The Antifungal Plant Defensin HsAFP1 from Heuchera sanguinea Induces Apoptosis in Candida albicans

Plant defensins are active against plant and human pathogenic fungi (such as Candida albicans) and baker's yeast. However, they are non-toxic to human cells, providing a possible source for treatment of fungal infections. In this study, we characterized the mode of action of the antifungal plant defensin HsAFP1 from coral bells by screening the Saccharomyces cerevisiae deletion mutant library for mutants with altered HsAFP1 sensitivity and verified the obtained genetic data by biochemical assays in S. cerevisiae and C. albicans. We identified 84 genes, which when deleted conferred at least fourfold hypersensitivity or resistance to HsAFP1. A considerable part of these genes were found to be implicated in mitochondrial functionality. In line, sodium azide, which blocks the respiratory electron transport chain, antagonized HsAFP1 antifungal activity, suggesting that a functional respiratory chain is indispensable for HsAFP1 antifungal action. Since mitochondria are the main source of cellular reactive oxygen species (ROS), we investigated the ROS-inducing nature of HsAFP1. We showed that HsAFP1 treatment of C. albicans resulted in ROS accumulation. As ROS accumulation is one of the phenotypic markers of apoptosis in yeast, we could further demonstrate that HsAFP1 induced apoptosis in C. albicans. These data provide novel mechanistic insights in the mode of action of a plant defensin

Topological and Functional Analysis of Pen-2, the fourth subunit of the gamma-secretase complex

Alzheimer s Disease (AD) is the most common form of dementia in elderly people and is characterized by the deposition of extracellular amyloid plaques and intracellular neurofibrillary tangles in the brains. The amyloid plaques are mainly composed of Abeta (Ab) peptides, resulting from proteolytic processing of the Amyloid Precursor Protein (APP). According to the amyloid cascade hypothesis the abnormal production and aggregation of these peptides is the trigger for the cascade of events ultimately resulting in AD.The final step in the production of Ab is catalyzed by gamma-secretase, a large membrane-bound aspartic acid protease, which is composed of four subunits: Presenilin (PS), Nicastrin (NCT), Aph-1 and Pen-2. Gamma-secretase is an attractive drug target to tackle AD. However, since the enzyme has, besides APP, many other substrates, one of which is Notch, a very important signal transduction molecule, caution is necessary upon designing gamma-secretase inhibitor/modulators PS harbors the catalytic aspartates of the enzyme complex, which are present in a water containing cavity. The role of the three other subunits is less clear. In this thesis, we focused on Pen-2, the smallest subunit of gamma-secretase. It is generally accepted that Pen-2 is involved in the endoproteolytic cleavage of PS, which is necessary to obtain active gamma-secretase complexes. However, neither the mode of action in the endoproteolysis nor possible other roles for Pen-2 in gamma-secretase are clear. We addressed the role of Pen-2 role in vivo by characterizing Pen-2-/- embryos. We observed an embryonic lethal phenotype that was very similar to the Notch-deficiency phenotype observed in mouse embryos knockout for the other gamma-secretase subunits. Because Pen-2 protein levels are strongly depending on the expression levels of the other gamma-secretase subunits, we concluded that the main function of Pen-2 is its role in the gamma-secretase complex.Next, we moved to the in vitro study of the function of Pen-2. For this study, we took advantage of the Pen-2-/- embryos from which we generated Pen-2-/- fibroblasts, allowing us to investigate the function of Pen-2 without interference of endogenous Pen-2 levels. We characterized the Pen-2-/- fibroblasts and observed a deficiency in gamma-secretase maturation and activity. Furthermore, we detected a trimeric gamma-secretase subcomplex composed of full length PS, NCT and Aph-1, which was devoid of gamma-secretase activity, even if we expressed a PS mutant that is active without endoproteolytical cleavage. These results suggest that the role of Pen-2 in the gamma-secretase complex extends beyond PS endoproteolysis.To further study this role, we applied a systematic cysteine scanning mutagenesis approach, replacing amino acids in Pen-2 one by one for a cysteine in a cysteine-less Pen-2 background. We showed that in most cases, single pointmutations in Pen-2 do not interfere with complex assembly, maturationandactivity, except for two residues in hydrophobic domain 1 of Pen-2, Glycine22 and Proline27. We demonstrated that Glycine22 is probably involved in the interaction of Pen-2 with other subunits, while Proline27 is important for the secondary structure of Pen-2.In a next step, we used the single cysteine mutants to analyze the exposure of the cysteines to water. To our surprise, we found that the N-terminal half of what is predicted to be hydrophobic domain 1, is in fact present in a constricted, hydrophilic environment. Furthermore, the loop of Pen-2 was accessible from the extracellular side, while the existing model for Pen-2 places this loop in the cytosol. Taking everything together, we propose a new model for Pen-2 in which the N-terminal part of hydrophobic domain 1 and the loop of Pen-2 are present in a water-accessible cavity in the membrane. Cryo-electron microscopy studies have demonstrated the presence of two low density areas in the gamma-secretase complex, which were predicted to be water cavities. One of them is proposed to be the catalytic cavity. To learn more about the putative cavity to which Pen-2 belongs, we performed cross-linking assays with a cysteine in the loop of Pen-2. We were able to cross-link the loop of Pen-2 to PS1 CTF indicating its close proximity to this part of gamma-secretase, which has been shown to be critical for the function of the protease. Finally, we verified whether the loop of Pen-2 was involved in the catalytic mechanism by changing its length or sequence. Modifying the loop indeed decreased overall gamma-secretase activity levels, but not drastic enough to attribute a direct key role in gamma-secretase activity to the loop of Pen-2. Interestingly, we observed a certain degree of substrate specificity in the loop mutants.Taking everything together, our data reveal a more dynamic conformation for Pen-2 that suggests participation in the function (and not only in the structure) of the enzyme complex.status: publishe

Analysis of the gamma-secretase interactome and validation of its association with tetraspanin-enriched microdomains

gamma-Secretase, an aspartyl protease that belongs to the iCLiPs (intramembrane cleaving proteases) family, is a multiprotein complex that consists of presenilin (PS), nicastrin (NCT), Aph-1 and Pen-2 (ref. 1). It is responsible for generation of the beta-amyloid peptide (A beta), the primary component of senile plaques in the brains of patients with Alzheimer's disease. Although the four components are necessary and sufficient for gamma-secretase activity(2-4), additional proteins are possibly involved in its regulation. Consequently, we purified proteins associated with the active gamma-secretase complex from reconstituted PS deficient fibroblasts, using tandem affinity purification (TAP)(5) and identified a series of proteins that transiently interact with the gamma-secretase complex and are probably involved in complex maturation, membrane trafficking and, importantly, the tetraspanin web. Tetraspanins form detergent-resistant microdomains in the cell membrane and regulate cell adhesion, cell signalling and proteolysis(6,7). Association of the gamma-secretase complex with tetraspanin-enriched microdomains provides an explanation for the previously documented localization of gamma-secretase to raft-like domains(8). Thus, these studies suggest that maintenance of the integrity of tetraspanin microdomains contributes to the refinement of proteolytic activity of the gamma-secretase complex.status: publishe

Functional and Topological Analysis of Pen-2, the Fourth Subunit of the γ-Secretase Complex*♦

The γ-secretase complex is a member of the family of intramembrane cleaving proteases, involved in the generation of the Aβ peptides in Alzheimer disease. One of the four subunits of the complex, presenilin, harbors the catalytic site, although the role of the other three subunits is less well understood. Here, we studied the role of the smallest subunit, Pen-2, in vivo and in vitro. We found a profound Notch-deficiency phenotype in Pen-2−/− embryos confirming the essential role of Pen-2 in the γ-secretase complex. We used Pen-2−/− fibroblasts to investigate the structure-function relation of Pen-2 by the scanning cysteine accessibility method. We showed that glycine 22 and proline 27 in hydrophobic domain 1 of Pen-2 are essential for complex formation and stability of γ-secretase. We also demonstrated that hydrophobic domain 1 and the loop domain of Pen-2 are located in a water-containing cavity and are in short proximity to the presenilin C-terminal fragment. We finally demonstrated the essential role of Pen-2 for the proteolytic activity of the complex. Our study supports the hypothesis that Pen-2 is more than a structural component of the γ-secretase complex and may contribute to the catalytic mechanism of the enzyme

Analysis of the gamma-secretase interactome and validation of its association with tetraspanin-enriched microdomains

gamma-Secretase, an aspartyl protease that belongs to the iCLiPs (intramembrane cleaving proteases) family, is a multiprotein complex that consists of presenilin (PS), nicastrin (NCT), Aph-1 and Pen-2 (ref. 1). It is responsible for generation of the beta-amyloid peptide (A beta), the primary component of senile plaques in the brains of patients with Alzheimer's disease. Although the four components are necessary and sufficient for gamma-secretase activity(2-4), additional proteins are possibly involved in its regulation. Consequently, we purified proteins associated with the active gamma-secretase complex from reconstituted PS deficient fibroblasts, using tandem affinity purification (TAP)(5) and identified a series of proteins that transiently interact with the gamma-secretase complex and are probably involved in complex maturation, membrane trafficking and, importantly, the tetraspanin web. Tetraspanins form detergent-resistant microdomains in the cell membrane and regulate cell adhesion, cell signalling and proteolysis(6,7). Association of the gamma-secretase complex with tetraspanin-enriched microdomains provides an explanation for the previously documented localization of gamma-secretase to raft-like domains(8). Thus, these studies suggest that maintenance of the integrity of tetraspanin microdomains contributes to the refinement of proteolytic activity of the gamma-secretase complex