Protein Palmitoylation Research Articles

Detection of Protein Palmitoylation Via Click Chemistry and Flow Cytometry

Palmitoylation is the process of attaching palmitate groups to protein cysteine residues. Many proteins have dynamic palmitoylation modifications that are added and removed to regulate protein function. Techniques to study protein palmitoylation, however, are slow and difficult to perform. We are in the process of developing a rapid technique for measuring protein palmitoylation using the palmitate analogue17‐ODYA and click chemistry followed by flow cytometry. To work out assay conditions, the Rho family GTPase TC10 is being used as a positive palmitoylation control, as it is known to be palmitoylated on its C‐terminal tail. To perform the analysis, 17‐ODYA is added to cells transfected with YFP‐tagged versions of TC10, and lysates from the cells are treated with click chemistry reagents to selectively conjugate the terminal alkyne group of 17‐ODYA with Alexa‐647 azide. The protein is isolated with magnetic anti‐FLAG beads which then are analyzed for YFP and Alexa‐647 fluorescence using flow cytometry. The current results indicate the method may be effectively detecting palmitoylation of TC10; however, the results will need to be validated using Western blotting as well as TC10 mutants that are incapable of being palmitoylated. Overall, the method will significantly enhance the time required to detect protein palmitoylation compared to traditional blot‐based techniques.Support or Funding InformationThis work was supported by Bemidji State University Biology Department and the College of Business, Mathematics, and Science. Support was also provided through the Neilson Foundation, Bemidji, MN and Regenerative Medicine Minnesota (RMM‐2017‐EP‐04).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Protein Palmitoylation Contributes to Extracellular Vesicle Production and Effect on Vascular Barrier Dysfunction during Systemic Inflammation

Extracellular vesicles (EVs) are membrane‐encapsulated particles released from various types of cells, ranging from 30–1000 nm in diameter. EVs facilitate intercellular communication by transferring proteins and lipid among cells not in direct contact. Recent studies suggest the involvement of EVs in the progression of systemic inflammatory response syndrome (SIRS) associated with trauma or infection. However, there is limited information about EV production, cargo content, or pathological function in systemic inflammation. Protein palmitoylation, the covalent attachment of palmitic acid to cysteine residues, catalyzed by a family of palmitoyl acyltransferases containing a zinc finger aspartate‐histidine‐histidine‐cysteine (DHHC) motif. Palmitoylation has recently been recognized as a novel signaling mechanism in regulating endothelial barrier function and microvascular permeability. In this study, we sought to examine whether protein palmitoylation affects EV production or function in microvascular barrier in response to systemic inflammation. EVs were isolated from the blood of wild‐type (WT) and DHHC21 deficient (Zdhhc21dep/dep) mice with SIRS. Nanoparticle tracking analysis showed an increased plasma level of EVs in WT mice subjected to SIRS compared to sham‐treated, but this response was diminished in Zdhhc21dep/dep mice. Next, isolated EVs from SIRS mice were injected into healthy mice intravenously to assess their effects on microvascular permeability. Intravital microscopic imaging analysis revealed that SIRS EVs from WT mice caused a massive leakage of plasma albumin into extravascular space, whereas SIRS EVs from Zdhhc21dep/dep mice produced less leakage. Furthermore, we examined the cargo contents of EVs produced during SIRS and identified an array of pro‐inflammatory molecules, including C‐reactive protein, interleukins and tumor necrosis factor‐a. Of particular note, SIRS EVs contained a high level of histones, which has been implicated in endothelial barrier injury. Such an EV profile was not observed in EVs from Zdhhc21dep/dep mice. Taken together, these data suggest systemic inflammation stimulates the production of pro‐inflammatory EVs that are capable of inducing microvascular leakage. This response is greatly attenuated in Zdhhc21 functional deficiency, indicating the involvement of palmitoylation in EV production or function.Support or Funding InformationThis work was supported by the National Institutes of Health Grants GM097270, HL070752, and HL126646.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Purification of Palmitoyl Protein Thioesterase and Acyl Protein Thioesterase for use in In Vitro Depalmitoylation Reactions

Palmitoylation refers to the attachment of palmitate to protein cysteine residues and assists with protein localization to the plasma membrane. Like protein phosphorylation, palmitoylation is often a dynamic process, and it involves the attachment of the palmitate by palmitoyl transferases and removal by palmitoyl protein thioesterase (PPTs) or acyl protein thioesterases (APTs). Currently, the techniques to study palmitoylation are difficult to perform and involve the use of harsh chemical reagents like hydroxylamine (HAM) to depalmitate proteins, often leading to destruction of the proteins being studied. Recombinant, purified PPT/APTs could be used as an alternative reagent to depalmitate proteins and also to investigate their individual specificities in depalmitoylation reactions. To purify APTs and PPTs, the coding sequences of human variants were PCR amplified, cloned and expressed to produce either GST or MBP‐tagged fusion proteins. Expressed proteins have been analyzed using SDS‐PAGE and bands of expected molecular mass have been identified. Enzyme activity is currently being tested with the substrate 4‐methylumbelliferyl‐6‐thiopalmitoyl‐b‐glucoside (MU‐6S‐palm‐bGlc) in a fluorogenic enzyme assay, with the goal of eventually using the fusion proteins in depalmitolyation reactions involving palmitoylated protein substrates.Support or Funding InformationThis work was supported by Bemidji State University Biology Department and the College of Business, Mathematics, and Science. Support was also provided through the Neilson Foundation, Bemidji, MN and Regenerative Medicine Minnesota (RMM‐2017‐EP‐04).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mutations in the zDHHC9 protein palmitoyltransferase result in X‐Linked Intellectual Disability (XLID) by distinct mechanisms

The zDHHC9 gene, which encodes a Ras palmitoyltransferase is highly expressed in the brain and the protein is enriched in the hippocampus, a region known to be involved with learning and memory. Loss of function mutations in the zDHHC9 result in lead X‐Linked Intellectual Disabilities (XLID). Analysis of the disease causing mutations has begun to provide insights into the molecular mechanisms underlining the neuronal function of protein palmitoylation. Previously we published that two missense mutations in zDHHC9 (P150S and R148W) result in a decrease in enzyme autopalmitoylation, the first of the two step reaction mechanism (JBC 289:18582 (2014)). Here we show that a nonsense mutation, R298* that truncates the C‐terminal 66‐amino acids of zDHHC9, results in an enzymatically active, but trafficking defective protein. Specifically, wild type zDHHC9 is localized in the Golgi and traffics via a microtubules based mechanism to distal sites of the neuron. In contrast, the truncated R298* protein is restricted to the Golgi. This suggests that subcellular localization is required to engage specific protein substrates involved with neuronal function. Since it is known that zDHHC9 palmitoylates Ras proteins, we investigated whether the loss of zDHHC9 function changes Ras dependent signaling. Wild type HAP1 cells were compared with HAP1 cells in which the zDHHC9 gene has been deleted. In cells lacking zDHHC9 there was a decrease in S6K and AKT1 phosphorylation, consistent with a reduction in Ras signaling. We are in the process of analyzing additional disease mutations of zDHHC9 using cell lines, rat hippocampal neurons and a yeast palmitoylation functional assay. In addition, Acyl‐RAC and ABE methods, coupled with mass spectrometry are being used to identify zDHHC9 substrates in neurons. These studies will provide insight into the role of palmitoylation in the pathophysiology of XLID.Support or Funding InformationThis study was supported by a grant from NIH R21NS090160 to RJD.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Membrane-Bound Selenoprotein Regulates its Activity by Autoproteolysis

Selenoprotein K (SELENOK) is an intrinsically disordered membrane protein that assists palmitoylation of proteins using its selenocysteine at position 92 (Sec92) and also contributes to the degradation of misfolded proteins in response to cellular stress. While only a small group of intrinsically disordered proteins are enzymes, the presence of Sec suggests that SELENOK may function as an enzyme.We discovered that SELENOK undergoes autoproteolysis, releasing its C terminal disordered fragment that contains Sec92, the first such observation for a selenoprotein. We demonstrate that SELENOK has multiple cleavage sites located around its Src homology 3 (SH3) binding segment. The cleavage shifts to adjacent sites when its conformation is altered by mutations and the use of different detergents. Mutation of potential catalytic residues such as Ser, His, Asp and Thr to Ala led to slower cleavage rates. The presence of Sec92 accelerated autoproteolysis but, interestingly, cleavage was retained in the absence of this amino acid. This indicates that Sec92 is not the nucleophile for cleavage but rather might be influencing the protein conformation. We suggest that SELENOK autoproteolyis regulates its association with different protein complexes in response to ER stress and other cellular signals.

Role of Palmitoylation of Postsynaptic Proteins in Promoting Synaptic Plasticity.

Many postsynaptic proteins undergo palmitoylation, the reversible attachment of the fatty acid palmitate to cysteine residues, which influences trafficking, localization, and protein interaction dynamics. Both palmitoylation by palmitoyl acyl transferases (PAT) and depalmitoylation by palmitoyl-protein thioesterases (PPT) is regulated in an activity-dependent, localized fashion. Recently, palmitoylation has received attention for its pivotal contribution to various forms of synaptic plasticity, the dynamic modulation of synaptic strength in response to neuronal activity. For instance, palmitoylation and depalmitoylation of the central postsynaptic scaffold protein postsynaptic density-95 (PSD-95) is important for synaptic plasticity. Here, we provide a comprehensive review of studies linking palmitoylation of postsynaptic proteins to synaptic plasticity.

SREBP-dependent lipidomic reprogramming as a broad-spectrum antiviral target

Viruses are obligate intracellular microbes that exploit the host metabolic machineries to meet their biosynthetic demands, making these host pathways potential therapeutic targets. Here, by exploring a lipid library, we show that AM580, a retinoid derivative and RAR-α agonist, is highly potent in interrupting the life cycle of diverse viruses including Middle East respiratory syndrome coronavirus and influenza A virus. Using click chemistry, the overexpressed sterol regulatory element binding protein (SREBP) is shown to interact with AM580, which accounts for its broad-spectrum antiviral activity. Mechanistic studies pinpoint multiple SREBP proteolytic processes and SREBP-regulated lipid biosynthesis pathways, including the downstream viral protein palmitoylation and double-membrane vesicles formation, that are indispensable for virus replication. Collectively, our study identifies a basic lipogenic transactivation event with broad relevance to human viral infections and represents SREBP as a potential target for the development of broad-spectrum antiviral strategies.

SwissPalm 2: Protein S-Palmitoylation Database.

Protein S-palmitoylation is increasingly recognized as an important posttranslational modification, present in all eukaryotic organisms, involved in the regulation of many biological processes. The SwissPalm database centralizes the large and increasing number of published palmitoyl-proteome datasets, provides tools to compare them, and includes curated data from the literature on the identification and analysis of palmitoylated proteins. SwissPalm 2 provides an updated version, with 38 palmitoyl-proteomes at the time of release, from 17 different species, and new features such as the inclusion of orthologs.

DHHC4 and DHHC5 Facilitate Fatty Acid Uptake by Palmitoylating and Targeting CD36 to the Plasma Membrane

Fatty acid uptake is the first step in fatty acid utilization, but it remains unclear how the process is regulated. Protein palmitoylation is a fatty acyl modification that plays a key regulatory role in protein targeting and trafficking; however, its function in regulating fatty acid metabolism is unknown. Here, we show that two of the Asp-His-His-Cys (DHHC) motif-containing palmitoyl acyltransferases, DHHC4 and DHHC5, regulate fatty acid uptake. DHHC4 and DHHC5 function at different subcellular localizations to control the palmitoylation, plasma membrane localization, and fatty acid uptake activity of the scavenger receptor CD36. Depletion of either DHHC4 or DHHC5 in cells disrupts CD36-dependent fatty acid uptake. Furthermore, both Dhhc4-/- and adipose-specific Dhhc5 knockout mice show decreased fatty acid uptake activity in adipose tissues and develop severe hypothermia upon acute cold exposure. These findings demonstrate a critical role of DHHC4 and DHHC5 in regulating fatty acid uptake.

Spatial organization of palmitoyl acyl transferases governs substrate localization and function.

Protein palmitoylation is a critical posttranslational modification that regulates protein trafficking, localization, stability, sorting and function. In mammals, addition of this lipid modification onto proteins is mediated by a family of 23 palmitoyl acyl transferases (PATs). PATs often palmitoylate substrates in a promiscuous manner, precluding our understanding of how these enzymes achieve specificity for their substrates. Despite generous efforts to identify consensus motifs defining PAT-substrate specificity, it remains to be determined whether additional factors beyond interaction motifs, such as local palmitoylation, participate in PAT-substrate selection. In this review, we emphasize the role of local palmitoylation, in which substrates are palmitoylated and trapped in the same subcellular compartments as their PATs, as a mechanism of enzyme-substrate specificity. We focus here on non-Golgi-localized PATs, as physical proximity to their substrates enables them to engage in local palmitoylation, compared to Golgi PATs, which often direct trafficking of their substrates elsewhere. PAT subcellular localization may be an under-recognized, yet important determinant of PAT-substrate specificity that may work in conjunction or completely independently of interaction motifs. We also discuss some current hypotheses about protein motifs that contribute to localization of non-Golgi-localized PATs, important for the downstream targeting of their substrates.

In Vitro Assays to Monitor the Enzymatic Activities of zDHHC Protein Acyltransferases.

A family of zDHHC protein acyltransferase (PAT) enzymes catalyze the S-palmitoylation of target proteins via a two-step mechanism. The first step involves transfer of palmitate from the palmitoyl-CoA donor to the active site cysteine of the zDHHC PAT enzyme, releasing reduced CoA (CoASH). In the second step, the palmitoyl-PAT intermediate thioester reacts with a cysteine side chain within the target substrate to produce the palmitoylated substrate product or, in the absence of a protein substrate, the palmitoyl-PAT intermediate thioester is hydrolyzed and releases palmitate. Formation and resolution of the palmitoyl-PAT intermediate complex (autopalmitoylation) is measured using a coupled enzyme system that monitors the production of CoASH via reduction of NAD+ by the α-ketoglutarate dehydrogenase complex. This assay can be used to isolate and characterize modulators of autopalmitoylation and is scalable to high-throughput screening (HTS). A second fluorescence-based assay is described that monitors the hydrolysis of the palmitoyl-PAT thioester linked intermediate by thin-layer chromatography using a palmitoyl-CoA analog, BODIPY®-C12:0-CoA, as a substrate. These two assays provide a methodology to quantify the first enzymatic step of the two-step zDHHC PAT reaction.

Measuring S-Depalmitoylation Activity In Vitro and In Live Cells with Fluorescent Probes.

S-palmitoylation is a reversible lipid posttranslational modification (PTM) that can mediate protein localization, trafficking, interaction with membranes, and a host of other biophysical characteristics. Over the past decade, a suite of chemoproteomic strategies have uncovered the breadth of S-palmitoylation, revealing widespread susceptibility to modification by this PTM throughout the human proteome. A focal point of research toward understanding the role of S-palmitoylation in varied cellular processes has focused on understanding how "writer" and "eraser" proteins function together to control the levels of S-palmitoylation of target proteins. The spatial and temporal regulation of S-palmitoylation by its "erasers"-acyl protein thioesterases (APTs)-is not fully understood. Tools which enable monitoring of the activity levels of the APTs in real-time in live cells illuminate how spatial control of these enzymes redecorate the lipidation state of the local proteome. To this end, we have developed fluorescence-based depalmitoylation probes (DPPs), which report S-depalmitoylase activity in live cells. Using DPPs, we have demonstrated that S-depalmitoylase activity changes in response to growth factor stimulation, unveiling potential regulation of cell growth and metabolism by APTs. Additionally, we recently discovered APTs in mitochondria using targeted DPPs, indicating new roles for S-depalmitoylation in this critical cellular compartment. Here, we present detailed protocols on how to carry out in vitro S-depalmitoylase activity assays and live cell fluorescence imaging employing the growing DPP toolbox.

Higher Levels of Protein Palmitoylation in the Frontal Cortex across Aging Were Associated with Reference Memory and Executive Function Declines.

Cognitive decline with aging is often due to altered levels of protein expression. The NMDA receptor (NMDAR) and the complex of proteins surrounding the receptor are susceptible to age-related changes in expression. In the frontal cortex of aged mice, there is a significant loss of expression of the GluN2B subunit of the NMDAR, an increase in Fyn expression, and no change in PSD-95. Studies have also found that, in the frontal cortex, phosphorylation of GluN2B subunits and palmitoylation of GluN2 subunits and NMDAR complex proteins are affected by age. In this study, we examined some of the factors that may lead to the differences in the palmitoylation levels of NMDAR complex proteins in the frontal cortex of aged animals. The Morris water maze was used to test spatial learning in 3- and 24-month-old mice. The acyl–biotinyl exchange method was used to precipitate palmitoylated proteins from the frontal cortices and hippocampi of the mice. Additionally, brain lysates from old and young mice were probed for the expression of fatty acid transporter proteins. An age-related increase of palmitoylated GluN2A, GluN2B, Fyn, PSD-95, and APT1 (acyl protein thioesterase 1) in the frontal cortex was associated with poorer reference memory and/or executive functions. These data suggest that there may be a perturbation in the palmitoylation cycle in the frontal cortex of aged mice that contributes to age-related cognitive declines.

S-Acylation of Proteins.

Palmitoylation or S-acylation is the posttranslational attachment of fatty acids to cysteine residues and is common among integral and peripheral membrane proteins. Palmitoylated proteins have been found in every eukaryotic cell type examined (yeast, insect, and vertebrate cells), as well as in viruses grown in these cells. The exact functions of protein palmitoylation are not well understood. Intrinsically hydrophilic proteins, especially signaling molecules, are anchored by long-chain fatty acids to the cytoplasmic face of the plasma membrane. Palmitoylation may also promote targeting to membrane subdomains enriched in glycosphingolipids and cholesterol or affect protein-protein interactions.This chapter describes (1) a standard protocol for metabolic labeling of palmitoylated proteins and also the procedures to prove a covalent and ester-type linkage of the fatty acids, (2) a simple method to analyze the fatty acid content of S-acylated proteins, (3) two methods to analyze dynamic palmitoylation for a given protein, and (4) protocols to study cell-free palmitoylation of proteins.

Enrichment of S-Palmitoylated Proteins for Mass Spectrometry Analysis.

As the 10-year anniversary of their first introduction approaches, alkynyl fatty acids have revolutionized the analysis of S-palmitoylation dynamics, acting as functional mimics incorporated into native modification sites in cultured cells. The alkyne functional group provides a robust handle for bioorthogonal Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) to reporter-linked azides, forming a stable conjugate for enrichment for mass spectrometry analysis or in-gel fluorescence. Importantly, metabolic labeling enables time-dependent analysis of S-palmitoylation dynamics, which can be used to profile incorporation and turnover rates across the proteome. Here we present a protocol for cell labeling, click chemistry conjugation, enrichment, and isobaric tandem mass tag labeling for quantitative mass spectrometry analysis of protein S-palmitoylation.

Probing Substrate Preferences of Depalmitoylases

Depalmitoylases play a crucial role in regulating dynamic protein palmitoylation. In this issue of Cell Chemical Biology, Amara etal. (2019) present fluorogenic peptide probes to analyze the activity and substrate specificity of depalmitoylases and uncover that the amino acid residues distal to the palmitoylation site could regulate depalmitoylases activities.

Purification of Recombinant DHHC Proteins Using an Insect Cell Expression System.

DHHC enzymes are a family of integral membrane proteins that catalyze the posttranslational addition of palmitate, a 16-carbon fatty acid, onto a cysteine residue of a protein. While the library of identified palmitoylated proteins has grown tremendously over the years, biochemical and mechanistic studies on DHHC proteins are challenged by the innate difficulty of purifying the enzyme in large amounts. Here we describe our protocol for preparing recombinant DHHC proteins tagged with a hexahistidine sequence and a FLAG epitope that aid in the purification. This procedure has been tested successfully in purifying several members of the enzyme family; DHHC3 and its catalytically inactive cysteine mutant, DHHS3 are used as examples. The recombinant protein is extracted from whole cell lysates using the detergent dodecylmaltoside (DDM) and is subjected to a two-column purification. Homogeneity and monodispersity of the purified protein are checked by size exclusion chromatography (SEC). A preparation from a 400-mL infection of Sf9 insect cell culture typically yields 0.5mg of DHHC3 and 1.0mg of catalytically inactive DHHS3. Both forms appear monodisperse up to a concentration of 1mg/mL by SEC.

Batten disease: biochemical and molecular characterization revealing novel PPT1 and TPP1 gene mutations in Indian patients

BackgroundNeuronal ceroid lipofuscinoses type I and type II (NCL1 and NCL2) also known as Batten disease are the commonly observed neurodegenerative lysosomal storage disorder caused by mutations in the PPT1 and TPP1 genes respectively. Till date, nearly 76 mutations in PPT1 and approximately 140 mutations, including large deletion/duplications, in TPP1 genes have been reported in the literature. The present study includes 34 unrelated Indian patients (12 females and 22 males) having epilepsy, visual impairment, cerebral atrophy, and cerebellar atrophy.MethodsThe biochemical investigation involved measuring the palmitoyl protein thioesterase 1 and tripeptidy peptidase l enzyme activity from the leukocytes. Based on the biochemical analysis all patients were screened for variations in either PPT1 gene or TPP1 gene using bidirectional Sanger sequencing. In cases where Sanger sequencing results was uninformative Multiplex Ligation-dependent Probe Amplification technique was employed. The online tools performed the protein homology modeling and orthologous conservation of the novel variants.ResultsOut of 34 patients analyzed, the biochemical assay confirmed 12 patients with NCL1 and 22 patients with NCL2. Molecular analysis of PPT1 gene in NCL1 patients revealed three known mutations (p.Val181Met, p.Asn110Ser, and p.Trp186Ter) and four novel variants (p.Glu178Asnfs*13, p.Pro238Leu, p.Cys45Arg, and p.Val236Gly). In the case of NCL2 patients, the TPP1 gene analysis identified seven known mutations and eight novel variants. Overall these 15 variants comprised seven missense variants (p.Met345Leu, p.Arg339Trp, p.Arg339Gln, p.Arg206Cys, p.Asn286Ser, p.Arg152Ser, p.Tyr459Ser), four frameshift variants (p.Ser62Argfs*19, p.Ser153Profs*19, p.Phe230Serfs*28, p.Ile484Aspfs*7), three nonsense variants (p.Phe516*, p.Arg208*, p.Tyr157*) and one intronic variant (g.2023_2024insT). No large deletion/duplication was identified in three NCL1 patients where Sanger sequencing study was normal.ConclusionThe given study reports 34 patients with Batten disease. In addition, the study contributes four novel variants to the spectrum of PPT1 gene mutations and eight novel variants to the TPP1 gene mutation data. The novel pathogenic variant p.Pro238Leu occurred most commonly in the NCL1 cohort while the occurrence of a known pathogenic mutation p.Arg206Cys dominated in the NCL2 cohort. This study provides an insight into the molecular pathology of NCL1 and NCL2 disease for Indian origin patients.

Nutrient-Dependent Changes of Protein Palmitoylation: Impact on Nuclear Enzymes and Regulation of Gene Expression.

Diet is the main environmental stimulus chronically impinging on the organism throughout the entire life. Nutrients impact cells via a plethora of mechanisms including the regulation of both protein post-translational modifications and gene expression. Palmitoylation is the most-studied protein lipidation, which consists of the attachment of a molecule of palmitic acid to residues of proteins. S-palmitoylation is a reversible cysteine modification finely regulated by palmitoyl-transferases and acyl-thioesterases that is involved in the regulation of protein trafficking and activity. Recently, several studies have demonstrated that diet-dependent molecules such as insulin and fatty acids may affect protein palmitoylation. Here, we examine the role of protein palmitoylation on the regulation of gene expression focusing on the impact of this modification on the activity of chromatin remodeler enzymes, transcription factors, and nuclear proteins. We also discuss how this physiological phenomenon may represent a pivotal mechanism underlying the impact of diet and nutrient-dependent signals on human diseases.

Proteomic Analysis of S-Palmitoylated Proteins in Ocular Lens Reveals Palmitoylation of AQP5 and MP20.

PurposeThe purpose of this study was to characterize the palmitoyl-proteome in lens fiber cells. S-palmitoylation is the most common form of protein S-acylation and the reversible nature of this modification functions as a molecular switch to regulate many biological processes. This modification could play important roles in regulating protein functions and protein–protein interactions in the lens.MethodsThe palmitoyl-proteome of bovine lens fiber cells was investigated by combining acyl-biotin exchange (ABE) chemistry and mass-spectrometry analysis. Due to the possibility of false-positive results from ABE experiment, a method was also developed for direct detection of palmitoylated peptides by mass spectrometry for validating palmitoylation of lens proteins MP20 and AQP5. Palmitoylation levels on AQP5 in different regions of the lens were quantified after iodoacetamide (IAA)-palmitate exchange.ResultsThe ABE experiment identified 174 potential palmitoylated proteins. These proteins include 39 well-characterized palmitoylated proteins, 92 previously reported palmitoylated proteins in other tissues, and 43 newly identified potential palmitoylated proteins including two important transmembrane proteins in the lens, AQP5 and MP20. Further analysis by direct detection of palmitoylated peptides confirmed palmitoylation of AQP5 on C6 and palmitoylation of MP20 on C159. Palmitoylation of AQP5 was found to only occur in a narrow region of the inner lens cortex and does not occur in the lens epithelium, in the lens outer cortex, or in the lens nucleus.ConclusionsAQP5 and MP20 are among 174 palmitoylated proteins found in bovine lens fiber cells. This modification to AQP5 and MP20 may play a role in their translocation from the cytoplasm to cell membranes during fiber cell differentiation.