Regulation of Dishevelled DEP domain swapping by conserved phosphorylation sites.

Wnt signals bind to Frizzled receptors to trigger canonical and noncanonical signaling responses that control cell fates during animal development and tissue homeostasis. All Wnt signals are relayed by the hub protein Dishevelled. During canonical (ß-catenin-dependent) signaling, Dishevelled assembles signalosomes via dynamic head-to-tail polymerization of its Dishevelled and Axin (DIX) domain, which are cross-linked by its Dishevelled, Egl-10, and Pleckstrin (DEP) domain through a conformational switch from monomer to domain-swapped dimer. The domain-swapped conformation of DEP masks the site through which Dishevelled binds to Frizzled, implying that DEP domain swapping results in the detachment of Dishevelled from Frizzled. This would be incompatible with noncanonical Wnt signaling, which relies on long-term association between Dishevelled and Frizzled. It is therefore likely that DEP domain swapping is differentially regulated during canonical and noncanonical Wnt signaling. Here, we use NMR spectroscopy and cell-based assays to uncover intermolecular contacts in the DEP dimer that are essential for its stability and for Dishevelled function in relaying canonical Wnt signals. These contacts are mediated by an intrinsically structured sequence spanning a conserved phosphorylation site upstream of the DEP domain that serves to clamp down the swapped N-terminal α-helix onto the structural core of a reciprocal DEP molecule in the domain-swapped configuration. Mutations of this phosphorylation site and its cognate surface on the reciprocal DEP core attenuate DEP-dependent dimerization of Dishevelled and its canonical signaling activity in cells without impeding its binding to Frizzled. We propose that phosphorylation of this crucial residue could be employed to switch off canonical Wnt signaling.

Towards accurate exclusion of neonatal bacterial meningitis: a feasibility study of a novel 16S rDNA PCR assay.

BACKGROUND: PCRctic is an innovative assay based on 16S rDNA PCR technology that has been designed to detect a single intact bacterium in a specimen of cerebro-spinal fluid (CSF). The assay's potential for accurate, fast and inexpensive discrimination of bacteria-free CSF makes it an ideal adjunct for confident exclusion of bacterial meningitis in newborn babies where the negative predictive value of bacterial culture is poor. This study aimed to stress-test and optimize PCRctic in the "field conditions" to attain a clinically useful level of specificity. METHODS: The specificity of PCRctic was evaluated in CSF obtained from newborn babies investigated for meningitis on a tertiary neonatal unit. Following an interim analysis, the method of skin antisepsis was changed to increase bactericidal effect, and snap-top tubes (Eppendorf™) replaced standard universal containers for collection of CSF to reduce environmental contamination. RESULTS: The assay's specificity was 90.5% in CSF collected into the snap-top tubes - up from 60% in CSF in the universal containers. The method of skin antisepsis had no effect on the specificity. All CSF cultures were negative and no clinical cases of neonatal bacterial meningitis occurred during the study. CONCLUSIONS: A simple and inexpensive optimization of CSF collection resulted in a high specificity output. The low prevalence of neonatal bacterial meningitis means that a large multi-centre study will be required to validate the assay's sensitivity and its negative predictive value.

Substrate clustering potently regulates the activity of WW-HECT domain-containing ubiquitin ligases.

The Nedd4 family of HECT domain-containing E3 ligases ubiquitinate many transcription factors and signaling proteins, and their activity is tightly regulated. Normally, intramolecular interactions curb the catalytic activity of the HECT domain, but these can be broken by the binding of PY motifs, found on substrate molecules and adaptors, to the WW domains characteristic of this E3 ligase family. This raises the prospect of substrates automatically activating the ligases, frustrating the purpose of ligase regulation. Here we show that soluble protein substrates and adaptors such as α arrestins, even with multiple PY elements, cannot activate ligase activity efficiently. However, we found that polymerization or membrane tethering of these substrates dramatically increases the ligase activity both in vivo and in vitro Aggregation of luciferase-containing substrates upon heat shock had a similar effect and could also expose cryptic PY elements in the substrates. We inferred that ligase activation critically requires a substantial array of clustered PY motifs and that the formation of such arrays on membranes or in polymeric aggregates may be an essential step in this mode of ligase regulation. We conclude that recruitment of α arrestins to membrane receptors and aggregation of unstable proteins after heat shock may be physiologically relevant mechanisms for triggering ubiquitination by Nedd4 family HECT domain-containing E3 ligases.

Peptide and small molecule inhibitors of HECT-type ubiquitin ligases.

The human genome encodes several hundred E3 ubiquitin ligases containing RING domains, and around 28 containing HECT domains. These enzymes catalyze the transfer of ubiquitin from E2 enzyme thioesters to a huge range of substrates and play crucial roles in many cellular functions. This makes them attractive potential therapeutic targets. However, they have proven difficult to inhibit: very few good inhibitors exist for RING domain ligases, and none have been described for HECT ligases. Here we show that bicyclic peptides isolated by phage display [Heinis C, Rutherford T, Freund S, Winter G (2009) Nat Chem Biol. 5(7):502-507] can target the E2 binding sites on the HECT domains of Smurf2, Nedd4, Mule/Huwe1, and WWP1, and thus act as specific inhibitors of these enzymes in vitro. By screening for displacement of one of these peptides from Smurf2, we were able to identify a small molecule, heclin (HECT ligase inhibitor), which inhibits several HECT ligases in tissue culture cells. In vitro, heclin does not block E2 binding but causes a conformational change that results in oxidation of the active site Cys. This demonstrates that HECT domains are potentially druggable and provides molecules that may be of experimental use. Heclin kills HEK293 cells growing in culture, consistent with an essential role for HECT ligase activity in mammalian cells.

Regulation of PTEN/Akt and MAP kinase signaling pathways by the ubiquitin ligase activators Ndfip1 and Ndfip2.

Ndfip1 and Ndfip2 are related endosomal membrane proteins that bind to and activate members of the Nedd4 family of E3 ubiquitin ligases. These ligases in turn affect receptor tyrosine kinase signaling by ubiquitinating several key components of the signaling pathways. Here we investigate the role of the Ndfip proteins in EGF signaling. We show that they associate with the EGF receptor and PTEN, and control the ubiquitination and abundance of PTEN, c-Cbl, and Src family kinases. Ndfip2, but not Ndfip1, also binds to and is phosphorylated by Src and Lyn, and can act as a scaffold for Src phosphorylation of Ndfip1 and potentially other substrates. Depletion of Ndfip1 inhibits Akt activation in EGF-stimulated HeLa cells, stimulates activation of Jnk, and enhances cell multiplication. Thus Ndfip1 and Ndfip2 are physically and functionally associated with multiple components of the EGF signaling cascade, and their levels modulate the relative output of different signaling pathways.

Arrestin-mediated endocytosis of yeast plasma membrane transporters.

Many plasma membrane transporters in yeast are endocytosed in response to excess substrate or certain stresses and degraded in the vacuole. Endocytosis invariably requires ubiquitination by the HECT domain ligase Rsp5. In the cases of the manganese transporter Smf1 and the amino acid transporters Can1, Lyp1 and Mup1 it has been shown that ubiquitination is mediated by arrestin-like adaptor proteins that bind to Rsp5 and recognize specific transporters. As yeast contains a large family of arrestins, this has been suggested as a general model for transporter regulation; however, analysis is complicated by redundancy amongst the arrestins. We have tested this model by removing all the arrestins and examining the requirements for endocytosis of four more transporters, Itr1 (inositol), Hxt6 (glucose), Fur4 (uracil) and Tat2 (tryptophan). This reveals functions for the arrestins Art5/Ygr068c and Art4/Rod1, and additional roles for Art1/Ldb19, Art2/Ecm21 and Art8/Csr2. It also reveals functional redundancy between arrestins and the arrestin-like adaptors Bul1 and Bul2. In addition, we show that delivery to the vacuole often requires multiple additional ubiquitin ligases or adaptors, including the RING domain ligase Pib1, and the adaptors Bsd2, Ear1 and Ssh4, some acting redundantly. We discuss the similarities and differences in the requirements for regulation of different transporters.

Insights from yeast endosomes.

The endosomal system of yeast is simpler than that of animal cells, but as it is mapped more similarities are emerging. A key role for ubiquitin in sorting proteins to and into multivesicular bodies has been demonstrated. The finding that Phox homology domains recognise phosphatidylinositol 3-phosphate explains how sorting nexins are recruited to endosomes, where they mediate the retrieval of membrane proteins from the endocytic pathway.

A transmembrane ubiquitin ligase required to sort membrane proteins into multivesicular bodies.

Membrane proteins with transmembrane domains (TMDs) that contain polar residues exposed to the lipid bilayer are selectively sorted into multivesicular bodies (MVBs) and delivered to the yeast vacuole. Sorting of some, although not all, proteins into these structures is mediated by ubiquitination. We have identified a transmembrane ubiquitin ligase, Tul1, that is resident in the Golgi apparatus and is required for the ubiquitination of proteins with polar TMDs, including vacuolar proteins such as carboxypeptidase S. We suggest that Tul1 provides quality control, identifying misfolded membrane proteins and marking them for transport to endosomes and degradation in the vacuole.

Control of the activity of WW-HECT domain E3 ubiquitin ligases by NDFIP proteins.

HECT domain E3 ubiquitin ligases of the NEDD4 family control many cellular processes, but their regulation is poorly understood. They contain multiple WW domains that recognize PY elements. Here, we show that the small PY-containing membrane proteins, NDFIP1 and NDFIP2 (NEDD4 family-interacting proteins), activate the catalytic activity of ITCH and of several other HECT ligases by binding to them. This releases them from an autoinhibitory intramolecular interaction, which seems to be characteristic of these enzymes. Activation of ITCH requires multiple PY-WW interactions, but little else. Binding of NDFIP proteins is highly dynamic, potentially allowing activated ligases to access other PY-containing substrates. In agreement with this, NDFIP proteins promote ubiquitination in vivo both of Jun proteins, which have a PY motif, and of endophilin, which does not.

Maturation of Golgi cisternae directly observed.

Newly synthesized secretory proteins pass through the Golgi apparatus, which consists of multiple cisternae containing distinct populations of enzymes. Are the cargo proteins shuttled between cisternae in vesicles or do they remain in a cisterna while it is the Golgi enzymes that are removed and replaced? As predicted by the latter model--the cisternal maturation hypothesis--two groups have directly observed the replacement of one Golgi protein with another in individual cisternae, thus answering the question. However, its solution raises many more unknowns.

Arrestin-like proteins mediate ubiquitination and endocytosis of the yeast metal transporter Smf1.

Many plasma membrane proteins in yeast are ubiquitinated and endocytosed, but how they are recognized for modification has remained unknown. Here, we show that the manganese transporter Smf1 is endocytosed when cells are exposed to cadmium ions, that this endocytosis depends on Rsp5-dependent ubiquitination of specific lysines and that it also requires phosphorylation at nearby sites. This phosphorylation is, however, constitutive rather than stress-induced. Efficient ubiquitination requires Ecm21 or Csr2, two members of a family of arrestin-like yeast proteins that contain several PY motifs and bind to Rsp5. Ecm21 also binds to phosphorylated Smf1, providing a link between Rsp5 and its substrate. PY motif-containing arrestin-like proteins are found in many species, including humans, and might have a general role as ubiquitin ligase adaptors.

Multiple interactions drive adaptor-mediated recruitment of the ubiquitin ligase rsp5 to membrane proteins in vivo and in vitro.

Recognition of membrane proteins by the Nedd4/Rsp5 ubiquitin ligase family is a critical step in their targeting to the multivesicular body pathway. Some substrates contain "PY" motifs (PPxY), which bind to WW domains in the ligase. Others lack PY motifs and instead rely on adaptors that recruit the ligase to them. To investigate the mechanism of adaptor-mediated ubiquitination, we have characterized the interactions between the adaptor Bsd2, the ubiquitin ligase Rsp5, and the membrane proteins Cps1, Tre1, and Smf1 from Saccharomyces cerevisiae. We have reconstituted adaptor-mediated modification of Cps1 and Tre1 in vitro, and we show that two PY motifs in Bsd2 and two WW domains (WW2 and WW3) in Rsp5 are crucial for this. The binding of a weak noncanonical DMAPSY motif in Bsd2 to WW3 is an absolute requirement for Bsd2 adaptor function. We show that sorting of the manganese transporter Smf1, which requires both Bsd2 and Tre1, depends upon two PY motifs in Bsd2 and one motif in Tre1 but only two WW domains in Rsp5. We suggest that sequential assembly of first a Bsd2/Rsp5 complex, then a Tre1/Bsd2/Rsp5 complex followed by a rearrangement of PY-WW interactions is required for the ubiquitination of Smf1.

Ubiquitination of alpha-synuclein filaments by Nedd4 ligases.

Alpha-synuclein can form beta-sheet filaments, the accumulation of which plays a key role in the development of Parkinson's disease, dementia with Lewy bodies and multiple system atrophy. It has previously been shown that alpha-synuclein is a substrate for the HECT domain-containing ubiquitin ligase Nedd4, and is subject to ubiquitin-mediated endosomal degradation. We show here that alpha-synuclein filaments are much better substrates for ubiquitination in vitro than monomeric alpha-synuclein, and that this increased susceptibility cannot be mimicked by the mere clustering of monomers. Recognition by Nedd4 family enzymes is not through the conventional binding of PPxY-containing sequences to WW domains of the ligase, but it also involves C2 and HECT domains. The disease-causing alpha-synuclein mutant A53T is a much less efficient substrate for Nedd4 ligases than the wild-type protein. We suggest that preferential recognition, ubiquitination and degradation of beta-sheet-containing filaments may help to limit toxicity, and that A53T alpha-synuclein may be more toxic, at least in part because it avoids this fate.

Membrane traffic: GGAs sort ubiquitin.

Membrane proteins that are tagged with ubiquitin are diverted from the secretory pathway into lysosomes. New work shows that it is the GGA proteins that initiate sorting in the Golgi, and suggests that similar principles apply to multiple sorting steps.

Slow diffusion of proteins in the yeast plasma membrane allows polarity to be maintained by endocytic cycling.

Many cells show a polarized distribution of some plasma membrane proteins, which may be maintained either by a diffusion barrier or kinetically: as first demonstrated in fibroblasts, locally exocytosed proteins will remain polarized if they are endocytosed and recycled before they can diffuse to equilibrium. In yeast, actin cables direct exocytosis to the bud and to the tips of polarized mating intermediates termed shmoos. A septin ring at the bud neck retains some proteins, but shmoos lack this. Here, we show that the exocytic SNARE Snc1 is kinetically polarized. It is concentrated at bud and shmoo tips, and this requires its endocytosis. Kinetic polarization is possible in these small cells because proteins diffuse much more slowly in the yeast plasma membrane than would be expected from measurements in animal cells. Slow diffusion requires neither the cell wall nor polymerized actin, but it is affected in the ergosterol synthesis mutant erg6. Other proteins also require endocytosis for efficient polarization, and the plasma membrane SNARE Sso1 can be polarized merely by appending an endocytic signal. Thus, despite their small size, yeast cells can use localized exocytosis and endocytic recycling as a simple mechanism to maintain polarity.

Disinhibition of the HECT E3 ubiquitin ligase WWP2 by polymerized Dishevelled.

Dishevelled is a pivot in Wnt signal transduction, controlling both ß-catenin-dependent transcription to specify proliferative cell fates, and cell polarity and other non-nuclear events in post-mitotic cells. In response to Wnt signals, or when present at high levels, Dishevelled forms signalosomes by dynamic polymerization. Its levels are controlled by ubiquitylation, mediated by various ubiquitin ligases, including NEDD4 family members that bind to a conserved PPxY motif in Dishevelled (mammalian Dvl1-3). Here, we show that Dvl2 binds to the ubiquitin ligase WWP2 and unlocks its ligase activity from autoinhibition. This disinhibition of WWP2 depends on several features of Dvl2 including its PPxY motif and to a lesser extent its DEP domain, but crucially on the ability of Dvl2 to polymerize, indicating that WWP2 is activated in Wnt signalosomes. We show that Notch intracellular domains are substrates for Dvl-activated WWP2 and their transcriptional activity is consequently reduced, providing a molecular mechanism for cross-talk between Wnt and Notch signalling. These regulatory interactions are conserved in Drosophila whose WWP2 orthologue, Suppressor-of-deltex, downregulates Notch signalling upon activation by Dishevelled in developing wing tissue. Attentuation of Notch signalling by Dishevelled signalosomes could be important during the transition of cells from the proliferative to the post-mitotic state.

TMF is a golgin that binds Rab6 and influences Golgi morphology.

BACKGROUND: Golgins are coiled-coil proteins associated with the Golgi apparatus, that are believed to be involved in the tethering of vesicles and the stacking of cisternae, as well as other functions such as cytoskeletal association. Many are peripheral membrane proteins recruited by GTPases. Several have been described in animal cells, and some in yeast, but the relationships between golgins from different species can be hard to define because although they share structural features, their sequences are not well conserved. RESULTS: We show here that the yeast protein Sgm1, previously shown to be recruited to the Golgi by the GTPase Ypt6, binds to Ypt6:GTP via a conserved 100-residue coiled-coil motif that can be identified in a wide range of eukaryotes. The mammalian equivalent of Sgm1 is TMF/ARA160, a protein previously identified in various screens as a putative transcription or chromatin remodelling factor. We show that it is a Golgi protein, and that it binds to the three known isoforms of the Ypt6 homologue Rab6. Depletion of the protein by RNA interference in rat NRK cells results in a modest dispersal of Golgi membranes around the cell, suggesting a role for TMF in the movement or adherence of Golgi stacks. CONCLUSION: We have identified TMF as an evolutionarily conserved golgin that binds Rab6 and contributes to Golgi organisation in animal cells.

Inefficient quality control of thermosensitive proteins on the plasma membrane.

BACKGROUND: Misfolded proteins are generally recognised by cellular quality control machinery, which typically results in their ubiquitination and degradation. For soluble cytoplasmic proteins, degradation is mediated by the proteasome. Membrane proteins that fail to fold correctly are subject to ER associated degradation (ERAD), which involves their extraction from the membrane and subsequent proteasome-dependent destruction. Proteins with abnormal transmembrane domains can also be recognised in the Golgi or endosomal system and targeted for destruction in the vacuole/lysosome. It is much less clear what happens to membrane proteins that reach their destination, such as the cell surface, and then suffer damage. METHODOLOGY/PRINCIPAL FINDINGS: We have tested the ability of yeast cells to degrade membrane proteins to which temperature-sensitive cytoplasmic alleles of the Ura3 protein or of phage lambda repressor have been fused. In soluble form, these proteins are rapidly degraded upon temperature shift, in part due to the action of the Doa10 and San1 ubiquitin ligases and the proteasome. When tethered to the ER protein Use1, they are also degraded. However, when tethered to a plasma membrane protein such as Sso1 they escape degradation, either in the vacuole or by the proteasome. CONCLUSIONS/SIGNIFICANCE: Membrane proteins with a misfolded cytoplasmic domain appear not to be efficiently recognised and degraded once they have escaped the ER, even though their defective domains are exposed to the cytoplasm and potentially to cytoplasmic quality controls. Membrane tethering may provide a way to reduce degradation of unstable proteins.

Structural analysis of the interaction between the SNARE Tlg1 and Vps51.

Membrane fusion in cells involves the interaction of SNARE proteins on apposing membranes. Formation of SNARE complexes is preceded by tethering events, and a number of protein complexes that are thought to mediate this have been identified. The VFT or GARP complex is required for endosome-Golgi traffic in yeast. It consists of four subunits, one of which, Vps51, has been shown to bind specifically to the SNARE Tlg1, which participates in the same fusion event. We have determined the structure of the N-terminal domain of Tlg1 bound to a peptide from the N terminus of Vps51. Binding depends mainly on residues 18-30 of Vps51. These form a short helix which lies in a conserved groove in the three-helix bundle formed by Tlg1. Surprisingly, although both Vps51 and Tlg1 are required for transport to the late Golgi from endosomes, removal of the Tlg1-binding sequences from Vps51 does not block such traffic in vivo. Thus, this particular interaction cannot be crucial to the process of vesicle docking or fusion.

Transferrin receptor-like proteins control the degradation of a yeast metal transporter.

Plasma membrane transporters are often downregulated by their substrates. The yeast manganese transporter Smf1 is subject to two levels of regulation: heavy metals induce its sequestration within the cell, and also its ubiquitination and degradation in the vacuole. Degradation requires Bsd2, a membrane protein with a PPxY motif that recruits the ubiquitin ligase Rsp5, and which has a role in the quality control of membrane proteins, that expose hydrophilic residues to the lipid bilayer. We show that degradation of Smf1 requires in addition one of a pair of related yeast proteins, Tre1 and Tre2, that also contain PPxY motifs. Tre1 can partially inhibit manganese uptake without Bsd2, but requires Bsd2 to induce Smf1 degradation. It has a relatively hydrophilic transmembrane domain and binds to Bsd2. We propose that the Tre proteins specifically link Smf1 to the Bsd2-dependent quality control system. Their luminal domains are related to the transferrin receptor, but these are dispensable for Smf1 regulation. Tre proteins and the transferrin receptors appear to have evolved independently from the same family of membrane-associated proteases.