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. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(1):31-38, 2018.

N-terminal entrance loop of yeast Yps1 and O-glycosylation of substrates are determinant factors controlling the shedding activity of this GPI-anchored endopeptidase.

BACKGROUND: S. cerevisiae Yps1 is the prototypical aspartic endopeptidase of the fungal yapsin family. This glycosylphosphatidylinositol (GPI) anchored enzyme was recently shown to be involved in the shedding of the GPI proteins Utr2, Gas1 and itself. It was also proposed to be part of a novel quality control mechanism that eliminates excess and/or misfolded GPI proteins. What regulates its shedding activity at the cell surface is however poorly understood. Yps1 is initially synthesized as a zymogen requiring proteolytic activation to remove a pro-peptide and further processing within a large insertion loop (N-entrance loop) generates a two-subunit endopeptidase. To investigate the role of this loop on its shedding activity, which typically takes place within Ser/Thr-rich domains, it was replaced with the short peptide found at the analogous position in Yps3. We also tested whether O-glycosylation might protect against proteolytic processing by Yps1. RESULTS: We show here that replacement of the N-entrance loop (N-ent loop) of Yps1 generates a single chain endopeptidase that undergoes partial (pH 6.0) or complete (pH 3.0) pro-peptide removal. At both pH, the shedding activity of the chimeric endopeptidase (Yps1-DL) toward Gas1 and itself is strongly and drastically increased, respectively. A direct correlation between endoproteolytic cleavage of this loop in native Yps1 and its shedding is observed. The Yps1-dependent shedding of two model GPI proteins (Gas1 and Yps1) is also stimulated by the absence of the O-mannosyltransferases, Pmt4 and Pmt2 respectively, involved in O-glycosylation of their Ser/Thr-rich domains. Under these conditions, some Yps1-independent shedding is also observed. CONCLUSIONS: Partial pro-peptide removal is essential to produce a functional Yps1 endopeptidase. The Yps1 N-ent loop plays a major role in regulating the shedding activity of the endopeptidase, most likely by limiting access to the active site, and its cleavage in native Yps1 is associated with its shedding. O-glycosylation protects against Yps1-dependent and -independent shedding of GPI proteins. It is postulated that hypoglycosylation of cell surface proteins, which may occur for misfolded proteins that escaped the ER-associated degradation, might target their elimination through shedding by Yps1 and possibly other yapsin members.

Structural and antimicrobial properties of human pre-elafin/trappin-2 and derived peptides against Pseudomonas aeruginosa.

BACKGROUND: Pre-elafin/trappin-2 is a human innate defense molecule initially described as a potent inhibitor of neutrophil elastase. The full-length protein as well as the N-terminal "cementoin" and C-terminal "elafin" domains were also shown to possess broad antimicrobial activity, namely against the opportunistic pathogen P. aeruginosa. The mode of action of these peptides has, however, yet to be fully elucidated. Both domains of pre-elafin/trappin-2 are polycationic, but only the structure of the elafin domain is currently known. The aim of the present study was to determine the secondary structures of the cementoin domain and to characterize the antibacterial properties of these peptides against P. aeruginosa. RESULTS: We show here that the cementoin domain adopts an α-helical conformation both by circular dichroism and nuclear magnetic resonance analyses in the presence of membrane mimetics, a characteristic shared with a large number of linear polycationic antimicrobial peptides. However, pre-elafin/trappin-2 and its domains display only weak lytic properties, as assessed by scanning electron micrography, outer and inner membrane depolarization studies with P. aeruginosa and leakage of liposome-entrapped calcein. Confocal microscopy of fluorescein-labeled pre-elafin/trappin-2 suggests that this protein possesses the ability to translocate across membranes. This correlates with the finding that pre-elafin/trappin-2 and elafin bind to DNA in vitro and attenuate the expression of some P. aeruginosa virulence factors, namely the biofilm formation and the secretion of pyoverdine. CONCLUSIONS: The N-terminal cementoin domain adopts α-helical secondary structures in a membrane mimetic environment, which is common in antimicrobial peptides. However, unlike numerous linear polycationic antimicrobial peptides, membrane disruption does not appear to be the main function of either cementoin, elafin or full-length pre-elafin/trappin-2 against P. aeruginosa. Our results rather suggest that pre-elafin/trappin-2 and elafin, but not cementoin, possess the ability to modulate the expression of some P.aeruginosa virulence factors, possibly through acting on intracellular targets.

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 alpha/beta 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 kex2Delta mutant, loop mutants that interfere with conversion into the two-subunit enzyme restore the kex2Delta 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.

Human pre-elafin inhibits a Pseudomonas aeruginosa-secreted peptidase and prevents its proliferation in complex media.

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.

Characterization of human pre-elafin mutants: full antipeptidase activity is essential to preserve lung tissue integrity in experimental emphysema.

Pre-elafin is a tight-binding inhibitor of neutrophil elastase and myeloblastin; two enzymes thought to contribute to tissue damage in lung emphysema. Previous studies have established that pre-elafin is also an effective anti-inflammatory molecule. However, it is not clear whether both functions are linked to the antipeptidase activity of pre-elafin. As a first step toward elucidating the structure/function relationship of this protein, we describe here the construction and characterization of pre-elafin variants with attenuated antipeptidase potential. In these mutants, the P1' methionine residue of the inhibitory loop is replaced by either a lysine (pre-elafinM25K) or a glycine (pre-elafinM25G) residue. Both mutated variants are stable and display biochemical properties undistinguishable from WT (wild-type) pre-elafin. However, compared with WT pre-elafin, their inhibitory constants are increased by one to four orders of magnitude toward neutrophil elastase, myeloblastin and pancreatic elastase, depending on the variants and enzymes tested. As suggested by molecular modelling, this attenuated inhibitory potential correlates with decreased van der Waals interactions between the variants and the enzymes S1' subsite. In elastase-induced experimental emphysema in mice, only WT pre-elafin protected against tissue destruction, as assessed by the relative airspace enlargement measured using lung histopathological sections. Pre-elafin and both mutants prevented transient neutrophil alveolitis. However, even the modestly affected pre-elafinM25K mutant, as assayed in vitro with small synthetic substrates, was a poor inhibitor of the neutrophil elastase and myeloblastin elastolytic activity measured with insoluble elastin. We therefore conclude that full antipeptidase activity of pre-elafin is essential to protect against lung tissue lesions in this experimental model.

Fungal yapsins and cell wall: a unique family of aspartic peptidases for a distinctive cellular function.

A novel class of aspartic peptidases known as fungal yapsins, whose first member ScYps1p was identified more than a decade ago in Saccharomyces cerevisiae, is characteristically modified by the addition of a glycophosphatidylinositol moiety and has a preference for cleaving substrates C-terminally to mono- and paired-basic residues. Over the years, several other members, first in S. cerevisiae and then in other fungi, have been identified. The implication of fungal yapsins in cell-wall assembly and/or remodelling had been suspected for many years. However, it is only very recently that studies performed on S. cerevisae and Candida albicans have confirmed their importance for cell-wall integrity. Here, we review 16 years of research, covering all fundamental aspects of these unique enzymes, in an effort to track their functional significance. We also propose a nomenclature for fungal yapsins based on their sequence identity with the founding members of this family, the S. cerevisiae yapsins.

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

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.

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.

Manganese activation of superoxide dismutase 2 in the mitochondria of Saccharomyces cerevisiae.

Manganese-dependent superoxide dismutase 2 (SOD2) in the mitochondria plays a key role in protection against oxidative stress. Here we probed the pathway by which SOD2 acquires its manganese catalytic cofactor. We found that a mitochondrial localization is essential. A cytosolic version of Saccharomyces cerevisiae Sod2p is largely apo for manganese and is only efficiently activated when cells accumulate toxic levels of manganese. Furthermore, Candida albicans naturally produces a cytosolic manganese SOD (Ca SOD3), yet when expressed in the cytosol of S. cerevisiae, a large fraction of Ca SOD3 also remained manganese-deficient. The cytosol of S. cerevisae cannot readily support activation of Mn-SOD molecules. By monitoring the kinetics for metalation of S. cerevisiae Sod2p in vivo, we found that prefolded Sod2p in the mitochondria cannot be activated by manganese. Manganese insertion is only possible with a newly synthesized polypeptide. Furthermore, Sod2p synthesis appears closely coupled to Sod2p import. By reversibly blocking mitochondrial import in vivo, we noted that newly synthesized Sod2p can enter mitochondria but not a Sod2p polypeptide that was allowed to accumulate in the cytosol. We propose a model in which the insertion of manganese into eukaryotic SOD2 molecules is driven by the protein unfolding process associated with mitochondrial import.

Anti-inflammatory activity of neutrophil elastase inhibitors.

Neutrophil elastase is a protease that is involved in the tissue destruction and inflammation that characterize numerous diseases, including hereditary emphysema, chronic obstructive pulmonary disease, cystic fibrosis, adult respiratory distress syndrome, ischemic-reperfusion injury and rheumatoid arthritis. Thus, elastase has been the object of extensive research to develop potent inhibitors that target its destructive and pro-inflammatory action. This review focuses on the anti-inflammatory activity of inhibitors that are currently, or were until recently in development, including purified or recombinantly produced endogenous inhibitors, genetically modified recombinant protein inhibitors and synthetic small-molecule inhibitors.

Anti-inflammatory effect of pre-elafin in lipopolysaccharide-induced acute lung inflammation.

The aim of the present study was to evaluate the anti-inflammatory activity of pre-elafin, an elastase-specific inhibitor, in lipopolysaccharide (LPS)-induced acute lung inflammation. C57BL/6 mice were pre-treated intranasally with recombinant human pre-elafin or vehicle only. One hour later, they were instilled intranasally with LPS (2 microg/mouse). Animals were sacrificed 6 hours after LPS instillation and bronchoalveolar lavage (BAL) was performed with three 1-ml aliquots of saline. LPS induced a lung inflammation characterised by a 100-fold increase in BAL neutrophils compared to control animals (265.8 +/- 54.5 x 10(3) and 2.4 +/- 1.3 x 10(3) neutrophils/ml, respectively). Pre-elafin dose-dependently reduced the neutrophil influx in the lung alveolar spaces by up to 84%. No elastase activity was detectable in all BAL fluids tested. Pre-elafin also reduced significantly LPS-induced gelatinase activity, as shown by zymography, and BAL macrophage inflammatory protein-2 (MIP-2) and KC levels, two potent neutrophil attractants and activators. Moreover, pre-elafin also significantly reduced mRNA levels of the three members of the IL-1 ligand family, namely IL-1alpha, IL-1beta and IL-1 receptor antagonist (IL-1Ra), type II IL-1 receptor, and TNFalpha as assessed in whole lung tissue by RNase protection assay. Thus, pre-elafin may be considered as a potent anti-inflammatory mediator.

Inhibition of human neutrophil elastase-induced acute lung injury in hamsters by recombinant human pre-elafin (trappin-2).

STUDY OBJECTIVES: Pre-elafin, also known as trappin-2, is an elastase-specific inhibitor that could be an ideal candidate for the treatment of neutrophil elastase-driven lung diseases. The inhibitory activity of pre-elafin resides in the COOH-terminal region that can be released as mature elafin. The NH(2)-terminal moiety of pre-elafin is characterized by the presence of a specific repeating sequence, termed cementoin, believed to immobilize the inhibitor to lung protein components and restrict its diffusion from the desired sites of action. This property should confer an advantage to pre-elafin compared to elafin in the treatment of neutrophil elastase-driven lung diseases. MEASUREMENTS: The inhibitory effect of recombinant human pre-elafin was assessed in a human neutrophil elastase-induced acute lung injury model in Golden Syrian hamsters. BAL fluid hemoglobin content was used as a marker of lung injury. RESULTS: Recombinant human pre-elafin administered intratracheally 1 h prior to neutrophil elastase dose-dependently inhibited the lung hemorrhage with a calculated half-effective dose of 8.1 microg/kg (0.7 nmol/kg). Pre-elafin was equally efficient when administered 3 h before neutrophil elastase. In contrast to pre-elafin, commercial synthetic elafin was ineffective in inhibiting neutrophil elastase-induced lung hemorrhage even at a dose of 4.45 nmol/kg. CONCLUSIONS: Our results suggest that pre-elafin may be eventually used in the treatment of neutrophil elastase-driven lung diseases.