To each its own: Mechanisms of cross-talk between GPI biosynthesis and cAMP-PKA signaling in Candida albicans versus Saccharomyces cerevisiae.

Candida albicans is an opportunistic fungal pathogen that can switch between yeast and hyphal morphologies depending on the environmental cues it receives. The switch to hyphal form is crucial for the establishment of invasive infections. The hyphal form is also characterized by the cell surface expression of hyphae-specific proteins, many of which are GPI-anchored and important determinants of its virulence. The coordination between hyphal morphogenesis and the expression of GPI-anchored proteins is made possible by an interesting cross-talk between GPI biosynthesis and the cAMP-PKA signaling cascade in the fungus; a parallel interaction is not found in its human host. On the other hand, in the nonpathogenic yeast, Saccharomyces cerevisiae, GPI biosynthesis is shut down when filamentation is activated and vice versa. This too is achieved by a cross-talk between GPI biosynthesis and cAMP-PKA signaling. How are diametrically opposite effects obtained from the cross-talk between two reasonably well-conserved pathways present ubiquitously across eukarya? This Review attempts to provide a model to explain these differences. In order to do so, it first provides an overview of the two pathways for the interested reader, highlighting the similarities and differences that are observed in C. albicans versus the well-studied S. cerevisiae model, before going on to explain how the different mechanisms of regulation are effected. While commonalities enable the development of generalized theories, it is hoped that a more nuanced approach, that takes into consideration species-specific differences, will enable organism-specific understanding of these processes and contribute to the development of targeted therapies.

The caspase-like Gpi8 subunit of Candida albicans GPI transamidase is a metal-dependent endopeptidase.

GPI anchored proteins (GPI-APs) act at the frontiers of cells, decoding environmental cues and determining host-pathogen interactions in several lower eukaryotes. They are also essential for viability in lower eukaryotes. The GPI biosynthetic pathway begins at the ER and follows a roughly linear pathway to generate the complete precursor (CP) glycolipid. The GPI transamidase (GPIT) transfers this glycolipid to the C-terminal end of newly translated proteins after removing their GPI attachment signal sequence (SS). The GPIT subunit that cleaves SS is Gpi8, a protein with a conserved Cys/His catalytic dyad typical of cysteine proteases. A CaGPI8 heterozygous mutant accumulates CPs and has reduced cell surface GPI-APs. Using a simple cell-free assay, we demonstrate that the heterozygous CaGPI8 strain has low endopeptidase activity as well. The revertant strain is restored in all these phenotypes. CaGpi8 is also shown to be a metalloenzyme, whose protease activity is sensitive to agents that modify Cys/His residues.

Saccharomyces cerevisiae Ras2 restores filamentation but cannot activate the first step of GPI anchor biosynthesis in Candida albicans.

Ras proteins are highly conserved small GTPases in eukaryotes. GTP-bound Ras binds to effectors to trigger signaling cascades. In order to understand how extensive is the functional homology between the highly homologous proteins, S. cerevisiae Ras2 and C. albicans Ras1, we examined whether ScRas2 could functionally complement CaRas1 in activating hyphal morphogenesis as well as GPI anchor biosynthesis. We show that ScRas2 functionally complements CaRas1 in rescuing growth as well as activating hyphal growth, a process that involves plasma membrane localized Ras activating cAMP/PKA signaling via Cyr1. However, ScRas2 is unable to activate the GPI-N-acetylglucosaminyl transferase (GPI-GnT) which catalyzes the first step of GPI biosynthesis. That CaRas1 alone activates GPI-GnT and not ScRas2 suggests that this process is cAMP independent. Interestingly, CaRas1 transcriptionally activates CaGPI2, encoding a GPI-GnT subunit that has been shown to interact with CaRas1 physically. In turn, CaGPI2 downregulates CaGPI19, encoding another GPI-GnT subunit. This has direct consequences for expression of CaERG11, encoding the target of azole antifungals. This effect too is specific to CaRas1 and ScRas2 is unable to replicate it.

Modulation of azole sensitivity and filamentation by GPI15, encoding a subunit of the first GPI biosynthetic enzyme, in Candida albicans.

Glycosylphosphatidylinositol (GPI)-anchored proteins are important for virulence of many pathogenic organisms including the human fungal pathogen, Candida albicans. GPI biosynthesis is initiated by a multi-subunit enzyme, GPI-N-acetylglucosaminyltransferase (GPI-GnT). We showed previously that two GPI-GnT subunits, encoded by CaGPI2 and CaGPI19, are mutually repressive. CaGPI19 also co-regulates CaERG11, the target of azoles while CaGPI2 controls Ras signaling and hyphal morphogenesis. Here, we investigated the role of a third subunit. We show that CaGpi15 is functionally homologous to Saccharomyces cerevisiae Gpi15. CaGPI15 is a master activator of CaGPI2 and CaGPI19. Hence, CaGPI15 mutants are azole-sensitive and hypofilamentous. Altering CaGPI19 or CaGPI2 expression in CaGPI15 mutant can elicit alterations in azole sensitivity via CaERG11 expression or hyphal morphogenesis, respectively. Thus, CaGPI2 and CaGPI19 function downstream of CaGPI15. One mode of regulation is via H3 acetylation of the respective GPI-GnT gene promoters by Rtt109. Azole sensitivity of GPI-GnT mutants is also due to decreased H3 acetylation at the CaERG11 promoter by Rtt109. Using double heterozygous mutants, we also show that CaGPI2 and CaGPI19 can independently activate CaGPI15. CaGPI15 mutant is more susceptible to killing by macrophages and epithelial cells and has reduced ability to damage either of these cell lines relative to the wild type strain, suggesting that it is attenuated in virulence.

Biophysical Characterization of SG2NA Variants and their Interaction with DJ-1 and Calmodulin in vitro.

SG2NA was first discovered as nuclear autoantigen in lung and bladder cancer patient. It was named SG2NA as its expression increases during S to G2 phase of cell cycle. SG2NA/Striatin3 was classified as a member of Striatin family along with Straitin and Zinedin due to its structural and functional relatedness. At the molecular level, SG2NA is characterized by the presence of multiple protein-protein interaction domains viz., a caveolin binding motif, a coiled coil structure, Ca2+-calmodulin binding domain and a large WD-40 repeat domain in the same order from amino to the carboxyl termini. Analysis of secondary structures of 87 and 78 kDa SG2NA isoforms showed characteristic combinations of α-helix, ß-structure, ß-turns and random coil; suggesting of effective refolding after denaturation. This study for the first time establishes the structural differences between the two prevalent isoforms of SG2NA. Recently we observed that DJ-1 interacts with variants of SG2NA both in vitro and in vivo. The SG2NA isoforms purified from inclusion bodies showed the different secondary structure conformations, stability and interaction pattern for their interacting partners (DJ-1 and calmodulin) which imparts functional diversity of SG2NA. The SG2NA isoforms showed significant differential binding affinity to DJ-1 and Calmodulin.

Ligand-induced conformation changes drive ATP hydrolysis and function in SMARCAL1.

Mutations and deletions in SMARCAL1, an SWI2/SNF2 protein, cause Schimke immuno-osseous dysplasia (SIOD). SMARCAL1 preferentially binds to DNA molecules possessing double-stranded to single-stranded transition regions and mediates annealing helicase activity. The protein is critical for alleviating replication stress and maintaining genome integrity. In this study, we have analysed the ATPase activity of three mutations ­ A468P, I548N and S579L ­ present in SIOD patients. These mutations are present in RecA-like domain I of the protein. Analysis using active DNA-dependent ATPase A domain (ADAAD), an N-terminal deleted construct of bovine SMARCAL1, showed that all three mutants were unable to hydrolyse ATP. Conformational studies indicated that the α-helix and ß-sheet content of the mutant proteins was altered compared to the wild-type protein. Molecular simulation studies confirmed that major structural changes had occurred in the mutant proteins. These changes included alteration of a loop region connecting motif Ia and II. As motif Ia has been implicated in DNA binding, ligand binding studies were done using fluorescence spectroscopy. These studies revealed that the Kd for protein-DNA interaction in the presence of ATP was indeed altered in the case of mutant proteins compared to the wild-type. Finally, in vivo studies were done to complement the in vitro and in silico studies. The results from these experiments demonstrate that mutations in human SMARCAL1 that result in loss in ATPase activity lead to increased replication stress and therefore possibly manifestation of SIOD.

First step of glycosylphosphatidylinositol (GPI) biosynthesis cross-talks with ergosterol biosynthesis and Ras signaling in Candida albicans.

Candida albicans is a leading cause of fungal infections worldwide. It has several glycosylphosphatidylinositol (GPI)-anchored virulence factors. Inhibiting GPI biosynthesis attenuates its virulence. Building on our previous work, we explore the interaction of GPI biosynthesis in C. albicans with ergosterol biosynthesis and hyphal morphogenesis. This study is also the first report of transcriptional co-regulation existing between two subunits of the multisubunit enzyme complex, GPI-N-acetylglucosaminyltransferase (GPI-GnT), involved in the first step of GPI anchor biosynthesis in eukaryotes. Using mutational analysis, we show that the accessory subunits, GPI2 and GPI19, of GPI-GnT exhibit opposite effects on ergosterol biosynthesis and Ras signaling (which determines hyphal morphogenesis). This is because the two subunits negatively regulate one another; GPI19 mutants show up-regulation of GPI2, whereas GPI2 mutants show up-regulation of GPI19. Two different models were examined as follows. First, the two GPI-GnT subunits independently interact with ergosterol biosynthesis and Ras signaling. Second, the two subunits mutually regulate one another and thereby regulate sterol levels and Ras signaling. Analysis of double mutants of these subunits indicates that GPI19 controls ergosterol biosynthesis through ERG11 levels, whereas GPI2 determines the filamentation by cross-talk with Ras1 signaling. Taken together, this suggests that the first step of GPI biosynthesis talks to and regulates two very important pathways in C. albicans. This could have implications for designing new antifungal strategies.

Motifs Q and I are required for ATP hydrolysis but not for ATP binding in SWI2/SNF2 proteins.

Active DNA-dependent ATPase A Domain (ADAAD) is a SWI2/SNF2 protein that hydrolyzes ATP in the presence of stem-loop DNA that contains both double-stranded and single-stranded regions. ADAAD possesses the seven helicase motifs that are a characteristic feature of all the SWI2/SNF2 proteins present in yeast as well as mammalian cells. In addition, these proteins also possess the Q motif ~17 nucleotides upstream of motif I. Using site-directed mutagenesis, we have sought to define the role of motifs Q and I in ATP hydrolysis mediated by ADAAD. We show that in ADAAD both motifs Q and I are required for ATP catalysis but not for ATP binding. In addition, the conserved glutamine present in motif Q also dictates the catalytic rate. The ability of the conserved glutamine present in motif Q to dictate the catalytic rate has not been observed in helicases. Further, the SWI2/SNF2 proteins contain a conserved glutamine, one amino acid residue downstream of motif I. This conserved glutamine, Q244 in ADAAD, also directs the rate of catalysis but is not required either for hydrolysis or for ligand binding. Finally, we show that the adenine moiety of ATP is sufficient for interaction with SWI2/SNF2 proteins. The γ-phosphate of ATP is required for inducing the conformational change that leads to ATPase activity. Thus, the SWI2/SNF2 proteins despite sequence conservation with helicases appear to behave in a manner distinct from that of the helicases.

Targeting efflux pumps-In vitro investigations with acridone derivatives and identification of a lead molecule for MDR modulation.

To search multi drug resistance modulators, acridones carrying hydroxyl amine substituent at N-10 and COOH/Cl at C-4 were investigated for their interactions with the three components of efflux pump viz. P-gp, ATP, and Mg(2+). Experimental and theoretical results indicated that the compounds with COOH group at C-4 interact with P-gp and Mg(2+) while other set of compounds with Cl at C-4 interact with ATP and Mg(2+). Spot assay and R6G influx/efflux assay of compound 3 using Candida albicans showed decrease in the fungal growth and efflux of R6G, respectively, in presence of compound 3 suggesting the suitability of this compound for MDR modulation.

Specific interaction of jacalin with phycocyanin, a fluorescent phycobiliprotein.

Recent research has shown that, like porphyrins, phycocyanin (PC) too can produce singlet oxygen upon excitation with the appropriate radiation and hence could be useful in photodynamic therapy (PDT) for cancer. Unlike porphyrins, PC has the advantage of being a non-toxic, non-carcinogenic, soluble protein. However, the challenge would be to target the fluorescent phycobiliprotein to malignant cells. We report here that the tumor-specific lectin, jacalin, binds PC specifically in a carbohydrate-independent manner and with affinities better than that for porphyrins. Hence the lectin could prove to be a useful carrier for targeted delivery of PC. The interaction involves both ionic and hydrophobic interactions and more than one contact site.

Elucidating the mechanism of DNA-dependent ATP hydrolysis mediated by DNA-dependent ATPase A, a member of the SWI2/SNF2 protein family.

The active DNA-dependent ATPase A domain (ADAAD), a member of the SWI2/SNF2 family, has been shown to bind DNA in a structure-specific manner, recognizing DNA molecules possessing double-stranded to single-stranded transition regions leading to ATP hydrolysis. Extending these studies we have delineated the structural requirements of the DNA effector for ADAAD and have shown that the single-stranded and double-stranded regions both contribute to binding affinity while the double-stranded region additionally plays a role in determining the rate of ATP hydrolysis. We have also investigated the mechanism of interaction of DNA and ATP with ADAAD and shown that each can interact independently with ADAAD in the absence of the other. Furthermore, the protein can bind to dsDNA as well as ssDNA molecules. However, the conformation change induced by the ssDNA is different from the conformational change induced by stem-loop DNA (slDNA), thereby providing an explanation for the observed ATP hydrolysis only in the presence of the double-stranded:single-stranded transition (i.e. slDNA).

A novel catalytic mechanism for ATP hydrolysis employed by the N-terminal nucleotide-binding domain of Cdr1p, a multidrug ABC transporter of Candida albicans.

Although essentially conserved, the N-terminal nucleotide-binding domain (NBD) of Cdr1p and other fungal transporters has some unique substitutions of amino acids which appear to have functional significance for the drug transporters. We have previously shown that the typical Cys193 in Walker A as well as Trp326 and Asp327 in the Walker B of N-terminal NBD (NBD-512) of Cdr1p has acquired unique roles in ATP binding and hydrolysis. In the present study, we show that due to spatial proximity, fluorescence resonance energy transfer (FRET) takes place between Trp326 of Walker B and MIANS [2-(4-maleimidoanilino) naphthalene-6-sulfonic acid] on Cys193 of Walker A motif. By exploiting FRET, we demonstrate how these critical amino acids are positioned within the nucleotide-binding pocket of NBD-512 to bind and hydrolyze ATP. Our results show that both Mg2+ coordination and nucleotide binding contribute to the formation of the active site. The entry of Mg2+ into the active site causes the first large conformational change that brings Trp326 and Cys193 in close proximity to each other. We also show that besides Trp326, typical Glu238 in the Q-loop also participates in coordination of Mg2+ by NBD-512. A second conformational change is induced when ATP, but not ADP, docks into the pocket. Asn328 does sensing of the gamma-phosphate of the substrate in the extended Walker B motif, which is essential for the second conformational change that must necessarily precede ATP hydrolysis. Taken together our results imply that the uniquely placed residues in NBD-512 have acquired critical roles in ATP catalysis, which drives drug extrusion.

Beyond carbohydrate binding: new directions in plant lectin research.

Although for a long time carbohydrate binding property has been used as the defining feature of lectins, studies carried out mostly during the last two decades or so demonstrate that many plant lectins exhibit specific interactions with small molecules that are predominantly hydrophobic in nature. Such interactions, in most cases, appear to be at specific sites that do not interfere with the ability of the lectins to recognise and bind carbohydrates. Further, several of these ligands have binding affinities comparable to those for the binding of specific carbohydrates to the lectins. Given the ability of lectins to specifically recognise the glycocode (carbohydrate code) on different cell surfaces and distinguish between diseased and normal tissues, these additional sites may be viewed as potential drug carrying sites that could be exploited for targeted delivery to sites of choice. Porphyrin-lectin complexes are especially suited for such targeting since porphyrins are already under investigation in photodynamic therapy for cancer. This review will provide an update on the interactions of plant lectins with non-carbohydrate ligands, with particular emphasis on porphyrin ligands. The implications and potential applications of such studies will also be discussed.

Functional characterization of N-terminal nucleotide binding domain (NBD-1) of a major ABC drug transporter Cdr1p of Candida albicans: uncommon but conserved Trp326 of Walker B is important for ATP binding.

Using purified N-terminal NBD (NBD-512) domain of Cdr1p, a major multidrug extrusion pump of human pathogenic yeast Candida albicans, we show the relevance of the unique positioning of an atypical Trp326 residue. Similar to Cys193 in Walker A, Trp326 in the Walker B motif of Cdr1p is also a conserved feature of other fungal ATP Binding Cassette (ABC) transporters. By employing fluorescence spectroscopy, chemical modification, and site-directed mutagenesis, we demonstrate that of the five Trp residues in the NBD-512 domain, Trp326 alone is important for nucleotide binding and subsequent conformational changes within the domain. Furthermore, mutation of Trp326 to Ala results in an increased K(M) without appreciably affecting V(max) of ATPase activity. Thus, Trp326 in NBD-512 appears to be important for nucleotide binding and not for its hydrolysis. Additionally, the role of Trp326 in ATP binding is independent of the presence of the adjacent well-conserved Asp327 residue which, like Cys193, has a catalytic role in ATP hydrolysis. Considering that Trp326 of Cdr1p is a typical feature of fungal transporters alone, our study suggests that these ABC transporters may reflect mechanistic differences with regard to nucleotide binding and hydrolysis as compared to their counterparts of non-fungal origin.

Physicochemical and saccharide-binding studies on the galactose-specific seed lectin from Trichosanthes cucumerina.

Physicochemical and saccharide-binding studies have been performed on Trichosanthes cucumerina seed lectin (TCSL). The agglutination activity of TCSL is highest in the pH range 8.0-11.0, whereas below pH 7.0 it decreases quite rapidly, which is consistent with the involvement of imidazole side chains of His residues, which titrate in this pH range, in the sugar-binding activity of the lectin. The lectin activity is unaffected between 0 and 60 degrees C, but a sharp decline occurs at higher temperatures. Isoelectric focusing studies show that TCSL has three isoforms with pI values of 5.3, 6.2, and 7.1, with the isoform of pI 6.2 being the most abundant. Circular dichroism spectroscopic studies reveal that TCSL contains about 28.4% beta-sheet, 10.6% beta-turns, 7% polyproline type 2 structure, with the remainder comprising unordered structure; the alpha-helix content is negligible. Binding of 4-methylumbelliferyl-beta-D-galactopyranoside (MeUmbbetaGal) to TCSL results in a significant increase in the fluorescence intensity of the ligand, and this change has been used to obtain the association constant for the interaction. At 25 degrees C, the association constant, K(a), for the TCSL-MeUmbbetaGal interaction was determined as 6.9 x 10(4)M(-1). Binding of nonfluorescent, inhibitory sugars was studied by monitoring their ability to reverse the fluorescence changes observed when MeUmbbetaGal was titrated with TCSL.