A Label-free Quantitative Proteomics Strategy to Identify E3 Ubiquitin Ligase Substrates Targeted to Proteasome Degradation

The ubiquitin-proteasome system is a central mechanism for controlled proteolysis that regulates numerous cellular processes in eukaryotes. As such, defects in this system can contribute to disease pathogenesis. In this pathway, E3 ubiquitin ligases provide platforms for binding specific substrates, thereby coordinating their ubiquitylation and subsequent degradation by the proteasome. Despite the identification of many E3 ubiquitin ligases, the identities of their specific substrates are still largely unresolved. The ankyrin repeat-containing protein with a suppressor of cytokine signaling box 2 (ASB2) gene that we initially identified as a retinoic acid-response gene in acute promyelocytic leukemia cells encodes the specificity subunit of an E3 ubiquitin ligase complex that is involved in hematopoietic cell differentiation. We have recently identified filamin A and filamin B as the first ASB2 targets and shown that ASB2 triggers ubiquitylation and proteasome-mediated degradation of these proteins. Here a global quantitative proteomics strategy is provided to identify substrates of E3 ubiquitin ligases targeted to proteasomal degradation. Indeed we used label-free methods for quantifying proteins identified by shotgun proteomics in extracts of cells expressing wild-type ASB2 or an E3 ubiquitin ligase-defective mutant of ASB2 under the control of an inducible promoter. Measurements of spectral count and mass spectrometric signal intensity demonstrated a drastic decrease of filamin A and filamin B in myeloid leukemia cells expressing wild-type ASB2 compared with cells expressing an E3 ubiquitin ligase-defective mutant of ASB2. Altogether we provide an original strategy that enables identification of E3 ubiquitin ligase substrates that have to be degraded.The ubiquitin-proteasome system (UPS)¹ plays an essential role in the regulation of protein stability in eukaryotic cells. Degradation of a protein by the UPS entails two successive steps: the covalent attachment of multiple ubiquitin molecules to the protein substrate and its degradation by the 26 S proteasome (1, 2). Ubiquitylation of protein substrates occurs through the sequential action of distinct enzymes: a ubiquitin-activating enzyme, E1; a ubiquitin-conjugating enzyme, E2; and a ubiquitin ligase, E3, responsible for the specific recognition of substrates. Increasing attention has been recently given to the UPS leading to the identification of hundreds of E3 ubiquitin ligases (E3s). Two major classes of E3s have been described: (i) E3s of the HECT (homologous to the E6-associated protein carboxyl terminus) domain family that function as ubiquitin carriers (3, 4) and (ii) E3s of the RING (really interesting new gene) or of the U box families that have no inherent catalytic activity but recruit an E2 enzyme toward substrates (5–7).Classical approaches to identify substrates of E3s are based on the identification of interacting proteins. Although these have successfully led to the identification of a number of substrates of monomeric E3s, identification of substrates of multimeric E3s is very challenging because of the weak affinity of substrates for their requisite specificity subunit and because of the labile nature of the substrate complexed with the specificity subunit (8).Acute promyelocytic leukemia (APL) is associated with six reciprocal translocations always involving the retinoic acid receptor α (RARα) gene (9–11). The RARα protein is a member of the nuclear receptor superfamily that stimulates myeloid differentiation in the presence of its ligand, all-trans-retinoic acid (RA). In more than 95% of APL, the t(15;17) translocation between the promyelocytic leukemia (PML) gene on chromosome 15 and the RARα gene on chromosome 17 produces the PML-RARα fusion protein (12). The PML-RARα protein enhances the repression of RARα target genes by increasing associations with corepressors (13–15) and by recruiting DNA methyltransferases (16). These complexes dissociate from the PML-RARα fusion protein in the presence of pharmacological concentrations of RA perhaps explaining why APL cells are sensitive to RA treatment. Indeed at pharmacological concentrations, RA induces complete remission in a high percentage of APL patients (17–19). By studying RA-induced differentiation of APL cells we have attempted to identify some of the genes that may be up-regulated during this process to further understand the control of growth and differentiation in leukemia (20). One gene identified in this manner, ASB2 (ankyrin repeat-containing protein with a suppressor of cytokine signaling box 2) is an RA-response gene involved in induced differentiation of myeloid leukemia cells (21–23).The ASB2 protein is a subunit of a multimeric E3 ubiquitin ligase of the cullin-RING ligase family (24, 25). The ASB2 suppressor of cytokine signaling box can be divided into a BC box that defines a binding site for the Elongin BC complex and a Cul5 box that determines the binding specificity for Cullin5 (24, 26). Indeed the ASB2 protein, by interacting with the Elongin BC complex, can assemble with a Cullin5/Rbx1 or -2 module to reconstitute an active E3 ubiquitin ligase complex (23–25). Within this complex, the ASB2 protein is the specificity subunit involved in the recruitment of specific substrate(s). Furthermore endogenous ASB2 protein was copurified with ubiquitin ligase activity in RA-treated APL cells suggesting that, during induced differentiation of leukemia cells, the ASB2 protein may target proteins involved in blocking differentiation to destruction by the proteasome machinery (24). We recently identified actin-binding proteins filamin A (FLNa) and filamin B (FLNb) as ASB2 targets and showed that ASB2 triggers ubiquitylation and drives proteasome-mediated degradation of these proteins during RA-induced differentiation of myeloid leukemia cells (23).With the aim to develop a strategy to identify E3 substrates that are degraded by the proteasome, we used an MS approach to identify ASB2 substrates in physiologically relevant settings. Indeed we used label-free quantitative proteomics to identify proteins that are absent or less abundant in cells that express wild-type ASB2 but that accumulate in cells expressing an ASB2 E3 ligase-defective mutant. Application of label-free MS methods that have the advantage to be simple, fast, and cheap enabled the identification of FLNa and FLNb as ASB2 substrates. This study provides a new strategy for the identification of E3 substrates that have to be degraded.     

Desmocollin 3-mediated Binding Is Crucial for Keratinocyte Cohesion and Is Impaired in Pemphigus

Desmocollin (Dsc) 1–3 and desmoglein (Dsg) 1–4, transmembrane proteins of the cadherin family, form the adhesive core of desmosomes. Here we provide evidence that Dsc3 homo- and heterophilic trans-interaction is crucial for epidermal integrity. Single molecule atomic force microscopy (AFM) revealed homophilic trans-interaction of Dsc3. Dsc3 displayed heterophilic interaction with Dsg1 but not with Dsg3. A monoclonal antibody targeted against the extracellular domain reduced homophilic and heterophilic binding as measured by AFM, caused intraepidermal blistering in a model of human skin, and a loss of intercellular adhesion in cultured keratinocytes. Because autoantibodies against Dsg1 are associated with skin blistering in pemphigus, we characterized the role of Dsc3 binding for pemphigus pathogenesis. In contrast to AFM experiments, laser tweezer trapping revealed that pemphigus autoantibodies reduced binding of Dsc3-coated beads to the keratinocyte cell surface. These data indicate that loss of heterophilic Dsc3/Dsg1 binding may contribute to pemphigus skin blistering.Desmogleins (Dsg)² and desmocollins (Dsc) are members of the Ca²⁺-dependent cadherin family of adhesion molecules that extend with their outer domains into the extracellular core of desmosomes. Desmosomal cadherins include four Dsg (Dsg1–4) and three Dsc3 isoforms (Dsc1–3) (1, 2). Desmosomal cadherins share a common domain organization with five N-terminally located extracellular subdomains (EC1–5). The membrane-distal EC1 domain is thought to contain the adhesive interface necessary for trans-interaction as could be concluded from structural analysis and blocking studies using peptides and antibodies (3–5). By establishing trans- and cis-interacting adhesive complexes, desmosomal cadherins participate in providing mechanical strength to stratified epithelia (6). In human epidermis Dsg1 and Dsc1 expression decreases from the outermost granular layer toward deeper layers, whereas Dsg3 and Dsc3 are primarily found in the basal layer and display an inverse expression gradient (7, 8). In contrast to classical cadherins present in adherens junctions that primarily undergo homophilic trans-interaction, desmosomal cadherins are generally believed to mediate both homo- and heterophilic binding (9). Recently, an important role of Dsc3 for integrity of murine epidermis was demonstrated in animals with conditional epidermal Dsc3 deficiency that suffered from severe intraepidermal blister formation (10) comparable with the phenotype of the autoimmune bullous skin disease pemphigus vulgaris (PV) (11). PV is associated with antibodies (Abs) against Dsg3, in part combined with Abs targeting Dsg1, whereas Dsg1 Abs alone are associated with pemphigus foliaceus (PF). However, PV and PF sera usually do not contain autoantibodies targeting Dsc3 (12). In view of the apparently important role of Dsc3 in epidermal adhesion, we addressed whether Dsg1 and Dsg3 might heterophilically interact with Dsc3 and whether Abs in pemphigus might interfere with such type of interaction.     

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.     

Synthesis of Free and Proliferating Cell Nuclear Antigen-bound Polyubiquitin Chains by the RING E3 Ubiquitin Ligase Rad5

In replicating yeast, lysine 63-linked polyubiquitin (polyUb) chains are extended from the ubiquitin moiety of monoubiquitinated proliferating cell nuclear antigen (monoUb-PCNA) by the E2-E3 complex of (Ubc13-Mms2)-Rad5. This promotes error-free bypass of DNA damage lesions. The unusual ability of Ubc13-Mms2 to synthesize unanchored Lys⁶³-linked polyUb chains in vitro allowed us to resolve the individual roles that it and Rad5 play in the catalysis and specificity of PCNA polyubiquitination. We found that Rad5 stimulates the synthesis of free polyUb chains by Ubc13-Mms2 in part by enhancing the reactivity of the Ubc13∼Ub thiolester bond. Polyubiquitination of monoUb-PCNA was further enhanced by interactions between the N-terminal domain of Rad5 and PCNA. Thus, Rad5 acts both to align monoUb-PCNA with Ub-charged Ubc13 and to stimulate Ub transfer onto Lys⁶³ of a Ub acceptor. We also found that Rad5 interacts with PCNA independently of the number of monoubiquitinated subunits in the trimer and that it binds to both unmodified and monoUb-PCNA with similar affinities. These findings indicate that Rad5-mediated recognition of monoUb-PCNA in vivo is likely to depend upon interactions with additional factors at stalled replication forks.DNA is susceptible to chemical alteration by many endogenous and exogenous agents. To counter this threat and maintain genome integrity, eukaryotic cells employ three main strategies: DNA repair pathways that directly reverse DNA damage, cell cycle checkpoints that allow time to repair the damage prior to replication, and DNA damage tolerance (DDT),² which is a method of bypassing DNA damage lesions during the DNA replication phase of the cell cycle.Proliferating cell nuclear antigen (PCNA) is a key regulatory protein in DNA replication and repair (1). At the replication fork, DNA is encircled by PCNA, a homotrimeric protein that promotes processive movement of the replicative DNA polymerase. Upon DNA damage and subsequent stalling of the replicative polymerase, Ub modifications of PCNA signal DDT, which allows a cell to bypass the lesion and proceed past this potential block in replication (2–4).In the DDT pathway, as in other Ub-dependent pathways, Ub is conjugated to a substrate by the actions of three enzymes, an E1 activating enzyme, an E2 conjugating enzyme, and an E3 ligase (5). The E1 enzyme initiates the pathway in a two-step reaction that utilizes ATP hydrolysis to activate the C terminus of Ub, culminating in the formation of an E1∼Ub thiolester. Subsequent transthiolation to the active site cysteine of the E2 generates an E2∼Ub thiolester. An E3 ligase then brings a substrate into close proximity to the E2∼Ub intermediate, thereby catalyzing the formation of an isopeptide bond between the amino group of a substrate lysine and the C-terminal glycine of Ub. Polyubiquitination occurs when this substrate is another Ub, either free or as part of a Ub-protein conjugate.The DDT pathway is characterized by distinct ubiquitination events on PCNA that occur in two stages (3, 4, 6). The first of these is monoubiquitination of lysine 164 on one or more of the PCNA subunits by the E2-E3 complex of Rad6-Rad18 in Saccharomyces cerevisiae (3, 4, 7). monoUb-PCNA can serve either as a signal for error-prone bypass of the DNA lesion by recruiting translesion polymerases or as a substrate for subsequent polyubiquitination by the E2 heterodimer Ubc13-Mms2 and the E3 ligase Rad5 (3, 4, 8, 9). The polyUb chain extended from the initial Ub moiety on monoUb-PCNA is linked specifically through Ub Lys⁶³ residues. This Lys⁶³-linked chain is thought to enable a template switch mechanism that allows for error-free bypass of the DNA lesion, in part by utilizing the single-strand DNA-dependent helicase activity of Rad5 (3, 4, 10, 11). Both PCNA ubiquitination events promote bypass of the DNA lesion rather than direct removal or repair of the lesion.We have been interested in the mechanism by which the yeast (Ubc13-Mms2)-Rad5 complex catalyzes the formation of Lys⁶³-linked polyUb on PCNA. Previous studies have shown that heterodimerization of the Ubc13-Mms2 E2 is essential for Lys⁶³-specific Ub-Ub conjugation in vitro and in vivo (12–15). Ubc13 is a canonical E2 enzyme with an active site cysteine that receives activated Ub by transthiolation from the E1∼Ub complex (12, 13). This Ub is referred to as the “donor Ub.” Mms2 is a Ub E2 variant protein that lacks the active site cysteine (12, 15); rather, Mms2 binds to a second Ub, the “acceptor Ub,” and positions it to facilitate nucleophilic attack on the Ubc13∼Ub thiolester bond by the ϵ-amine of Lys⁶³ (15, 16). The positioning of the acceptor Ub by Mms2 controls the specificity of polyUb assembly such that only Lys⁶³-linked chains can be formed (16).Ubc13-Mms2 can synthesize Lys⁶³-linked chains in vitro in the absence of a PCNA substrate or an E3 ligase (12, 13). However, unlike the synthesis of free Lys⁶³-linked polyUb chains by Ubc13-Mms2, little is known about the polyubiquitination of PCNA or the role of the Rad5 E3 ligase in these reactions. Rad5 can bind PCNA and Rad18, and it contains a catalytic RING domain that characterizes the largest class of E3 ligases (17–21). There is evidence that RING E3s like Rad5 may play a more active role in ubiquitination than simply bringing the substrate into close proximity with the E2∼Ub. Several RING E3s have been shown to stimulate the synthesis of unanchored polyUb chains or autoubiquitination of their cognate E2s in the absence of substrates (22–24). This stimulation may be related to the ability of RING E3s to enhance reactivity of the E2∼Ub thiolester bond through allosteric effects (25, 26).Using purified recombinant forms of Ubc13, Mms2, and Rad5, we have explored the assembly of free Lys⁶³-linked polyUb chains as well as the extension of a polyUb chain on a synthetic analog of monoUb-PCNA. We show that Rad5 facilitates ubiquitination in part by increasing the reactivity of the Ubc13∼Ub thiolester bond. With monoUb-PCNA substrates, Rad5 also stimulated polyubiquitination through direct interactions with PCNA and recruitment of Ub-charged Ubc13-Mms2. Surprisingly, Rad5 recognition of monoUb-PCNA appeared to depend on interactions only with the PCNA moiety of the conjugate, which suggests that substrate selectivity in vivo is likely to depend on additional factors.     

In Vitro and In Vivo Biology of Recombinant Adenovirus Vectors with E1, E1/E2A,or E1/E4 Deleted

Isogenic, E3-deleted adenovirus vectors defective in E1, E1 and E2A, or E1 and E4 were generated in complementation cell lines expressing E1, E1 and E2A, or E1 and E4 and characterized in vitro and in vivo. In the absence of complementation, deletion of both E1 and E2A completely abolished expression of early and late viral genes, while deletion of E1 and E4 impaired expression of viral genes, although at a lower level than the E1/E2A deletion. The in vivo persistence of these three types of vectors was monitored in selected strains of mice with viral genomes devoid of transgenes to exclude any interference by immunogenic transgene-encoded products. Our studies showed no significant differences among the vectors in the short-term maintenance and long-term (4-month) persistence of viral DNA in liver and lung cells of immunocompetent and immunodeficient mice. Furthermore, all vectors induced similar antibody responses and comparable levels of adenovirus-specific cytotoxic T lymphocytes. These results suggest that in the absence of transgenes, the progressive deletion of the adenovirus genome does not extend the in vivo persistence of the transduced cells and does not reduce the antivirus immune response. In addition, our data confirm that, in the absence of transgene expression, mouse cellular immunity to viral antigens plays a minor role in the progressive elimination of the virus genome.Replication-deficient human adenoviruses (Ad) have been widely investigated as ex vivo and in vivo gene delivery systems for human gene therapy. The ability of these vectors to mediate the efficient expression of candidate therapeutic or vaccine genes in a variety of cell types, including postmitotic cells, is considered an advantage over other gene transfer vectors (3, 28, 49). However, the successful application of currently available E1-defective Ad vectors in human gene therapy has been hampered by the fact that transgene expression is only transient in vivo (2, 15, 16, 33, 36, 46). This short-lived in vivo expression of the transgene has been explained, at least in part, by the induction in vivo of cytotoxic immune responses to cells infected with the Ad vector. Studies with rodent systems have suggested that cytotoxic T lymphocytes (CTLs) directed against virus antigens synthesized de novo in the transduced tissues play a major role in eliminating cells containing the E1-deleted viral genome (56–58, 61). Consistent with the concept of cellular antiviral immunity, expression of transgenes is significantly extended in experimental rodent systems that are deficient in various components of the cellular immune system or that have been rendered immunocompromised by administration of pharmacological agents (2, 33, 37, 48, 60, 64).Based on the assumption that further reduction of viral antigen expression may lower the immune response and thus extend persistence of transgene expression, previous studies have investigated the consequences of deleting both E1 and an additional viral regulatory region, such as E2A or E4. The E2A region encodes a DNA binding protein (DBP) with specific affinity for single-stranded Ad DNA. The DNA binding function is essential for the initiation and elongation of viral DNA synthesis during the early phase of Ad infection. During the late phase of infection, DBP plays a central role in the activation of the major late promoter (MLP) (for a recent review, see reference 44). The E4 region, located at the right end of the viral genome, encodes several regulatory proteins with pleiotropic functions which are involved in the accumulation, splicing, and transport of early and late viral mRNAs, in DNA replication, and in virus particle assembly (reviewed in reference 44). The simultaneous deletion of E1 and E2A or of E1 and E4 should therefore further reduce the replication of the virus genome and the expression of early and late viral genes. Such multidefective vectors have been generated and tested in vitro and in vivo (9, 12, 17, 19–21, 23, 24, 26, 34, 40, 52, 53, 59, 62, 63). Recombinant vectors with E1 deleted and carrying an E2A temperature-sensitive mutation (E2Ats) have been shown in vitro to express much smaller amounts of virus proteins, leading to extended transgene expression in cotton rats and mice (19, 20, 24, 59). To eliminate the risks of reversion of the E2Ats point mutation to a wild-type phenotype, improved vectors with both E1 and E2A deleted were subsequently generated in complementation cell lines coexpressing E1 and E2A genes (26, 40, 63). In vitro analysis of human cells infected by these viruses demonstrated that the double deletion completely abolished viral DNA replication and late protein synthesis (26). Similarly, E1/E4-deleted vectors have been generated in various in vitro complementation systems and tested in vitro and in vivo (9, 17, 23, 45, 52, 53, 62). These studies showed that deletion of both E1 and E4 did indeed reduce significantly the expression of early and late virus proteins (17, 23), leading to a decreased anti-Ad host immune response (23), reduced hepatotoxicity (17, 23, 52), and improved in vivo persistence of the transduced liver cells (17, 23, 52).Interpretation of these results is difficult, however, since all tested E1- and E1/E4-deleted vectors encoded the bacterial β-galactosidase (βgal) marker, whose strong immunogenicity is known to influence the in vivo persistence of Ad-transduced cells (32, 37). Moreover, the results described above are not consistent with the conclusions from other studies showing, in various immunocompetent mouse models, that cellular immunity to Ad antigens has no detectable impact on the persistence of the transduced cells (37, 40, 50, 51). Furthermore, in contrast to results of earlier studies (19, 20, 59), Fang et al. (21) demonstrated that injection of E1-deleted/E2Ats vectors into immunocompetent mice and hemophilia B dogs did not lead to an improvement of the persistence of transgene expression compared to that with isogenic E1-deleted vectors. Similarly, Morral et al. (40) did not observe any difference in persistence of transgene expression in mice injected with either vectors deleted in E1 only or vectors deleted in both E1 and E2A. Finally, the demonstration that some E4-encoded products can modulate transgene expression (1, 17, 36a) makes the evaluation of E1- and E1/E4-deleted vectors even more complex when persistence of transgene expression is used for direct comparison of the in vivo persistence of cells transduced by the two types of vectors.The precise influence of the host immune response to viral antigens on the in vivo persistence of the transduced cells, and hence the impact of further deletions in the virus genome, therefore still remains unclear. To investigate these questions, we generated a set of isogenic vectors with single deletions (AdE1°) and double deletions (AdE1°E2A° and AdE1°E4°) and their corresponding complementation cell lines and compared the biologies and immunogenicities of these vectors in vitro and in vivo. To eliminate any possible influence of transgene-encoded products on the interpretation of the in vivo results, we used E1-, E1/E2A-, and E1/E4-deleted vectors with no transgenes.     

Conserved Glutamate Residues Glu-343 and Glu-519 Provide Mechanistic Insights into Cation/Nucleoside Cotransport by Human Concentrative Nucleoside Transporter hCNT3

Human concentrative nucleoside transporter 3 (hCNT3) utilizes electrochemical gradients of both Na⁺ and H⁺ to accumulate pyrimidine and purine nucleosides within cells. We have employed radioisotope flux and electrophysiological techniques in combination with site-directed mutagenesis and heterologous expression in Xenopus oocytes to identify two conserved pore-lining glutamate residues (Glu-343 and Glu-519) with essential roles in hCNT3 Na⁺/nucleoside and H⁺/nucleoside cotransport. Mutation of Glu-343 and Glu-519 to aspartate, glutamine, and cysteine severely compromised hCNT3 transport function, and changes included altered nucleoside and cation activation kinetics (all mutants), loss or impairment of H⁺ dependence (all mutants), shift in Na⁺:nucleoside stoichiometry from 2:1 to 1:1 (E519C), complete loss of catalytic activity (E519Q) and, similar to the corresponding mutant in Na⁺-specific hCNT1, uncoupled Na⁺ currents (E343Q). Consistent with close-proximity integration of cation/solute-binding sites within a common cation/permeant translocation pore, mutation of Glu-343 and Glu-519 also altered hCNT3 nucleoside transport selectivity. Both residues were accessible to the external medium and inhibited by p-chloromercuribenzene sulfonate when converted to cysteine.Physiologic nucleosides and the majority of synthetic nucleoside analogs with antineoplastic and/or antiviral activity are hydrophilic molecules that require specialized plasma membrane nucleoside transporter (NT)³ proteins for transport into or out of cells (1–4). NT-mediated transport is required for nucleoside metabolism by salvage pathways and is a critical determinant of the pharmacologic actions of nucleoside drugs (3–6). By regulating adenosine availability to purinoreceptors, NTs also modulate a diverse array of physiological processes, including neurotransmission, immune responses, platelet aggregation, renal function, and coronary vasodilation (4, 6, 7). Two structurally unrelated NT families of integral membrane proteins exist in human and other mammalian cells and tissues as follows: the SLC28 concentrative nucleoside transporter (CNT) family and the SLC29 equilibrative nucleoside transporter (ENT) family (3, 4, 6, 8, 9). ENTs are normally present in most, possibly all, cell types (4, 6, 8). CNTs, in contrast, are found predominantly in intestinal and renal epithelia and other specialized cell types, where they have important roles in absorption, secretion, distribution, and elimination of nucleosides and nucleoside drugs (1–3, 5, 6, 9).The CNT protein family in humans is represented by three members, hCNT1, hCNT2, and hCNT3. Belonging to a CNT subfamily phylogenetically distinct from hCNT1/2, hCNT3 utilizes electrochemical gradients of both Na⁺ and H⁺ to accumulate a broad range of pyrimidine and purine nucleosides and nucleoside drugs within cells (10, 11). hCNT1 and hCNT2, in contrast, are Na⁺-specific and transport pyrimidine and purine nucleosides, respectively (11–13). Together, hCNT1–3 account for the three major concentrative nucleoside transport processes of human and other mammalian cells. Nonmammalian members of the CNT protein family that have been characterized functionally include hfCNT, a second member of the CNT3 subfamily from the ancient marine prevertebrate the Pacific hagfish Eptatretus stouti (14), CeCNT3 from Caenorhabditis elegans (15), CaCNT from Candida albicans (16), and the bacterial nucleoside transporter NupC from Escherichia coli (17). hfCNT is Na⁺- but not H⁺-coupled, whereas CeCNT3, CaCNT, and NupC are exclusively H⁺-coupled. Na⁺:nucleoside coupling stoichiometries are 1:1 for hCNT1 and hCNT2 and 2:1 for hCNT3 and hfCNT3 (11, 14). H⁺:nucleoside coupling ratios for hCNT3 and CaCNT are 1:1 (11, 16).Although much progress has been made in molecular studies of ENT proteins (4, 6, 8), studies of structurally and functionally important regions and residues within the CNT protein family are still at an early stage. Topological investigations suggest that hCNT1–3 and other eukaryote CNT family members have a 13 (or possibly 15)-transmembrane helix (TM) architecture, and multiple alignments reveal strong sequence similarities within the C-terminal half of the proteins (18). Prokaryotic CNTs lack the first three TMs of their eukaryotic counterparts, and functional expression of N-terminally truncated human and rat CNT1 in Xenopus oocytes has established that these three TMs are not required for Na⁺-dependent uridine transport activity (18). Consistent with this finding, chimeric studies involving hCNT1 and hfCNT (14) and hCNT1 and hCNT3 (19) have demonstrated that residues involved in Na⁺- and H⁺-coupling reside in the C-terminal half of the protein. Present in this region of the transporter, but of unknown function, is a highly conserved (G/A)XKX₃NEFVA(Y/M/F) motif common to all eukaryote and prokaryote CNTs.By virtue of their negative charge and consequent ability to interact directly with coupling cations and/or participate in cation-induced and other protein conformational transitions, glutamate and aspartate residues play key functional and structural roles in a broad spectrum of mammalian and bacterial cation-coupled transporters (20–30). Little, however, is known about their role in CNTs. This study builds upon a recent mutagenesis study of conserved glutamate and aspartate residues in hCNT1 (31) to undertake a parallel in depth investigation of corresponding residues in hCNT3. By employing the multifunctional capability of hCNT3 as a template for these studies, this study provides novel mechanistic insights into the molecular mechanism(s) of CNT-mediated cation/nucleoside cotransport, including the role of the (G/A)XKX₃NEFVA(Y/M/F) motif.