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.

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.

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.