Dataset Information

Unexpected role for a serine/threonine-rich domain in the Candida albicans Iff protein family.

ABSTRACT: Glycosylphosphatidylinositol (GPI)-anchored proteins are an important class of cell wall proteins in Candida albicans because of their localization and their function, even if more than half of them have no characterized homolog in the databases. In this study, we focused on the IFF protein family, investigating their exposure on the cell surface and the sequences that determine their subcellular localization. Protein localization and surface exposure were monitored by the addition of a V5 tag on all members of the family. The data obtained using the complete proteins showed for Iff3 (or -9), Iff5, Iff6, and Iff8 a covalent linkage to the ?-1,6-glucan network but, remarkably, showed that Iff2/Hyr3 was linked through disulfide bridges or NaOH-labile bonds. However, since some proteins of the Iff family were undetectable, we designed chimeric constructions using the last 60 amino acids of these proteins to test the localization signal. These constructions showed a ?-1,6-glucan linkage for Iff1/Rbr3, Iff2/Hyr3, Iff4 and Iff7/Hyr4 C-terminal-Iff5 fusion proteins, and a membrane localization for the Iff10/Flo9 C terminus-Iff5 fusion protein. Immunofluorescence analyses coupled to these cell fraction data confirmed the importance of the length of the central serine/threonine-rich region for cell surface exposure. Further analysis of the Iff2/Hyr3 linkage to the cell surface showed for the first time that a serine/threonine central region of a GPI-anchored protein may be responsible for the disulfide and the NaOH bonds to the glucan and glycoproteins network and may also override the signal of the proximal ? site region.

SUBMITTER: Boisrame A

PROVIDER: S-EPMC3187068 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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Unexpected role for a serine/threonine-rich domain in the Candida albicans Iff protein family.

Boisramé Anita A Cornu Amandine A Da Costa Grégory G Richard Mathias L ML

Eukaryotic cell 20110812 10

Glycosylphosphatidylinositol (GPI)-anchored proteins are an important class of cell wall proteins in Candida albicans because of their localization and their function, even if more than half of them have no characterized homolog in the databases. In this study, we focused on the IFF protein family, investigating their exposure on the cell surface and the sequences that determine their subcellular localization. Protein localization and surface exposure were monitored by the addition of a V5 tag o ...[more]

PMID: 21841123

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Project description:The opportunistic pathogen Candida is one of the most common causes of nosocomial bloodstream infections. Because candidemia is associated with high mortality rates and because the incidences of multidrug-resistant Candida are increasing, efforts to identify novel targets for the development of potent antifungals are warranted. Here, we describe the structure and function of the first member of a family of protein phosphatases that is specific to fungi, protein phosphatase Z1 (PPZ1) from Candida albicans We show that PPZ1 not only is active but also is as susceptible to inhibition by the cyclic peptide inhibitor microcystin-LR as its most similar human homolog, protein phosphatase 1α (PP1α [GLC7 in the yeast Saccharomyces cerevisiae]). Unexpectedly, we also discovered that, despite its 66% sequence identity to PP1α, the catalytic domain of PPZ1 contains novel structural elements that are not present in PP1α. We then used activity and pulldown assays to show that these structural differences block a large subset of PP1/GLC7 regulatory proteins from effectively binding PPZ1, demonstrating that PPZ1 does not compete with GLC7 for its regulatory proteins. Equally important, these unique structural elements provide new pockets suitable for the development of PPZ1-specific inhibitors. Together, these studies not only reveal why PPZ1 does not negatively impact GLC7 activity in vivo but also demonstrate that the family of fungus-specific phosphatases-especially PPZ1 from C. albicans-are highly suitable targets for the development of novel drugs that specifically target C. albicans without cross-reacting with human phosphatases. Candida albicans is a medically important human pathogen that is the most common cause of fungal infections in humans. In particular, approximately 46,000 cases of health care-associated candidiasis occur each year in the United States. Because these infections are associated with high mortality rates and because multiple species of Candida are becoming increasingly resistant to antifungals, there are increasing efforts to identify novel targets that are essential for C. albicans virulence. Here we use structural and biochemical approaches to elucidate how a member of a fungus-specific family of enzymes, serine/threonine phosphatase PPZ1, functions in C. albicans We discovered multiple unique features of PPZ1 that explain why it does not cross-react with, and in turn compete for, PP1-specific regulators, a long-standing question in the field. Most importantly, however, these unique features identified PPZ1 as a potential target for the development of novel antifungal therapeutics that will provide new, safe, and potent treatments for candidiasis in humans.

Project description:Background: Fungal extracellular vesicles (EVs) have been implicated in host-pathogen and pathogen-pathogen communication in some fungal diseases. In depth research into fungal EVs has been hindered by the lack of specific protein markers such as those found in mammalian EVs that have enabled sophisticated isolation and analysis techniques. Despite their role in fungal EV biogenesis, ESCRT proteins such as Vps23 (Tsg101) and Bro1 (ALIX) are not present as fungal EV cargo. Furthermore, tetraspanin homologs are yet to be identified in many fungi including the model yeast S. cerevisiae. Objective: We performed de novo identification of EV protein markers for the major human fungal pathogen Candida albicans with adherence to MISEV2018 guidelines. Materials and methods: EVs were isolated by differential ultracentrifugation from DAY286, ATCC90028 and ATCC10231 yeast cells, as well as DAY286 biofilms. Whole cell lysates (WCL) were also obtained from the EV-releasing cells. Label-free quantitative proteomics was performed to determine the set of proteins consistently enriched in EVs compared to WCL. Results: 47 proteins were consistently enriched in C. albicans EVs. We refined these to 22 putative C. albicans EV protein markers including the claudin-like Sur7 family (Pfam: PF06687) proteins Sur7 and Evp1 (orf19.6741). A complementary set of 62 EV depleted proteins was selected as potential negative markers. Conclusions: The marker proteins for C. albicans EVs identified in this study will be useful tools for studies on EV biogenesis and cargo loading in C. albicans and potentially other fungal species and will also assist in elucidating the role of EVs in C. albicans pathogenesis. Many of the proteins identified as putative markers are fungal specific proteins indicating that the pathways of EV biogenesis and cargo loading may be specific to fungi, and that assumptions made based on studies in mammalian cells could be misleading. Abbreviations: A1 - ATCC10231; A9 - ATCC90028; DAY B - DAY286 biofilm; DAY Y - DAY286 yeast; EV - extracellular vesicle; Evp1 - extracellular vesicle protein 1 (orf19.6741); GO - gene ontology; Log2(FC) - log2(fold change); MCC - membrane compartment of Can1; MDS - multidimensional scaling; MISEV - minimal information for studies of EVs; sEVs - small EVs; SP - signal peptide; TEMs - tetraspanin enriched microdomains; TM - transmembrane; VDM - vesicle-depleted medium; WCL - whole cell lysate.

Dataset Information

Unexpected role for a serine/threonine-rich domain in the Candida albicans Iff protein family.

Publications

Unexpected role for a serine/threonine-rich domain in the Candida albicans Iff protein family.

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