Ubiquitination Regulates Proteolytic Processing of G Protein-coupled Receptors after Their Sorting to Lysosomes

Ubiquitination is essential for the endocytic sorting of various G protein-coupled receptors to lysosomes. Here we identify a distinct function of this covalent modification in controlling the later proteolytic processing of receptors. Mutation of all cytoplasmic lysine residues in the murine δ-opioid receptor blocked receptor ubiquitination without preventing ligand-induced endocytosis of receptors or their subsequent delivery to lysosomes, as verified by proteolysis of extramembrane epitope tags and down-regulation of radioligand binding to the transmembrane helices. Surprisingly, a functional screen revealed that the E3 ubiquitin ligase AIP4 specifically controls down-regulation of wild type receptors measured by radioligand binding without detectably affecting receptor delivery to lysosomes defined both immunochemically and biochemically. This specific AIP4-dependent regulation required direct ubiquitination of receptors and was also regulated by two deubiquitinating enzymes, AMSH and UBPY, which localized to late endosome/lysosome membranes containing internalized δ-opioid receptor. These results identify a distinct function of AIP4-dependent ubiquitination in controlling the later proteolytic processing of G protein-coupled receptors, without detectably affecting their endocytic sorting to lysosomes. We propose that ubiquitination or ubiquitination/deubiquitination cycling specifically regulates later proteolytic processing events required for destruction of the receptor''s hydrophobic core.A fundamental cellular mechanism contributing to homeostatic regulation of receptor-mediated signal transduction involves ligand-induced endocytosis of receptors followed by proteolysis in lysosomes. The importance of such proteolytic down-regulation has been documented extensively for a number of seven-transmembrane or G protein-coupled receptors (GPCRs),³ which comprise the largest known family of signaling receptors expressed in animals, as well as for other important signaling receptors, such as the epidermal growth factor receptor tyrosine kinase (1–5).One GPCR that is well known to undergo endocytic trafficking to lysosomes is the δ-opioid peptide receptor (DOR or DOP-R) (6). Following endocytosis, DOR traffics efficiently to lysosomes in both neural and heterologous cell models (6–8), whereas many membrane proteins, including various GPCRs, recycle rapidly to the plasma membrane (9–12). Such molecular sorting of internalized receptors between divergent recycling and degradative pathways is thought to play a fundamental role in determining the functional consequences of regulated endocytosis (2, 3, 13, 14). The sorting process that directs internalized DOR to lysosomes is remarkably efficient and appears to occur rapidly (within several min) after receptor endocytosis (11). Nevertheless, biochemical mechanisms that control lysosomal trafficking and proteolysis of DOR remain poorly understood.A conserved mechanism that promotes lysosomal trafficking of a number of membrane proteins, including various signaling receptors, is mediated by covalent modification of cytoplasmic lysine residues with ubiquitin (4, 15–17). Ubiquitination was first identified as an endocytic sorting determinant in studies of vacuolar trafficking of the yeast GPCR Ste2p (18). Subsequent studies have established numerous examples of lysyl-ubiquitination being required for sorting endocytic cargo to lysosomes and have identified conserved machinery responsible for the targeting of ubiquitinated cargo to lysosomes (3, 17, 19–22).The CXCR4 chemokine receptor provides a clear example of ubiquitin-dependent lysosomal sorting of a mammalian GPCR. Ubiquitination of the carboxyl-terminal cytoplasmic domain of the CXCR4 receptor, mediated by the E3 ubiquitin ligase AIP4, is specifically required for the HRS- and VPS4-dependent trafficking of internalized receptors to lysosomes. Blocking this ubiquitination event by Lys → Arg mutation of the receptor specifically inhibits trafficking of internalized receptors to lysosomes, resulting in recycling rather than lysosomal proteolysis of receptors after ligand-induced endocytosis (23–25).Lysosomal trafficking of DOR, in contrast, is not prevented by mutation of cytoplasmic lysine residues (26) and can be regulated by ubiquitination-independent protein interaction(s) (27, 28). Nevertheless, both wild type and lysyl-mutant DORs traffic to lysosomes via a similar pathway as ubiquitin-dependent membrane cargo and require both HRS and active VPS4 to do so (29). These observations indicate that DOR engages the same core endocytic mechanism utilized by ubiquitination-directed membrane cargo but leave unresolved whether ubiquitination of DOR plays any role in this important cellular mechanism of receptor down-regulation.There is no doubt that DOR can undergo significant ubiquitination in mammalian cells, including HEK293 cells (30–32), where lysosomal trafficking of lysyl-mutant receptors was first observed (26). Ubiquitination was shown previously to promote proteolysis of DOR by proteasomes and to function in degrading misfolded receptors from the biosynthetic pathway (30, 31). A specific role of ubiquitination in promoting proteasome- but not lysosome-mediated proteolysis of DOR has been emphasized (32) and proposed to contribute to proteolytic down-regulation of receptors also from the plasma membrane (33).To our knowledge, no previous studies have determined if DOR ubiquitination plays any role in controlling receptor proteolysis mediated by lysosomes, although this represents a predominant pathway by which receptors undergo rapid down-regulation following ligand-induced endocytosis in a number of cell types, including HEK293 cells (8). In the present study, we have taken two approaches to addressing this fundamental question. First, we have investigated in greater detail the effects of lysyl-mutation on DOR ubiquitination and trafficking. Second, we have independently investigated the role of ubiquitination in controlling lysosomal proteolysis of wild type DOR. Our results clearly establish the ability of DOR to traffic efficiently to lysosomes in the absence of any detectable ubiquitination. Further, they identify a distinct and unanticipated function of AIP4-dependent ubiquitination in regulating the later proteolytic processing of receptors and show that this distinct ubiquitin-dependent regulatory mechanism operates effectively downstream of the sorting decision that commits internalized receptors for delivery to lysosomes.     

A Novel Strategy to Isolate Ubiquitin Conjugates Reveals Wide Role for Ubiquitination during Neural Development

Ubiquitination has essential roles in neuronal development and function. Ubiquitin proteomics studies on yeast and HeLa cells have proven very informative, but there still is a gap regarding neuronal tissue-specific ubiquitination. In an organism context, direct evidence for the ubiquitination of neuronal proteins is even scarcer. Here, we report a novel proteomics strategy based on the in vivo biotinylation of ubiquitin to isolate ubiquitin conjugates from the neurons of Drosophila melanogaster embryos. We confidently identified 48 neuronal ubiquitin substrates, none of which was yet known to be ubiquitinated. Earlier proteomics and biochemical studies in non-neuronal cell types had identified orthologs to some of those but not to others. The identification here of novel ubiquitin substrates, those with no known ubiquitinated ortholog, suggests that proteomics studies must be performed on neuronal cells to identify ubiquitination pathways not shared by other cell types. Importantly, several of those newly found neuronal ubiquitin substrates are key players in synaptogenesis. Mass spectrometry results were validated by Western blotting to confirm that those proteins are indeed ubiquitinated in the Drosophila embryonic nervous system and to elucidate whether they are mono- or polyubiquitinated. In addition to the ubiquitin substrates, we also identified the ubiquitin carriers that are active during synaptogenesis. Identifying endogenously ubiquitinated proteins in specific cell types, at specific developmental stages, and within the context of a living organism will allow understanding how the tissue-specific function of those proteins is regulated by the ubiquitin system.Posttranslational modification of proteins by ubiquitin is involved in a wide range of cellular processes (1). Ubiquitination is linked to the turnover of an ever growing number of proteins; it regulates protein trafficking and is also widely used to transiently facilitate protein-protein interactions (2, 3). As the number of known ubiquitinated proteins keeps growing, the focus is turning toward identifying when, where, and how those proteins are ubiquitinated in vivo with the aim of understanding how protein function is being regulated within the context of a whole organism. The ubiquitin pathway is essential for brain development and function, and its failure is associated with a number of neurodegenerative diseases, including Parkinson and Alzheimer diseases (4–6). Ubiquitin conjugation is carried out by the sequential action of ubiquitin-activating (E1), -conjugating (E2), and -ligating (E3) enzymes and can be reversed by deubiquitinating enzyme (DUB)1 proteases. The involvement of a number of those enzymes in synaptogenesis has been documented in several model systems (7–12). In Drosophila, for example, synaptogenesis is dependent on the E3 ligase Highwire and on the DUB fat facets (13). A few proteins involved in synaptogenesis have been shown to be ubiquitin substrates, including the postsynaptic proteins Shank, GKAP, and AKAP79/150 in cultured neurons (14) and the Caenorhabditis elegans synaptic protein DLK-1 kinase, which was shown to be ubiquitinated when overexpressed in HEK293T kidney cells (9). Most neuronal targets of the ubiquitin pathway, however, remain undiscovered. Yeast and HeLa cell-based proteomics approaches have failed to provide significant insights into the neuronal mechanisms regulated by ubiquitination. With the exception of a polyubiquitin affinity-based purification that successfully identified by Western blotting three ubiquitin substrates in cultured neurons (14), no proteomics approach has been described that can identify ubiquitinated neuronal proteins. Because neuronal function and activity are highly context-dependent, rather than working on neuronal culture, we have aimed to identify which proteins are ubiquitinated in vivo within the neurons of a living organism.Herein, we describe a novel strategy for the efficient isolation of neuronal ubiquitin conjugates from flies. The approach is based on the in vivo biotinylation of ubiquitin by ectopically expressing the Escherichia coli BirA enzyme to attach a biotin molecule to a specific BirA recognition sequence (15, 16) added at the N terminus of each ubiquitin chain. With the purpose of isolating ubiquitin conjugates uniquely from the nervous system of Drosophila melanogaster, we used the GAL4/UAS system for tissue-targeted expression (17). To increase the biotinylation efficiency, we took advantage of the processing activity of endogenous DUBs to digest a linear polypeptide precursor containing six copies of the tagged ubiquitin and the BirA enzyme, which are then present in the same cellular microenvironment. Because of the strength and the specificity of the avidin-biotin interaction, we were able to isolate and enrich the neuronal ubiquitinated proteins from a multicellular organism up to levels not achieved previously by any other approach. This allowed us to identify by mass spectrometry those neuronal proteins that are ubiquitinated and to resolve by Western blotting whether they are mono- or polyubiquitinated. This was achieved in the absence of proteasome inhibitors; therefore, physiological ubiquitination levels are reported. We focused on identifying the proteins that are ubiquitinated within the neurons in the period from neurite outgrowth and axonal pathfinding to target recognition and synapse formation (18). For that purpose, we applied our strategy on postmitotic neurons during embryonic stages 13–17 (19), a 12-h period during which embryos undergo synaptogenesis. Our strategy could be used to isolate ubiquitin conjugates from other tissues from the fruit flies, from different developmental stages, and in different mutant backgrounds, and it is likely to be applicable to other model organisms.     

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.     

Occludin Phosphorylation and Ubiquitination Regulate Tight Junction Trafficking and Vascular Endothelial Growth Factor-induced Permeability

Vascular endothelial growth factor (VEGF) alters tight junctions (TJs) and promotes vascular permeability in many retinal and brain diseases. However, the molecular mechanisms of barrier regulation are poorly understood. Here we demonstrate that occludin phosphorylation and ubiquitination regulate VEGF-induced TJ protein trafficking and concomitant vascular permeability. VEGF treatment induced TJ fragmentation and occludin trafficking from the cell border to early and late endosomes, concomitant with increased occludin phosphorylation on Ser-490 and ubiquitination. Furthermore, both co-immunoprecipitation and immunocytochemistry demonstrated that VEGF treatment increased the interaction between occludin and modulators of intracellular trafficking that contain the ubiquitin interacting motif, including Epsin-1, epidermal growth factor receptor pathway substrate 15 (Eps15), and hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs). Inhibiting occludin phosphorylation by mutating Ser-490 to Ala suppressed VEGF-induced ubiquitination, inhibited trafficking of TJ proteins, and prevented the increase in endothelial permeability. In addition, an occludin-ubiquitin chimera disrupted TJs and increased permeability without VEGF. These data demonstrate a novel mechanism of VEGF-induced occludin phosphorylation and ubiquitination that contributes to TJ trafficking and subsequent vascular permeability.Under normal physiological conditions the blood-brain barrier and blood-retinal barrier regulate the transport of water, ions, amino acids, and waste products, between the neural parenchyma and blood (1). A high degree of well developed tight junctions (TJs)² in the vascular endothelium, in association with adherens junctions, contribute to both the blood-brain and blood-retinal barriers (2). Accumulating evidence suggests that a number of pathological eye diseases such as diabetes, retinopathy of prematurity, age-related macular degeneration, inflammation, and infectious diseases disrupt the TJs altering the blood-retinal barrier. Common mediators of vascular permeability and TJ deregulation are growth factors and cytokines that may induce macular edema and lead to loss of vision (1). Vascular endothelial growth factor (VEGF), in particular, induces vascular permeability and stimulates angiogenesis, contributing to disease pathogenesis in diabetic retinopathy and retinopathy of prematurity (3). VEGF also contributes to blood-brain barrier disruption with subsequent edema and angiogenesis in brain tumors and stroke (4). Recent advances in biomedical research have provided therapeutic approaches to neutralize VEGF; however, these strategies have not yet demonstrated effective resolution of diabetic macular edema (5, 6).TJs control the paracellular flux of solutes and fluids across the blood-brain and blood-retinal barriers. Several transmembrane proteins including occludin, tricellulin, the claudin family, and junction adhesion molecules are thought to confer adhesion to the TJ barrier and to be organized by members of the zonula occludens family (ZO-1, -2, or -3) (7–9). Experimental evidence has established that the claudins confer barrier properties and claudin-5 specifically contributes to the vascular component of the blood-brain barrier demonstrated by gene deletion studies (10). In contrast, the function of occludin in paracellular flux has remained less clear. Mice with occludin gene deletion continue to form TJs in gut epithelia with normal barrier properties (11). However, studies have also demonstrated that diabetes reduces occludin content in rat retina (12) and alters its distribution from continuous cell border localization to intracellular puncta (13). These observations suggest that the intracellular trafficking of TJ proteins promotes paracellular flux and vascular permeability in diabetic animals (12, 14).VEGF was originally identified as a vascular permeability factor as well as a pro-angiogenic growth factor (15, 16). Both biological effects exacerbate the pathology of retinal vascular diseases (17), and they are mediated via intracellular signal transduction, especially based on the phosphorylation of Src, protein kinase C, and so on (18). Additionally, VEGF treatment and diabetes induce occludin phosphorylation in rat retinal vasculature and endothelial cell culture coincident with increased permeability (19). Recently, using mass spectrometry five occludin phosphorylation sites were identified in retinal endothelial cell culture after VEGF treatment (20). Among these sites, phosphorylation at Ser-490 was shown to increase in response to VEGF treatment. However, no evidence has directly demonstrated the contribution of occludin phosphorylation to VEGF-induced endothelial permeability or defined the mechanism by which phosphorylation of occludin alters paracellular flux.Modification of proteins with monomeric or polymeric ubiquitin chains contributes to control of multiple biological functions including protein degradation, intracellular trafficking, translational regulation, and DNA repair (21). Phosphorylation of receptor tyrosine kinases, such as epidermal growth factor receptor or vascular endothelial growth factor receptor-2, is followed by ubiquitination and regulated trafficking to endosomes. This endocytosis process depends on the interaction between the ubiquitinated receptors and carrier proteins that possess a ubiquitin interacting motif (UIM) such as Epsin, epidermal growth factor receptor pathway substrate 15 (Eps15), and hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) (21–24). Recent publications have demonstrated that occludin can be ubiquitinated targeting the protein for degradation through the ubiquitin-proteasome system in epithelial cell types (25, 26). Here we demonstrate that phosphorylation of occludin at Ser-490 is necessary for occludin ubiquitination in response to VEGF in endothelial cells. Furthermore, the ubiquitination promotes interaction of occludin with UIM containing modulators of trafficking and regulates the internalization of TJ proteins altering endothelial permeability. Together, these results suggest that occludin phosphorylation and subsequent ubiquitination are necessary for VEGF-induced TJ trafficking and endothelial permeability.     

Refined Preparation and Use of Anti-diglycine Remnant (K-ε-GG) Antibody Enables Routine Quantification of 10,000s of Ubiquitination Sites in Single Proteomics Experiments

Detection of endogenous ubiquitination sites by mass spectrometry has dramatically improved with the commercialization of anti-di-glycine remnant (K-ε-GG) antibodies. Here, we describe a number of improvements to the K-ε-GG enrichment workflow, including optimized antibody and peptide input requirements, antibody cross-linking, and improved off-line fractionation prior to enrichment. This refined and practical workflow enables routine identification and quantification of ∼20,000 distinct endogenous ubiquitination sites in a single SILAC experiment using moderate amounts of protein input.The commercialization of antibodies that recognize lysine residues modified with a di-glycine remnant (K-ε-GG)¹ has significantly transformed the detection of endogenous protein ubiquitination sites by mass spectrometry (1–5). Prior to the development of these highly specific reagents, proteomics experiments were limited to identification of up to only several hundred ubiquitination sites, which severely limited the scope of global ubiquitination studies (6). Recent proteomic studies employing anti-K-ε-GG antibodies have enhanced our understanding of ubiquitin biology through the identification of thousands of ubiquitination sites and the analysis of the change in relative abundance of these sites after chemical or biological perturbation (1–3, 5, 7). Use of stable isotope labeling by amino acids in cell culture (SILAC) for quantification has enabled researchers to better understand the extent of ubiquitin regulation upon proteasome inhibition and precisely identify those protein classes, such as newly synthesized proteins or chromatin-related proteins, that see overt changes in their ubiquitination levels upon drug treatment (2, 3, 5). Emanuel et al. (1) have combined genetic and proteomics assays implementing the anti-K-ε-GG antibody to identify hundreds of known and putative Cullin-RING ligase substrates, which has clearly demonstrated the extensive role of Cullin-RING ligase ubiquitination on cellular protein regulation.Despite the successes recently achieved with the use of the anti-K-ε-GG antibody, increased sample input (up to ∼35 mg) and/or the completion of numerous experimental replicates have been necessary to achieve large numbers of K-ε-GG sites (>5,000) in a single SILAC-based experiment (1–3, 5). For example, it has been recently shown that detection of more than 20,000 unique ubiquitination sites is possible from the analysis of five different murine tissues (8). However, as the authors indicate, only a few thousands sites are detected in any single analysis of an individual tissue sample (8). It is recognized that there is need for further improvements in global ubiquitin technology to increase the depth-of-coverage attainable in quantitative proteomic experiments using moderate amounts of protein input (9). Through systematic study and optimization of key pre-analytical variables in the preparation and use of the anti-K-ε-GG antibody as well as the proteomic workflow, we have now achieved, for the first time, routine quantification of ∼20,000 nonredundant K-ε-GG sites in a single SILAC triple encoded experiment starting with 5 mg of protein per SILAC channel. This represents a 10-fold improvement over our previously published method (3).     

POSH Stimulates the Ubiquitination and the Clathrin-independent Endocytosis of ROMK1 Channels

POSH (plenty of SH3) is a scaffold protein that has been shown to act as an E3 ubiquitin ligase. Here we report that POSH stimulates the ubiquitination of Kir1.1 (ROMK) and enhances the internalization of this potassium channel. Immunostaining reveals the expression of POSH in the renal cortical collecting duct. Immunoprecipitation of renal tissue lysate with ROMK antibody and glutathione S-transferase pulldown experiments demonstrated the association between ROMK and POSH. Moreover, immunoprecipitation of lysates of HEK293T cells transfected with ROMK1 or with constructs encoding the ROMK-N terminus or ROMK1-C-Terminus demonstrated that POSH binds to ROMK1 on its N terminus. To study the effect of POSH on ROMK1 channels, we measured potassium currents with electrophysiological methods in HEK293T cells and in oocytes transfected or injected with ROMK1 and POSH. POSH decreased potassium currents, and the inhibitory effect of POSH on ROMK channels was dose-dependent. Biotinylation assay further showed that POSH decreased surface expression of ROMK channels in HEK293T cells transfected with ROMK1 and POSH. The effect of POSH on ROMK1 channels was specific because POSH did not inhibit sodium current in oocytes injected with ENaC-α, β, and γ subunits. Moreover, POSH still decreased the potassium current in oocytes injected with a ROMK1 mutant (R1Δ373–378), in which a clathrin-dependent tyrosine-based internalization signal residing between amino acid residues 373 and 378 is deleted. However, the inhibitory effect of POSH on ROMK channels was absent in cells expressing with dominant negative dynamin and POSHΔRING, in which the RING domain was deleted. Expression of POSH also increased the ubiquitination of ROMK1, whereas expression of POSHΔRING diminished its ubiquitination in HEK293T cells. The notion that POSH may serve as an E3 ubiquitin ligase is also supported by in vitro ubiquitination assays in which adding POSH increased the ROMK ubiquitination. We conclude that POSH inhibits ROMK channels by enhancing dynamin-dependent and clathrin-independent endocytosis and by stimulating ubiquitination of ROMK channels.ROMK channels (Kir1.1) are located in the apical membrane of the epithelial cells of the renal thick ascending limb (TAL)² and the CCD, where they are responsible for potassium recycling across the apical membrane in the TAL and potassium secretion in the CCD (1, 2). The expression of ROMK channels in the plasma membrane in the CCD is regulated by a variety of factors including protein kinases and dietary potassium intake (3–9). For instance, with-no-lysine kinase 4 (WNK4) and Src family protein-tyrosine kinase (PTK) reduce the expression of ROMK channels in the plasma membrane by stimulating dynamin-dependent endocytosis (10, 11). Several studies have demonstrated that potassium restriction decreased, and high potassium intake increased, the ROMK channel expression in the apical membrane of CCD epithelial cells (12, 13). Although the mechanism by which dietary potassium intake regulates surface expression is not completely understood, one possible mechanism is through modulating the ubiquitination of ROMK channels. The role for ubiquitination in regulating channel surface expression and endocytosis is best demonstrated by the observation that NEDD-4, an E3 ligase that contains the HECT domain (homologous to E6-AP C-terminal), regulates the ubiquitination of epithelial sodium channels (ENaC) (14–16). It has been shown that Nedd4 binds to ENaC on a PY motif (XPPXY) and causes channel internalization (17). Nedd-4 has also been reported to be responsible for ubiquitination of channels other than ENaC (18–21). We have previously demonstrated that ROMK1 channels can be monoubiquitinated and ubiquitinated ROMK channels were subjected to endocytosis (22). However, because ROMK channels lack a PY motif, it is unlikely that Nedd4 regulates ROMK channels in this fashion. POSH is a RING (really interesting new gene)-containing scaffold protein and has been suggested to be an E3 ligase for Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and Herp (homocystein-induced ER protein), and it has been shown to play an obligate role in cellular production of the human immunodeficiency virus, type 1 virus (23–25). Thus, the aim of the present study is to test whether POSH may act as an E3 ubiquitin ligase for the ubiquitination of ROMK channels.     

CD4 Glycoprotein Degradation Induced by Human Immunodeficiency Virus Type 1 Vpu Protein Requires the Function of Proteasomes and the Ubiquitin-Conjugating Pathway

The human immunodeficiency virus type 1 (HIV-1) vpu gene encodes a type I anchored integral membrane phosphoprotein with two independent functions. First, it regulates virus release from a post-endoplasmic reticulum (ER) compartment by an ion channel activity mediated by its transmembrane anchor. Second, it induces the selective down regulation of host cell receptor proteins (CD4 and major histocompatibility complex class I molecules) in a process involving its phosphorylated cytoplasmic tail. In the present work, we show that the Vpu-induced proteolysis of nascent CD4 can be completely blocked by peptide aldehydes that act as competitive inhibitors of proteasome function and also by lactacystin, which blocks proteasome activity by covalently binding to the catalytic β subunits of proteasomes. The sensitivity of Vpu-induced CD4 degradation to proteasome inhibitors paralleled the inhibition of proteasome degradation of a model ubiquitinated substrate. Characterization of CD4-associated oligosaccharides indicated that CD4 rescued from Vpu-induced degradation by proteasome inhibitors is exported from the ER to the Golgi complex. This finding suggests that retranslocation of CD4 from the ER to the cytosol may be coupled to its proteasomal degradation. CD4 degradation mediated by Vpu does not require the ER chaperone calnexin and is dependent on an intact ubiquitin-conjugating system. This was demonstrated by inhibition of CD4 degradation (i) in cells expressing a thermally inactivated form of the ubiquitin-activating enzyme E₁ or (ii) following expression of a mutant form of ubiquitin (Lys₄₈ mutated to Arg₄₈) known to compromise ubiquitin targeting by interfering with the formation of polyubiquitin complexes. CD4 degradation was also prevented by altering the four Lys residues in its cytosolic domain to Arg, suggesting a role for ubiquitination of one or more of these residues in the process of degradation. The results clearly demonstrate a role for the cytosolic ubiquitin-proteasome pathway in the process of Vpu-induced CD4 degradation. In contrast to other viral proteins (human cytomegalovirus US2 and US11), however, whose translocation of host ER molecules into the cytosol occurs in the presence of proteasome inhibitors, Vpu-targeted CD4 remains in the ER in a transport-competent form when proteasome activity is blocked.

The human immunodeficiency virus type 1 (HIV-1)-specific accessory protein Vpu performs two distinct functions in the viral life cycle (11, 12, 29, 34, 46, 47, 50–52; reviewed in references 31 and 55): enhancement of virus particle release from the cell surface, and the selective induction of proteolysis of newly synthesized membrane proteins. Known targets for Vpu include the primary virus receptor CD4 (63, 64) and major histocompatibility complex (MHC) class I molecules (28). Vpu is an oligomeric class I integral membrane phosphoprotein (35, 48, 49) with a structurally and functionally defined domain architecture: an N-terminal transmembrane anchor and C-terminal cytoplasmic tail (20, 34, 45, 47, 50, 65). Vpu-induced degradation of endoplasmic reticulum (ER) membrane proteins involves the phosphorylated cytoplasmic tail of the protein (50), whereas the virion release function is mediated by a cation-selective ion channel activity associated with the membrane anchor (19, 31, 45, 47).CD4 is a 55-kDa class I integral membrane glycoprotein that serves as the primary coreceptor for HIV entry into cells. CD4 consists of a large lumenal domain, a transmembrane peptide, and a 38-residue cytoplasmic tail. It is expressed on the surface of a subset of T lymphocytes that recognize MHC class II-associated peptides, and it plays a pivotal role in the development and maintenance of the immune system (reviewed in reference 30). Down regulation of CD4 in HIV-1-infected cells is mediated through several independent mechanisms (reviewed in references 5 and 55): intracellular complex formation of CD4 with the HIV envelope protein gp160 (8, 14), endocytosis of cell surface CD4 induced by the HIV-1 nef gene product (1, 2), and ER degradation induced by the HIV-1 vpu gene product (63, 64).Vpu-induced degradation of CD4 is an example of ER-associated protein degradation (ERAD). ERAD is a common outcome when proteins in the secretory pathway are unable to acquire their native structure (4). Although it was thought that ERAD occurs exclusively inside membrane vesicles of the ER or other related secretory compartments, this has gained little direct experimental support. Indeed, there are several recent reports that ERAD may actually represent export of the target protein to the cytosol, where it is degraded by cytosolic proteases. It was found that in yeast, a secreted protein, prepro-α-factor (pαF), is exported from microsomes and degraded in the cytosol in a proteasome-dependent manner (36). This process was dependent on the presence of calnexin, an ER-resident molecular chaperone that interacts with N-linked oligosaccharides containing terminal glucose residues (3). In mammalian cells, two human cytomegalovirus (HCMV) proteins, US2 and US11, were found to cause the retranslocation of MHC class I molecules from the ER to the cytosol, where they are destroyed by proteasomes (61, 62). In the case of US2, class I molecules were found to associate with a protein (Sec61) present in the channel normally used to translocate newly synthesized proteins into the ER (termed the translocon), leading to the suggestion that the ERAD substrates are delivered to the cytosol by retrograde transport through the Sec61-containing pore (61). Fujita et al. (24) reported that, similar to these findings, the proteasome-specific inhibitor lactacystin (LC) partially blocked CD4 degradation in transfected HeLa cells coexpressing CD4, Vpu, and HIV-1 Env glycoproteins. In the present study, we show that Vpu-induced CD4 degradation can be completely blocked by proteasome inhibitors, does not require the ER chaperone calnexin, but requires the function of the cytosolic polyubiquitination machinery which apparently targets potential ubiquitination sites within the CD4 cytoplasmic tail. Our findings point to differences between the mechanism of Vpu-mediated CD4 degradation and ERAD processes induced by the HCMV proteins US2 and US11 (61, 62).     

Using in Vivo Biotinylated Ubiquitin to Describe a Mitotic Exit Ubiquitome from Human Cells

Mitotic division requires highly regulated morphological and biochemical changes to the cell. Upon commitment to exit mitosis, cells begin to remove mitotic regulators in a temporally and spatially controlled manner to bring about the changes that reestablish interphase. Ubiquitin-dependent pathways target these regulators to generate polyubiquitin-tagged substrates for degradation by the 26S proteasome. However, the lack of cell-based assays to investigate in vivo ubiquitination limits our knowledge of the identity of substrates of ubiquitin-mediated regulation in mitosis. Here we report an in vivo ubiquitin tagging system used in human cells that allows efficient purification of ubiquitin conjugates from synchronized cell populations. Coupling purification with mass spectrometry, we have identified a series of mitotic regulators targeted for polyubiquitination in mitotic exit. We show that some are new substrates of the anaphase-promoting complex/cyclosome and validate KIFC1 and RacGAP1/Cyk4 as two such targets involved respectively in timely mitotic spindle disassembly and cell spreading. We conclude that in vivo biotin tagging of ubiquitin can provide valuable information about the role of ubiquitin-mediated regulation in processes required for rebuilding interphase cells.Ubiquitination has emerged as a major post-translational modification determining the fate of cellular proteins. One of these fates is proteolysis, whereby the assembly of polyubiquitin chains creates signatures on target proteins that specify delivery to the 26S proteasome for proteolytic destruction. Targeted proteolysis is critical to the control of cell division. For example, the universally conserved mechanism of mitotic exit depends upon rapid proteolysis of mitotic cyclins and securins to drive the transition from mitosis to interphase. This transition is under surveillance by the spindle assembly checkpoint (SAC),¹ which controls the activity of a multi-subunit ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C) (1, 2).Much of the known specificity in the ubiquitin-proteasome system (UPS) is mediated at the level of substrate targeting by ubiquitin ligase (E3) enzymes, of which there are more than 600 in human cells. Given these facts, it is perhaps surprising that the APC/C is almost the only known engineer of the protein landscape after anaphase onset, targeting mitotic regulators for destruction with high temporal specificity (2–4). Some roles for nondegradative ubiquitination in regulating the localization of mitotic kinases Aurora B and Plk1 have been described (5–9), and a growing list of reported ubiquitin interactors can modulate ubiquitin-dependent events during mitosis (10). However, the majority of ubiquitination events that have so far been described as occurring at the transition from mitosis to interphase are APC/C-dependent.Two co-activator subunits, Cdc20 and Cdh1, play vital roles in APC/C-dependent substrate recognition (11) by recognizing two widely characterized degrons, the D-box and the KEN motif (12, 13). Computational approaches that have been used to calculate the total number of APC/C substrates from the prevalence of degrons in the human proteome estimate that there are between 100 and 200 substrates (14), and experiments using in vitro ubiquitination of protein arrays have given rise to estimates in the same range (15). Most of the mitotic regulators targeted by the APC/C during mitotic exit in human cells have been identified via in vitro degradation assays or ubiquitination assays on in vitro–expressed pools of substrates (15–18). These approaches have identified several important substrates, but in the absence of in vivo parameters they may not identify substrates whose targeting depends on post-translational modifications or substrates that are only recognized in vivo as components of higher-order complexes. Not all substrates identified in this way have been validated as polyubiquitinated proteins in vivo. Multiple recent proteomic studies have identified large numbers of in vivo ubiquitin-modified sites from yeast (19–21) and human cells (22–29). None of these studies have used synchronized cell populations to provide information on the timing or regulation of substrate ubiquitination.We reasoned that a better view of ubiquitin-mediated processes that regulate mitotic exit would come from identifying proteins that are ubiquitinated in vivo during mitotic exit. With this goal in mind we adopted a system for in vivo tagging of ubiquitin chains with biotin, previously used to identify ubiquitin-conjugated proteins from the Drosophila neural system (30), and applied it to a human cell line (U2OS) that can be tightly synchronized at mitosis. In contrast to several recent studies that employed antibodies specific to the diGly-Lys remnant that marks ubiquitination sites following trypsin digestion (19, 25), an in vivo ubiquitin tagging strategy allows direct validation of candidate ubiquitinated proteins (whether mono- or polyubiquitinated) through immunoblotting of samples. Moreover, in contrast to other methods for affinity tagging of ubiquitin, or affinity purification via ubiquitin-binding domains, the use of the biotin tag enables purification under highly denaturing conditions for stringent isolation of ubiquitin-conjugated material from higher eukaryotes. His₆-tagged ubiquitin is also available for use under denaturing conditions, but it is not generally useful in higher eukaryotic cells, where a high frequency of proteins containing multiple histidine residues confounds the specificity of nickel-affinity pulldowns (as discussed in detail in Ref. 30). Therefore, in this paper we describe the reproducible identification and validation of mitoticphase-specific polyubiquitinated proteins via the in vivo biotinylation of ubiquitin. A large number of polyubiquitinated proteins that we identified are specific to mitotic exit, when the APC/C is active, and we expect that many of them are substrates for the APC/C. We formally identified KIFC1/HSET and Cyk4/RACGAP1 as targets of APC/C-dependent ubiquitin-mediated proteolysis after anaphase onset and investigated the role of their ubiquitination in the regulation of mitotic exit. Cell cycle phase-specific information on protein ubiquitination and the generation of ubiquitinated protein networks provides a framework for further investigation of ubiquitin-controlled processes occurring during the rebuilding of interphase cells.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]