Protein Farnesyltransferase Catalyzes Unanticipated Farnesylation and Geranylgeranylation of Shortened Target Sequences

Ashok, Sudhat; Hildebrandt, Emily R.; Ruiz, Colby S.; Hardgrove, Daniel S.; Coreno, David W; Schmidt, W.; Hougland, James L.

doi:10.1021/acs.biochem.0c00081

Cited by 20 publications

(23 citation statements)

References 70 publications

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“…The differences in the prenylated proteins identified may be attributed to the differences in the cell line employed, labeling conditions, the enzymatic parameters associated with the probe used or the specific MS instrumentation employed for analysis. While extended C(X) 3 X-box containing proteins and Cxx peptides have been demonstrated to be in vitro prenylation substrates 5 , 6 , none of those proteins were found to be enriched in a statistically significant manner in our analysis. Several of these polypeptides do exist in mammalian proteomes, however their endogenous levels or efficiency of prenylation may be too low to be detected using the current chemical proteomic strategies.…”

Section: Discussionmentioning

confidence: 54%

“…1 A) 3 , 4 . Interestingly, recent studies have shown that extended C(X) 3 X motifs in model proteins can be farnesylated within cells 5 , while shortened Cxx peptides are also acceptable FTase substrates in vitro 6 . Dual geranylgeranylation on a family of Rab proteins also occurs on C-terminal CCXX, CXC, or CC motifs catalyzed by rab geranylgeranyltransferase (GGTase-II or RabGGTase) 7 .…”

Section: Introductionmentioning

confidence: 99%

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Metabolic labeling with an alkyne probe reveals similarities and differences in the prenylomes of several brain-derived cell lines and primary cells

Suazo

Jeong

Ahmadi

et al. 2021

Sci Rep

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Protein prenylation involves the attachment of one or two isoprenoid group(s) onto cysteine residues positioned near the C-terminus. This modification is essential for many signal transduction processes. In this work, the use of the probe C15AlkOPP for metabolic labeling and identification of prenylated proteins in a variety of cell lines and primary cells is explored. Using a single isoprenoid analogue, 78 prenylated protein groups from the three classes of prenylation substrates were identified including three novel prenylation substrates in a single experiment. Applying this method to three brain-related cell lines including neurons, microglia, and astrocytes showed substantial overlap (25%) in the prenylated proteins identified. In addition, some unique prenylated proteins were identified in each type. Eight proteins were observed exclusively in neurons, five were observed exclusively in astrocytes and three were observed exclusively in microglia, suggesting their unique roles in these cells. Furthermore, inhibition of farnesylation in primary astrocytes revealed the differential responses of farnesylated proteins to an FTI. Importantly, these results provide a list of 19 prenylated proteins common to all the cell lines studied here that can be monitored using the C15AlkOPP probe as well as a number of proteins that were observed in only certain cell lines. Taken together, these results suggest that this chemical proteomic approach should be useful in monitoring the levels and exploring the underlying role(s) of prenylated proteins in various diseases.

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Section: Discussionmentioning

confidence: 54%

Section: Introductionmentioning

confidence: 99%

Metabolic labeling with an alkyne probe reveals similarities and differences in the prenylomes of several brain-derived cell lines and primary cells

Suazo

Jeong

Ahmadi

et al. 2021

Sci Rep

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“…The yeast strains used in this study have been previously described ( Table S5 ) [ 30 ]. Yeast strains were propagated at rt in either YPD or appropriate selective media when plasmid transformed.…”

Section: Methodsmentioning

confidence: 99%

MALDI-MS Analysis of Peptide Libraries Expands the Scope of Substrates for Farnesyltransferase

Schey

Buttery

Hildebrandt

et al. 2021

IJMS

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Protein farnesylation is a post-translational modification where a 15-carbon farnesyl isoprenoid is appended to the C-terminal end of a protein by farnesyltransferase (FTase). This modification typically causes proteins to associate with the membrane and allows them to participate in signaling pathways. In the canonical understanding of FTase, the isoprenoids are attached to the cysteine residue of a four-amino-acid CaaX box sequence. However, recent work has shown that five-amino-acid sequences can be recognized, including the pentapeptide CMIIM. This paper describes a new systematic approach to discover novel peptide substrates for FTase by combining the combinatorial power of solid-phase peptide synthesis (SPPS) with the ease of matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). The workflow consists of synthesizing focused libraries containing 10–20 sequences obtained by randomizing a synthetic peptide at a single position. Incubation of the library with FTase and farnesyl pyrophosphate (FPP) followed by mass spectrometric analysis allows the enzymatic products to be clearly resolved from starting peptides due to the increase in mass that occurs upon farnesylation. Using this method, 30 hits were obtained from a series of libraries containing a total of 80 members. Eight of the above peptides were selected for further evaluation, reflecting a mixture that represented a sampling of diverse substrate space. Six of these sequences were found to be bona fide substrates for FTase, with several meeting or surpassing the in vitro efficiency of the benchmark sequence CMIIM. Experiments in yeast demonstrated that proteins bearing these sequences can be efficiently farnesylated within live cells. Additionally, a bioinformatics search showed that a variety of pentapeptide CaaaX sequences can be found in the mammalian genome, and several of these sequences display excellent farnesylation in vitro and in yeast cells, suggesting that the number of farnesylated proteins within mammalian cells may be larger than previously thought.

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“…140,141 Recent efforts to expand the existing accepted paradigm of protein S-prenylation have shown that proteins terminating in longer (CXXXX) or shorter (CXX) C-terminal motifs can be efficiently modified by the prenyltransferase enzymes. 142,143 Although these sequences have been shown to be substrates in vitro and within cells, there is no reported evidence of these types of modifications on endogenous proteins to date. Singly prenylated proteins often undergo additional processing steps to increase their hydrophobicity.…”

Section: S-prenylationmentioning

confidence: 99%

“…In addition, the same group reported that peptide models with shortened CXX sequences can also be recognized as S-prenylation substrates in vitro. 143 These studies highlight the promiscuity of prenyltransferase enzymes, and may expand our current understanding of their substrate recognition and potentially enlarge the scope of S-prenylation substrates. However, it has been noted that no endogenous proteins terminating in such unconventional prenylation motifs have been identified.…”

Section: S-prenylation Substratesmentioning

confidence: 99%

A Not-So-Ancient Grease History: Click Chemistry and Protein Lipid Modifications

2021

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Protein lipid modification involves the attachment of hydrophobic groups to proteins via ester, thioester, amide, or thioether linkages. In this review, the specific click chemical reactions that have been employed to study protein lipid modification and their use for specific labeling applications are first described. This is followed by an introduction to the different types of protein lipid modifications that occur in biology. Next, the roles of click chemistry in elucidating specific biological features including the identification of lipid-modified proteins, studies of their regulation, and their role in diseases are presented. A description of the use of protein–lipid modifying enzymes for specific labeling applications including protein immobilization, fluorescent labeling, nanostructure assembly, and the construction of protein–drug conjugates is presented next. Concluding remarks and future directions are presented in the final section.

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Protein Farnesyltransferase Catalyzes Unanticipated Farnesylation and Geranylgeranylation of Shortened Target Sequences

Cited by 20 publications

References 70 publications

Metabolic labeling with an alkyne probe reveals similarities and differences in the prenylomes of several brain-derived cell lines and primary cells

Metabolic labeling with an alkyne probe reveals similarities and differences in the prenylomes of several brain-derived cell lines and primary cells

MALDI-MS Analysis of Peptide Libraries Expands the Scope of Substrates for Farnesyltransferase

A Not-So-Ancient Grease History: Click Chemistry and Protein Lipid Modifications

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