Pharmacological properties of acid N-thiazolylamide FFA2 agonists.

FFA2 is a receptor for short-chain fatty acids. Propionate (C3) and 4-chloro-α-(1-methylethyl)-N-2-thiazolyl-benzeneacetamide (4-CMTB), the prototypical synthetic FFA2 agonist, evoke calcium mobilization in neutrophils and inhibit lipolysis in adipocytes via this G-protein-coupled receptor. 4-CMTB contains an N-thiazolylamide motif but no acid group, and 4-CMTB and C3 bind to different sites on FFA2 and show allosteric cooperativity. Recently, FFA2 agonists have been described that contain both N-thiazolylamide and carboxylate groups, reminiscent of bitopic ligands. These are thought to engage the carboxylate-binding site on FFA2, but preliminary evidence suggests they do not bind to the same site as 4-CMTB even though both contain N-thiazolylamide. Here, we describe the characterization of four FFA2 ligands containing both N-thiazolylamide and carboxylate. (R)-3-benzyl-4-((4-(2-chlorophenyl)thiazol-2-yl)(methyl)amino)-4-oxobutanoic acid (compound 14) exhibits allosteric agonism with 4-CMTB but not C3. Three other compounds agonize FFA2 in [(35)S]GTPγS-incorporation or cAMP assays but behave as inverse agonists in yeast-based gene-reporter assays, showing orthosteric antagonism of C3 responses but allosteric antagonism of 4-CMTB responses. Thus, the bitopic-like FFA2 ligands engage the orthosteric site but do not compete at the site of 4-CMTB binding on an FFA2 receptor molecule. Compound 14 activates FFA2 on human neutrophils and mouse adipocytes, but appears not to inhibit lipolysis upon treatment of human primary adipocytes in spite of the presence of a functional FFA2 receptor in these cells. Hence, these new ligands may reveal differences in coupling of FFA2 between human and rodent adipose tissues.

The human PDZome: a gateway to PSD95-Disc large-zonula occludens (PDZ)-mediated functions.

Protein-protein interactions organize the localization, clustering, signal transduction, and degradation of cellular proteins and are therefore implicated in numerous biological functions. These interactions are mediated by specialized domains able to bind to modified or unmodified peptides present in binding partners. Among the most broadly distributed protein interaction domains, PSD95-disc large-zonula occludens (PDZ) domains are usually able to bind carboxy-terminal sequences of their partners. In an effort to accelerate the discovery of PDZ domain interactions, we have constructed an array displaying 96% of the human PDZ domains that is amenable to rapid two-hybrid screens in yeast. We have demonstrated that this array can efficiently identify interactions using carboxy-terminal sequences of PDZ domain binders such as the E6 oncoviral protein and protein kinases (PDGFRß, BRSK2, PCTK1, ACVR2B, and HER4); this has been validated via mass spectrometry analysis. Taking advantage of this array, we show that PDZ domains of Scrib and SNX27 bind to the carboxy-terminal region of the planar cell polarity receptor Vangl2. We also have demonstrated the requirement of Scrib for the promigratory function of Vangl2 and described the morphogenetic function of SNX27 in the early Xenopus embryo. The resource presented here is thus adapted for the screen of PDZ interactors and, furthermore, should facilitate the understanding of PDZ-mediated functions.

Protein tyrosine kinase 7 has a conserved role in Wnt/ß-catenin canonical signalling.

The receptor protein tyrosine kinase 7 (PTK7) was recently shown to participate in noncanonical Wnt/planar cell polarity signalling during mouse and frog embryonic development. In this study, we report that PTK7 interacts with ß-catenin in a yeast two-hybrid assay and mammalian cells. PTK7-deficient cells exhibit weakened ß-catenin/T-cell factor transcriptional activity on Wnt3a stimulation. Furthermore, Xenopus PTK7 is required for the formation of Spemann's organizer and for Siamois promoter activation, events that require ß-catenin transcriptional activity. Using epistatic assays, we demonstrate that PTK7 functions upstream from glycogen synthase kinase 3. Taken together, our data reveal a new and conserved role for PTK7 in the Wnt canonical signalling pathway.

Structure of the yeast polarity protein Sro7 reveals a SNARE regulatory mechanism.

Polarized exocytosis requires coordination between the actin cytoskeleton and the exocytic machinery responsible for fusion of secretory vesicles at specific sites on the plasma membrane. Fusion requires formation of a complex between a vesicle-bound R-SNARE and plasma membrane Qa, Qb and Qc SNARE proteins. Proteins in the lethal giant larvae protein family, including lethal giant larvae and tomosyn in metazoans and Sro7 in yeast, interact with Q-SNAREs and are emerging as key regulators of polarized exocytosis. The crystal structure of Sro7 reveals two seven-bladed WD40 beta-propellers followed by a 60-residue-long 'tail', which is bound to the surface of the amino-terminal propeller. Deletion of the Sro7 tail enables binding to the Qbc SNARE region of Sec9 and this interaction inhibits SNARE complex assembly. The N-terminal domain of Sec9 provides a second, high-affinity Sro7 interaction that is unaffected by the tail. The results suggest that Sro7 acts as an allosteric regulator of exocytosis through interactions with factors that control the tail. Sequence alignments indicate that lethal giant larvae and tomosyn have a two-beta-propeller fold similar to that of Sro7, but only tomosyn appears to retain the regulatory tail.

The yeast lgl family member Sro7p is an effector of the secretory Rab GTPase Sec4p.

Rab guanosine triphosphatases regulate intracellular membrane traffic by binding specific effector proteins. The yeast Rab Sec4p plays multiple roles in the polarized transport of post-Golgi vesicles to, and their subsequent fusion with, the plasma membrane, suggesting the involvement of several effectors. Yet, only one Sec4p effector has been documented to date: the exocyst protein Sec15p. The exocyst is an octameric protein complex required for tethering secretory vesicles, which is a prerequisite for membrane fusion. In this study, we describe the identification of a second Sec4p effector, Sro7p, which is a member of the lethal giant larvae tumor suppressor family. Sec4-GTP binds to Sro7p in cell extracts as well as to purified Sro7p, and the two proteins can be coimmunoprecipitated. Furthermore, we demonstrate the formation of a ternary complex of Sec4-GTP, Sro7p, and the t-SNARE Sec9p. Genetic data support our conclusion that Sro7p functions downstream of Sec4p and further imply that Sro7p and the exocyst share partially overlapping functions, possibly in SNARE regulation.

Lethal giant larvae proteins interact with the exocyst complex and are involved in polarized exocytosis.

The tumor suppressor lethal giant larvae (Lgl) plays a critical role in epithelial cell polarization. However, the molecular mechanism by which Lgl carries out its functions is unclear. In this study, we report that the yeast Lgl proteins Sro7p and Sro77p directly interact with Exo84p, which is a component of the exocyst complex that is essential for targeting vesicles to specific sites of the plasma membrane for exocytosis, and that this interaction is important for post-Golgi secretion. Genetic analyses demonstrate a molecular pathway from Rab and Rho GTPases through the exocyst and Lgl to SNAREs, which mediate membrane fusion. We also found that overexpression of Lgl and t-SNARE proteins not only improves exocytosis but also rescues polarity defects in exocyst mutants. We propose that, although Lgl is broadly distributed in the cells, its localized interaction with the exocyst and kinetic activation are important for the establishment and reenforcement of cell polarity.

Structurally conserved interaction of Lgl family with SNAREs is critical to their cellular function.

The Lethal giant larvae (Lgl) tumor suppressor family is conserved from yeast to mammals and plays a critical yet controversial role in cell polarity. Studies on Drosophila Lgl suggest that its function in polarity is through regulation of the acto-myosin cytoskeleton. In contrast, studies on the yeast Lgl homologs, Sro7/Sro77, suggest a function in exocytosis through interaction with the t-SNARE Sec9. Using yeast/mammalian Lgl chimeras, we demonstrate that the overall architecture of Lgl proteins is highly conserved and that the C-terminal domain is the major site of SNARE interaction within both yeast and mammalian homologs. Importantly, we find that the ability of Lgl chimeras to function as the only source of Lgl in yeast correlates precisely with the ability to interact with the yeast t-SNARE. We report a novel interaction between Sro7 and the yeast myosin V, Myo2. However, we find that interactions with either Myo2 or Myo1 (myosin II) cannot account for the dramatic functional differences observed for these chimeras in yeast. These results provide the first demonstration that the interaction of an Lgl family member with a specific effector is critical to its function in vivo. These data support the model that the Lgl family functions in cell polarity, at least in part, by regulating SNARE-mediated membrane delivery events at the cell surface.

Alteration in the cytosolic triacylglycerol biosynthetic machinery leads to decreased cell growth and triacylglycerol synthesis in oleaginous yeast.

Altered nutrient content (levels of glucose) caused a drastic reduction in cell growth and triacylglycerol (TAG) production in the wild-type (WT) Rhodotorula glutinis. This was due to the decreased level of synthesis of TAG biosynthetic enzymes, reflected by a reduction in enzyme activity. A similar observation was made in the case of non-lethal mutants of TAG-deficient oleaginous yeast, namely TAG1 and TAG2, which were generated by ethyl methane sulphonate mutagenesis. Metabolic labelling of TAG-deficient cells with [(14)C]acetate, [(32)P]orthophosphate and [(14)C]mevalonate showed a negligible TAG formation with minimal alterations in phospholipid and sterol compositions. Assays on the activities of cytosolic TAG biosynthetic enzymes revealed that lysophosphatidic acid and diacylglycerol acyltransferases (ATs) were defective in TAG1 and TAG2 respectively. The activity of membrane-bound isoforms of TAG biosynthetic enzymes remains unaltered in the mutants. Analysis of cytosolic TAG biosynthetic enzymes by immunoblotting and immunoprecipitation indicated that the defective ATs were a part of the TAG biosynthetic multienzyme complex. Quantitatively, the cytosolic lysophosphatidic acid-AT was comparable between TAG1 and the WT. However, diacylglycerol-AT was relatively less in TAG2 than the WT. These results demonstrated that either by decreasing the nutrient content or mutating the enzymes of the soluble TAG biosynthetic pathway, TAG production was decreased with concomitant reduction in the cell growth.