Yves Bourbonnais

Proteins with whey-acidic-protein motifs and cancer.

The importance of early diagnosis to reduce the morbidity and mortality from cancer has led to a search for new sensitive and specific tumour markers. Molecular techniques developed over the past few years allow simultaneous screening of thousands of genes, and have been applied to different cancers to identify many genes that are modulated in various cancers. Of these, attention has focused on genes coding for a family of proteins with whey-acidic-protein (WAP) motifs. Most notably, the genes coding for elafin, antileukoproteinase 1 (previously called secretory leucocyte proteinase inhibitor, SLPI), WAP four disulphide core domain protein 1 (previously called prostate stromal protein 20 kDa, PS20), and WAP four disulphide core domain protein 2 (previously called major human epididymis-specific protein E4, HE4), have been identified as candidate molecular markers for several cancers. In this review, we assess data for an association between cancer and human WAP proteins, and discuss their potential role in tumour progression. We also propose a new mechanism by which WAP proteins might have a role in carcinogenesis.

Expression cloning of the Candida albicans CSA1 gene encoding a mycelial surface antigen by sorting of Saccharomyces cerevisiae transformants with monoclonal antibody-coated magnetic beads

The mycelial surface antigen recognized by monoclonal antibody (mAb) 4E1 has previously been shown to be present predominantly in the terminal third of the hyphal structures in Candida albicans. We report here the expression cloning of the corresponding gene (CSA1 ) by mAb 4E1‐coated magnetic beads sorting of Saccharomyces cerevisiae transformants expressing a C. albicans genomic library. The strategy is both highly selective and highly sensitive and provides an additional genetic tool for the cloning and characterization of C. albicans genes encoding surface proteins. CSA1 is an intronless gene encoding a 1203‐residue protein composed of repetitive motifs and domains. Northern analysis indicates that CSA1 is preferentially expressed during the mycelial growth phase, although a low level of CSA1 mRNA can be detected in the yeast form. As evidenced by indirect immunofluorescence microscopy with mAb 4E1, Csa1p is not randomly distributed over the surface of yeast cells, but localizes predominantly in the growing buds. This suggests that the distribution of Csa1p may be restricted to sites of cell surface elongation. Both heterozygous and homozygous C. albicans csa1Δ mutants are viable. Upon induction of mycelial growth, the number and size of hyphal structures derived from the mutants are similar to those observed in the parental wild‐type strain. The physiological role of Csa1p has yet to be determined. However, the presence in Csa1p of repeated cysteine‐rich hydrophobic domains with significant sequence similarity to motifs found in surface proteins (Ag2 and Pth11) from two distantly related fungal pathogens (Coccidioides immitis and Magnaporthe grisea respectively) suggests a common function in host interaction.

Activation mechanism, functional role and shedding of glycosylphosphatidylinositol‐anchored Yps1p at the Saccharomyces cerevisiae cell surface

Yeast cell wall assembly is a highly regulated and dynamic process. A class of cell surface aspartic peptidases anchored by a glycosylphosphatidylinositol (GPI) group, collectively known as yapsins, was proposed to be involved in cell wall construction. The Saccharomyces cerevisiae Yps1p, the prototypal yapsin, is processed internally within a loop region to produce an α/β two‐subunit enzyme. Here we investigated the activation mechanism of GPI‐anchored Yps1p and identified some of its substrates. We report that all activation steps of GPI‐Yps1p take place at the cell surface and are regulated by the environmental pH. GPI‐Yps1p is active in vivo at pH 6.0 and pH 3.0 and functions as a sheddase for a subset of GPI‐anchored enzymes, including itself and the Gas1 glucanosyltransferase. Importantly, while native GPI‐Yps1p weakly suppresses many phenotypes associated with the yeast kex2Δ mutant, loop mutants that interfere with conversion into the two‐subunit enzyme restore the kex2Δ phenotypes to near wild type level. We propose that cleavage of this internal loop region plays an important regulatory function through stimulating its shedding activity. Collectively, our data provide a direct link between the pH regulation of yeast cell wall assembly and the activity of a yapsin.

Multiple cellular processes affected by the absence of the Rpb4 subunit of RNA polymerase II contribute to the deficiency in the stress response of the yeast rpb4 Δ mutant

Abstract. We previously described the isolation of yeast mutants (sex mutants) that secrete reduced amounts of mature α-factor when it is synthesized as part of a fusion with prosomatostatin. In the present study we show that the sex3-1 mutant displays pleiotropic phenotypes. These include an abnormal morphology, an osmoremediable caffeine sensitivity, reduced secretion of mature α-factor, a weakened cell wall and a marked deficiency in halotolerance. Cloning of the SEX3 gene revealed that it is identical to the RPB4 gene. This gene encodes the fourth largest subunit of yeast RNA polymerase II, which has been postulated to play a major role in the response to stress. We show that transcriptional activation in response to either a cell wall stress or to growth in the presence of elevated salt concentrations is minimally affected by the loss of RPB4 function. However, whereas the levels of several mRNAs are similarly reduced (by about 30%) in rpb4 mutants grown in rich medium at moderate temperature, some transcripts, in particular ZDS1, are more abundant. An increase dosage of ZDS1, or of genes involved in cell wall assembly and in secretion (RHO1 and SRO77, respectively), partially suppresses the sensitivity of rpb4Δ cells to high temperature, heat shock and stationary phase. Collectively, our results indicate that the loss of Rpb4p perturbs several cellular functions that contribute to the inappropriate stress response of rpb4Δ yeast. We therefore conclude that this RNA polymerase II subunit is not specifically involved in the stress response.

Human Pre-Elafin Inhibits a Pseudomonas aeruginosa-Secreted Peptidase and Prevents Its Proliferation in Complex Media

ABSTRACT Pseudomonas aeruginosa is a life-threatening opportunist human pathogen frequently associated with lung inflammatory diseases, namely, cystic fibrosis. Like other species, this gram-negative bacteria is increasingly drug resistant. During the past decade, intensive research efforts have been focused on the identification of natural innate defense molecules with broad antimicrobial activities, collectively known as antimicrobial peptides. Human pre-elafin, best characterized as a potent inhibitor of neutrophil elastase with anti-inflammatory properties, was also shown to possess antimicrobial activity against both gram-positive and gram-negative bacteria, including P. aeruginosa. Its mode of action was, however, not known. Using full-length pre-elafin, each domain separately, and mutated variants of pre-elafin with attenuated antipeptidase activity toward neutrophil elastase, we report here that both pre-elafin domains contribute, through distinct mechanisms, to its antibacterial activity against Pseudomonas aeruginosa. Most importantly, we demonstrate that the whey acidic protein (WAP) domain specifically inhibits a secreted peptidase with the characteristics of arginyl peptidase (protease IV). This is the first demonstration that a human WAP-motif protein inhibits a secreted peptidase to prevent bacterial growth in vitro. Since several WAP-motif proteins from various species demonstrate antimicrobial function with variable activities toward bacterial species, we suggest that this mechanism may be more common than initially anticipated.

Overexpression of MID2 suppresses the profilin‐deficient phenotype of yeast cells

Profilin‐deficient Saccharomyces cerevisiae cells show abnormal growth, actin localization, chitin deposition, bud formation and cytokinesis. Previous studies have also revealed a synthetic lethality between pfy1 and late secretory mutants, suggesting a role for profilin in intracellular transport. In this work, we document further the secretion defect associated with the pfy1Δ mutant. Electron microscopic observations reveal an accumulation of glycoproteins in the bud and in the mother cell. The MATa, pfy1Δ cells mate as well as wild‐type cells, while the mating efficiency of MATα, pfy1Δ cells is reduced. Pulse‐chase experiments demonstrate an accumulation of the 19 kDa α‐factor precursor and delayed secretion of the mature α‐factor. The TGN protein Kex2p is the principal enzyme responsible for the endoproteolytic cleavage of the α‐factor precursor. An immunofluorescence detection of Kex2p shows an altered localization in pfy1Δ cells. Instead of a discrete punctate distribution, the enzyme is dispersed throughout the cytoplasm. A high‐copy‐number plasmid containing MID2, which encodes a potential transmembrane protein involved in cell cycle control, suppresses the abnormal growth, actin distribution, α‐factor maturation and the accumulation of intracellular membranous structures in pfy1Δ cells.

Increased local levels of granulocyte colony-stimulating factor are associated with the beneficial effect of pre-elafin (SKALP/trappin-2/WAP3) in experimental emphysema.

Abstract Few therapeutic options are offered to treat inflammation and alveolar wall destruction in emphysema. The effect of recombinant human pre-elafin, an elastase inhibitor, was evaluated in porcine pancreatic elastase (PPE)-induced emphysema in C57BL/6 mice. In a first protocol, mice received a single instillation of pre-elafin (17.5 pmol/mouse) at 1 h post-PPE and were sacrificed up to 72 h post-PPE. A single instillation of pre-elafin significantly reduced PPE-induced neutrophil accumulation in lungs, as assessed by bronchoalveolar lavage (BAL), by 51%, 71% and 67% at 24, 48 and 72 h, respectively. In a second protocol, mice also received a single dose of PPE, but pre-elafin three times a week for 2 weeks. After 2 weeks, pre-elafin significantly reduced the PPE-induced increase in BAL macrophage numbers, airspace dimensions and lung hysteresivity by 74%, 62% and 52%, respectively. Since G-CSF was previously shown to reduce emphysematous changes in mice, the BAL levels of this mediator were measured 6 h post-PPE in animals treated as described in the first protocol. Pre-elafin significantly increased G-CSF levels in PPE-exposed mice compared to sham- and PPE only-exposed animals. This suggests that the beneficial effects of pre-elafin could be mediated, at least in part, by its ability to increase G-CSF levels in the lung.

Implementing a web-based introductory bioinformatics course for non-bioinformaticians that incorporates practical exercises

A recent scientific discipline, bioinformatics, defined as using informatics for the study of biological problems, is now a requirement for the study of biological sciences. Bioinformatics has become such a powerful and popular discipline that several academic institutions have created programs in this field, allowing students to become specialized. However, biology students who are not involved in a bioinformatics program also need a solid toolbox of bioinformatics software and skills. Therefore, we have developed a completely online bioinformatics course for non‐bioinformaticians, entitled “BIF‐1901 Introduction à la bio‐informatique et à ses outils (Introduction to bioinformatics and bioinformatics tools),” given by the Department of Biochemistry, Microbiology, and Bioinformatics of Université Laval (Quebec City, Canada). This course requires neither a bioinformatics background nor specific skills in informatics. The underlying main goal was to produce a completely online up‐to‐date bioinformatics course, including practical exercises, with an intuitive pedagogical framework. The course, BIF‐1901, was conceived to cover the three fundamental aspects of bioinformatics: (1) informatics, (2) biological sequence analysis, and (3) structural bioinformatics. This article discusses the content of the modules, the evaluations, the pedagogical framework, and the challenges inherent to a multidisciplinary, fully online course.