TBC1D3, a Hominoid-Specific Gene, Delays IRS-1 Degradation and Promotes Insulin Signaling by Modulating p70 S6 Kinase Activity

Insulin/IGF-1 signaling plays a pivotal role in the regulation of cellular homeostasis through its control of glucose metabolism as well as due to its effects on cell proliferation. Aberrant regulation of insulin signaling has been repeatedly implicated in uncontrolled cell growth and malignant transformations. TBC1D3 is a hominoid specific gene previously identified as an oncogene in breast and prostate cancers. Our efforts to identify the molecular mechanisms of TBC1D3-induced oncogenesis revealed the role of TBC1D3 in insulin/IGF-1 signaling pathway. We document here that TBC1D3 intensifies insulin/IGF-1-induced signal transduction through intricate, yet elegant fine-tuning of signaling mechanisms. We show that TBC1D3 expression substantially delayed ubiquitination and degradation of insulin receptor substrate-1 (IRS-1). This effect is achieved through suppression of serine phosphorylation at S636/639, S307 and S312 of IRS-1, which are key phosphorylation sites required for IRS-1 degradation. Furthermore, we report that the effect of TBC1D3 on IRS-1:S636/639 phosphorylation is mediated through TBC1D3-induced activation of protein phosphatase 2A (PP2A), followed by suppression of T389 phosphorylation on p70 S6 kinase (S6K). TBC1D3 specifically interacts with PP2A regulatory subunit B56γ, indicating that TBC1D3 and PP2A B56γ operate jointly to promote S6K:T389 dephosphorylation. These findings suggest that TBC1D3 plays an unanticipated and potentially unique role in the fine-tuning of insulin/IGF-1 signaling, while providing novel insights into the regulation of tumorigenesis by a hominoid-specific protein

The Ubiquitous Dermokine Delta Activates Rab5 Function in the Early Endocytic Pathway

The expression of the recently identified dermokine (Dmkn) gene leads to four families of proteins with as yet unknown functions. The secreted α, β and γ isoforms share an epidermis-restricted expression pattern, whereas the δ isoform is intracellular and ubiquitous. To get an insight into Dmknδ function, we performed yeast two-hybrid screening and identified the small GTPases Rab5 as partners for Dmknδ. The Rab5 proteins are known to regulate membrane docking and fusion in the early endocytic pathway. GST pull-down assays confirmed the direct interaction between Rab5 and Dmknδ. Transient expression of Dmknδ in HeLa cells led to the formation of punctate structures colocalized with endogenous Rab5 and clathrin, indicating Dmknδ involvement in the early steps of endocytosis. Dmknδ indeed colocalized with transferrin at early stages of endocytosis, but did not modulate its endocytosis or recycling kinetics. We also showed that Dmknδ was able to bind both inactive (GDP-bound) and active (GTP-bound) forms of Rab5 in vitro but preferentially targeted GDP-bound form in HeLa cells. Interestingly, Dmknδ expression rescued the Rab5S34N-mediated inhibition of endosome fusion. Moreover, Dmknδ caused the enlargement of vesicles positive for Rab5 by promoting GTP loading onto the small GTPase. Together our data reveal that Dmknδ activates Rab5 function and thus is involved in the early endosomal trafficking

Interleukin-4 Alters Early Phagosome Phenotype by Modulating Class I PI3K Dependent Lipid Remodeling and Protein Recruitment

Phagocytosis is a complex process that involves membranelipid remodeling and the attraction and retention of key effector proteins. Phagosome phenotype depends on the type of receptor engaged and can be influenced by extracellular signals. Interleukin 4 (IL-4) is a cytokine that induces the alternative activation of macrophages (MΦs) upon prolonged exposure, triggering a different cell phenotype that has an altered phagocytic capacity. In contrast, the direct effects of IL-4 during phagocytosis remain unknown. Here, we investigate the impact of short-term IL-4 exposure (1 hour) during phagocytosis of IgG-opsonized yeast particles by MΦs. By time-lapse confocal microscopy of GFP-tagged lipid-sensing probes, we show that IL-4 increases the negative charge of the phagosomal membrane by prolonging the presence of the negatively charged second messenger PI(3,4,5)P3. Biochemical assays reveal an enhanced PI3K/Akt activity upon phagocytosis in the presence of IL-4. Blocking the specific class I PI3K after the onset of phagocytosis completely abrogates the IL-4-induced changes in lipid remodeling and concomitant membrane charge. Finally, we show that IL-4 direct signaling leads to a significantly prolonged retention profile of the signaling molecules Rac1 and Rab5 to the phagosomal membrane in a PI3K-dependent manner. This protracted early phagosome phenotype suggests an altered maturation, which is supported by the delayed phagosome acidification measured in the presence of IL-4. Our findings reveal that molecular differences in IL-4 levels, in the extracellular microenvironment, influence the coordination of lipid remodeling and protein recruitment, which determine phagosome phenotype and, eventually, fate. Endosomal and phagosomal membranes provide topological constraints to signaling molecules. Therefore, changes in the phagosome phenotype modulated by extracellular factors may represent an additional mechanism that regulates the outcome of phagocytosis and could have significant impact on the net biochemical output of a cell

A Novel Method for Inducing Amastigote-To-Trypomastigote Transformation In Vitro in Trypanosoma cruzi Reveals the Importance of Inositol 1,4,5-Trisphosphate Receptor

Background Trypanosoma cruzi is a parasitic protist that causes Chagas disease, which is prevalent in Latin America. Because of the unavailability of an effective drug or vaccine, and because about 8 million people are infected with the parasite worldwide, the development of novel drugs demands urgent attention. T. cruzi infects a wide variety of mammalian nucleated cells, with a preference for myocardial cells. Non-dividing trypomastigotes in the bloodstream infect host cells where they are transformed into replication-capable amastigotes. The amastigotes revert to trypomastigotes (trypomastigogenesis) before being shed out of the host cells. Although trypomastigote transformation is an essential process for the parasite, the molecular mechanisms underlying this process have not yet been clarified, mainly because of the lack of an assay system to induce trypomastigogenesis in vitro. Methodology/Principal Findings Cultivation of amastigotes in a transformation medium composed of 80% RPMI-1640 and 20% Grace\u27s Insect Medium mediated their transformation into trypomastigotes. Grace\u27s Insect Medium alone also induced trypomastigogenesis. Furthermore, trypomastigogenesis was induced more efficiently in the presence of fetal bovine serum. Trypomastigotes derived from in vitro trypomastigogenesis were able to infect mammalian host cells as efficiently as tissue-culture-derived trypomastigotes (TCT) and expressed a marker protein for TCT. Using this assay system, we demonstrated that T. cruzi inositol 1,4,5-trisphosphate receptor (TcIP3R) - an intracellular Ca2+ channel and a key molecule involved in Ca2+ signaling in the parasite - is important for the transformation process. Conclusion/Significance Our findings provide a new tool to identify the molecular mechanisms of the amastigote-totrypomastigote transformation, leading to a new strategy for drug development against Chagas disease

Active transcription and ultrastructural changes during Trypanosoma cruzi metacyclogenesis

The differentiation of proliferating epimastigote forms of Trypanosoma cruzi , the protozoan parasite that causes Chagas’ disease, into the infective and non-proliferating metacyclic forms can be reproduced in the laboratory by incubating the cells in a chemically-defined medium that mimics the urine of the insect vector. Epimastigotes have a spherical nucleus, a flagellum protruding from the middle of the protozoan cell, and a disk-shaped kinetoplast - an organelle that corresponds to the mitochondrial DNA. Metacyclic trypomastigotes have an elongated shape with the flagellum protruding from the posterior portion of the cell and associated with a spherical kinetoplast. Here we describe the morphological events of this transformation and characterize a novel intermediate stage by three-dimensional reconstruction of electron microscope serial sections. This new intermediate stage is characterized by a kinetoplast compressing an already elongated nucleus, indicating that metacyclogenesis involves active movements of the flagellar structure relative to the cell body. As transcription occurs more intensely in proliferating epimastigotes than in metacyclics, we also examined the presence of RNA polymerase II and measured transcriptional activity during the differentiation process. Both the presence of the enzyme and transcriptional activity remain unchanged during all steps of metacyclogenesis. RNA polymerase II levels and transcriptional activity only decrease after metacyclics are formed. We suggest that transcription is required during the epimastigote-to-metacyclic trypomastigote differentiation process, until the kinetoplast and flagellum reach the posterior position of the parasites in the infective form.<br>A diferenciação de formas epimastigotas (proliferativas) do Trypanosoma cruzi, parasita protozoário causador da doença de Chagas, em formas metacíclicas tripomastigotas (infectivas e não proliferativas), pode ser reproduzida em laboratório incubando-se as células em um meio quimicamente definido que imita a urina do inseto vetor deste parasita. Os epimastigotas têm um núcleo esférico, o flagelo se projeta da metade do corpo do protozoário e o cinetoplasto (organela que possui o DNA mitocondrial) possui formato de disco. Os tripomastigotas metacíclicos têm um núcleo alongado com o flagelo emergindo da extremidade posterior da célula associado ao cinetoplasto esférico. Neste trabalho descrevemos as mudanças morfológicas que ocorrem durante essa transformação e caracterizamos uma nova forma intermediária do parasita usando reconstrução tridimensional de cortes seriados, visualizados por microscopia eletrônica de transmissão. Essa nova forma intermediária é caracterizada pela compressão do cinetoplasto contra o núcleo alongado, indicando que a metaciclogênese envolve movimentos ativos do cinetoplasto associado à estrutura flagelar em relação ao corpo celular. Como tripomastigotas metacíclicos transcrevem menos que as formas epimastigotas proliferativas, verificamos a presença da RNA polimerase II e medimos a atividade transcricional durante o processo de diferenciação. A presença da enzima e a atividade transcricional permanecem inalteradas durante todas as etapas da metaciclogênese, desaparecendo apenas quando as formas metacíclicas são formadas. Sugerimos que a diferenciação requer uma atividade transcricional, necessária para uma intensa remodelação da célula, que acontece até o cinetoplasto e o flagelo atingirem uma posição posterior do corpo do tripomastigota metacíclico