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

Protein prenylation is a posttranslational modification involving the attachment of a C15 or C20 isoprenoid group to a cysteine residue near the C-terminus of the target substrate by protein farnesyltransferase (FTase) or protein geranylgeranyltransferase type I (GGTase-I), respectively. Both of these protein prenyltransferases recognize a C-terminal "CaaX" sequence in their protein substrates, but recent studies in yeast-and mammalian-based systems have demonstrated FTase

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“…Rat FTase and GGTase-I were expressed in BL21(DE3) Escherichia coli and purified as previously described. 6,40…”

Section: Expression and Purification Of Ftase And Ggtase-imentioning

confidence: 99%

Protein Farnesyltransferase Catalyzes Unanticipated Farnesylation and Geranylgeranylation of Shortened Target Sequences

et al. 2020

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“…In contrast, GGTase-II catalyzes the addition of two geranylgeranyl groups to two cysteine residues in sequences including CXC and CC. In addition to the known canonical CaaX motif that can be recognized by FTase, recent studies have showed that a longer C(x) 3 X motif can also recognized by both yeast and mammalian FTases [6]. Protein prenylation by FTase and GGTase-I typically is followed by the removal of the aaX residues promoted by Ras converting enzyme (Rce1) or Ste24 [7,8].…”

Section: Introductionmentioning

confidence: 99%

Optimization of Metabolic Labeling with Alkyne-Containing Isoprenoid Probes

Ahmadi

Suazo

Distefano

2019

Methods in Molecular Biology

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Protein prenylation, found in eukaryotes, is a post translational modification in which one or two isoprenoid groups are added to the C terminus of selected proteins using either a farnesyl group or a geranylgeranyl group. Prenylation facilitates protein localization mainly to the plasma membrane where the prenylated proteins, including small GTPases, mediate signal transduction pathways. Changes in the level of prenylated proteins may serve a critical function in a variety of diseases. Metabolic labeling using modified isoprenoid probes followed by enrichment and proteomic analysis allows the identities and levels of prenylated proteins to be investigated. In this protocol, we illustrate how the conditions for metabolic labeling are optimized to maximize probe incorporation in HeLa cells through a combination of in-gel fluorescence and densitometric analysis.

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“…The contributions of protein prenylation in eukaryotic cell biology are continuously under investigation, and the demonstration that CaaaX-residues can also be prenylated [4] expanded even more the repertoire of possible prenylated proteins. Prenylation controls the localization of several proteins that are crucial for the viability of fungal pathogens.…”

Section: Discussionmentioning

confidence: 99%

“…In contrast, protein substrates that are prenylated by GGTase-II harbor CXC or CC recognition motifs [1][2][3]. Recently, it has been demonstrated that proteins bearing a C-terminus CaaaX sequence may also be subject to prenylation by Ftase enzymes [4].…”

Section: Introductionmentioning

confidence: 99%

Differential requirements of protein geranylgeranylation for the virulence of human pathogenic fungi

et al. 2019

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Protein prenylation is a crucial post-translational modification largely mediated by two heterodimeric enzyme complexes, farnesyltransferase and geranylgeranyltransferase type-I (GGTase-I), each composed of a shared α-subunit and a unique β-subunit. GGTase-I enzymes are validated drug targets that contribute to virulence in Cryptococcus neoformans and to the yeast-to-hyphal transition in Candida albicans. Therefore, we sought to investigate the importance of the αsubunit, RamB, and the β-subunit, Cdc43, of the A. fumigatus GGTase-I complex to hyphal growth and virulence. Deletion of cdc43 resulted in impaired hyphal morphogenesis and thermosensitivity, which was exacerbated during growth in rich media. The Δcdc43 mutant also displayed hypersensitivity to cell wall stress agents and to cell wall synthesis inhibitors, suggesting alterations of cell wall biosynthesis or stress signaling. In support of this, analyses of cell wall content revealed decreased amounts of β-glucan in the Δcdc43 strain. Despite strong in vitro phenotypes, the Δcdc43 mutant was fully virulent in two models of murine invasive aspergillosis, similar to the control strain. We further found that a strain expressing the α-subunit gene, ramB, from a tetracycline-inducible promoter was inviable under non-inducing in vitro growth conditions and was virtually avirulent in both mouse models. Lastly, virulence studies using C. albicans strains with tetracycline-repressible RAM2 or CDC43 expression revealed reduced pathogenicity associated with downregulation of either gene in a murine model of disseminated infection. Together, these findings indicate a differential requirement for protein geranylgeranylation for fungal virulence, and further inform the selection of specific prenyltransferases as promising antifungal drug targets for each pathogen.

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

Cited by 41 publications

References 99 publications

Protein Farnesyltransferase Catalyzes Unanticipated Farnesylation and Geranylgeranylation of Shortened Target Sequences

Protein Farnesyltransferase Catalyzes Unanticipated Farnesylation and Geranylgeranylation of Shortened Target Sequences

Optimization of Metabolic Labeling with Alkyne-Containing Isoprenoid Probes

Differential requirements of protein geranylgeranylation for the virulence of human pathogenic fungi

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