Metabolic Labeling of Ras with Tritiated Palmitate to Monitor Palmitoylation and Depalmitoylation

Tsai, Frederick D.; Wynne, Joseph; Ahearn, Ian M.; Philips, Mark R.

doi:10.1007/978-1-62703-791-4_3

Cited by 9 publications

(13 citation statements)

References 19 publications

(19 reference statements)

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“…Later, a more energetic radioactive palmitate analog [ 125 I]iodohexadecanoic acid ( 125 I-IC16) was adapted to this method. [ 125 I] generates gamma rays that are more penetrating, thus greatly shortening the exposure time to 24 h (Tsai et al, 2014). Other advances in radioactive labeling involve improving the sensitivity of X-ray films, which enables visualization of [ 3 H] palmitate-labeled proteins in 1-3 days (Tsai et al, 2014).…”

Section: Radioactive Metabolic Incorporationmentioning

confidence: 99%

“…Moreover, acyl-exchange assays are unique among all the palmitoylation detecting methods for their capability in providing a snapshot of the palmitoylated protein profile in cells. The major disadvantage of acyl-exchange assays lies within the fact that they are not compatible with pulse-chase experiments, as the entire acyl-exchange procedure is performed in protein lysates (Tsai et al, 2014).…”

Section: Hydroxylamine-based Acyl-exchange Assaymentioning

confidence: 99%

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Protein Palmitoylation in Leukocyte Signaling and Function

Yang

Chatterjee

et al. 2020

Front. Cell Dev. Biol.

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Palmitoylation is a post-translational modification (PTM) based on thioester-linkage between palmitic acid and the cysteine residue of a protein. This covalent attachment of palmitate is reversibly and dynamically regulated by two opposing sets of enzymes: palmitoyl acyltransferases containing a zinc finger aspartate-histidine-histidine-cysteine motif (PAT-DHHCs) and thioesterases. The reversible nature of palmitoylation enables fine-tuned regulation of protein conformation, stability, and ability to interact with other proteins. More importantly, the proper function of many surface receptors and signaling proteins requires palmitoylation-meditated partitioning into lipid rafts. A growing number of leukocyte proteins have been reported to undergo palmitoylation, including cytokine/chemokine receptors, adhesion molecules, pattern recognition receptors, scavenger receptors, T cell co-receptors, transmembrane adaptor proteins, and signaling effectors including the Src family of protein kinases. This review provides the latest findings of palmitoylated proteins in leukocytes and focuses on the functional impact of palmitoylation in leukocyte function related to adhesion, transmigration, chemotaxis, phagocytosis, pathogen recognition, signaling activation, cytotoxicity, and cytokine production.

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Section: Radioactive Metabolic Incorporationmentioning

confidence: 99%

Section: Hydroxylamine-based Acyl-exchange Assaymentioning

confidence: 99%

Protein Palmitoylation in Leukocyte Signaling and Function

Yang

Chatterjee

et al. 2020

Front. Cell Dev. Biol.

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“…DHHC9 is restricted to the Golgi and may account for the localization of NRAS and HRAS on this compartment in addition to the PM (73). Interestingly, KRAS4A does not accumulate on Golgi suggesting that this RAS protein is a substrate for another PAT (74). …”

Section: Constitutive C-terminal Modifications: Membrane Association mentioning

confidence: 99%

“…However, accurate measurement of the palmitate half-like on endogenous RAS has been limited by the poor sensitivity of detecting radiolabeled [ 3 H] palmitoyl modification (74). Iodinated derivatives of palmitate have been used to enhance sensitivity (97), but these reagents are not commercially available.…”

Section: Constitutive C-terminal Modifications: Membrane Association mentioning

confidence: 99%

Posttranslational Modifications of RAS Proteins

Ahearn

Zhou

Philips

2018

Cold Spring Harb Perspect Med

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The three human RAS genes encode four proteins that play central roles in oncogenesis by acting as binary molecular switches that regulate signaling pathways for growth and differentiation. Each is subject to a set of post-translational modifications (PTMs) that modify their activity or are required for membrane targeting. The enzymes that catalyze the various PTMs are potential targets for anti-RAS drug discovery. The PTMs of RAS proteins are the focus of this review.

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“…Therefore, multiple strategies, including mass spectrometric characterization, 26 metabolic labeling and click chemistry prob, 27,28 etc. were developed to recognize palmitoylation sites so as to further study and elucidate the molecular mechanisms and dynamics of 4 palmitoylation.…”

Section: Introductionmentioning

confidence: 99%

In Silico Identification of Protein S-Palmitoylation Sites and Their Involvement in Human Inherited Disease

Li¹,

Li²,

Ning³

et al. 2015

J. Chem. Inf. Model.

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S-Palmitoylation is a key regulatory mechanism controlling protein targeting, localization, stability, and activity. Since increasing evidence shows that its disruption is implicated in many human diseases, the identification of palmitoylation sites is attracting more attention. However, the computational methods that are published so far for this purpose have suffered from a poor balance of sensitivity and specificity; hence, it is difficult to get a good generalized prediction ability on an external validation set, which holds back the further analysis of associations between disruption of palmitoylation and human inherited diseases. In this work, we present a reliable identification method for protein S-palmitoylation sites, called SeqPalm, based on a series of newly composed features from protein sequences and the synthetic minority oversampling technique. With only 16 extracted key features, this approach achieves the most favorable prediction performance up to now with sensitivity, specificity, and Matthew's correlation coefficient values of 95.4%, 96.3%, and 0.917, respectively. Then, all known disease-associated variations are studied by SeqPalm. It is found that 243 potential loss or gain of palmitoylation sites are highly associated with human inherited disease. The analysis presents several potential therapeutic targets for inherited diseases associated with loss or gain of palmitoylation function. There are even biological evidence that are coordinate with our prediction results. Therefore, this work presents a novel approach to discover the molecular basis of pathogenesis associated with abnormal palmitoylation. SeqPalm is now available online at http://lishuyan.lzu.edu.cn/seqpalm , which can not only annotate the palmitoylation sites of proteins but also distinguish loss or gain of palmitoylation sites by protein variations.

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Metabolic Labeling of Ras with Tritiated Palmitate to Monitor Palmitoylation and Depalmitoylation

Cited by 9 publications

References 19 publications

Protein Palmitoylation in Leukocyte Signaling and Function

Protein Palmitoylation in Leukocyte Signaling and Function

Posttranslational Modifications of RAS Proteins

In Silico Identification of Protein S-Palmitoylation Sites and Their Involvement in Human Inherited Disease

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