUB NS protease failed to demonstrate trans cleavage activity (J.-P. Chen et al., J. Virol. 70:4707–4713, 1996). To study whether trans cleavage could be detected in vitro, a protease catalytic site mutant and a mutant in which the C-terminal 31 amino acids of P90 were deleted were independently introduced into plasmid constructs that express the complete NSP-ORF. Cotranslation of these mutants in vitro yielded both the native and the mutated forms of P90, indicating that the protease present in the mutated construct cleaved the catalytic-site mutant precursor. Thus, RUB NS protease can function in trans.     

Cytoplasmic expression of mouse prion protein causes severe toxicity in Caenorhabditis elegans

To test if Caenorhabditis elegans could be established as a model organism for prion study, we created transgenic C. elegans expressing the cytosolic form of the mouse prion protein, MoPrP(23-231), which lacks the N-terminal signal sequence and the C-terminal glycosylphosphatidylinisotol (GPI) anchor site. We report here that transgenic worms expressing MoPrP(23-231)-CFP exhibited a wide range of distinct phenotypes: from normal growth and development, reduced mobility and development delay, complete paralysis and development arrest, to embryonic lethality. Similar levels of MoPrP(23-231)-CFP were produced in animals exhibiting these distinct phenotypes, suggesting that MoPrP(23-231)-CFP might have misfolded into distinct toxic species. In combining with the observation that mutations in PrP that affect prion pathogenesis also affect the toxic phenotypes in C. elegans, we conclude that the prion protein-folding mechanism is similar in mammals and C. elegans. Thus, C. elegans can be a useful model organism for prion research.