Dissecting protein loops with a statistical scalpel suggests a functional implication of some structural motifs

<p>Abstract</p> <p>Background</p> <p>One of the strategies for protein function annotation is to search particular structural motifs that are known to be shared by proteins with a given function.</p> <p>Results</p> <p>Here, we present a systematic extraction of structural motifs of seven residues from protein loops and we explore their correspondence with functional sites. Our approach is based on the structural alphabet HMM-SA (Hidden Markov Model - Structural Alphabet), which allows simplification of protein structures into uni-dimensional sequences, and advanced pattern statistics adapted to short sequences. Structural motifs of interest are selected by looking for structural motifs significantly over-represented in SCOP superfamilies in protein loops. We discovered two types of structural motifs significantly over-represented in SCOP superfamilies: (i) ubiquitous motifs, shared by several superfamilies and (ii) superfamily-specific motifs, over-represented in few superfamilies. A comparison of ubiquitous words with known small structural motifs shows that they contain well-described motifs as turn, niche or nest motifs. A comparison between superfamily-specific motifs and biological annotations of Swiss-Prot reveals that some of them actually correspond to functional sites involved in the binding sites of small ligands, such as ATP/GTP, NAD(P) and SAH/SAM.</p> <p>Conclusions</p> <p>Our findings show that statistical over-representation in SCOP superfamilies is linked to functional features. The detection of over-represented motifs within structures simplified by HMM-SA is therefore a promising approach for prediction of functional sites and annotation of uncharacterized proteins.</p

SA-Mot: a web server for the identification of motifs of interest extracted from protein loops

The detection of functional motifs is an important step for the determination of protein functions. We present here a new web server SA-Mot (Structural Alphabet Motif) for the extraction and location of structural motifs of interest from protein loops. Contrary to other methods, SA-Mot does not focus only on functional motifs, but it extracts recurrent and conserved structural motifs involved in structural redundancy of loops. SA-Mot uses the structural word notion to extract all structural motifs from uni-dimensional sequences corresponding to loop structures. Then, SA-Mot provides a description of these structural motifs using statistics computed in the loop data set and in SCOP superfamily, sequence and structural parameters. SA-Mot results correspond to an interactive table listing all structural motifs extracted from a target structure and their associated descriptors. Using this information, the users can easily locate loop regions that are important for the protein folding and function. The SA-Mot web server is available at http://sa-mot.mti.univ-paris-diderot.fr

RHO Exchange Factors in the Regulation of Squamous Cell Stemness and Carcinogenesis

[EN] The squamous cell carcinoma (SCC) is a major cause of cancer mortality. The carcinogenesis of this tumor is linked to a series of molecular derangements that frequently features the deregulation of RHO GTPase signaling. However, the events and agents underpinning such deregulation are yet to be defined. RHO exchange factors (GEFs), the proteins that catalyze the activation of RHO GTPases, have been traditionally contemplated as potential protumorigenic players in this context, but their large numbers and the lack of appropriate models has precluded the elucidation of their true functions in cancer settings. To address this issue, in this thesis we have focused on VAV2 to spearhead the characterization of RHO GEFs as key mediators of tumorigenesis and, more importantly, as candidate targets for novel SCC-directed therapeutic approaches. Here we demonstrate that VAV2 becomes upregulated in cutaneous and head-and-neck SCCs, where it engages a transcriptional program involved in the induction of stem cell-like regenerative proliferation and undifferentiation. Significantly, we show that VAV2 activity predicts disease outcome and that its inhibition within specific catalytic thresholds provides antitumoral benefits without disturbing organismal homeostasis. This work also exposes non-oncogenic roles for this GEF in the physiological maintenance of the cutaneous squamous epithelium, where it regulates the abundance, activity and responsiveness of hair follicle stem cells through the control of their transcriptomic circuits. Lastly, by extending these studies to the whole family of RHO GEFs, our research shows that VAV2 belongs to a small collection of exchange factors with pivotal roles in either the promotion or impairment of SCC tumorigenesis-associated processes. Taken together, our findings unveil hitherto unknown regulators of SCC fitness whose activity can be harnessed to modulate tumor growth and malignancy

A yeast model of Bloom's syndrome.

Bloom's syndrome (BS) is a rare inherited disorder associated with growth retardation, immunodeficiency and a predisposition to cancers of all types. At a cellular level BS is associated with marked chromosomal instability, as manifest by interchanges between homologous chromosomes and sister chromatids. BS arises as a consequence of defects in a DNA helicase, BLM, which is a member of the RecQ family of helicases. Defects in other RecQ family helicases, derived from prokaryotic and lower eukaryotic cells, also result in a loss of genomic integrity. This suggests that this group of helicase subfamily share a degree of functional conservation. In this thesis, the validity of using the budding yeast, S.cerevisiae, as a model system for BS has been assessed. In this organism, the structural homologue of the BLM protein is Sgs1p. Deletion of Sgs1p is associated with mitotic hyperrecombination and defective chromosome segregation. Deletion of SGS1 also suppresses the slow growth and hyperrecombination phenotype of top3 mutants, which are defective in topoisomerase III. Ectopic expression of a wild type SGS1 gene in sgs1Δtop3Δ mutants reinduced the slow growth defect, and this effect was reproduced when the human BLM gene was expressed. This result suggested firstly, that the yeast and human proteins share functional similarities and secondly, that the interaction between the RecQ family helicases and topoisomerase III is highly conserved. On this basis, the remainder of this thesis focussed upon the role of topoisomerase III. Deletion of the S.cerevisiae TOP3 gene leads to a slow growth phenotype accompanied by an accumulation of cells with a late S/G2 content of DNA. top3Δ mutants exhibit a RAD24/RAD9-dependent delay in the G2 phase, suggesting a role for topoisomerase III in the resolution of abnormal DNA structures/damage arising during S-phase. Consistent with this notion, top3Δ strains are defective in the intra-S-phase checkpoint that slows the rate of S-phase progression following exposure to DNA damaging agents and are sensitive to killing by a variety of DNA damaging agents, including ultra-violet light, ɣ-rays and the alkylating agent MMS. This S-phase checkpoint defect was not associated with a failure to induce expression of DNA damage response genes. Consistent with an S-phase specific role for topoisomerase III, expression of the TOP3 mRNA is activated in the late G1 phase, and DNA damage checkpoints operating outside of S-phase are unaffected by deletion of TOP3. All of the phenotypic consequences of loss of topoisomerase III function were suppressed by deletion of SGS1. These data implicate topoisomerase III and, by inference, Sgs1p in a checkpoint role in response to DNA damage arising during S-phase. A model to explain the role of these proteins during DNA replication is proposed

Investigating Haspin-dependent phosphorylation of histones during mitosis

La protéine Haspine est une sérine / thréonine kinase mitotique conservée, connue pour fonctionner par la phosphorylation de l'histone H3 (H3pT3). Bien que H3T3 soit le seul substrat bien connu de Haspine, il se peut que H3pT3 ne suffise pas à expliquer toutes les fonctions de Haspine au cours de la mitose. Fait intéressant, l’homologie de la portion N-terminale de H3 avec la portion Cterminale de H2B suggère que la thréonine 119 de H2B (H2BT119) est un candidat potentiel fort pour être un substrat majeur de Haspine. Ainsi, l’objectif de ce projet était d’étudier la phosphorylation de H2BT119 dépendante de Haspine pendant la mitose. La phosphorylation de H2B recombinant sur la position T119 par Haspine a été confirmée par un dosage de kinase en utilisant la radioactivité. En outre, le signal H2BpT119 sur la protéine recombinante H2B a été détecté par un anticorps anti-H2BpT119, confirmant la phosphorylation de H2B dépendante de Haspine sur ce site dans le test de kinase in vitro. En outre, une augmentation de la taille moléculaire de H2B a été observée après le dosage de la kinase. Les résultats des expériences in vivo, incluant l'analyse par Western-blot d'extrait d'histones mitotiques et la microscopie à fluorescence avec l'anti-H2BpT119, suggèrent une phosphorylation de H2B au site T119, qui s'est également avérée dépendant de l'activité de Haspine. Cependant, en raison de la potentielle réactivité croisée de l’anti-H2BpT119 avec H3pT3, une forme marquée de H2B a été utilisée lors de l’étude du signal H2BpT119 au cours de la mitose. Le marquage augmente la taille moléculaire de H2B et aide à reconnaître H2BpT119 loin de H3pT3. Plusieurs systèmes de marquage ont été utilisés, mais toutes les tentatives ont échoué, en raison du faible niveau de H2B exogène. Cependant, les résultats de ce projet suggèrent que le signal H2BpT119 pendant la mitose pourrait révéler un nouveau mécanisme dépendant de Haspine pour la régulation de la ségrégation des chromosomes. Par conséquent, il reste important d'étudier cette marque d'histone au cours de la mitose.Haspin is a conserved mitotic serine/threonine kinase that is known to function through histone H3 phosphorylation (H3pT3). Despite this, H3T3 is the only well-known substrate for Haspin, H3pT3 may not be enough to explain all Haspin functions during mitosis. Interestingly, homology of H3 N-terminus with H2B C-terminus suggests that H2BT119 is a strong potential candidate to be a major substrate of Haspin. Thus, the aim was to investigate Haspin-dependent phosphorylation of H2BT119 during mitosis. Phosphorylation of recombinant H2B at T119 by Haspin was confirmed by a radioactivity-based kinase assay. Also, H2BpT119 signal on recombinant H2B was detected by an anti-H2BpT119 antibody, confirming Haspin-dependent phosphorylation of H2B at this site in the in vitro kinase assay. Also, an upshift of H2B was observed following the kinase assay. Results from in vivo experiments, including Western blot analysis of mitotic histone extract and immunofluorescence microscopy with the anti-H2BpT119, support phosphorylation of T119 in H2B, which also was found to depend on Haspin activity. However, due to the potential anti-H2BpT119 cross-reactivity with H3pT3, while exploring H2BpT119 signal during mitosis, tagged H2B was used. The tag increases H2B molecular size and helps to distinguish H2BpT119 from H3pT3. Several tagging systems was used, but all attempts failed, because of the low level of the exogenous tagged H2B. However, the results of this project suggest H2BpT119 signal during mitosis that may reveal a novel Haspindependent mechanism for chromosome segregation regulation. Therefore, it remains important to study this histone mark during mitosis

Functional and Mechanical Role of Splice Variant of Mucin4 (MUC4/X) and Trefoil Factors in Pancreatic Cancer Pathogenesis

Pancreatic Cancer (PC) is one of the vicious cancers as it ranks third in the race of leading cause of cancer-related death. Lack of early diagnostic marker, poor understanding of molecular mechanism of the disease and failure to conventional chemotherapy makes this disease dreadful. Mucin 4 (MUC4), a high molecular weight glycoprotein is one of the top differentially expressed molecules in PC while not expressed in normal pancreas. Accumulating evidence from our lab suggested its tumorigenic role in PC by increasing cell proliferation, invasion, chemotherapy resistance, tumor growth, and metastasis. Previously, our lab and other has identified 24 different splice variant of MUC4 among them MUC4/X is devoid of exon 2 and 3 and MUC4/Y is devoid of exon 2. Exon 2 encodes for the largest domain of MUC4 suggesting that MUC4/X is devoid of the largest domain of MUC4 which variable tandem repeat. Though lots of effort has been made to identify its role in PC, there is still a gap on understanding its splice variant in PC as splice variant has an invaluable role in tumor pathogenesis. Recently splice variant has emerged as one of the key players for tumorigenesis and MUC4 is one of the key players for PC pathogenesis, we aim to identify the functional and mechanical role of MUC4/X, a splice variant which is devoid of the largest domain of MUC4 yet contains all other functional domain, in PC pathogenesis. Thus, in this part of dissertation, we sought to identify the role of splice variant MUC4/X, a unique splice variant of wild-type MUC4 which contain all functional domain except largest tandem repeat. First, we identified that, MUC4/X in aberrantly expressed in poorly differentiated PC clinical sample. Then our invitro experimental evidence suggested overexpression of MUC4/X in PC cells is involved in increased cell proliferation, invasion and metastasis. Moreover, our orthotopic transplantation system also corroborated our in-vitro findings which showed increased volume of tumor and metastasis to distant organ. Using inducible tet-on system to overexpress MUC4/X in the presence of WT-MUC4 in CAPAN-1 cells, we identified that MUC4/X has increased cell proliferation and invasion suggesting their role as tumorigenic alone as well as in the presence of WT-MUC4. Our mechanical investigation indicate that overexpression of MUC4/X led to upregulation of integrin β1-FAK-ERK pathway which might be potential mechanism for MUC4/X mediated PC tumorigenesis. Lack of early effective diagnostic marker and resistance to chemotherapy are the major reasons for poor PC patient outcome. There is a pressing need to identify highly specific and sensitive biomarker as well as precise understanding of chemoresistance of PC. Trefoil factors (TFFs) are small secretory molecules mostly associated with mucin. Their primary role is to protect gastrointestinal tract partnering with mucin. Report on aberrant expression, potential as biomarker and role in tumorigenicity has conveyed for many cancers, however, their role in PC is still elusive. Recently they have emerged as a part of gene signature of classical subtype of PC, a subtype which showed gemcitabine resistance towards PC. As it is high time to identify effective biomarker and understanding the role of chemoresistance in PC, in this part of my thesis, we focused to evaluate TFFs diagnostic potential using a training and validation cohort of PC clinical sample. Here, we comprehensively investigated the diagnostic potential of all the member of trefoil family, i.e., TFF1, TFF2, and TFF3 (TFFs) in combination with CA19.9 for detection of PC. In silico analysis of publicly available datasets and expression analysis from human and spontaneous PC mouse model revealed a significantly increased expression of TFFs in precursor lesions and PC cases. Additionally, we performed a comprehensive analysis in the sample set (n= 377) comprising of independent training and validation set using ELISA consisted of benign controls (BC), chronic pancreatitis (CP), and various stages of PC. Our analysis revealed that TFF1 and TFF2 were significantly elevated in early stages of PC in comparison to BC (P Additionally, we also aim to identify the molecular landscape of TFFs role in gemcitabine resistance of PC which integrates analyzing publicly available cancer genome dataset, dissecting transcriptomic and signaling pathways and identification of biochemical interaction. From TCGA database analysis revealed a significant positive correlation between TFF1 and GR predictor of PC (P=0.0001). Our in vitro studies showed that SW1990-TFF1-KD cells induced apoptosis, reduced colony formation capacity and modulated many apoptotic regulators such increase of cleaved caspases and decrease of CIAP in the presence of gemcitabine. Furthermore, TFF1 was observed to be colocalized with MUC5AC, in human and mouse PC tissues suggesting their partnering are critical for PC pathogenesis. Interestingly, our chromatin immunoprecipitation indicates that 16 fold enrichment of GATA-6, an overexpressed transcription factor in classical subtype of PC, was observed on two distinct TFF1 promoter sites and GATA-6-siRNA repressed expression of TFF1. Moreover, protein-protein docking studies revealed the interaction of TFF1 with CXCR4 at Phe-172, Ser-122 and Glu-1 and TFF1 recombinant protein treatment in SW1990 cells increased CXCR4 mediated downstream signaling critical for GR. In this part, our overall data demonstrate that TFF1 may play a crucial role in gemcitabine resistance which is regulated by GATA6 and by interacting with MUC5AC