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Synthetic Fluorogenic Peptides Reveal Dynamic Substrate Specificity of Depalmitoylases.

ABSTRACT: Palmitoylation is a post-translational modification involving the thioesterification of cysteine residues with a 16-carbon-saturated fatty acid. Little is known about rates of depalmitoylation or the parameters that dictate these rates. Here we report a modular strategy to synthesize quenched fluorogenic substrates for the specific detection of depalmitoylase activity and for mapping the substrate specificity of individual depalmitoylases. We demonstrate that human depalmitoylases APT1 and APT2, and TgPPT1 from the parasite Toxoplasma gondii, have distinct specificities that depend on amino acid residues distal to the palmitoyl cysteine. This information informs the design of optimal and non-optimal substrates as well as isoform-selective substrates to detect the activity of a specific depalmitoylase in complex proteomes. In addition to providing tools for studying depalmitoylases, our findings identify a previously unrecognized mechanism for regulating steady-state levels of distinct palmitoylation sites by sequence-dependent control of depalmitoylation rates.

SUBMITTER: Amara N

PROVIDER: S-EPMC6338520 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Synthetic Fluorogenic Peptides Reveal Dynamic Substrate Specificity of Depalmitoylases.

Amara Neri N Foe Ian T IT Onguka Ouma O Garland Megan M Bogyo Matthew M

Cell chemical biology 20181101 1

Palmitoylation is a post-translational modification involving the thioesterification of cysteine residues with a 16-carbon-saturated fatty acid. Little is known about rates of depalmitoylation or the parameters that dictate these rates. Here we report a modular strategy to synthesize quenched fluorogenic substrates for the specific detection of depalmitoylase activity and for mapping the substrate specificity of individual depalmitoylases. We demonstrate that human depalmitoylases APT1 and APT2, ...[more]

PMID: 30393067

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Project description:The fourth member of Ca2+-dependent mammalian secretory subtilase, PC4 (proprotein convertase 4), is primarily expressed in testicular germ cell and ovarian macrophage. Its role in sperm fertilization and in early embryonic development has been demonstrated earlier through several studies, including those with PC4 null mice. A number of physiological substrates found in reproductive tissues have been postulated or identified for PC4 by various biochemical studies. These include growth factors IGF-1 (insulin-like growth factor-1) and IGF-2, hormonal polypeptide proPACAP (where PACAP stands for pituitary adenylate cyclase-activating polypeptide) and a number of surface proteins of ADAM (ADisintegrin And Metalloproteinase-like) family such as ADAM-1 (fertilin a), ADAM-2 (fertilin b), ADAM-3 (procyritestin) and ADAM-5. To provide further evidence in support of this notion and also to study the substrate specificity and bioassay of PC4, a series of intramolecularly quenched fluorogenic peptides containing the cleavage sites and several mutants were prepared. A comparative kinetic analysis and measurement of Vmax (app)/Km (app) ratio of these fluorogenic substrates against PC4 and PC7 revealed that the mutant variants of h (human) proPACAP and m (mouse) ADAM-5 derived peptides Q-PACAP141-151-mutant [Abz-141RVKNKGRRI150P151SY(NO2)-A-CONH2] (150A151Y replaced by PS) and Q-ADAM-5380-388-mutant [Abz-380E381PKPARRP388RY(NO2)A-CONH2] (381R replaced by P) are most efficiently and selectively cleaved by PC4. Using these two and Q-IGF-263-71 peptides, we showed that the sperm extract of normal adult mice is much higher when compared with that of PC4-null mice. This suggests that these fluorogenic peptides are useful for specific bioassay of PC4 activity. In addition, kinetic studies with various peptidyl-MCA indicate that the hexapeptide Ac-KTKQLR-MCA (where MCA stands for 4-methyl coumaryl-7-amide) is most efficiently and selectively cleaved by PC4 at RMCA, making it another effective agent for bioassay of PC4 activity. The study concludes that the most probable sequence motif for recognition by PC4 is KXKXXR or KXXR, where X is any amino acid other than cysteine and that it prefers proline at P3, P5 and/or P2' positions. It was also revealed that PC4 is a good candidate processing enzyme for growth factors IGF-1 and -2, neuropeptide proPACAP and several ADAM proteins such as ADAM-1, -2, -3 and -5.

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Synthetic Fluorogenic Peptides Reveal Dynamic Substrate Specificity of Depalmitoylases.

Publications

Synthetic Fluorogenic Peptides Reveal Dynamic Substrate Specificity of Depalmitoylases.

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