Physical interaction between bacterial heat shock protein (Hsp) 90 and Hsp70 chaperones mediates their cooperative action to refold denatured proteins.

In eukaryotes, heat shock protein 90 (Hsp90) is an essential ATP-dependent molecular chaperone that associates with numerous client proteins. HtpG, a prokaryotic homolog of Hsp90, is essential for thermotolerance in cyanobacteria, and in vitro it suppresses the aggregation of denatured proteins efficiently. Understanding how the non-native client proteins bound to HtpG refold is of central importance to comprehend the essential role of HtpG under stress. Here, we demonstrate by yeast two-hybrid method, immunoprecipitation assays, and surface plasmon resonance techniques that HtpG physically interacts with DnaJ2 and DnaK2. DnaJ2, which belongs to the type II J-protein family, bound DnaK2 or HtpG with submicromolar affinity, and HtpG bound DnaK2 with micromolar affinity. Not only DnaJ2 but also HtpG enhanced the ATP hydrolysis by DnaK2. Although assisted by the DnaK2 chaperone system, HtpG enhanced native refolding of urea-denatured lactate dehydrogenase and heat-denatured glucose-6-phosphate dehydrogenase. HtpG did not substitute for DnaJ2 or GrpE in the DnaK2-assisted refolding of the denatured substrates. The heat-denatured malate dehydrogenase that did not refold by the assistance of the DnaK2 chaperone system alone was trapped by HtpG first and then transferred to DnaK2 where it refolded. Dissociation of substrates from HtpG was either ATP-dependent or -independent depending on the substrate, indicating the presence of two mechanisms of cooperative action between the HtpG and the DnaK2 chaperone system.

The Arabidopsis bHLH transcription factors MYC3 and MYC4 are targets of JAZ repressors and act additively with MYC2 in the activation of jasmonate responses.

Jasmonates (JAs) trigger an important transcriptional reprogramming of plant cells to modulate both basal development and stress responses. In spite of the importance of transcriptional regulation, only one transcription factor (TF), the Arabidopsis thaliana basic helix-loop-helix MYC2, has been described so far as a direct target of JAZ repressors. By means of yeast two-hybrid screening and tandem affinity purification strategies, we identified two previously unknown targets of JAZ repressors, the TFs MYC3 and MYC4, phylogenetically closely related to MYC2. We show that MYC3 and MYC4 interact in vitro and in vivo with JAZ repressors and also form homo- and heterodimers with MYC2 and among themselves. They both are nuclear proteins that bind DNA with sequence specificity similar to that of MYC2. Loss-of-function mutations in any of these two TFs impair full responsiveness to JA and enhance the JA insensitivity of myc2 mutants. Moreover, the triple mutant myc2 myc3 myc4 is as impaired as coi1-1 in the activation of several, but not all, JA-mediated responses such as the defense against bacterial pathogens and insect herbivory. Our results show that MYC3 and MYC4 are activators of JA-regulated programs that act additively with MYC2 to regulate specifically different subsets of the JA-dependent transcriptional response.

A molecular framework for light and gibberellin control of cell elongation.

Cell elongation during seedling development is antagonistically regulated by light and gibberellins (GAs). Light induces photomorphogenesis, leading to inhibition of hypocotyl growth, whereas GAs promote etiolated growth, characterized by increased hypocotyl elongation. The mechanism underlying this antagonistic interaction remains unclear. Here we report on the central role of the Arabidopsis thaliana nuclear transcription factor PIF4 (encoded by PHYTOCHROME INTERACTING FACTOR 4) in the positive control of genes mediating cell elongation and show that this factor is negatively regulated by the light photoreceptor phyB (ref. 4) and by DELLA proteins that have a key repressor function in GA signalling. Our results demonstrate that PIF4 is destabilized by phyB in the light and that DELLAs block PIF4 transcriptional activity by binding the DNA-recognition domain of this factor. We show that GAs abrogate such repression by promoting DELLA destabilization, and therefore cause a concomitant accumulation of free PIF4 in the nucleus. Consistent with this model, intermediate hypocotyl lengths were observed in transgenic plants over-accumulating both DELLAs and PIF4. Destabilization of this factor by phyB, together with its inactivation by DELLAs, constitutes a protein interaction framework that explains how plants integrate both light and GA signals to optimize growth and development in response to changing environments.

The degradation of HFR1, a putative bHLH class transcription factor involved in light signaling, is regulated by phosphorylation and requires COP1.

All developmental transitions throughout the life cycle of a plant are influenced by light. In Arabidopsis, multiple photoreceptors including the UV-A/blue-sensing cryptochromes (cry1-2) and the red/far-red responsive phytochromes (phyA-E) monitor the ambient light conditions. Light-regulated protein stability is a major control point of photomorphogenesis. The ubiquitin E3 ligase COP1 (constitutively photomorphogenic 1) regulates the stability of several light-signaling components. HFR1 (long hypocotyl in far-red light) is a putative transcription factor with a bHLH domain acting downstream of both phyA and the cryptochromes. HFR1 is closely related to PIF1, PIF3, and PIF4 (phytochrome interacting factor 1, 3 and 4), but in contrast to the latter three, there is no evidence for a direct interaction between HFR1 and the phytochromes. Here, we show that the protein abundance of HFR1 is tightly controlled by light. HFR1 is an unstable phosphoprotein, particularly in the dark. The proteasome and COP1 are required in vivo to degrade phosphorylated HFR1. In addition, HFR1 can interact with COP1, consistent with the idea of COP1 directly mediating HFR1 degradation. We identify a domain, conserved among several bHLH class proteins involved in light signaling , as a determinant of HFR1 stability. Our physiological experiments indicate that the control of HFR1 protein abundance is important for a normal de-etiolation response.

E2F activation of S phase promoters via association with HCF-1 and the MLL family of histone H3K4 methyltransferases.

E2F transcriptional regulators control human-cell proliferation by repressing and activating the transcription of genes required for cell-cycle progression, particularly the S phase. E2F proteins repress transcription in association with retinoblastoma pocket proteins, but less is known about how they activate transcription. Here, we show that the human G1 phase regulator HCF-1 associates with both activator (E2F1 and E2F3a) and repressor (E2F4) E2F proteins, properties that are conserved in insect cells. Human HCF-1-E2F interactions are versatile: their associations and binding to E2F-responsive promoters are cell-cycle selective, and HCF-1 displays coactivator properties when bound to the E2F1 activator and corepressor properties when bound to the E2F4 repressor. During the G1-to-S phase transition, HCF-1 recruits the mixed-lineage leukemia (MLL) and Set-1 histone H3 lysine 4 methyltransferases to E2F-responsive promoters and induces histone methylation and transcriptional activation. These results suggest that HCF-1 induces cell-cycle-specific transcriptional activation by E2F proteins to promote cell proliferation.

zyg-11and cul-2 promote the metaphase to anaphase transition and M phase exit of meiosis II and prevent polarity establishment in C.elegans

Résumé Les mécanismes qui coordonnent la progression du cycle cellulaire lors de la méiose avec les événements du développement embryonnaire précoce, y compris la formation des axes de polarité embryonnaire, sont peu compris. Dans le zygote du vers Caenorhabditis elegans, les premiers signes de polarité Antéro-Postérieur (A-P) embryonnaire apparaissent après que la méiose soit terminée. La nature des protéines et des mécanismes moléculaires qui cassent la symétrie du zygote n'est pas connue. Nous démontrons que zyg-11 et cul-2 promeuvent la transition métaphase - anaphase et la sortie de la phase M lors de la seconde division méiotique. Nos résultats indiquent que ZYG-11 agit comme unité recrutant le substrat d'une ligase E3 comprennant CUL-2. Nos résultats montrent aussi que le délai de sortie de la phase M dépend de l'accumulation de la Cyclin B, CYB-3. Nous démontrons que dans des embryons zyg-11(RNAi) ou cul-2(RNAi), une polarité inversée est établie lors du délai de méiosis II. Enfin nous montrons que les défauts de cycle cellulaire et ceux de polarité peuvent être séparés. De plus, nous faisons apparaitre que l'établissement d'une polarité inversée pendant le délai de méiose II des embryons zyg-11(RNAi), comme l'établissement de la A-P polarité des embryons sauvage ne semblent pas requérir les microtubules. Nous montrons également les premiers résultats d'un crible deux hybrides ainsi qu'un crible génomique qui vise à identifier des gènes dont l'inactivation augmente ou supprime les défauts de mutants pour le gène zyg-11, afin d'identifier les gènes qui intéragissent avec ZYG-11 pour assumer ses deux fonctions séparables. Par conséquent, nos trouvailles suggèrent un modèle selon lequel ZYG-11 est une sous-unité qui recrute les substrats d'une ligase E3 basée sur CUL-2 qui promeut la progression du cycle cellulaire et empêche l'établissement de la polarité pendant la méiose II, et où le centrosome agit comme la clé qui polarise l'embryon à la fin de la méiose. Summary The mechanisms that couple meiotic cell cycle progression to subsequent developmental events, including specification of embryonic axes, are poorly understood. In the one cell stage embryos of Caenorhabditis elegans, the first signs of Antero-Posterior (A-P) polarity appear after meiosis completion. A centrosome ¬derived component breaks symmetry of the embryo, but the molecular nature of this polarity signal is not known. We established that zyg-11 and cul-2 promote the metaphase to anaphase transition and M phase exit at meiosis II. Our results indicate that ZYG-11 acts as a substrate recruitment subunit of a CUL-2-based E3 ligase. Moreover, we find that the delayed meiosis II exit of embryos lacking zyg-11 is caused by accumulation of the B-type cyclin, CYB-3. We demonstrate that inverted A-P polarity is established during the meiosis II delay in zyg-11(RNAi) and cul¬2(RNAi) embryos. Importantly, we demonstrate that the polarity defects following zyg-11 or cul-2 inactivation can be uncoupled from the cell cycle defects. Furthermore, we found that microtubules appear dispensable for inverted polarity during the meiosis II delay in zyg-11(RNAi) embryos, as well as for A-P polarity during the first mitotic cell cycle in wild-type embryos. We also show the initial results from a comprehensive yeast two hybrid, as well as an RNAi-based functional genomic enhancer and suppressor screen, that may lead to identification of proteins that interact with zyg-11 to ensure the two functions. Our findings suggest a model in which ZYG-11 is a substrate recruitment subunit of an CUL-2-based E3 ligase that promotes cell cycle progression and prevents polarity establishment during meiosis II, and in which the centrosome acts as a cue to polarize the embryo along the AP axis after exit from the meiotic cell cycle.

" Leishmania major " metacaspase in programmed cell death

Abstract: The human protozoan parasite Leishmania major has been shown to exhibit several morphological and biochemical features characteristic of a programmed cell death (PCD) when differentiating into infectious stages and under a variety of stress conditions. In mammalian cells, the principal effector molecules of PCD or apoptosis are caspases. Although some caspase-like peptidase activity has been reported in dying parasites, no caspase gene is present in the L. major genome. However, a single metacaspase gene is present in L. major whose encoded protein harbors the predicted secondary structure and the catalytic dyad histidine/cysteine described for caspases and other metacaspases identified in plants and yeast. Metacaspases are also present in other protozoan parasites such as Trypanosoma and Plasmodium species and are not present in mammalian cells, which make them a possible drug target for the treatment of the parasitic diseases they cause. The Saccharomyces cerevisiae metacaspase YCA1 has been implicated in the death of aging cells, cells defective in some biological functions, and cells exposed to different environmental stresses. In this study, we evaluated the functional heterologous complementation of a S. cerevisiae ycal null mutant with the L. major metacaspase (LmjMCA} in cell death induced by oxidative stress. We show that LmjMCA is involved in yeast cell death, similar to YCA1, and that this function depends on its catalytic activity. LmjMCA was found to be auto-processed as occurs for caspases, however, LmjMCA did not exhibit any activity with caspase substrates. In contrast, LmjMCA was active towards substrates with arginine in the P1 position, with the activity being abolished following H147A and C202A catalytic site mutations and addition of the arginal inhibitor leupeptin. In order to identify the L. major proteins that may function as substrates, inhibitors, or may bind and recruit LmjMCA, a yeast two-hybrid screening with cDNA libraries from different life cycle stages of the parasite was conducted. Proteins putatively involved in PCD were identified as interacting with LmjMCA, however, the interaction of LmjMCA with proteins involved in other physiological processes such as vesicle transport, suggests that LmjMCA could have additional roles in the different life cycle stages of the parasite. Résumé: Plusieurs caractéristiques morphologiques et biochimiques rappelant la mort cellulaire programmée ont été identifiées dans les stades infectieux et sous des conditions de stress, chez le parasite protozoaire humain, Leishmania major. Dans les cellules de mammifères, les caspases sont les molécules effectrices principales impliquées dans la mort cellulaire programmée et l'apoptose. Bien qu'une activité caspase ait été retrouvée dans des parasites en mon` cellulaire, le génome de Leishmania ne contient aucun gène qui pourrait coder pour une caspase. À la place, on retrouve un gène unique codant pour une métacaspase. Une prédiction de la structure secondaire de la métacaspase montre que cette métacaspase a un domaine catalytique contenant la dyade histidine/cystéine présente dans les caspases et les autres métacaspases décrites chez les plantes et la levure. Les métacaspases sont aussi présentes dans d'autres parasites protozoaires tels que Trypanosome et Plasmodium, mais ne sont pas présentes dans les cellules de mammifères, ce qui en fait des cibles intéressantes pour le développement de drogue. Dans la levure, Saccharomyces cerevisiae, la métacaspase YCA1 est impliquée dans la mort des cellules âgées, la mort des cellules défectueuses dans certaines fonctions biologiques et dans les cellules exposées à différents stress environnementaux. Dans cette étude, une complémentation hétérologue fonctionnelle d'un mutant de la levure déficient en YCA1 par le gène LmjMCA de L. major lors de l'induction de ta mort par stress oxydatif a été évaluée. Nos résultats montrent que LmjMCA peut remplacer YGA1 dans le programme de mort cellulaire chez la levure et que celte fonction dépend de son activité catalytique. De plus, LmjMCA subit une auto clivage comme les caspases mais n'exhibe aucune spécificité pour les substrats des caspases. Au contraire, LmjMCA est active envers des substrats ayant une arginine en position P1, son activité étant détruite suite à des changements de son domaine catalytique par les mutations H147A et C202A ou suite à une inhibition para la leupeptine. Afin d'identifier quels pourraient être les substrats, les inhibiteurs ou les molécules interagissant avec LmjMCA, nous avons entrepris un criblage double-hybride en utilisant des librairies de d'ADNc provenant de différents stades du cycle parasitaire. Plusieurs protéines potentiellement impliquées dans un programme de mort cellulaire ont été identifiées comme interagissant avec LmjMCA. Cependant, l'identification de protéines impliquées dans le transport vésiculaire suggère aussi que LmjMCA pourrait avoir un rôle additionnel dans les différents stades du cycle parasitaire. Résumé destiné à un large public: De nos jours, la leishmaniose est la deuxième plus importante maladie parasitaire après la malaria. Malgré les avancées accomplies dans les stratégies de contrôle, près de deux millions de nouveaux cas apparaissent chaque année. Actuellement, la principale stratégie pour faire face à ce problème épidémiologique consiste en un traitement pharmacologique des personnes infectées. Pourtant, seule une dizaine de médicaments, dont la plupart sont toxiques, est disponible pour traiter la leishmaniose et des cas de résistance émergent dans certains pays endémiques. Il devient donc urgent de mettre au point de nouveaux traitements anti-leishmaniens capables d'éliminer le parasite sans effets indésirables sur le patient. Récemment, des caractéristiques morphologiques et biochimiques de la mort cellulaire programmée (MCP) semblables au processus de l'apoptose chez les mammifères ont été décrites dans Leishmania. Cependant, des gènes codant pour des protéines similaires à celles qui sont impliquées dans l'apoptose, comme les caspases, ne se retrouvent pas dans le génome de Leishmanía major. Néanmoins, les espèces de Leishmanía, aussi bien que d'autres parasites protozoaires responsables des trypanosomiases et de la malaria, possèdent des métacaspases qui sont des protéines homologues aux caspases mais qui ne sont pas présentes chez les mammifères. C'est pourquoi, la caractérisation de la métacaspase de Leishmania (LmjMCA) ainsi que ses mécanismes d'activation pourrait être une piste d'investigation intéressante dans l'identification de nouvelles cibles thérapeutiques dans les voies de signalisation de la MCP des parasites protozoaires. Dans la levure, Saccharomyces cerevisiae, la métacaspase YCA1 est impliquée dans la mort des cellules âgées, la mort des cellules défectueuses dans certaines fonctions biologiques et dans les cellules exposées à différents stress environnementaux. Dans cette étude, une complémentation hétérologue fonctionnelle d'un mutant de la levure déficient en YCA1 par le gène LmjMCA de L major lors de l'induction de la mort par stress oxydatif a été évaluée. Nos résultats montrent que LmjMCA peut remplacer YCA1 dans le programme de mort cellulaire chez la levure et que cette fonction dépend de son activité catalytique. De plus, LmjMCA subit une auto clivage comme les caspases mais n'exhibe aucune spécificité pour les substrats des caspases. Au contraire, LmjMCA est active envers des substrats ayant une arginine en position P1, son activité étant détruite suite à des changements de son domaine catalytique par les mutations H147A et C202A ou suite à une inhibition para la leupeptine. Afin d'identifier quels pourraient être les substrats, les inhibiteurs ou les molécules interagissant avec LmjMCA, nous avons entrepris un criblage double-hybride en utilisant des librairies de d'ADNe provenant de différents stades du cycle parasitaire. Plusieurs protéines potentiellement impliquées dans un programme de mort cellulaire ont été identifiées comme interagissant avec LmjMCA. Cependant, l'identification de protéines impliquées dans le transport vésiculaire suggère aussi que LmjMCA pourrait avoir un rôle additionnel dans les différents stades du cycle parasitaire. Resumen destinado al público en general: La leishmaniasis es la segunda enfermedad parasitaria más importante en el mundo actual. Aproximadamente 2 millones de nuevos casos ocurren cada año a pesar de los avances logrados en el desarrollo de nuevos métodos de control. El tratamiento farmacológico de las personas infectadas es actualmente la principal estrategia de control, sin embargo, menos de una decena de medicamentos se encuentran disponibles en el mercado, la mayoría de ellos son tóxicos, y ya empiezan a encontrarse parásitos resistentes en algunos países endémicos para la leishmaniasis. El desarrollo de nuevos medicamentos capaces de eliminar los parásitos sin producir efectos indeseables en los humanos, es una necesidad inminente. Recientemente, algunas de las características morfológicas y bioquímicas de la muerte celular programada (MCP) similares al proceso de la apoptosis en mamíferos, han sido descritas en parasitos de Leishmania. Sin embargo, genes que codifiquen proteínas similares a aquellas involucradas en la apoptosis, como las caspasas, no se encuentran en el genoma de Leishmania major. AI contrario, las especies de Leishmania, así como de los otros parásitos responsables de la tripanosomiasis y de la malaria, poseen metacaspases, proteínas homologas a las caspases pero que no están presentes en las células de mamíferos. La caracterización de la metacaspasa de L. major y de sus mecanismos de activación constituye, por lo tanto, un área de investigación interesante para la identificación de nuevos blancos terapéuticos en el proceso de MCP de los parásitos protozoarios. En la levadura Saccharomyces cerevisiae, la metacaspasa YCA1 ha sido descrita como implicada en la muerte de células envejecidas, células defectuosas en algunas funciones biológicas, y en células expuestas a diferentes tipos de estrés ambiental. En el presente estudio se evaluó la complementación heteróloga funcional de una levadura mutante deficiente en YCA1 con el gen de metacaspase de L. major (LmjMCA) en la MCP inducida por estrés oxidativo. Nuestros resultados muestran que la LmjMCA puede reemplazarla YCA1 en la MCP de la levadura dependiente de su actividad catalítica y que la LmjMCA se auto-procesa similar a las caspasas. Sin embargo, LmjMCA no reconoce los substratos de caspasas sino substratos con una arginina en ta posición P1. Dicha actividad enzimática fue abolida con la inducción de las mutaciones puntuales H147A y C202A en la díada catalítica de LmjMCA y con la adición de leupeptina, un inhibidor con arginina. Con el fin de identificar proteínas que pudieran funcionar como substratos, inhibidores o moléculas modificadoras de LmjMCA, se aplicó el método de doble-híbrido en levadura con bibliotecas de ADNc provenientes de diferentes estadios del ciclo de vida del parásito. Algunas proteínas potencialmente implicadas. en la MCP del parásito fueron identiñcadas interactuando con LmjMCA. La identificación de otras proteínas involucradas en transporte vesicular sugiere que la LmjMCA podría tener una función biológica adicional en los diferentes estadios del ciclo de vida dei parásito.

Interaction of Fas(Apo-1/CD95) with proteins implicated in the ubiquitination pathway.

Fas(Apo-1/CD95), a receptor belonging to the tumor necrosis factor receptor family, induces apoptosis when triggered by Fas ligand. Upon its activation, the cytoplasmic domain of Fas binds several proteins which transmit the death signal. We used the yeast two-hybrid screen to isolate Fas-associated proteins. Here we report that the ubiquitin-conjugating enzyme UBC9 binds to Fas at the interface between the death domain and the membrane-proximal region of Fas. This interaction is also seen in vivo. UBC9 transiently expressed in HeLa cells bound to the co-expressed cytoplasmic segment of Fas. FAF1, a Fas-associated protein that potentiates apoptosis (Chu et al. (1996) Proc. Natl. Acad. Sci. USA 92, 11894-11898), was found to contain sequences similar to ubiquitin. These results suggest that proteins related to the ubiquitination pathway may modulate the Fas signaling pathway.

The serine-rich N-terminal region of Arabidopsis phytochrome A is required for protein stability.

Deletion or substitution of the serine-rich N-terminal stretch of grass phytochrome A (phyA) has repeatedly been shown to yield a hyperactive photoreceptor when expressed under the control of a constitutive promoter in transgenic tobacco or Arabidopsis seedlings retaining their native phyA. These observations have lead to the proposal that the serine-rich region is involved in negative regulation of phyA signaling. To re-evaluate this conclusion in a more physiological context we produced transgenic Arabidopsis seedlings of the phyA-null background expressing Arabidopsis PHYA deleted in the sequence corresponding to amino acids 6-12, under the control of the native PHYA promoter. Compared to the transgenic seedlings expressing wild-type phyA, the seedlings bearing the mutated phyA showed normal responses to pulses of far-red (FR) light and impaired responses to continuous FR light. In yeast two-hybrid experiments, deleted phyA interacted normally with FHY1 and FHL, which are required for phyA accumulation in the nucleus. Immunoblot analysis showed reduced stability of deleted phyA under continuous red or FR light. The reduced physiological activity can therefore be accounted for by the enhanced destruction of the mutated phyA. These findings do not support the involvement of the serine-rich region in negative regulation but they are consistent with a recent report suggesting that phyA turnover is regulated by phosphorylation.

Vasopressin-inducible ubiquitin-specific protease 10 increases ENaC cell surface expression by deubiquitylating and stabilizing sorting nexin 3

Adjustment of Na+ balance in extracellular fluids is achieved by regulated Na+ transport involving the amiloride-sensitive epithelial Na+ channel (ENaC) in the distal nephron. In this context, ENaC is controlled by a number of hormones, including vasopressin, which promotes rapid translocation of water and Na+ channels to the plasma membrane and long-term effects on transcription of vasopressin-induced and -reduced transcripts. We have identified a mRNA encoding the deubiquitylating enzyme ubiquitin-specific protease 10 (Usp10), whose expression is increased by vasopressin at both the mRNA and the protein level. Coexpression of Usp10 in ENaC-transfected HEK-293 cells causes a more than fivefold increase in amiloride-sensitive Na+ currents, as measured by whole cell patch clamping. This is accompanied by a three- to fourfold increase in surface expression of alpha- and gamma-ENaC, as shown by cell surface biotinylation experiments. Although ENaC is well known to be regulated by its direct ubiquitylation, Usp10 does not affect the ubiquitylation level of ENaC, suggesting an indirect effect. A two-hybrid screen identified sorting nexin 3 (SNX3) as a novel substrate of Usp10. We show that it is a ubiquitylated protein that is degraded by the proteasome; interaction with Usp10 leads to its deubiquitylation and stabilization. When coexpressed with ENaC, SNX3 increases the channel's cell surface expression, similarly to Usp10. In mCCD(cl1) cells, vasopressin increases SNX3 protein but not mRNA, supporting the idea that the vasopressin-induced Usp10 deubiquitylates and stabilizes endogenous SNX3 and consequently promotes cell surface expression of ENaC

FAM161A, associated with retinitis pigmentosa, is a component of the cilia-basal body complex and interacts with proteins involved in ciliopathies.

Retinitis pigmentosa (RP) is a retinal degenerative disease characterized by the progressive loss of photoreceptors. We have previously demonstrated that RP can be caused by recessive mutations in the human FAM161A gene, encoding a protein with unknown function that contains a conserved region shared only with a distant paralog, FAM161B. In this study, we show that FAM161A localizes at the base of the photoreceptor connecting cilium in human, mouse and rat. Furthermore, it is also present at the ciliary basal body in ciliated mammalian cells, both in native conditions and upon the expression of recombinant tagged proteins. Yeast two-hybrid analysis of binary interactions between FAM161A and an array of ciliary and ciliopathy-associated proteins reveals direct interaction with lebercilin, CEP290, OFD1 and SDCCAG8, all involved in hereditary retinal degeneration. These interactions are mediated by the C-terminal moiety of FAM161A, as demonstrated by pull-down experiments in cultured cell lines and in bovine retinal extracts. As other ciliary proteins, FAM161A can also interact with the microtubules and organize itself into microtubule-dependent intracellular networks. Moreover, small interfering RNA-mediated depletion of FAM161A transcripts in cultured cells causes the reduction in assembled primary cilia. Taken together, these data indicate that FAM161A-associated RP can be considered as a novel retinal ciliopathy and that its molecular pathogenesis may be related to other ciliopathies.

FAM161A, associated with autosomal recessive retinitis pigmentosa,localizes at the level of the photoreceptor cilium and interacts with proteins involved in ciliopathies

Purpose: We have previously demonstrated that mutations in the FAM161A gene, encoding a protein with unknown function and no similarities with other characterized sequences, cause autosomal recessive retinitis pigmentosa (RP). The purpose of this work is to investigate the functional role of FAM161A within the retina and its relationship with other proteins involved in RP. Methods: The subcellular localization of FAM161A in the retina was assessed by immunohistochemistry of retinal sections and dissociated photoreceptors from mice, which were stained using antibodies against FAM161A and antibodies against cilium markers. The function of FAM161A was further assessed in ciliated mammalian cell lines by expression of recombinant FAM161A with various fusion tags. The binary interaction between FAM161A and a collection of ciliary and ciliopathy-associated proteins was analyzed using a yeast two-hybrid assay. The results obtained with this technique were validated using independent protein-protein interaction assays (GST-pull downs, co-transfection and co-immunoprecipitation). Results: Native FAM161A localized at the connecting cilium of photoreceptor cells, as demonstrated by immunofluorescence in both dissociated photoreceptors and retinal sections of mice. More specifically, co-staining with markers for ciliary sub-structures (RPGRIP1L, Centrin, RP1, GT335) demonstrated that FAM161A decorated the basal body and the very apical part of the connecting cilium. Upon overexpression in ciliated cultured mammalian cells, FAM161A localized to the ciliary basal body. Yeast two-hybrid analysis of the binary interaction of FAM161A and an array of ciliary proteins revealed the direct interaction of FAM161A with three proteins of which the cognate genes are mutated in retinal ciliopathies. The confirmation of these interactions using different biochemical assays is currently in progress. Conclusions: FAM161A is a ciliary basal body protein of the photoreceptor connecting cilium, rendering the associated RP as a novel retinal ciliopathy. The confined expression of FAM161A in the retina and the direct interaction of FAM161A with other retinal ciliopathy-associated proteins may explain the retinal phenotype of this specific subset of mechanistically and phenotypically connected retinal disorders.

Interactome mapping of the phosphatidylinositol 3-kinase-mammalian target of rapamycin pathway identifies deformed epidermal autoregulatory factor-1 as a new glycogen synthase kinase-3 interactor.

The phosphatidylinositol 3-kinase-mammalian target of rapamycin (PI3K-mTOR) pathway plays pivotal roles in cell survival, growth, and proliferation downstream of growth factors. Its perturbations are associated with cancer progression, type 2 diabetes, and neurological disorders. To better understand the mechanisms of action and regulation of this pathway, we initiated a large scale yeast two-hybrid screen for 33 components of the PI3K-mTOR pathway. Identification of 67 new interactions was followed by validation by co-affinity purification and exhaustive literature curation of existing information. We provide a nearly complete, functionally annotated interactome of 802 interactions for the PI3K-mTOR pathway. Our screen revealed a predominant place for glycogen synthase kinase-3 (GSK3) A and B and the AMP-activated protein kinase. In particular, we identified the deformed epidermal autoregulatory factor-1 (DEAF1) transcription factor as an interactor and in vitro substrate of GSK3A and GSK3B. Moreover, GSK3 inhibitors increased DEAF1 transcriptional activity on the 5-HT1A serotonin receptor promoter. We propose that DEAF1 may represent a therapeutic target of lithium and other GSK3 inhibitors used in bipolar disease and depression.

The ubiquitin-like protein LC3 regulates the Rho-GEF activity of AKAP-Lbc.

AKAP-Lbc is a member of the A-kinase anchoring protein (AKAP) family that has been recently associated with the development of pathologies, such as cardiac hypertrophy and cancer. We have previously demonstrated that, at the molecular level, AKAP-Lbc functions as a guanine nucleotide exchange factor (GEF) that promotes the specific activation of RhoA. In the present study, we identified the ubiquitin-like protein LC3 as a novel regulatory protein interacting with AKAP-Lbc. Mutagenesis studies revealed that LC3, through its NH(2)-terminal alpha-helical domain, interacts with two binding sites located within the NH(2)-terminal regulatory region of AKAP-Lbc. Interestingly, LC3 overexpression strongly reduced the ability of AKAP-Lbc to interact with RhoA, profoundly impairing the Rho-GEF activity of the anchoring protein and, as a consequence, its ability to promote cytoskeletal rearrangements associated with the formation of actin stress fibers. Moreover, AKAP-Lbc mutants that fail to interact with LC3 show a higher basal Rho-GEF activity as compared with the wild type protein and become refractory to the inhibitory effect of LC3. This suggests that LC3 binding maintains AKAP-Lbc in an inactive state that displays a reduced ability to promote downstream signaling. Collectively, these findings provide evidence for a previously uncharacterized role of LC3 in the regulation of Rho signaling and in the reorganization of the actin cytoskeleton.

The cAMP response element binding protein-2 (CREB-2) can interact with the C/EBP-homologous protein (CHOP).

cAMP response element binding protein-2 (CREB-2) is a basic leucine zipper (bZIP) factor that was originally described as a repressor of CRE-dependent transcription but that can also act as a transcriptional activator. Moreover, CREB-2 is able to function in association with the viral Tax protein as an activator of the human T-cell leukemia virus type I (HTLV-I) promoter. Here we show that CREB-2 is able to interact with C/EBP-homologous protein (CHOP), a bZIP transcription factor known to inhibit CAAT/enhancer-dependent transcription. Cotransfection of CHOP with CREB-2 results in decreased activation driven by the cellular CRE motif or the HTLV-I proximal Tax-responsive element, confirming that CREB-2 and CHOP can interact with each other in vivo.