Regulation of the Activity of the Dual-Function DnaA Protein in Caulobacter crescentus.

DnaA is a conserved essential bacterial protein that acts as the initiator of chromosomal replication as well as a master transcriptional regulator in Caulobacter crescentus. Thus, the intracellular levels of active DnaA need to be tightly regulated during the cell cycle. Our previous work suggested that DnaA may be regulated at the level of its activity by the replisome-associated protein HdaA. Here, we describe the construction of a mutant DnaA protein [DnaA(R357A)]. The R357 residue in the AAA+ domain of the C. crescentus DnaA protein is equivalent to the R334 residue of the E. coli DnaA protein, which is required for the Regulatory Inactivation of DnaA (RIDA). We found that the expression of the DnaA(R357A) mutant protein in C. crescentus, but not the expression of the wild-type DnaA protein at similar levels, causes a severe phenotype of over-initiation of chromosomal replication and that it blocks cell division. Thus, the mutant DnaA(R357A) protein is hyper-active to promote the initiation of DNA replication, compared to the wild-type DnaA protein. DnaA(R357A) could not replace DnaA in vivo, indicating that the switch in DnaA activity once chromosomal replication has started may be an essential process in C. crescentus. We propose that the inactivation of DnaA is the main mechanism ensuring that chromosomal replication starts only once per cell cycle. We further observed that the R357A substitution in DnaA does not promote the activity of DnaA as a direct transcriptional activator of four important genes, encoding HdaA, the GcrA master cell cycle regulator, the FtsZ cell division protein and the MipZ spatial regulator of cell division. Thus, the AAA+ domain of DnaA may play a role in temporally regulating the bifunctionality of DnaA by reallocating DnaA molecules from initiating DNA replication to transcribing genes within the unique DnaA regulon of C. crescentus.

Loss of Memo, a novel FGFR regulator, results in reduced lifespan.

Memo is a widely expressed 33-kDa protein required for heregulin (HRG)-, epidermal growth factor (EGF)-, and fibroblast growth factor (FGF)-induced cell motility. Studies in mouse embryonic fibroblasts, wild-type or knockout for Memo, were performed to further investigate the role of Memo downstream of FGFR. We demonstrated that Memo associates with the FGFR signalosome and is necessary for optimal activation of signaling. To uncover Memo's physiological role, Memo conditional-knockout mice were generated. These animals showed a reduced life span, increased insulin sensitivity, small stature, graying hair, alopecia, kyphosis, loss of subcutaneous fat, and loss of spermatozoa in the epididymis. Memo-knockout mice also have elevated serum levels of active vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D), and calcium compared to control littermates expressing Memo. In summary, the results from in vivo and in vitro models support the hypothesis that Memo is a novel regulator of FGFR signaling with a role in controlling 1,25(OH)2D production and normal calcium homeostasis.

The bZIP transcription factor Rca1p is a central regulator of a novel CO₂ sensing pathway in yeast.

Like many organisms the fungal pathogen Candida albicans senses changes in the environmental CO(2) concentration. This response involves two major proteins: adenylyl cyclase and carbonic anhydrase (CA). Here, we demonstrate that CA expression is tightly controlled by the availability of CO(2) and identify the bZIP transcription factor Rca1p as the first CO(2) regulator of CA expression in yeast. We show that Rca1p upregulates CA expression during contact with mammalian phagocytes and demonstrate that serine 124 is critical for Rca1p signaling, which occurs independently of adenylyl cyclase. ChIP-chip analysis and the identification of Rca1p orthologs in the model yeast Saccharomyces cerevisiae (Cst6p) point to the broad significance of this novel pathway in fungi. By using advanced microscopy we visualize for the first time the impact of CO(2) build-up on gene expression in entire fungal populations with an exceptional level of detail. Our results present the bZIP protein Rca1p as the first fungal regulator of carbonic anhydrase, and reveal the existence of an adenylyl cyclase independent CO(2) sensing pathway in yeast. Rca1p appears to regulate cellular metabolism in response to CO(2) availability in environments as diverse as the phagosome, yeast communities or liquid culture.

AMP-activated protein kinase mediates glucocorticoid-induced metabolic changes: a novel mechanism in Cushing's syndrome.

Chronic exposure to glucocorticoid hormones, resulting from either drug treatment or Cushing's syndrome, results in insulin resistance, central obesity, and symptoms similar to the metabolic syndrome. We hypothesized that the major metabolic effects of corticosteroids are mediated by changes in the key metabolic enzyme adenosine monophosphate-activated protein kinase (AMPK) activity. Activation of AMPK is known to stimulate appetite in the hypothalamus and stimulate catabolic processes in the periphery. We assessed AMPK activity and the expression of several metabolic enzymes in the hypothalamus, liver, adipose tissue, and heart of a rat glucocorticoid-excess model as well as in in vitro studies using primary human adipose and primary rat hypothalamic cell cultures, and a human hepatoma cell line treated with dexamethasone and metformin. Glucocorticoid treatment inhibited AMPK activity in rat adipose tissue and heart, while stimulating it in the liver and hypothalamus. Similar data were observed in vitro in the primary adipose and hypothalamic cells and in the liver cell line. Metformin, a known AMPK regulator, prevented the corticosteroid-induced effects on AMPK in human adipocytes and rat hypothalamic neurons. Our data suggest that glucocorticoid-induced changes in AMPK constitute a novel mechanism that could explain the increase in appetite, the deposition of lipids in visceral adipose and hepatic tissue, as well as the cardiac changes that are all characteristic of glucocorticoid excess. Our data suggest that metformin treatment could be effective in preventing the metabolic complications of chronic glucocorticoid excess.

Loss of the thyroid hormone-binding protein Crym renders striatal neurons more vulnerable to mutant huntingtin in Huntington's disease.

The mechanisms underlying preferential atrophy of the striatum in Huntington's disease (HD) are unknown. One hypothesis is that a set of gene products preferentially expressed in the striatum could determine the particular vulnerability of this brain region to mutant huntingtin (mHtt). Here, we studied the striatal protein µ-crystallin (Crym). Crym is the NADPH-dependent p38 cytosolic T3-binding protein (p38CTBP), a key regulator of thyroid hormone (TH) T3 (3,5,3'-triiodo-l-thyronine) transportation. It has been also recently identified as the enzyme that reduces the sulfur-containing cyclic ketimines, which are potential neurotransmitters. Here, we confirm the preferential expression of the Crym protein in the rodent and macaque striatum. Crym expression was found to be higher in the macaque caudate than in the putamen. Expression of Crym was reduced in the BACHD and Knock-in 140CAG mouse models of HD before onset of striatal atrophy. We show that overexpression of Crym in striatal medium-size spiny neurons using a lentiviral-based strategy in mice is neuroprotective against the neurotoxicity of an N-terminal fragment of mHtt in vivo. Thus, reduction of Crym expression in HD could render striatal neurons more susceptible to mHtt suggesting that Crym may be a key determinant of the vulnerability of the striatum. In addition our work points to Crym as a potential molecular link between striatal degeneration and the THs deregulation reported in HD patients.

Genomic roles of the HCF transcriptional regulator in "Drosophila"

Abstract : Transcriptional regulation is the result of a combination of positive and negative effectors, such as transcription factors, cofactors and chromatin modifiers. During my thesis project I studied chromatin association, and transcriptional and cell cycle regulatory functions of dHCF, the Drosophila homologue of the human protein HCF-1 (host cell factor-1). The human and Drosophila HCF proteins are synthesized as large polypeptides that are cleaved into two subunits (HCFN and HCFC), which remain associated with one another by non covalent interactions. Studies in mammalian cells over the past 20 years have been devoted to understanding the cellular functions of HCF-1 and have revealed that it is a key regulator of transcription and cell cycle regulation. In human cells, HCF-1 interacts with the histone methyltransferase Set1/Ash2 and MLL/Ash2 complexes and the histone deacetylase Sin3 complex, which are involved in transcriptional activation and repression, respectively. HCF-1 is also recruited to promoters to regulate G1 -to-S phase progression during the cell cycle by the activator transcription factors E2F1 and E2F3, and by the repressor transcription factor E2F4. HCF-1 protein structure and these interactions between HCP-1 and E2F transcriptional regulator proteins are also conserved in Drosophila. In this doctoral thesis, I use proliferating Drosophila SL2 cells to study both the genomic-binding sites of dHCF, using a combination of chromatin immunoprecipitation and ultra high throughput sequencing (ChIP-seq) analysis, and dHCF regulated genes, employing RNAi and microarray expression analysis. I show that dHCF is bound to over 7500 chromosomal sites in proliferating SL2 cells, and is located at +-200 bp relative to the transcriptional start sites of about 30% of Drosophila genes. There is also a direct relationship between dHCF promoter association and promoter- associated transcriptional activity. Thus, dHCF binding levels at promoters correlated directly with transcriptional activity. In contrast, expression studies showed that dHCF appears to be involved in both transcriptional activation and repression. Analysis of dHCF-binding sites identified nine dHCF-associated motifs, four of them linked dHCF to (i) two insulator proteins, GAGA and BEAF, (ii) the E-box motif, and (iii) a degenerated TATA-box. The dHCF-associated motifs allowed the organization of the dHCF-bound genes into five biological processes: differentiation, cell cycle and gene expression, regulation of endocytosis, and cellular localization. I further show that different mechanisms regulate dHCF association with chromatin. Despite that after dHCF cleavage the dHCFN and dHCFC subunits remain associated, the two subunits showed different affinities for chromatin and differential binding to a set of tested promoters, suggesting that dHCF could target specific promoters through each of the two subunits. Moreover, in addition to the interaction between dHCF and E2F transcription factors, the dHCF binding pattern is correlated with dE2F2 genomic 4 distribution. I show that dE2F factors are necessary for recruitment of dHCF to the promoter of a set of dHCF regulated genes. Therefore dHCF, as in mammals, is involved in regulation of G1 to S phase progression in collaboration with the dE2Fs transcription factors. In addition, gene expression arrays reveal that dHCF could indirectly regulate cell cycle progression by promoting expression of genes involved in gene expression and protein synthesis, and inhibiting expression of genes involved in cell-cell adhesion. Therefore, dHCF is an evolutionary conserved protein, which binds to many specific sites of the Drosophila genome via interaction with DNA of chromatin-binding proteins to regulate the expression of genes involved in many different cellular functions. Résumé : La regulation de la transcription est le résultat des effets positifs et négatifs des facteurs de transcription, cofacteurs et protéines effectrices qui modifient la chromatine. Pendant mon projet de thèse, j'ai étudié l'association a la chromatine, ainsi que la régulation de la transcription et du cycle cellulaire par dHCF, l'homologue chez la drosophile de la protéine humaine HCF-1 (host cell factor-1). Chez 1'humain et la V drosophile, les deux protéines HCF sont synthétisées sous la forme d'un long polypeptide, qui est ensuite coupé en deux sous-unités au centre de la protéine. Les deux sous-unités restent associées ensemble grâce a des interactions non-covalentes. Des études réalisées pendant les 20 dernières années ont permit d'établir que HCF-l et un facteur clé dans la régulation de la transcription et du cycle cellulaire. Dans les cellules humaines, HCF-1 active et réprime la transcription en interagissant avec des complexes de protéines qui activent la transcription en méthylant les histones (HMT), comme par Set1/Ash2 et MLL/Ash2, et d'autres complexes qui répriment la transcription et sont responsables de la déacétylation des histones (HDAC) comme la protéine Sin3. HCF-l est aussi recruté aux promoteurs par les activateurs de la transcription E2F l et E2F3a, et par le répresseur de la transcription E2F4 pour réguler la transition entre les phases G1 et S du cycle cellulaire. La structure de HCF-1 et les interactions entre HCF-l et les régulateurs de la transcription sont conservées chez la drosophile. Pendant ma these j'ai utilisé les cellules de la drosophile, SL2 en culture, pour étudier les endroits de liaisons de HCF-l à la chromatine, grâce a immunoprecipitation de la chromatine et du séquençage de l'ADN massif ainsi que les gènes régulés par dHCF 3 grâce a la technique de RNAi et des microarrays. Mes résultats on montré que dHCF se lie à environ 7565 endroits, et estimé a 1200 paire de bases autour des sites d'initiation de la transcription de 30% des gènes de la drosophile. J 'ai observe une relation entre dHCF et le niveau de la transcription. En effet, le niveau de liaison dHCF au promoteur corrèle avec l'activité de la transcription. Cependant, mes études d'expression ont montré que dHCF est implique dans le processus d'activation et mais aussi de répression de la transcription. L'analyse des séquences d'ADN liées par dHCF a révèle neuf motifs, quatre de ces motifs ont permis d'associer dl-ICF a deux protéines isolatrices GAGA et BEAF, au motif pour les E-boxes et a une TATA-box dégénérée. Les neuf motifs associes à dHCF ont permis d'associer les gènes lies par dHCF au promoteur a cinq processus biologiques: différentiation, cycle cellulaire, expression de gènes, régulation de l'endocytosis et la localisation cellulaire, J 'ai aussi montré qu'il y a plusieurs mécanismes qui régulent l'association de dHCF a la chromatine, malgré qu'après clivage, les deux sous-unites dHCFN and dHCFC, restent associées, elles montrent différentes affinités pour la chromatine et lient différemment un group de promoteurs, les résultats suggèrent que dHCF peut se lier aux promoteurs en utilisant chacune de ses sous-unitées. En plus de l'association de dHCF avec les facteurs de transcription dE2F s, la distribution de dHCF sur le génome corrèle avec celle du facteur de transcription dE2F2. J'ai aussi montré que les dE2Fs sont nécessaires pour le recrutement de dHCF aux promoteurs d'un sous-groupe de gènes régules par dHCF. Mes résultats ont aussi montré que chez la drosophile comme chez les humains, dl-ICF est implique dans la régulation de la progression de la phase G1 a la phase S du cycle cellulaire en collaboration avec dE2Fs. D'ailleurs, les arrays d'expression ont suggéré que dHCF pourrait réguler le cycle cellulaire de façon indirecte en activant l'expression de gènes impliqués dans l'expression génique et la synthèse de protéines, et en inhibant l'expression de gènes impliqués dans l'adhésion cellulaire. En conclusion, dHCF est une protéine, conservée dans l'évolution, qui se lie spécifiquement a beaucoup d'endroits du génome de Drosophile, grâce à l'interaction avec d'autres protéines, pour réguler l'expression des gènes impliqués dans plusieurs fonctions cellulaires.

Promoter recognition and activation by the global response regulator CbrB in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, the CbrA/CbrB two-component system is instrumental in the maintenance of the carbon-nitrogen balance and for growth on carbon sources that are energetically less favorable than the preferred dicarboxylate substrates. The CbrA/CbrB system drives the expression of the small RNA CrcZ, which antagonizes the repressing effects of the catabolite repression control protein Crc, an RNA-binding protein. Dicarboxylates appear to cause carbon catabolite repression by inhibiting the activity of the CbrA/CbrB system, resulting in reduced crcZ expression. Here we have identified a conserved palindromic nucleotide sequence that is present in upstream activating sequences (UASs) of promoters under positive control by CbrB and σ(54) RNA polymerase, especially in the UAS of the crcZ promoter. Evidence for recognition of this palindromic sequence by CbrB was obtained in vivo from mutational analysis of the crcZ promoter and in vitro from electrophoretic mobility shift assays using crcZ promoter fragments and purified CbrB protein truncated at the N terminus. Integration host factor (IHF) was required for crcZ expression. CbrB also activated the lipA (lipase) promoter, albeit less effectively, apparently by interacting with a similar but less conserved palindromic sequence in the UAS of lipA. As expected, succinate caused CbrB-dependent catabolite repression of the lipA promoter. Based on these results and previously published data, a consensus CbrB recognition sequence is proposed. This sequence has similarity to the consensus NtrC recognition sequence, which is relevant for nitrogen control.

Role of the GNOM gene in Arabidopsis apical-basal patterning--From mutant phenotype to cellular mechanism of protein action.

How the apical-basal axis of polarity is established in embryogenesis is still a mystery in plant development. This axis appeared specifically compromised by mutations in the Arabidopsis GNOM gene. Surprisingly, GNOM encodes an ARF guanine-nucleotide exchange factor (ARF-GEF) that regulates the formation of vesicles in membrane trafficking. In-depth functional analysis of GNOM and its closest relative, GNOM-LIKE 1 (GNL1), has provided a mechanistic explanation for the development-specific role of a seemingly mundane trafficking regulator. The current model proposes that GNOM is specifically involved in the endosomal recycling of the auxin-efflux carrier PIN1 to the basal plasma membrane in provascular cells, which in turn is required for the accumulation of the plant hormone auxin at the future root pole through polar auxin transport. Thus, the analysis of GNOM highlights the importance of cell-biological processes for a mechanistic understanding of development.

NPAS2 as a transcriptional regulator of non-rapid eye movement sleep: genotype and sex interactions.

Because the transcription factor neuronal Per-Arnt-Sim-type signal-sensor protein-domain protein 2 (NPAS2) acts both as a sensor and an effector of intracellular energy balance, and because sleep is thought to correct an energy imbalance incurred during waking, we examined NPAS2's role in sleep homeostasis using npas2 knockout (npas2-/-) mice. We found that, under conditions of increased sleep need, i.e., at the end of the active period or after sleep deprivation (SD), NPAS2 allows for sleep to occur at times when mice are normally awake. Lack of npas2 affected electroencephalogram activity of thalamocortical origin; during non-rapid eye movement sleep (NREMS), activity in the spindle range (10-15 Hz) was reduced, and within the delta range (1-4 Hz), activity shifted toward faster frequencies. In addition, the increase in the cortical expression of the NPAS2 target gene period2 (per2) after SD was attenuated in npas2-/- mice. This implies that NPAS2 importantly contributes to the previously documented wake-dependent increase in cortical per2 expression. The data also revealed numerous sex differences in sleep; in females, sleep need accumulated at a slower rate, and REMS loss was not recovered after SD. In contrast, the rebound in NREMS time after SD was compromised only in npas2-/- males. We conclude that NPAS2 plays a role in sleep homeostasis, most likely at the level of the thalamus and cortex, where NPAS2 is abundantly expressed.

Insight in protein S deficiency from mouse models

Protein S (ProS) is an important negative regulator of blood coagulation. Its physiological importance is evident in purpura fulminans and other life-threatening thrombotic disorders typical of ProS deficient patients. Our previous characterization of ProS deficiency in mouse models has shown similarities with the human phenotypes: heterozygous ProS-deficient mice (Pros+/-) had increased thrombotic risk whereas homozygous deficiency in ProS (Pros-/-) was incompatible with life (Blood 2009; 114:2307-2314). In tissues, ProS exerts cellular functions by binding to and activating tyrosine kinase receptors of the Tyro3 family (TAM) on the cell surface.To extend the analysis of coagulation defects beyond the Pros-/- phenotype and add new insights into the sites of synthesis ProS and its action, we generated mice with inactivated ProS in hepatocytes (Proslox/loxAlbCre+) as well as in endothelial and hematopoietic cells (Proslox/loxTie2Cre+). Both models resulted in significant reduction of circulating ProS levels and in a remarkable increased thrombotic risk in vivo. In a model of tissue factor (TF)-induced venous thromboembolism (VTE), only 17% of Proslox/loxAlbCre+ mice (n=12) and only 13% of Proslox/loxTie2Cre+ mice (n=14) survived, compared with 86% of Proslox/lox mice (n=14; P<0.001).To mimic a severe acquired ProS deficiency, ProS gene was inactivated at the adult stage using the polyI:C-inducible Mx1-Cre system (Proslox/loxMx1Cre+). Ten days after polyI:C treatment, Proslox/loxMx1Cre+ mice developed disseminated intravascular coagulation with extensive lung and liver thrombosis.It is worth noting that no skin lesions compatible with purpura fulminans were observed in any of the above-described models of partial ProS deficiency. In order to shed light on the pathogenesis of purpura fulminans, we exposed the different ProS-deficient mice to warfarin (0.2 mg/day). We observed that Pros+/-, Proslox/loxAlbCre+ and Proslox/loxTie2Cre+ mice developed retiform purpura (characterized by erythematous and necrotic lesions of the genital region and extremities) and died after 3 to 5 days after the first warfarin administration.In human, ProS is also synthesized by megakaryocytes and hence stored at high concentrations in circulating platelets (pProS). The role of pProS has been investigated by generating megakaryocyte ProS-deficient model using the PF4 promoter as Cre driver (Proslox/loxPf4Cre+). In the TF-induced VTE model, Proslox/loxPf4Cre+ (n=15) mice showed a significant increased risk of thrombosis compared to Proslox/lox controls (n=14; survival rate 47% and 86%, respectively; P<0.05). Furthermore, preliminary results suggest survival to be associated with higher circulating ProS levels. In order to evaluate the potential role of pProS in thrombus formation, we investigated the thrombotic response to intravenous injection of collagen-epinephrine in vivo and platelet function in vitro. Both in vivo and in vitro experiments showed similar results between Proslox/loxPf4Cre+ and Proslox/lox, indicating that platelet reactivity was not influenced by the absence of pProS. These data suggest that pProS is delivered at the site of thrombosis to inhibit thrombin generation.We further investigated the ability of ProS to function as a ligand of TAM receptors, by using homozygous and heterozygous deficient mice for both the TAM ligands ProS and Gas6. Gas6-/-Pros-/- mice died in utero and showed comparable dramatic bleeding and thrombotic phenotype as described for Pros-/- embryos.In conclusion, like complete ProS deficiency, double deficiency in ProS and Gas6 was lethal, whereas partial ProS deficiency was not. Mice partially deficient in ProS displayed a prothrombotic phenotype, including those with only deficiency in pProS. Purpura fulminans did not occur spontaneously in mice with partial Pros deficiency but developed upon warfarin administration.Thus, the use of different mice models of ProS deficiency can be instrumental in the study of its highly variable thrombotic phenotype and in the investigation of additional roles of ProS in inflammation and autoimmunity through TAM signaling.

Role of the JNK-interacting protein 1/islet brain 1 in cell degeneration in Alzheimer disease and diabetes.

Numerous epidemiological studies and some pharmacological clinical trials show the close connection between Alzheimer disease (AD) and type 2 diabetes (T2D) and thereby, shed more light into the existence of possible similar pathogenic mechanisms between these two diseases. Diabetes increases the risk of developing AD and sensitizers of insulin currently used as diabetes drugs can efficiently slow cognitive decline of the neurological disorder. Deposits of amyloid aggregate and hyperphosphorylation of tau, which are hallmarks of AD, have been also found in degenerating pancreatic islets beta-cells of patients with T2D. These events may have a causal role in the pathogenesis of the two diseases. Increased c-Jun NH(2)-terminal kinase (JNK) activity is found in neurofibrillary tangles (NFT) of AD and promotes programmed cell death of beta-cells exposed to a diabetic environment. The JNK-interacting protein 1 (JIP-1), also called islet brain 1 (IB1) because it is mostly expressed in the brain and islets, is a key regulator of the JNK pathway in neuronal and beta-cells. JNK, hyperphosphorylated tau and IB1/JIP-1 all co-localize with amyloids deposits in NFT and islets of AD and patients with T2D. This review discusses the role of the IB1/JIP-1 and the JNK pathway in the molecular pathogenesis of AD and T2D.

Oligomerization and DNA binding of Ler, a master regulator of pathogenicity of enterohemorrhagic and enteropathogenic Escherichia coli

Ler is a DNA-binding, oligomerizable protein that regulates pathogenicity islands in enterohemorrhagic and enteropathogenic Escherichia coli strains. Ler counteracts the transcriptional silencing effect of H-NS, another oligomerizable nucleoid-associated protein. We studied the oligomerization of Ler in the absence and presence of DNA by atomic force microscopy. Ler forms compact particles with a multimodal size distribution corresponding to multiples of 35 units of Ler. DNA wraps around Ler particles that contain more than 1516 Ler monomers. The resulting shortening of the DNA contour length is in agreement with previous measurements of the length of DNA protected by Ler in footprinting assays. We propose that the repetition unit corresponds to the number of monomers per turn of a tight helical Ler oligomer. While the repressor (H-NS) and anti-repressor (Ler) have similar DNA-binding domains, their oligomerization domains are unrelated. We suggest that the different oligomerization behavior of the two proteins explains the opposite results of their interaction with the same or proximal regions of DNA.

Yellow submarine of the Wnt/Frizzled signaling: submerging from the G protein harbor to the targets.

The Wnt/Frizzled signaling pathway plays multiple functions in animal development and, when deregulated, in human disease. The G-protein coupled receptor (GPCR) Frizzled and its cognate heterotrimeric Gi/o proteins initiate the intracellular signaling cascades resulting in cell fate determination and polarization. In this review, we summarize the knowledge on the ligand recognition, biochemistry, modifications and interacting partners of the Frizzled proteins viewed as GPCRs. We also discuss the effectors of the heterotrimeric Go protein in Frizzled signaling. One group of these effectors is represented by small GTPases of the Rab family, which amplify the initial Wnt/Frizzled signal. Another effector is the negative regulator of Wnt signaling Axin, which becomes deactivated in response to Go action. The discovery of the GPCR properties of Frizzled receptors not only provides mechanistic understanding to their signaling pathways, but also paves new avenues for the drug discovery efforts.

The fasting-induced adipose factor/angiopoietin-like protein 4 is physically associated with lipoproteins and governs plasma lipid levels and adiposity.

Proteins secreted from adipose tissue are increasingly recognized to play an important role in the regulation of glucose metabolism. However, much less is known about their effect on lipid metabolism. The fasting-induced adipose factor (FIAF/angiopoietin-like protein 4/peroxisome proliferator-activated receptor gamma angiopoietin-related protein) was previously identified as a target of hypolipidemic fibrate drugs and insulin-sensitizing thiazolidinediones. Using transgenic mice that mildly overexpress FIAF in peripheral tissues we show that FIAF is an extremely powerful regulator of lipid metabolism and adiposity. FIAF overexpression caused a 50% reduction in adipose tissue weight, partly by stimulating fatty acid oxidation and uncoupling in fat. In addition, FIAF overexpression increased plasma levels of triglycerides, free fatty acids, glycerol, total cholesterol, and high density lipoprotein (HDL)-cholesterol. Functional tests indicated that FIAF overexpression severely impaired plasma triglyceride clearance but had no effect on very low density lipoprotein production. The effects of FIAF overexpression were amplified by a high fat diet, resulting in markedly elevated plasma and liver triglycerides, plasma free fatty acids, and plasma glycerol levels, and impaired glucose tolerance in FIAF transgenic mice fed a high fat diet. Remarkably, in mice the full-length form of FIAF was physically associated with HDL, whereas truncated FIAF was associated with low density lipoprotein. In human both full-length and truncated FIAF were associated with HDL. The composite data suggest that via physical association with plasma lipoproteins, FIAF acts as a powerful signal from fat and other tissues to prevent fat storage and stimulate fat mobilization. Our data indicate that disturbances in FIAF signaling might be involved in dyslipidemia.

RAE-1, a novel PHR binding protein, is required for axon termination and synapse formation in Caenorhabditis elegans.

Previous studies in Caenorhabditis elegans showed that RPM-1 (Regulator of Presynaptic Morphology-1) regulates axon termination and synapse formation. To understand the mechanism of how rpm-1 functions, we have used mass spectrometry to identify RPM-1 binding proteins, and have identified RAE-1 (RNA Export protein-1) as an evolutionarily conserved binding partner. We define a RAE-1 binding region in RPM-1, and show that this binding interaction is conserved and also occurs between Rae1 and the human ortholog of RPM-1 called Pam (protein associated with Myc). rae-1 loss of function causes similar axon and synapse defects, and synergizes genetically with two other RPM-1 binding proteins, GLO-4 and FSN-1. Further, we show that RAE-1 colocalizes with RPM-1 in neurons, and that rae-1 functions downstream of rpm-1. These studies establish a novel postmitotic function for rae-1 in neuronal development.