Proteolytic Processing Causes Extensive Heterogeneity of Tissue Matrilin Forms

The matrilins are a family of multidomain extracellular matrix proteins with adapter functions. The oligomeric proteins have a bouquet-like structure and bind to a variety of different ligands whereby the avidity of their interactions is dependent on the number of subunits and domains present. Here we show the contribution of post-translational proteolytic processing to the heterogeneity of matrilins seen in tissue extracts and cell culture supernatants. A cleavage site after two glutamate residues in the hinge region close to the C-terminal coiled-coil oligomerization domain is conserved among the matrilins. Cleavage at this site yields molecules that lack almost complete subunits. The processing is least pronounced in matrilin-1 and particularly complex in matrilin-2, which contains additional cleavage sites. Replacement of the hinge region in matrilin-4 by the matrilin-1 hinge region had no marked effect on the processing. A detailed study revealed that matrilin-4 is processed already in the secretory pathway and that the activation of the responsible enzymes is dependent on proprotein convertase activity. Matrilin-3 and -4, but not matrilin-1 subunits present in matrilin-1/-3 hetero-oligomers, were identified as substrates for ADAMTS4 and ADAMTS5, whereas ADAMTS1 did not cleave any matrilin. A neo-epitope antibody raised against the N terminus of the C-terminal cleavage product of matrilin-4 detected processed matrilin-4 in cultures of primary chondrocytes as well as on cartilage sections showing that the conserved cleavage site is used in vivo.The matrilins form a four-member family of modular, multisubunit matrix proteins, which are expressed in cartilage and many other forms of extracellular matrix (for review, see Ref. 1). They participate in the formation of fibrillar or filamentous structures (2–7) and mediate interactions between collagen-containing fibrils (8, 9) and other matrix constituents like aggrecan (10), small leucine-rich proteoglycans (9), or COMP (11). Matrilins form homo- and hetero-oligomers by their C-terminal coiled-coil domain. In addition, the subunits contain epidermal growth factor-like and von Willebrand factor A (VWA)²-like domains, where the latter are presumably the major ligand binding domains (11). Mutations in matrilin-3 in humans cause different forms of chondrodysplasia (12–14) and are also linked to the development of hand osteoarthritis (15) and intervertebral disc degeneration (16).Proteolytic processing of extracellular matrix proteins plays both physiological and pathophysiological roles. Proteolysis is a major post-translational modification used to modify the function of proteins. Tissue homeostasis requires a well balanced synthesis and degradation of extracellular matrix proteins, specifically mediated by protease families like matrix metalloproteinases (17), ADAMs (18), or ADAMTSs (19). The development of degenerative diseases is often accompanied by an activation of such proteases. In addition, the cleavage sometimes releases protein fragments that have completely new functions (20, 21).Determination of which extracellular proteases cleave which substrates is crucial to understand the physiological function of both (22). Physiological cleavage has been described for most members of the matrilin family (4–6), but was not yet extensively studied. The adapter function of the matrilins may be modulated by physiological proteolysis that causes the loss of single subunits and thereby decreases the binding avidity (5). Interestingly, an earlier identified cleavage site in the hinge region of matrilin-4, N-terminal of the coiled-coil, is conserved throughout the matrilin family (5) and it was recently shown that matrilin-3 is cleaved by ADAMTS4 in vitro at this site (23). Here we studied matrilin processing in some detail and identified another member of the ADAMTS family, ADAMTS5, as being able to cleave matrilin-3 and -4. Such cleavage is likely to alter the cohesion of the extracellular matrix.     

An Oligomeric Signaling Platform Formed by the Toll-like Receptor Signal Transducers MyD88 and IRAK-4

Toll-like receptors (TLRs) mediate responses to pathogen-associated molecules as part of the vertebrate innate immune response to infection. Receptor dimerization is coupled to downstream signal transduction by the recruitment of a post-receptor complex containing the adaptor protein MyD88 and the IRAK protein kinases. In this work, we show that the death domains of human MyD88 and IRAK-4 assemble into closed complexes having unusual stoichiometries of 7:4 and 8:4, the Myddosome. Formation of the Myddosome is likely to be a key event for TLR4 signaling in vivo as we show here that pathway activation requires that the receptors cluster into lipid rafts. Taken together, these findings indicate that TLR activation causes the formation of a highly oligomeric signaling platform analogous to the death-inducing signaling complex of the Fas receptor pathway.In vertebrates, the initial responses of innate immunity are mediated by a family of pattern recognition receptors, which are able to sense the presence of a variety of microbial products such as lipids and non-self nucleic acid (1). One important family of pattern recognition receptors is the Toll-like receptors (TLRs)⁴ that are expressed by many immune system cell types such as macrophages and dendritic cells. TLRs are class one transmembrane receptors that are activated by a process of stimulus-induced dimerization of their extracellular domains. This in turn causes the cytoplasmic Toll/interleukin-1 (IL-1) domains (TIRs) to dimerize, forming a scaffold for the recruitment of downstream signaling components (2). TLRs use five signaling adaptor proteins to couple receptor activation to downstream signal transduction (3). All of these adaptors have TIRs and engage with the activated TLRs by TIR-TIR interactions.One of the adaptor proteins, MyD88, is of particular importance because it is used by all but one of the TLRs as well as by the IL-1 and interferon-γ receptors. MyD88-deficient mice have profoundly impaired innate immune responses and are susceptible to a wide range of infectious diseases. The MyD88 sequence is tripartite and is comprised of a death domain (DD) at the N terminus, a short (40-amino-acid) intermediate domain (ID) of unknown structure, and a C-terminal TIR. Evidence from yeast two-hybrid experiments suggests that MyD88 can self-associate with contacts in both the DD and the TIR (4). The current view of post-receptor signal transduction is that two MyD88 TIR domains bind to the activated TLR, and this enables the recruitment of the protein kinases IRAK-4 and IRAK-1 (5). These kinases have DDs at their N termini, and both are recruited into a complex with MyD88 after signal initiation. It appears that IRAK-4 is recruited first, and this binding requires the ID of MyD88 (6, 7). Thus MyD88s, a splice variant that lacks the ID, down-regulates TLR signaling and cannot recruit IRAK-4 into the post-receptor complex. In contrast, IRAK-1 interacts with MyD88s presumably by DD-DD rather than DD-ID interactions. The next step in the signaling process is for IRAK-4 to phosphorylate IRAK-1, causing activation of the latter and hyper-autophosphorylation. IRAK-1 then dissociates from the complex and interacts with the ubiquitin-protein isopeptide ligase (E3) TRAF6 (8, 9).DDs together with the structurally related caspase recruitment domains (CARDs) and death effector domains (DEDs) form the death domain superfamily (10). There are 215 proteins encoded by the human genome that are predicted to have this fold, and they are widely used in cellular signaling including the TLR and apoptotic pathways. Structurally, DDs contain six antiparallel α-helices, and they are predominantly involved in protein-protein interactions with other DDs. Three modes of DD-DD interaction, types 1, 2, and 3 (10), have been characterized and are illustrated by the structures of the Drosophila Tube-Pelle heterodimer (11), the Procaspase-9 homodimer (12), and most remarkably, by the PIDDosome (13). In the latter case, PIDD, RAIDD, and Caspase-2 form a complex, which results in the proximity-induced activation of Caspase-2 protease activity, which in turn leads to cytochrome c release and apoptotic cell death. The DDs of PIDD and RAIDD interact to produce a complex having a stoichiometry of 5:7, and the subunits are arranged in three layers with five PIDDs, five RAIDDs, and then two RAIDDs. The structure is stabilized by 25 DD-DD contacts of which six are type 2, nine are type 1, and 10 are type 3.In this study, we report that like PIDD and RAIDD, the DDs of human MyD88 and IRAK-4 assemble into defined structures having stoichiometries of 7:4 and 8:4. We propose that the structure has two layers with a ring of seven or eight MyD88 subunits and a second layer of four IRAK-4 subunits. The formation of these higher order assemblies provides insight into the complex regulation and cross-talk observed in the TLR signaling pathways.     

Regulation of Epithelial Na+ Transport by Soluble Adenylyl Cyclase in Kidney Collecting Duct Cells

Alkalosis impairs the natriuretic response to diuretics, but the underlying mechanisms are unclear. The soluble adenylyl cyclase (sAC) is a chemosensor that mediates bicarbonate-dependent elevation of cAMP in intracellular microdomains. We hypothesized that sAC may be an important regulator of Na⁺ transport in the kidney. Confocal images of rat kidney revealed specific immunolocalization of sAC in collecting duct cells, and immunoblots confirmed sAC expression in mouse cortical collecting duct (mpkCCD_c14) cells. These cells exhibit aldosterone-stimulated transepithelial Na⁺ currents that depend on both the apical epithelial Na⁺ channel (ENaC) and basolateral Na⁺,K⁺-ATPase. RNA interference-mediated 60-70% knockdown of sAC expression comparably inhibited basal transepithelial short circuit currents (I_sc) in mpkCCD_c14 cells. Moreover, the sAC inhibitors KH7 and 2-hydroxyestradiol reduced I_sc in these cells by 50-60% within 30 min. 8-Bromoadenosine-3′,5′-cyclic-monophosphate substantially rescued the KH7 inhibition of transepithelial Na⁺ current. Aldosterone doubled ENaC-dependent I_sc over 4 h, an effect that was abolished in the presence of KH7. The sAC contribution to I_sc was unaffected with apical membrane nystatin-mediated permeabilization, whereas the sAC-dependent Na⁺ current was fully inhibited by basolateral ouabain treatment, suggesting that the Na⁺,K⁺-ATPase, rather than ENaC, is the relevant transporter target of sAC. Indeed, neither overexpression of sAC nor treatment with KH7 modulated ENaC currents in Xenopus oocytes. ATPase and biotinylation assays in mpkCCD_c14 cells demonstrated that sAC inhibition decreases catalytic activity rather than surface expression of the Na⁺,K⁺-ATPase. In summary, these results suggest that sAC regulates both basal and agonist-stimulated Na⁺ reabsorption in the kidney collecting duct, acting to enhance Na⁺,K⁺-ATPase activity.Maintenance of intracellular pH depends in part on the extracellular to intracellular Na⁺ gradient, and elevation of intracellular [Na⁺] can lead to acidification of the cytoplasm. It has been shown that acidification of the cytoplasm of cells from frog skin and toad bladder by increased partial pressure of CO₂ reduces Na⁺ transport and permeability (1, 2). Conversely, the rise in plasma bicarbonate caused by metabolic alkalosis with chronic diuretic use has been shown to increase net renal Na⁺ reabsorption independently of volume status, electrolyte depletion, and/or increased aldosterone secretion (3, 4). However, the underlying mechanisms involved in these phenomena remain unclear.The soluble adenylyl cyclase (sAC)² is a chemosensor that mediates the elevation of cAMP in intracellular microdomains (5-7). Unlike transmembrane adenylyl cyclases (tmACs), sAC is insensitive to regulation by forskolin or heterotrimeric G proteins (8) and is directly activated by elevations of intracellular calcium (9, 10) and/or bicarbonate ions (11). Thus, sAC mediates localized intracellular increases in cAMP in response to variations in bicarbonate levels or its closely related parameters, partial pressure of CO₂ and pH. Mammalian sAC is more similar to bicarbonate-regulated cyanobacterial adenylyl cyclases than to other mammalian nucleotidyl cyclases, which may indicate that there is a unifying mechanism for the regulation of cAMP signaling by bicarbonate across biological systems. Although sAC appears to be encoded by a single gene, there is significant isoform diversity for this ubiquitously expressed enzyme (11, 12) generated by alternative splicing (reviewed in Ref. 13). sAC has been shown to regulate the subcellular localization and/or activity of membrane transport proteins such as the vacuolar H⁺-ATPase (V-ATPase) and cystic fibrosis transmembrane conductance regulator in epithelial cells (14, 15). Functional activity of sAC has been reported in the kidney (16), and sAC has been localized to epithelial cells in the distal nephron (14, 17).Given that natriuresis is decreased during metabolic alkalosis, when bicarbonate is elevated, and Na⁺ reabsorption is impaired by high partial pressure of CO₂, we hypothesized that bicarbonate-regulated sAC may play a key role in the regulation of transepithelial Na⁺ transport in the distal nephron. Reabsorption of Na⁺ in the kidney and other epithelial tissues is mediated by the parallel operation of apical ENaC and basolateral Na⁺,K⁺-ATPase, and both transport proteins can be stimulated by cAMP via the cAMP-dependent protein kinase (PKA) (18, 53). The aims of this study were to investigate the role of sAC in the regulation of transepithelial Na⁺ transport in the kidney through the use of specific sAC inhibitors and electrophysiological measurements. We found that sAC inhibition blocks transepithelial Na⁺ reabsorption in polarized mpkCCD_c14 cells under both basal and hormone-stimulated conditions. Selective membrane permeabilization studies revealed that although ENaC activity appears to be unaffected by sAC inhibition, flux through the Na⁺,K⁺-ATPase is sensitive to sAC modulation. Inhibiting sAC decreases ATPase activity without affecting plasma membrane expression of the pump; thus, tonic sAC activity appears to be required for Na⁺ reabsorption in kidney collecting duct.     

N-cadherin/p120 Catenin Association at Cell-Cell Contacts Occurs in Cholesterol-rich Membrane Domains and Is Required for RhoA Activation and Myogenesis

p120 catenin is a major regulator of cadherin stability at cell-cell contacts and a modulator of Rho GTPase activities. In C2C12 myoblasts, N-cadherin is stabilized at cell contacts through its association with cholesterol-rich membrane domains or lipid rafts (LR) and acts as an adhesion-activated receptor that activates RhoA, an event required for myogenesis induction. Here, we report that association of p120 catenin with N-cadherin at cell contacts occurs specifically in LR. We demonstrate that interaction of p120 catenin with N-cadherin is required for N-cadherin association with LR and for its stabilization at cell contacts. LR disruption inhibits myogenesis induction and N-cadherin-dependent RhoA activation as does the perturbation of the N-cadherin-p120 catenin complex after p120 catenin knockdown. Finally, we observe an N-cadherin-dependent accumulation of RhoA at phosphatidylinositol 4,5-bisphosphate-enriched cell contacts which is lost after LR disruption. Thus, a functional N-cadherin-catenin complex occurs in cholesterol-rich membrane microdomains which allows the recruitment of RhoA and the regulation of its activity during myogenesis induction.Skeletal myogenesis is a multistep process regulated by diffusible molecules and the interaction of muscle cell precursors with their neighbors and the extracellular matrix (1, 2). Particularly, N-cadherin-dependent intercellular adhesion has a major role in cell cycle exit and induction of skeletal muscle differentiation through activation of the Rho family GTPases. RhoA positively regulates MyoD expression and skeletal muscle differentiation because it is required for serum response factor-mediated activation of several muscle-specific gene promoters (3, 4).Dynamic association of cadherin complexes at the plasma membrane (PM)⁴ is crucial for cadherin-mediated signaling. Their extracellular domain mediates homophilic cell-cell adhesion, whereas the intracellular domain associates with catenins that produce attachment sites for the F-actin cytoskeleton (5–7). The juxtamembrane domain of the cadherin cytoplasmic tail binds to p120 catenin, which regulates cadherin stability at cell contacts and modulates Rho GTPase activities (8–11). Cadherin stability is directly dependent on p120 catenin, and in its absence most cadherins are internalized and often degraded, suggesting that p120 catenin controls cadherin turnover at the cell surface (11, 12). Moreover, mutations in the E-cadherin region that bind to p120 catenin dissociate the E-cadherin-p120 catenin complex and disrupt strong cell adhesion, although interaction with other catenins remains intact (13). Cadherin stability at cell-cell contacts is also regulated by homophilic binding between extracellular domains and association with the F-actin cytoskeleton (14, 15). Association of N-cadherin with cholesterol-enriched microdomains, called lipid rafts (LR), at cell contacts, also stabilizes N-cadherin (16). Because p120 catenin interaction with cadherins and N-cadherin association with LR at cell contact sites are both involved in cadherin stability at cell contact sites, we asked whether p120 catenin association with N-cadherin required LR. We observed that their association occurred specifically in these cholesterol-rich domains. Moreover, using an N-cadherin mutant unable to bind to p120 catenin, we showed that the N-cadherin/p120 catenin interaction was required for N-cadherin association with LR and its stabilization at cell contacts. Because N-cadherin is implicated in the commitment to myogenesis through RhoA activation, we questioned whether its association with p120 catenin in LR was a prerequisite for RhoA activation. LR disruption inhibited myogenesis induction, association of p120 catenin with N-cadherin, and N-cadherin-dependent RhoA activation, as did the perturbation of the N-cadherin-p120 catenin complex after p120 catenin knockdown. Together, these data suggest a crucial role for the N-cadherin/p120 catenin association in LR in the regulation of RhoA activity during myogenesis induction.     

Characterization of Wise Protein and Its Molecular Mechanism to Interact with both Wnt and BMP Signals

Cross-talk of BMP and Wnt signaling pathways has been implicated in many aspects of biological events during embryogenesis and in adulthood. A secreted protein Wise and its orthologs (Sostdc1, USAG-1, and Ectodin) have been shown to modulate Wnt signaling and also inhibit BMP signals. Modulation of Wnt signaling activity by Wise is brought about by an interaction with the Wnt co-receptor LRP6, whereas BMP inhibition is by binding to BMP ligands. Here we have investigated the mode of action of Wise on Wnt and BMP signals. It was found that Wise binds LRP6 through one of three loops formed by the cystine knot. The Wise deletion construct lacking the LRP6-interacting loop domain nevertheless binds BMP4 and inhibits BMP signals. Moreover, BMP4 does not interfere with Wise-LRP6 binding, suggesting separate domains for the physical interaction. Functional assays also show that the ability of Wise to block Wnt1 activity through LRP6 is not impeded by BMP4. In contrast, the ability of Wise to inhibit BMP4 is prevented by additional LRP6, implying a preference of Wise in binding LRP6 over BMP4. In addition to the interaction of Wise with BMP4 and LRP6, the molecular characteristics of Wise, such as glycosylation and association with heparan sulfate proteoglycans on the cell surface, are suggested. This study helps to understand the multiple functions of Wise at the molecular level and suggests a possible role for Wise in balancing Wnt and BMP signals.Wise is a secreted protein that was isolated from a functional screen of a chick cDNA library of embryonic tissues. It was identified as being able to alter the antero-posterior character of neuralized Xenopus animal caps by promoting activity of the Wnt pathway (1). Independently, the homologous protein was isolated from a functional screen to detect genes that are preferentially expressed in the rat endometrium, which had been maximally sensitized to implantation, and named USAG-1 (uterine sensitization-associated gene-1) (2). The protein was identified a third time from the GenBank^TM sequence data base of mouse as a putative secreted protein, shown to be a BMP antagonist, and named Ectodin (3). The gene has also been called Sostdc1 (Sclerostin domain-containing 1) or Sostl (Sclerostin-like) due to the homology with Sclerostin-encoding gene Sost (4, 5). USAG-1/Wise/Ectodin/Sostdc1 is expressed in various tissues, such as the surface ectoderm of the posterior axis (1, 6), branchial arches (3, 6), the dermal papilla in hair follicles (7), vibrissae (3), mammalian tooth cusps (3, 8), rat endometrium (2), developing testis (9–11), interdigital tissues (12), and embryonic and adult kidneys (13, 14).Wise appears to have a dual role in modulating the Wnt pathway. Injection of Wnt8 RNA into a ventral vegetal blastomere of Xenopus embryos at the four-cell stage induces a full secondary axis to form, and this is blocked by the addition of Wise RNA as well as other Wnt inhibitors (1). Activation of the Wnt/β-catenin pathway in hair follicles triggers regeneration of hair growth, and expression of Wise appears to have a defined role to inhibit this (15). In this context, Wise expression is repressed by the nuclear receptor co-repressor, Hairless, which results in activation of the Wnt pathway; thus, a model of periodic regeneration of hair follicles has been proposed (15, 16). In addition, Wise and its homologue USAG-1 have been shown to block Wnt1, Wnt3a, and Wnt10b activities in reporter assays (14, 15, 17). Wise was found to bind to the Wnt co-receptor, LRP6, sharing the binding domain with Wnt ligands. Importantly, Wise was found to compete with Wnt8 for binding to LRP6, therefore suggesting a mechanism for inhibition of the Wnt pathway whereby Wise blocks the binding of ligand and receptor (1). Wise may also be retained in the endoplasmic reticulum and inhibit the trafficking of LRP6 to the cell surface (18). Wise also binds LRP4 (19), a member of the LRP family functioning inhibitory to Wnt signals (20). It is noteworthy that Wise was isolated from a screen designed to detect the activation of the Wnt/β-catenin pathway, not inhibition. The exact mechanism of how Wise exerts such a context-dependent modulation on the Wnt pathway is yet to be clarified.Osteoblast differentiation of MC3T3-E1 cells, as measured by alkaline phosphatase activity, can be induced by a wide range of BMP molecules. In this assay, Ectodin, the mouse ortholog of Wise, was shown to inhibit differentiation induced by BMP2, -4, -6, or -7 in a dose-dependent manner (3). Similarly, Ectodin (also known as USAG-1) was also found to inhibit the bone differentiation induced by BMP2, -4, or -7 in C2C12 cells (14). Ectodin also inhibits BMP2- or BMP7-induced Msx2 expression in dissected mouse tooth buds in organ culture (3). In tooth buds, Ectodin expression is detected in the dental ectoderm and mesenchymal cells excluding from the enamel knot (3). Ectodin/USAG-1-deficient mice created by targeted-disruption show altered tooth morphology and extra teeth, indicating that Ectodin and BMP tightly control tooth development and patterning in mammals (8, 21–23). Furthermore, in mouse adult kidneys, the ability of BMP7 to repair established renal injury is blocked by USAG-1 (13). All of these findings indicate that USAG-1/Wise/Ectodin has a clear antagonistic effect on BMP signaling, where it binds BMP2, -4, -6, and -7 (3, 14) and presumably prevents BMP binding to its receptors.Analysis of the sequence of Wise reveals that it has the C₁X_nC₂XGXC₃X_nC₄X_nC₅XC₆ motif of a six-membered cystine knot, where C₁ forms a disulfide bond with C₄, C₂ with C₅, and C₃ with C₆ (for a review of the cystine knot, see Refs. 24–27). This arrangement results in a globular protein with three loops, “finger 1,” “heel,” and “finger 2,” held together with an eight-membered ring of C₂XGXC₃C₆XC₅C₂ (Fig. 1). BMP antagonists represent a subfamily in the cystine knot superfamily, and this is further subdivided into three subfamilies based on the size of the cystine knot. These are the CAN family (eight-membered ring), Twisted Gastrulation (nine-membered ring), and Chordin and Noggin (10-membered ring) (27). There is generally little sequence homology between family members in the heel, finger 1, and finger 2 regions, yet Wise does show a moderate homology with Sclerostin (28). Sclerostin is involved in regulating bone mass (4, 5) and also appears to antagonize both Wnt (29–32) and BMP (28, 33, 34) signals. This paper aims to analyze the dual role of Wise on Wnt and BMP pathways by probing the structural features of the protein and reconciling them to physiological properties. It also aims to reveal the molecular nature of the protein in view of possible glycosylation, secretion, and association with extracellular matrix.Open in a separate windowFIGURE 1.Structure of chick Wise protein. A, stereo ribbon representation of the chick Wise three-dimensional structural model (residues 68–186). Purple, β-strands; green, loop regions. Yellow, disulfide bonds in the cystine knot plus a further disulfide (cysteines 89 and 147) linking two fingers of the structure. N- and C-terminal ends are indicated. B, schematic drawing of the full-length chick Wise structure. Arrowhead, the predicted signal sequence cleavage site for secretion; black dot, asparagine at position 47 (N47), the glycosylated site revealed in this study. Six cysteine residues forming the “cystine knot” are shown in circles, and disulfide bonds for the knot formation are shown by dotted lines. Three loops (Finger 1, Heel, and Finger 2) are indicated. The scheme also shows the deleted parts of Wise constructs ΔN, Δheel, and ΔC.     

Mahogunin Ring Finger-1 (MGRN1) E3 Ubiquitin Ligase Inhibits Signaling from Melanocortin Receptor by Competition with G��s

Mahogunin ring finger-1 (MGRN1) is a RING domain-containing ubiquitin ligase mutated in mahoganoid, a mouse mutation causing coat color darkening, congenital heart defects, high embryonic lethality, and spongiform neurodegeneration. The melanocortin hormones regulate pigmentation, cortisol production, food intake, and body weight by signaling through five G protein-coupled receptors positively coupled to the cAMP pathway (MC1R–MC5R). Genetic analysis has shown that mouse Mgrn1 is an accessory protein for melanocortin signaling that may inhibit MC1R and MC4R by unknown mechanisms. These melanocortin receptors (MCRs) regulate pigmentation and body weight, respectively. We show that human melanoma cells express 4 MGRN1 isoforms differing in the C-terminal exon 17 and in usage of exon 12. This exon contains nuclear localization signals. MGRN1 isoforms decreased MC1R and MC4R signaling to cAMP, without effect on β₂-adrenergic receptor. Inhibition was independent on receptor plasma membrane expression, ubiquitylation, internalization, or stability and occurred upstream of Gα_s binding to/activation of adenylyl cyclase. MGRN1 co-immunoprecipitated with MCRs, suggesting a physical interaction of the proteins. Significantly, overexpression of Gα_s abolished the inhibitory effect of MGRN1 and decreased co-immunoprecipitation with MCRs, suggesting competition between MGRN1 and Gα_s for binding to MCRs. Although all MGRN1s were located in the cytosol in the absence of MCRs, exon 12-containing isoforms accumulated in the nuclei upon co-expression with the receptors. Therefore, MGRN1 inhibits MCR signaling by a new mechanism involving displacement of Gα_s, thus accounting for key features of the mahoganoid phenotype. Moreover, MGRN1 might provide a novel pathway for melanocortin signaling from the cell surface to the nucleus.     

The Flexible Motif V of Epstein-Barr Virus Deoxyuridine 5��-Triphosphate Pyrophosphatase Is Essential for Catalysis

Deoxyuridine 5′-triphosphate pyrophosphatases (dUTPases) are ubiquitous enzymes essential for hydrolysis of dUTP, thus preventing its incorporation into DNA. Although Epstein-Barr virus (EBV) dUTPase is monomeric, it has a high degree of similarity with the more frequent trimeric form of the enzyme. In both cases, the active site is composed of five conserved sequence motifs. Structural and functional studies of mutants based on the structure of EBV dUTPase gave new insight into the mechanism of the enzyme. A first mutant allowed us to exclude a role in enzymatic activity for the disulfide bridge involving the beginning of the disordered C terminus. Sequence alignments revealed two groups of dUTPases, based on the position in sequence of a conserved aspartic acid residue close to the active site. Single mutants of this residue in EBV dUTPase showed a highly impaired catalytic activity, which could be partially restored by a second mutation, making EBV dUTPase more similar to the second group of enzymes. Deletion of the flexible C-terminal tail carrying motif V resulted in a protein completely devoid of enzymatic activity, crystallizing with unhydrolyzed Mg²⁺-dUTP complex in the active site. Point mutations inside motif V highlighted the essential role of lid residue Phe²⁷³. Magnesium appears to play a role mainly in substrate binding, since in absence of Mg²⁺, the K_m of the enzyme is reduced, whereas the k_cat is less affected.Epstein-Barr virus, a human γ-herpesvirus, is the causative agent of infectious mononucleosis and establishes a lifelong persistent infection in over 90% of the world''s population. EBV³ is implicated in a number of cancers, such as Burkitt''s lymphoma, undifferentiated nasopharyngeal carcinoma, or Hodgkin disease. The large DNA genome of this virus codes for about 86 proteins implicated in a large number of functions related to viral latency or the lytic cycle, during which the virus replicates.One protein of the lytic cycle is deoxyuridine 5′-triphosphate pyrophosphatase, a ubiquitous enzyme catalyzing the cleavage of dUTP into dUMP and pyrophosphate (PP_i). This enzyme not only provides the precursor for the formation of dTMP by thymidylate synthase, but it also has a crucial role in maintaining a low dUTP/dTTP ratio in the cell in order to limit the incorporation of deoxyuridylate into DNA by DNA polymerases. Based on their oligomerization state, dUTPases can be divided into three families.The first family of dUTPases contains homodimeric enzymes with the prototype structure of Trypanosoma cruzi dUTPase (1).dUTPases of the second and largest family form homotrimers. Their structure is unrelated to dimeric dUTPases. Trimeric dUTPases are found in most eukaryotes, in prokaryotes, in some DNA viruses, such as poxvirus, and in a number of retroviruses. Several x-ray structures of dUTPases of this family are available: Escherichia coli (2), Homo sapiens (3), equine infectious anemia virus (4), feline immunodeficiency virus (5), Mason-Pfizer monkey virus (6), Mycobacterium tuberculosis (7), Plasmodium falciparum (8), Arabidopsis thaliana (9), and vaccinia virus (10). The active site is formed by five conserved motifs that are distributed over the entire sequence (11) (Fig. 1A). Each subunit of the trimer contributes to the formation of each of the three active sites (2). Whereas the first four motifs are well ordered, motif V is most often disordered and is only observed in some structures after inhibitor binding such as in the feline immunodeficiency virus dUTPase structure (Protein Data Bank entry 1f7r) (12) and the human dUTPase structure (Protein Data Bank entry 1q5h) (3), both in complex with dUDP. Recently, several structures showing motif V and α,β-imido-dUTP in a conformation close to the situation during catalysis became available: one of the human enzyme (Protein Data Bank entry 3ehw) (13) and one from M. tuberculosis (Protein Data Bank entry 2py4) (14). Together with kinetic information about the human enzyme (15), a model of enzymatic action became available: (i) rapid, probably diffusion-limited binding of the substrate; (ii) a substrate-induced structural change required for the formation of the catalytically competent conformation; (iii) the rate-limiting hydrolysis step; and (iv) a rapid release of the reaction products PP_i and dUMP. Hydrolysis occurs through a nucleophilic in-line attack on the α-phosphate by a water molecule activated by an aspartic acid residue (16).Open in a separate windowFIGURE 1.EBV dUTPase sequence and structure. A, sequence alignment of different dUTPases with known structures and the one from C. glutamaticum. Residue numbers are given above the sequence for the human enzyme and below the sequences for the mycobacterial and EBV enzymes. The linker region is highlighted with cyan letters. The five conserved motifs are highlighted. Key residues of motif V are printed in red. A pink background highlights residues of motif V where side chain hydroxyl and main chain amide contact γ-phosphate in the crystal structure (13, 14). Green background, main chain amide interaction with β- and γ-phosphate; residues that have been deleted in the ΔV mutant are underlined. The conserved residues interacting with the motif V arginine of human-like dUTPases are printed in blue; those interacting with the motif V arginine of mycobacterium-like dUTPases are shown in magenta. B, structure of EBV dUTPase in complex with α,β-imido-dUTP and Mg²⁺ (Protein Data Bank entry 2bt1). The catalytic residue Asp⁷⁶ is shown together with Asp¹³¹ next to it. The four visible sequence motifs of the active site are colored according to Fig. 1A, and the loop connecting the two domains (residues 114–125) is colored in cyan. The bound α,β-imido-dUTP molecule is shown as sticks and colored according to atom types. C, the ΔV structure (pink) in complex with dUTP (sticks, atom colors) and Mg²⁺. The disordered part of the connecting loop is located between the two arrows. Superposed is the dUMP-bound WT dUTPase structure (Protein Data Bank entry 2bsy; gray, connecting loop in cyan, motif I in green, disordered part dotted).The third family contains monomeric dUTPases encoded by avian and mammalian herpesviruses, which include important human pathogens, such as Epstein-Barr virus and herpes simplex virus. They have limited sequence homology to the trimeric enzymes; the five motifs forming the active site are present but reshuffled and spread out over a single polypeptide that is twice as long as the sequence of the subunit in trimeric enzymes. Working on the enzyme from EBV, we reported the first crystal structures of a monomeric dUTPase determined in complex with the product dUMP or the non-hydrolyzable substrate analogue α,β-imido-dUTP (17) (Fig. 1B). Like most dUTPase structures, those of the EBV enzyme showed four of the five conserved sequence motifs (Fig. 1A), with motif V invisible due to its flexibility, and revealed an active site that is extremely similar to those of trimeric dUTPases. EBV dUTPase furthermore exhibited similar kinetic parameters (17, 18). This therefore implies that the conclusions of studies of the enzymatic mechanism for trimeric dUTPases are also valid for the EBV enzyme and vice versa.In the previously published EBV dUTPase structures (17), the N and C termini of the ordered structure were linked by a disulfide bridge between Cys⁴ and Cys²⁴⁶. We therefore wished to check the influence of this bridge on activity and its possible regulatory role by studying mutant C4S.EBV dUTPase structures (17) revealed that residues 114–133 linking the two domains of monomeric dUTPase and containing part of motif I show different structures in the α,β-imido-dUTP-bound form (Fig. 1B) and in the dUMP-bound form (Fig. 1C). This leads indirectly to a non-productive conformation of catalytic residue Asp⁷⁶ of motif III (Fig. 1B). Motif I is always well ordered in trimeric dUTPases. We hypothesized first that the clustering of negative charges of two aspartic acid residues (catalytic residues Asp⁷⁶ and Asp¹³¹ of the linker region) and the γ-phosphate of the inhibitor leads to destabilization of this part of the structure. When dUTPase structures showing motif V in a productive conformation became available (13, 14), they showed an interaction of the residue corresponding to Asp¹³¹ with the conserved arginine residue of motif V. On the other hand, Asp¹³¹ is only partially conserved in sequence alignments. These revealed two classes of dUTPases, the first containing EBV dUTPase with a conserved aspartic acid residue in a position corresponding to Asp¹³¹ and the second group containing human and E. coli enzymes with a conserved aspartic acid residue at the position corresponding to residue 78 in EBV (Fig. 1A). We decided to study three single mutants of Asp¹³¹ (D131S, D131N, and D131E) along with a double mutant (D131S/G78D), making the EBV enzyme more similar to the second group.Finally, we report the structure of a dUTPase mutant with motif V deleted (ΔV), constructed in order to facilitate binding studies and crystallographic studies in view of potential antiviral drug design. This mutant showed the presence of intact Mg²⁺-dUTP in its active site, posing the question of why this mutant is completely inactive although most of the catalytic machinery is in place. In order to understand the precise mechanism of action of motif V, we decided to study two conserved residues of motif V, Arg²⁶⁸ and Phe²⁷³, since it has been shown that they interact with the bound substrate analogue α,β-imido-dUTP (13, 14).Since binding of an Mg²⁺-substrate complex appeared possible without catalysis, we wanted to further characterize the role of Mg²⁺. Previous studies carried out on E. coli dUTPase (16, 19, 20) indicated an important role of Mg²⁺ for catalytic activity, by promoting enzyme-substrate complex stabilization (21). Various dUTPase structures in the presence of α,β-imido-dUTP show the metal ion coordinating all three phosphate groups.     

Dissecting the Mechanisms of Tissue Transglutaminase-induced Cross-linking of α-Synuclein: IMPLICATIONS FOR THE PATHOGENESIS OF PARKINSON DISEASE*S?

Tissue transglutaminase (tTG) has been implicated in the pathogenesis of Parkinson disease (PD). However, exactly how tTG modulates the structural and functional properties of α-synuclein (α-syn) and contributes to the pathogenesis of PD remains unknown. Using site-directed mutagenesis combined with detailed biophysical and mass spectrometry analyses, we sought to identify the exact residues involved in tTG-catalyzed cross-linking of wild-type α-syn and α-syn mutants associated with PD. To better understand the structural consequences of each cross-linking reaction, we determined the effect of tTG-catalyzed cross-linking on the oligomerization, fibrillization, and membrane binding of α-syn in vitro. Our findings show that tTG-catalyzed cross-linking of monomeric α-syn involves multiple cross-links (specifically 2-3). We subjected tTG-catalyzed cross-linked monomeric α-syn composed of either wild-type or Gln → Asn mutants to sequential proteolysis by multiple enzymes and peptide mapping by mass spectrometry. Using this approach, we identified the glutamine and lysine residues involved in tTG-catalyzed intramolecular cross-linking of α-syn. These studies demonstrate for the first time that Gln⁷⁹ and Gln¹⁰⁹ serve as the primary tTG reactive sites. Mutating both residues to asparagine abolishes tTG-catalyzed cross-linking of α-syn and tTG-induced inhibition of α-syn fibrillization in vitro. To further elucidate the sequence and structural basis underlying these effects, we identified the lysine residues that form isopeptide bonds with Gln⁷⁹ and Gln¹⁰⁹. This study provides mechanistic insight into the sequence and structural basis of the inhibitory effects of tTG on α-syn fibrillogenesis in vivo, and it sheds light on the potential role of tTG cross-linking on modulating the physiological and pathogenic properties of α-syn.Parkinson disease (PD)² is a progressive movement disorder that is caused by the loss of dopaminergic neurons in the substantia nigra, the part of the brain responsible for controlling movement. Clinically, PD is manifested in symptoms that include tremors, rigidity, and difficulty in initiating movement (bradykinesia). Pathologically, PD is characterized by the presence of intraneuronal, cytoplasmic inclusions known as Lewy bodies (LB), which are composed primarily of the protein “α-synuclein” (α-syn) (1) and are seen in the post-mortem brains of PD patients with the sporadic or familial forms of the disease (2). α-Syn is a presynaptic protein of 140 residues with a “natively” unfolded structure (3). Three missense point mutations in α-syn (A30P, E46K, and A53T) are associated with the early-onset, dominant, inherited form of PD (4, 5). Moreover, duplication or triplication of the α-syn gene has been linked to the familial form of PD, suggesting that an increase in α-syn expression is sufficient to cause PD. Together, these findings suggest that α-syn plays a central role in the pathogenesis of PD.The molecular and cellular determinants that govern α-syn oligomerization and fibrillogenesis in vivo remain poorly understood. In vitro aggregation studies have shown that the mutations associated with PD (A30P, E46K, and A53T) accelerate α-syn oligomerization, but only E46K and A53T α-syn show higher propensity to fibrillize than wild-type (WT) α-syn (6-8). This suggests that oligomerization, rather than fibrillization, is linked to early-onset familial PD (9). Our understanding of the molecular composition and biochemical state of α-syn in LBs has provided important clues about protein-protein interactions and post-translational modifications that may play a role in modulating oligomerization, fibrillogenesis, and LB formation of the protein. In addition to ubiquitination (10), phosphorylation (11, 12), nitration (13, 14), and C-terminal truncation (15, 16), analysis of post-mortem brain tissues from PD and Lewy bodies in dementia patients has confirmed the colocalization of tissue transglutaminase (tTG)-catalyzed cross-linked α-syn monomers and higher molecular aggregates in LBs within dopaminergic neurons (17, 18). Tissue transglutaminase catalyzes a calcium-dependent transamidating reaction involving glutamine and lysine residues, which results in the formation of a covalent cross-link via ε-(γ-glutamyl) lysine bonds (Fig. 2F). To date, seven different isoforms of tTGs have been reported, of which only tTG2 seems to be expressed in the human brain (19), whereas tTG1 and tTG3 are more abundantly found in stratified squamous epithelia (20). Subsequent immuno-histochemical, colocalization, and immunoprecipitation studies have shown that the levels of tTG and cross-linked α-syn species are increased in the substantia nigra of PD brains (17). These findings, combined with the known role of tTG in cross-linking and stabilizing bimolecular assemblies, led to the hypothesis that tTG plays an important role in the initiation and propagation of α-syn fibril formation and that it contributes to fibril stability in LBs. This hypothesis was initially supported by in vitro studies demonstrating that tTG catalyzes the polymerization of the α-syn-derived non-amyloid component (NAC) peptide via intermolecular covalent cross-linking of residues Gln⁷⁹ and Lys⁸⁰ (21) and by other studies suggesting that tTG promotes the fibrillization of amyloidogenic proteins implicated in the pathogenesis of other neurodegenerative diseases such as Alzheimer disease, supranuclear palsy, Huntington disease, and other polyglutamine diseases (22-24). However, recent in vitro studies with full-length α-syn have shown that tTG catalyzes intramolecular cross-linking of monomeric α-syn and inhibits, rather than promotes, its fibrillization in vitro (25, 26). The structural basis of this inhibitory effect and the exact residues involved in tTG-mediated cross-linking of α-syn, as well as structural and functional consequences of these modifications, remain poorly understood.Open in a separate windowFIGURE 2.tTG-catalyzed cross-linking of α-syn involves one to three intramolecular cross-links. A-C, MALDI-TOF/TOF analysis of native (—) and cross-linked (- - -) α-syn, showing that most tTG-catalyzed cross-linking products of WT or disease-associated mutant forms of α-syn are intramolecularly linked (predominant peak with two cross-links), and up to three intramolecular cross-links can occur (left shoulder). The abbreviations M and m/cl are used to designate native and cross-linked α-synuclein, respectively. D and E, kinetic analysis of α-syn (A30P) cross-linking monitored by MALDI-TOF and SDS-PAGE. F, schematic depiction of the tTG-catalyzed chemical reaction (isodipeptide formation) between glutamine and lysine residues.In this study, we have identified the primary glutamine and lysine residues involved in tTG-catalyzed, intramolecularly cross-linked monomeric α-syn and investigated how cross-linking these residues affects the oligomerization, fibrillization, and membrane binding of α-syn in vitro. Using single-site mutagenesis and mass spectrometry applied to exhaustive proteolytic digests of native and cross-linked monomeric α-syn, we identified Gln¹⁰⁹ and Gln⁷⁹ as the major tTG substrates. We demonstrate that the altered electrophoretic mobility of the intramolecularly cross-linked α-syn in SDS-PAGE occurs as a result of tTG-catalyzed cross-linking of Gln¹⁰⁹ to lysine residues in the N terminus of α-syn, which leads to the formation of more compact monomers. Consistent with previous studies, we show that intramolecularly cross-linked α-syn forms off-pathway oligomers that are distinct from those formed by the wild-type protein and that do not convert to fibrils within the time scale of our experiments (3-5 days). We also show that membrane-bound α-syn is a substrate of tTG and that intramolecular cross-linking does not interfere with the ability of monomeric α-syn to adopt an α-helical conformation upon binding to synthetic membranes. These studies provide novel mechanistic insight into the sequence and structural basis of events that allow tTG to inhibit α-syn fibrillogenesis, and they shed light on the potential role of tTG-catalyzed cross-linking in modulating the physiological and pathogenic properties of α-syn.