Microbiology & Immunology/Baxter Laboratory

Background: Earlier we suggested the concept of the positive evolutionary role of tumors. According to this concept, tumors provide conditions for the expression of evolutionarily new and/ or sleeping genes in their cells. Thus, tumors are considered as evolutionary proving ground or reservoir of expression. To support this concept we have previously characterized in silico and experimentally a new class of human tumor-related transcribed sequences.

The functional characterization of the molecular components of biological systems is an outstanding task in the post-genomic era and is often addressed by gene knockdown screening which quantifies phenotypes in the form of multiparametric profiles. The identification of sets of genes producing similar phenotypic effects is fulfilled by the clustering of profiles. Here we address the question of how to quantify the biological significance of clusters of phenotypic profiles with respect to proteinprotein interaction (PPI) data.

Endocytosis is a complex process fulfilling many cellular and developmental functions. Understanding how it is regulated and integrated with other cellular processes requires a comprehensive analysis of its molecular constituents and general design principles. Here, we developed a new strategy to phenotypically profile the human genome with respect to transferrin (TF) and epidermal growth factor (EGF) endocytosis by combining RNA interference, automated high-resolution confocal microscopy, quantitative multiparametric image analysis and high-performance computing. We identified several novel components of endocytic trafficking, including genes implicated in human diseases. We found that signalling pathways such as Wnt, integrin/cell adhesion, transforming growth factor (TGF)-b and Notch regulate the endocytic system, and identified new genes involved in cargo sorting to a subset of signalling endosomes. A systems analysis by Bayesian networks further showed that the number, size, concentration of cargo and intracellular position of endosomes are not determined randomly but are subject to specific regulation, thus uncovering novel properties of the endocytic system.

ing coding sequences of proteins and other gene products, changes in gene expression (due to regulatory mutations) underlie many or even most phenotypic differences between species. Until recently, gene expression comparisons between species were largely restricted to closely related species such as humans and chimpanzees, owing to technological limitations (species-specific microarrays). However, recent technological developments (highthroughput sequencing of RNAs) now allow the comparison of transcriptomes between divergent mammals.

Leishmania are obligatory intracellular parasitic protozoa that cause a wide range of diseases in humans, cycling between extracellular promastigotes in the mid-gut of sand flies and intracellular amastigotes in the phagolysosomes of mammalian macrophages. Although many of the molecular mechanisms of development inside macrophages remain a mystery, the development of a host-free system that simulates phagolysosome conditions (37°C and pH 5.5) has provided new insights into these processes. The time course of promastigote-to-amastigote differentiation can be divided into four morphologically distinct phases: I, signal perception (0 -5 h after exposure); II, movement cessation and aggregation (5-10 h); III, amastigote morphogenesis (10 -24 h); and IV, maturation (24 -120 h). Transcriptomic and proteomic analyses have indicated that differentiation is a coordinated process that results in adaptation to life inside phagolysosomes. Recent phosphoproteomic analysis revealed extensive differences in phosphorylation between promastigotes and amastigotes and identified stage-specific phosphorylation motifs. We hypothesized that the differentiation signal activates a phosphorylation pathway that initiates Leishmania transformation, and here we used isobaric tags for relative and absolute quantitation to interrogate the dynamics of changes in the phosphorylation profile during Leishmania donovani promastigote-to-amastigote differentiation. Analysis of 163 phosphopeptides (from 106 proteins) revealed six distinct kinetic profiles; with increases in phosphorylation predominated during phases I and III, whereas phases II and IV were characterized by greater dephosphorylation. Several proteins (including a protein kinase) were phosphorylated in phase I after exposure to the complete differentiation signal (i.e. signalspecific; 37°C and pH 5.5), but not after either of the physical parameters separately. Several other protein kinases (including regulatory subunits) and phosphatases also showed changes in phosphorylation during differentiation. This work constitutes the first genome-scale interrogation of phosphorylation dynamics in a parasitic protozoa, revealing the outline of a signaling pathway during Leishmania differentiation.