Proteomic profiling of S-acylated macrophage proteins identifies a role for palmitoylation in mitochondrial targeting of phospholipid scramblase 3
ABSTRACT S-Palmitoylation, the reversible post-translational acylation of specific cysteine residues with the fatty acid palmitate, promotes the membrane tethering and subcellular localization of proteins in several biological pathways. Although inhibiting palmitoylation holds promise as a means for manipulating protein targeting, advances in the field have been hampered by limited understanding of palmitoylation enzymology and consensus motifs. In order to define the complement of S-acylated proteins in the macrophage, we treated RAW 264.7 macrophage membranes with hydroxylamine to cleave acyl thioesters, followed by biotinylation of newly exposed sulfhydryls and streptavidin-agarose affinity chromatography. Among proteins identified by LC-MS/MS, S-acylation status was established by spectral counting to assess enrichment under hydroxylamine versus mock treatment conditions. Of 1183 proteins identified in four independent experiments, 80 proteins were significant for S-acylation at false discovery rate = 0.05, and 101 significant at false discovery rate = 0.10. Candidate S-acylproteins were identified from several functional categories, including membrane trafficking, signaling, transporters, and receptors. Among these were 29 proteins previously biochemically confirmed as palmitoylated, 45 previously reported as putative S-acylproteins in proteomic screens, 24 not previously associated with palmitoylation, and three presumed false-positives. Nearly half of the candidates were previously identified by us in macrophage detergent-resistant membranes, suggesting that palmitoylation promotes lipid raft-localization of proteins in the macrophage. Among the candidate novel S-acylproteins was phospholipid scramblase 3 (Plscr3), a protein that regulates apoptosis through remodeling the mitochondrial membrane. Palmitoylation of Plscr3 was confirmed through (3)H-palmitate labeling. Moreover, site-directed mutagenesis of a cluster of five cysteines (Cys159-161-163-164-166) abolished palmitoylation, caused Plscr3 mislocalization from mitochondrion to nucleus, and reduced macrophage apoptosis in response to etoposide, together suggesting a role for palmitoylation at this site for mitochondrial targeting and pro-apoptotic function of Plscr3. Taken together, we propose that manipulation of protein palmitoylation carries great potential for intervention in macrophage biology via reprogramming of protein localization.
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- "It remained to be clarified whether suppression of exosomal secretion of Scr3 by 2-BP was due to inhibition of palmitoylation of either Scr3 itself or other palmitoylated proteins. To address this question, as shown in Figure 5(A), we constructed expression plasmids of untagged Scr3 mutants in which either three or four cysteine residues were substituted with alanine (Scr3_3CA or Scr3_4CA) in the corresponding sequence of the previously determined palmitoylation site of murine Scr3 . As shown in Figure 5(B), the WT Scr3 protein was consistently detected by WB in the exosomal fraction, but the amount of secreted Scr3_3CA protein markedly decreased, and secretion of the Scr3_4CA protein was barely detectable as in the case of the negative control (ctrl) in which an empty vector was used for transfection. "
ABSTRACT: Phospholipid scramblases (PLSCRs) are palmitoylated membrane-associating proteins. Regardless of the given names, their physiological functions are not clear and thought to be unrelated to phospholipid scrambling activities observed in vitro. Using a previously established cell line of HEK293 cells constitutively expressing human Scr3 (PLSCR3) that interacts with ALG-2 Ca2+-dependently, we found that Scr3 was secreted into the culture medium. Secretion of Scr3 was suppressed by 2-bromopalmitate (a palmitoylation inhibitor) and by GW4869 (an inhibitor of ceramide synthesis). Secreted Scr3 was recovered in exosomal fractions by sucrose density gradient centrifugation. Palmitoylation sites and the N-terminal Pro-rich region were necessary for efficient secretion, but ALG-2-binding sites were dispensable. Overexpression of GFP-fused VPS4BE235Q, a dominant negative mutant of an AAA ATPase with a defect in disassembling ESCRT (endosomal sorting complex required for transport)-III subunits, significantly reduced secretion of Scr3. Immunofluorescence microscopic analyses showed that Scr3 was largely localised to enlarged endosomes induced by overexpression of a GFP-fused constitutive active mutant of Rab5A (GFP-Rab5AQ79L). Secreted Scr3 was taken up by HeLa cells, suggesting that Scr3 functions as a cell-to-cell transferable modulator carried by exosomes in a paracrine manner.Bioscience Reports 01/2013; 33(2). DOI:10.1042/BSR20120123 · 2.85 Impact Factor
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- "Kang et al. extended this method to study whole rat brain, embryonic rat neurons or rat synaptosomes, confirming 21 novel palmitoylated proteins out of >200 candidates . Others have recently reported similar studies in african trypanosomes , human platelets , and macrophages . This approach was recently optimized to eliminate several manipulations by capturing hydroxylamine-sensitive cysteines using a thiol-reactive resin . "
ABSTRACT: S-palmitoylation is a reversible post-translational modification important for controlling the membrane targeting and function of numerous membrane proteins with diverse roles in signalling, scaffolding, and trafficking. We sought to identify novel palmitoylated proteins in B lymphocytes using acyl-biotin exchange chemistry, coupled with differential analysis by liquid-chromatography tandem mass spectrometry. In total, we identified 57 novel palmitoylated protein candidates from human EBV-transformed lymphoid cells. Two of them, namely CD20 and CD23 (low affinity immunoglobulin epsilon Fc receptor), are immune regulators that are effective/potential therapeutic targets for haematological malignancies, autoimmune diseases and allergic disorders. Palmitoylation of CD20 and CD23 was confirmed by heterologous expression of alanine mutants coupled with bioorthogonal metabolic labeling. This study demonstrates a new subset of palmitoylated proteins in B cells, illustrating the ubiquitous role of protein palmitoylation in immune regulation.PLoS ONE 05/2012; 7(5):e37187. DOI:10.1371/journal.pone.0037187 · 3.23 Impact Factor
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- "Four profiling studies (Martin and Cravatt, 2009; Yang et al, 2010; Yount et al, 2010; Merrick et al, 2011) identified, with high confidence, calnexin as an S-acylated protein in mammalian cells. By immunoprecipitation of either the endogenous or tagged calnexin from 3 H-palmitate-labelled cells, we validated these profiling studies: the protein indeed incorporated radiolabelled palmitate, which could be removed by hydroxylamine hydrochloride treatment, indicating the involvement of a thioester bond (Figure 1A). "
ABSTRACT: A third of the human genome encodes N-glycosylated proteins. These are co-translationally translocated into the lumen/membrane of the endoplasmic reticulum (ER) where they fold and assemble before they are transported to their final destination. Here, we show that calnexin, a major ER chaperone involved in glycoprotein folding is palmitoylated and that this modification is mediated by the ER palmitoyltransferase DHHC6. This modification leads to the preferential localization of calnexin to the perinuclear rough ER, at the expense of ER tubules. Moreover, palmitoylation mediates the association of calnexin with the ribosome-translocon complex (RTC) leading to the formation of a supercomplex that recruits the actin cytoskeleton, leading to further stabilization of the assembly. When formation of the calnexin-RTC supercomplex was affected by DHHC6 silencing, mutation of calnexin palmitoylation sites or actin depolymerization, folding of glycoproteins was impaired. Our findings thus show that calnexin is a stable component of the RTC in a manner that is exquisitely dependent on its palmitoylation status. This association is essential for the chaperone to capture its client proteins as they emerge from the translocon, acquire their N-linked glycans and initiate folding.The EMBO Journal 02/2012; 31(7):1823-35. DOI:10.1038/emboj.2012.15 · 10.75 Impact Factor