The Deubiquitinating Enzyme USP10 Regulates the Post-endocytic Sorting of Cystic Fibrosis Transmembrane Conductance Regulator in Airway Epithelial Cells

The cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC transporter superfamily, is a cyclic AMP-regulated chloride channel and a regulator of other ion channels and transporters. In epithelial cells CFTR is rapidly endocytosed from the apical plasma membrane and efficiently recycles back to the plasma membrane. Because ubiquitination targets endocytosed CFTR for degradation in the lysosome, deubiquitinating enzymes (DUBs) are likely to facilitate CFTR recycling. Accordingly, the aim of this study was to identify DUBs that regulate the post-endocytic sorting of CFTR. Using an activity-based chemical screen to identify active DUBs in human airway epithelial cells, we demonstrated that Ubiquitin Specific Protease-10 (USP10) is located in early endosomes and regulates the deubiquitination of CFTR and its trafficking in the post-endocytic compartment. small interference RNA-mediated knockdown of USP10 increased the amount of ubiquitinated CFTR and its degradation in lysosomes, and reduced both apical membrane CFTR and CFTR-mediated chloride secretion. Moreover, a dominant negative USP10 (USP10-C424A) increased the amount of ubiquitinated CFTR and its degradation, whereas overexpression of wt-USP10 decreased the amount of ubiquitinated CFTR and increased the abundance of CFTR. These studies demonstrate a novel function for USP10 in facilitating the deubiquitination of CFTR in early endosomes and thereby enhancing the endocytic recycling of CFTR.The endocytosis, endocytic recycling, and endosomal sorting of numerous transport proteins and receptors are regulated by ubiquitination (1–6). Ubiquitin, an 8-kDa protein, is conjugated to target proteins via a series of steps that includes ubiquitin-activating enzymes (E1),² ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3) (1). Proteins that are ubiquitinated in the plasma membrane are internalized and are either deubiquitinated and recycle back to the plasma membrane or, via interactions with the endosomal sorting complexes required for transport machinery, are delivered to the lysosome for degradation (1–7). Sorting of ubiquitinated plasma membrane proteins for either the lysosomal pathway or for the recycling pathway is regulated, in part, by the removal of ubiquitin by deubiquitinating enzymes (DUBs) (1–6). Thus, the balance between ubiquitination and deubiquitination regulates the plasma membrane abundance of several membrane proteins, including the epithelial sodium channel (ENaC), the epidermal growth factor receptor, the transforming growth factor-β receptor, and the cytokine receptor γ-c (8–14).CFTR is rapidly endocytosed from the plasma membrane and undergoes rapid and efficient recycling back to the plasma membrane in human airway epithelial cells, with >75% of endocytosed wild-type CFTR recycling back to the plasma membrane (15–18). A study published several years ago demonstrated that, although ubiquitination did not regulate CFTR endocytosis, ubiquitination reduced the plasma membrane abundance of CFTR in BHK cells by redirecting CFTR from recycling endosomes to lysosomes for degradation (19). However, neither the E3 ubiquitin ligase(s) responsible for the ubiquitination of CFTR nor the DUB(s) responsible for the deubiquitination of CFTR in the endocytic pathway have been identified in any cell type. Moreover, the effect of the ubiquitin status of CFTR on its endocytic sorting in human airway epithelial cells has not been reported. Thus, the goals of this study were to determine if the ubiquitin status regulates the post-endocytic sorting of CFTR in polarized airway epithelial cells, and to identify the DUBs that deubiquitinate CFTR.Approximately 100 DUBs have been identified in the human genome and are classified into five families based on sequence similarity and mechanism of action (1–6, 20, 21). To identify DUBs that regulate the deubiquitination of CFTR from this large class of enzymes, we chose an activity-based, chemical probe screening approach developed by Dr. Hidde Ploegh (4, 21, 22). This approach utilizes a hemagglutinin (HA)-tagged ubiquitin probe engineered with a C-terminal modification incorporating a thiol-reactive group that forms an irreversible, covalent bond with active DUBs. Using this approach we demonstrated in polarized human airway epithelial cells that ubiquitin-specific protease-10 (USP10) is located in early endosomes and regulates the deubiquitination of CFTR and thus its trafficking in the post-endocytic compartment. These studies demonstrate a novel function for USP10 in promoting the deubiquitination of CFTR in early endosomes and thereby enhancing the endocytic recycling of CFTR.     

Proteomic Analyses Reveal Divergent Ubiquitylation Site Patterns in Murine Tissues

Posttranslational modifications of proteins increase the complexity of the cellular proteome and enable rapid regulation of protein functions in response to environmental changes. Protein ubiquitylation is a central regulatory posttranslational modification that controls numerous biological processes including proteasomal degradation of proteins, DNA damage repair and innate immune responses. Here we combine high-resolution mass spectrometry with single-step immunoenrichment of di-glycine modified peptides for mapping of endogenous putative ubiquitylation sites in murine tissues. We identify more than 20,000 unique ubiquitylation sites on proteins involved in diverse biological processes. Our data reveals that ubiquitylation regulates core signaling pathways common for each of the studied tissues. In addition, we discover that ubiquitylation regulates tissue-specific signaling networks. Many tissue-specific ubiquitylation sites were obtained from brain highlighting the complexity and unique physiology of this organ. We further demonstrate that different di-glycine-lysine-specific monoclonal antibodies exhibit sequence preferences, and that their complementary use increases the depth of ubiquitylation site analysis, thereby providing a more unbiased view of protein ubiquitylation.Ubiquitin is a small 76-amino-acid protein that is conjugated to the ε-amino group of lysines in a highly orchestrated enzymatic cascade involving ubiquitin activating (E1), ubiquitin conjugating (E2), and ubiquitin ligase (E3) enzymes (1). Ubiquitylation is involved in the regulation of diverse cellular processes including protein degradation (2, 3, 4), DNA damage repair (5, 6), DNA replication (7), cell surface receptor endocytosis, and innate immune signaling (8, 9). Deregulation of protein ubiquitylation is implicated in the development of cancer and neurodegenerative diseases (10, 11). Inhibitors targeting the ubiquitin proteasome system are used in the treatment of hematologic malignancies such as multiple myeloma (12, 13).Recent developments in the mass spectrometry (MS)-based proteomics have greatly expedited proteome-wide analysis of posttranslational modifications (PTMs) (14–17). Large-scale mapping of ubiquitylation sites by mass spectrometry is based on the identification of the di-glycine remnant that results from trypsin digestion of ubiquitylated proteins and remains attached to ubiquitylated lysines (18). Recently, two monoclonal antibodies were developed that specifically recognize di-glycine remnant modified peptides enabling their efficient enrichment from complex peptide mixtures (19, 20). These antibodies have been used to identify thousands of endogenous ubiquitylation sites in human cells, and to quantify site-specific changes in ubiquitylation in response to different cellular perturbations (20–22). It should be noted that the di-glycine remnant is not specific for proteins modified by ubiquitin but also proteins modified by NEDD8 or ISG15 generate an identical di-glycine remnant on modified lysines making it impossible to distinguish between these modifications by mass spectrometry. However, expression of NEDD8 in mouse tissues was shown to be developmentally down-regulated (23), and ISG15 expression in bovine tissues is low in the absence of interferon stimulation (24). In cell culture experiments it was shown that a great majority of sites identified using di-glycine-lysine-specific antibodies stems from ubiquitylated peptides (20).The rates of cell proliferation and protein turnover in mammals vary dramatically between different tissues. Immortalized cell lines, often derived from cancer, are selected for high proliferation rates and fail to represent the complex conditions in tissues. Tissue proteomics can help to gain a more comprehensive understanding of physiological processes in multicellular organisms. Analysis of tissue proteome and PTMs can provide important insights into tissue-specific processes and signaling networks that regulate these processes (25–32). In addition, development of mass spectrometric methods for analysis of PTMs in diseased tissues might lead to the identification of biomarkers.In this study, we combined high-resolution mass spectrometry with immunoenrichment of di-glycine modified peptides to investigate endogenous ubiquitylation sites in murine tissues. We identified more than 20,000 ubiquitylation sites from five different murine tissues and report the largest ubiquitylation dataset obtained from mammalian tissues to date. Furthermore, we compared the performance of the two monoclonal di-glycine-lysine-specific antibodies available for enrichment of ubiquitylated peptides, and reveal their relative preferences for different amino acids flanking ubiquitylation sites.     

Protein N-terminal Acetyltransferases Act as N-terminal Propionyltransferases In Vitro and In Vivo

N-terminal acetylation (Nt-acetylation) is a highly abundant protein modification in eukaryotes catalyzed by N-terminal acetyltransferases (NATs), which transfer an acetyl group from acetyl coenzyme A to the alpha amino group of a nascent polypeptide. Nt-acetylation has emerged as an important protein modifier, steering protein degradation, protein complex formation and protein localization. Very recently, it was reported that some human proteins could carry a propionyl group at their N-terminus. Here, we investigated the generality of N-terminal propionylation by analyzing its proteome-wide occurrence in yeast and we identified 10 unique in vivo Nt-propionylated N-termini. Furthermore, by performing differential N-terminome analysis of a control yeast strain (yNatA), a yeast NatA deletion strain (yNatAΔ) or a yeast NatA deletion strain expressing human NatA (hNatA), we were able to demonstrate that in vivo Nt-propionylation of several proteins, displaying a NatA type substrate specificity profile, depended on the presence of either yeast or human NatA. Furthermore, in vitro Nt-propionylation assays using synthetic peptides, propionyl coenzyme A, and either purified human NATs or immunoprecipitated human NatA, clearly demonstrated that NATs are Nt-propionyltransferases (NPTs) per se. We here demonstrate for the first time that Nt-propionylation can occur in yeast and thus is an evolutionarily conserved process, and that the NATs are multifunctional enzymes acting as NPTs in vivo and in vitro, in addition to their main role as NATs, and their potential function as lysine acetyltransferases (KATs) and noncatalytic regulators.Modifications greatly increases a cell''s proteome diversity confined by the natural amino acids. As more than 80% of human proteins, more than 70% of plant and fly proteins and more than 60% of yeast proteins are N-terminally acetylated (Nt-acetylated),¹ this modification represents one of the most common protein modifications in eukaryotes (1–5). Recent studies have pointed to distinct functional consequences of Nt-acetylation (6): creating degradation signals recognized by a ubiquitin ligase of a new branch of the N-end rule pathway (7), preventing translocation across the endoplasmic reticulum membrane (8), and mediating protein complex formation (9). Nt-acetylation further appears to be essential for life in higher eukaryotes; for instance, a mutation in the major human N-terminal acetyltransferase (NAT), hNatA, was recently shown to be the cause of Ogden syndrome by which male infants are underdeveloped and die at infancy (10). Unlike lysine acetylation, Nt-acetylation is considered an irreversible process, and further, to mainly occur on the ribosome during protein synthesis (11–15). In yeast and humans, three NAT complexes are responsible for the majority of Nt-acetylation; NatA, NatB and NatC, each of which has a defined substrate specificity (16). NatA acetylates Ser-, Ala-, Gly-, Thr-, Val- and Cys- N-termini generated on removal of the initiator methionine (iMet) (1, 17–19). NatB and NatC acetylate N-termini in which the iMet is followed by an acidic (20–23) or a hydrophobic residue respectively (24–26). Naa40p/NatD was shown to acetylate the Ser-starting N-termini of histones H2A and H4 (27, 28). NatE, composed of the catalytic Naa50p (Nat5p) has substrate specificity toward iMet succeeded by a hydrophobic amino acid (29, 30). As largely the same Nt-acetylation patterns are found in yeast and humans, it was believed that the NAT-machineries were conserved in general (31). However, the recently discovered higher eukaryotic specific NAT, Naa60p/NatF, was found to display a partially distinct substrate specificity in part explaining the higher degree of Nt-acetylation in higher versus lower eukaryotes (4).Human NatA is composed of two main subunits: the catalytic subunit hNaa10p and the auxiliary subunit, hNaa15p that is presumably responsible for anchoring the complex to the ribosome (14, 19). The chaperone-like HYPK protein is also stably associated with the NatA subunits and may be essential for efficient NatA activity (32). In addition, hNaa50p was shown to be physically associated with hNatA, however it is believed not to affect NatA activity (14, 33, 34). hNaa50p was also shown to exhibit N^ε-acetyltransferase (KAT) activity (29), however, the structure of hNaa50p with its peptide substrate bound strongly indicates that the peptide binding pocket is specifically suited to accommodate N-terminal peptides, as opposed to lysine residues (35). The human NatA subunits are associated with ribosomes, but interestingly, significant fractions are also nonribosomal (19, 30, 32). Of further notice, the catalytic subunits, hNaa10p and hNaa50p, were also found to partially act independently of the hNatA complex (30, 36).Recent studies have identified novel in vivo acyl modifications of proteins. Mass spectrometry data of affinity-enriched acetyllysine-containing peptides from HeLa cells showed the presence of propionylated and butyrylated lysines in histone H4 peptides (37). Similar analyses also showed the presence of propionylated lysines in p53, p300 and CREB-binding protein (38) besides the yeast histones H2B, H3 and H4 (39). Propionylated or butyrylated residues differ by only one or two extra methyl moieties as compared with their acetylated counterparts, thereby adding more hydrophobicity and bulkiness to the affected residue. To date, no distinct propionyl- or butyryltransferases responsible for these modifications have been identified. However, by using propionyl coenzyme A (Prop-CoA) or butyryl coenzyme A (But-CoA) as donors in the enzyme reaction, it was shown that some of the previously characterized lysine acetyltransferases (KATs) are able to respectively catalyze propionylation and butyrylation of lysine residues both in vitro (37, 40–42) and in vivo (38, 41). Similarly, it has been shown that lysine deacetylases also are capable of catalyzing depropionylation (40, 41, 43, 44) and debutyrylation (44) (see review (45)).Interestingly, mass spectrometry data also suggested that propionylated N-termini are present in human cell lines (46, 47). Until today, an N-terminal propionyl transferase (NPT) catalyzing N-terminal propionylation (Nt-propionylation) has to our knowledge not been identified.In this study, we hypothesized that NATs might have the ability to act as NPTs. In vitro experiments using purified hNaa10p, hNaa50p or immunoprecipitated human NatA complex indeed confirmed their intrinsic capacity to catalyze Nt-propionylation toward synthetic peptides. NatA was also found capable of Nt-butyrylation in vitro. By means of N-terminomics, we further investigated the presence of yeast Nt-propionylated proteins in vivo. Indeed, we found evidence for Nt-propionylation being a naturally occurring modification in yeast. Interestingly, in a yeast strain lacking NatA, we observed a loss in Nt-propionylation and Nt-acetylation for several NatA substrates, as compared with a control yeast strain expressing endogenous NatA or a strain ectopically expressing hNatA. Thus, besides acting as NATs, yeast and human NatA can act as NPTs and we thus demonstrate for the first time that NATs have the capacity of both acetylating and propionylating protein N-termini in vivo and in vitro.