Cell-free identification of novel N-myristoylated proteins from complementary DNA resources using bioorthogonal myristic acid analogues

Takamitsu, Emi; Fukunaga, Kazuyoshi; Iio, Yusuke; Moriya, Koko; Utsumi, Toshihiko

doi:10.1016/j.ab.2014.07.006

Cited by 14 publications

(21 citation statements)

References 37 publications

(50 reference statements)

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“…Among these 13 proteins, 6 proteins (PPM1B, SAMM50, PLEKHN, STK32A, HID1, P2RX5) were recently reported to be N -myrsitoylated by cell-free and cellular metabolic labeling [ 25 , 29 ] or by bioorthogonal reaction followed by MS-based identification [ 19 , 20 ]. The analysis of the role of protein N -myristoylation on the intracellular localization or function has been performed on 4 proteins (PLEKHN, STK32A, PPM1B, HID1) [ 25 , 29 , 30 ] out of these 6 proteins.…”

Section: Resultsmentioning

confidence: 99%

“…As a result, the products of 13 cDNA clones (FBXL7, PPM1B, SAMM50, PLEKHN, AIFM3, C22orf42, STK32A, FAM131C, DRICH1, MCC1, HID1, P2RX5, STK32B) were found to be human N -myristoylated proteins ( Table 1 ). Among these 13 proteins, 6 proteins (PPM1B, SAMM50, PLEKHN, STK32A, HID1, P2RX5) were recently demonstrated to be N -myrsitoylated [ 19 , 20 , 25 , 29 ], and the analysis of the role of protein N -myristoylation on the intracellular localization or function were reported on 4 proteins (PLEKHN, STK32A, PPM1B, HID1) [ 25 , 29 , 30 ]. These human N -myristoylated proteins contained not only physiologically important proteins such as a protein kinase, E3-ubiquitin ligase component, cancer-related protein, apoptosis-related protein, but also integral transmembrane proteins that play critical roles in cellular functions.…”

Section: Discussionmentioning

confidence: 99%

“…Plasmid pTD1 (Shimadzu) was used for the insect cell-free protein synthesis system [ 24 ]. Plasmids pcDNA3-tActin-FLAG, pcDNA3-tActinG2A-FLAG, pTD1-tActin-FLAG, pTD1-tActinG2A-FLAG and pcDNA3-FLAG were constructed as described previously [ 7 , 25 ]. Construction of the expression vector pTD1-Δ10aa-tActin-Flag for screening N -myristoylated proteins is summarized in S2 Table .…”

Section: Methodsmentioning

confidence: 99%

“…The translation reaction was performed using an insect cell-free protein synthesis system (Shimadzu) in the presence of [ 3 H]leucine or [ 3 H]myristic acid as described previously [ 25 ]. The mixture (composed of 6.2 μL insect cell lysate, 3.7 μL reaction buffer, 0.5 μL 4 mM methionine, 1.0 μL [ 3 H]leucine [1 μCi] or 3.0 μL [ 3 H]myristic acid [20 μCi], and 2 μL mRNA [8 μg]) was incubated at 26°C for 6 h. The translation products were then analyzed by SDS-PAGE and fluorography.…”

Section: Methodsmentioning

confidence: 99%

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Identification of Human N-Myristoylated Proteins from Human Complementary DNA Resources by Cell-Free and Cellular Metabolic Labeling Analyses

et al. 2015

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To identify physiologically important human N-myristoylated proteins, 90 cDNA clones predicted to encode human N-myristoylated proteins were selected from a human cDNA resource (4,369 Kazusa ORFeome project human cDNA clones) by two bioinformatic N-myristoylation prediction systems, NMT-The MYR Predictor and Myristoylator. After database searches to exclude known human N-myristoylated proteins, 37 cDNA clones were selected as potential human N-myristoylated proteins. The susceptibility of these cDNA clones to protein N-myristoylation was first evaluated using fusion proteins in which the N-terminal ten amino acid residues were fused to an epitope-tagged model protein. Then, protein N-myristoylation of the gene products of full-length cDNAs was evaluated by metabolic labeling experiments both in an insect cell-free protein synthesis system and in transfected human cells. As a result, the products of 13 cDNA clones (FBXL7, PPM1B, SAMM50, PLEKHN, AIFM3, C22orf42, STK32A, FAM131C, DRICH1, MCC1, HID1, P2RX5, STK32B) were found to be human N-myristoylated proteins. Analysis of the role of protein N-myristoylation on the intracellular localization of SAMM50, a mitochondrial outer membrane protein, revealed that protein N-myristoylation was required for proper targeting of SAMM50 to mitochondria. Thus, the strategy used in this study is useful for the identification of physiologically important human N-myristoylated proteins from human cDNA resources.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Identification of Human N-Myristoylated Proteins from Human Complementary DNA Resources by Cell-Free and Cellular Metabolic Labeling Analyses

et al. 2015

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“…We generated a C-terminal DDK-tagged version of HK1S (HK1S DDK ) and a mutant for the Gly 2 myristoylation site, HK1S (G2A)DDK . The full-length proteins were synthesized in a cell-free system employing rabbit reticulocyte lysates, an established system for co-translational N-myristoylation ( Takamitsu et al, 2014 ). We employed TNT in vitro coupled transcription-translational system in conjugation with metabolic incorporation of the bio-orthogonal myristic acid analogue.…”

Section: Resultsmentioning

confidence: 99%

Novel myristoylation of the sperm-specific hexokinase 1 isoform regulates its atypical localization

2015

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The hexokinase 1 variant in mammalian spermatozoa (HK1S) has a unique N-terminus and this isoform atypically localizes to the plasma membrane. However, the mechanism of this process currently remains ambiguous. In this report, we show that fatty acylation underlies the specific sorting of HK1S. Employing chimeric reporter constructs, we first established that compartmentalization of HK1S does not function exclusively in sperm cells and that this feature is swappable to somatic HEK293 cells. Although the N-terminus lacks the classical consensus signature for myristoylation and the sequence-based predictions fail to predict myristoylation of HK1S, complementary experimental approaches confirmed that HK1S is myristoylated. Using live-cell confocal microscopy, we show that the mutation of a single amino acid, the myristoyl recipient Gly2, impedes the prominent feature of plasma membrane association and relocates the enzyme to the cytosol but not the nucleus. Additionally, substitutions of the putatively palmitoylated Cys5 is also reflected in a similar loss of compartmentalization of the protein. Taken together, our findings conclusively demonstrate that the N-terminal ‘MGQICQ’ motif in the unique GCS domain of HK1S acquires hydrophobicity by dual lipidic modifications, N-myristoylation and palmitoylation, to serve the requirements for membranous associations and thus its compartmentalization.

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Immobilization of azide-functionalized proteins to micro- and nanoparticles directly from cell lysate

Saini,

Parasa,

Clayton

et al. 2023

Microchim Acta

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Immobilization of proteins and enzymes on solid supports has been utilized in a variety of applications, from improved protein stability on supported catalysts in industrial processes to fabrication of biosensors, biochips, and microdevices. A critical requirement for these applications is facile yet stable covalent conjugation between the immobilized and fully active protein and the solid support to produce stable, highly bio-active conjugates. Here, we report functionalization of solid surfaces (gold nanoparticles and magnetic beads) with bio-active proteins using site-specific and biorthogonal labeling and azide-alkyne cycloaddition, a click chemistry. Specifically, we recombinantly express and selectively label calcium-dependent proteins, calmodulin and calcineurin, and cAMP-dependent protein kinase A (PKA) with N-terminal azide-tags for efficient conjugation to nanoparticles and magnetic beads. We successfully immobilized the proteins on to the solid supports directly from the cell lysate with click chemistry, forgoing the step of purification. This approach is optimized to yield low particle aggregation and high levels of protein activity post-conjugation. The entire process enables streamlined workflows for bioconjugation and highly active conjugated proteins. Graphical Abstract

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Cell-free identification of novel N-myristoylated proteins from complementary DNA resources using bioorthogonal myristic acid analogues

Cited by 14 publications

References 37 publications

Identification of Human N-Myristoylated Proteins from Human Complementary DNA Resources by Cell-Free and Cellular Metabolic Labeling Analyses

Identification of Human N-Myristoylated Proteins from Human Complementary DNA Resources by Cell-Free and Cellular Metabolic Labeling Analyses

Novel myristoylation of the sperm-specific hexokinase 1 isoform regulates its atypical localization

Immobilization of azide-functionalized proteins to micro- and nanoparticles directly from cell lysate

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