Restricted Substrate Specificity for the Geranylgeranyltransferase-I Enzyme in Cryptococcus neoformans: Implications for Virulence

Abstract

Proper cellular localization is required for the function of many proteins. The CaaX prenyltransferases (where CaaX indicates a cysteine followed by two aliphatic amino acids and a variable amino acid) direct the subcellular localization of a large group of proteins by catalyzing the attachment of hydrophobic isoprenoid moieties onto C-terminal CaaX motifs, thus facilitating membrane association. This group of enzymes includes farnesyltransferase (Ftase) and geranylgeranyltransferase-I (Ggtase-1). Classically, the variable (X) amino acid determines whether a protein will be an Ftase or Ggtase-I substrate, with Ggtase-I substrates often containing CaaL motifs. In this study, we identify the gene encoding the ␤ subunit of Ggtase-I (CDC43) and demonstrate that Ggtase-mediated activity is not essential. However, Cryptococcus neoformans CDC43 is important for thermotolerance, morphogenesis, and virulence. We find that Ggtase-I function is required for full membrane localization of Rho10 and the two Cdc42 paralogs (Cdc42 and Cdc420). Interestingly, the related Rac and Ras proteins are not mislocalized in the cdc43⌬ mutant even though they contain similar CaaL motifs. Additionally, the membrane localization of each of these GTPases is dependent on the prenylation of the CaaX cysteine. These results indicate that C. neoformans CaaX prenyltransferases may recognize their substrates in a unique manner from existing models of prenyltransferase specificity. It also suggests that the C. neoformans Ftase, which has been shown to be more important for C. neoformans proliferation and viability, may be the primary prenyltransferase for proteins that are typically geranylgeranylated in other species.

Efficient farnesylation of an extended C-terminal C(x)3X sequence motif expands the scope of the prenylated proteome

“…Similar expansions of potentially farnesylated proteins could be expected in pathogenic organisms that employ either endogenous or host-mediated farnesylation, such as Plasmodium falciparum, Candida albicans, and Legionella pneumophila. (75,(80)(81)(82)(83)(84)(85) Our expansion of the potential substrates recognized by FTase, supported by the reactivity of human-derived C(x)3X peptide sequences, highlights the importance of continuing studies towards identifying additional new and biologically relevant protein substrates and determining their prenylation state within the cell. In particular, identification and isolation of endogenously prenylated C(x)3X proteins will be essential to understanding the biological impact of prenylation of non-canonical FTase substrates.…”

Advantages and Challenges of Phenotypic Screens: The Identification of Two Novel Antifungal Geranylgeranyltransferase I Inhibitors

“…29 However, no inhibition of growth by the tested compounds could be observed ( Table 1). There are reports that geranylgeranylation is not essential in those pathogens, as it can be compensated by FTase activity.…”

All about that fat: Lipid modification of proteins in Cryptococcus neoformans

“…Manumycin A, an inhibitor of the human FTase for Ras, was found to kill fungal cells rapidly (<4 h) with minimum inhibitory concentrations close to those for AmB (Hast et al , 2011). The subunit common to cryptococcal FTase and GGTase I is encoded by PFT1 (Lin et al , 2010), while CDC43 encodes the unique subunit of GGTaseI (Selvig et al , 2013). The latter gene is not essential, suggesting that farnesylation is the dominant form of prenylation in C. neoformans.…”

“…Other small G proteins in C. neoformans , such as Cdc42 and its paralog Cdc420, Rho1 and its paralog Rho10, and Rac2, also have CAAL motifs and their homologs are GGTaseI substrates in other organisms. However, only Cdc42 was completely mislocalized in response to deletion of cryptococcal CDC43 , while Rho10 was partially affected (Selvig et al , 2013). This suggests that in C. neoformans the CAAL motif is insufficient to provide prenylation specificity, consistent with structural and biochemical analyses of C. neoformans FTase that show differences in the substrate binding pocket and high affinity for CAAL motifs in vitro (Hast et al , 2011).…”

“…Their occurrence in pathogenic microbes had been relatively unexplored, until recent reports detailing lipid modifications of proteins from bacteria (Hicks and Galan, 2013; Ivanov and Roy, 2013), parasites (Goldston et al , 2014), and fungi. The last group includes medically important organisms such as Candida albicans (Richard et al , 2002; Piispanen et al , 2011), Aspergillus fumigatus (Fontaine et al , 2003; Fortwendel et al , 2012; Fang et al , 2015), and Cryptococcus neoformans (Selvig et al , 2013; Nichols et al , 2015; Santiago-Tirado et al , 2015). …”