Intact Ubiquitin Research Articles

Utilizing Precursor Ion Connectivity of Different Charge States to Improve Peptide and Protein Identification in MS/MS Analysis.

Tandem mass spectrometry (MS/MS) has become a key method for the structural analysis of biomolecules such as peptides and proteins. A pervasive problem in MS/MS analyses, especially for top-down proteomics, is the occurrence of chimeric spectra, when two or more precursor ions are co-isolated and fragmented, thus leading to complex MS/MS spectra that are populated with fragment ions originating from different precursor ions. This type of convoluted data typically results in low sequence database search scores due to the vast number of mixed-source fragment ions, of which only a fraction originates from a specific precursor ion. Herein, we present a novel workflow that deconvolutes the data of chimeric MS/MS spectra, improving the protein search scores and sequence coverages in database searching and thus providing a more confident peptide and protein identification. Previously misidentified proteins or proteins with insignificant search scores can be correctly and significantly identified following the presented data acquisition and analysis workflow with search scores increasing by a factor of 3-4 for smaller precursor ions (peptides) and >6 for larger precursor ions such as intact ubiquitin and cytochrome C.

Generation of site-specific ubiquitinated histones through chemical ligation to probe the specificities of histone deubiquitinases

The attachment of mono-ubiquitin to histones as a post-translational modification plays important roles in regulating chromatin structure and function. Like other epigenetic modifications, the site of ubiquitin attachment is critically important in determining its functional outcome. Depending on the type of histone and the specific lysine residue that is modified, ubiquitination acts in diverse pathways including DNA damage repair, transcription elongation, and transcription repression. Specific reader, writer and eraser activities have evolved to distinguish nucleosomes by ubiquitination of different sites. To facilitate biochemical studies of ubiquitinated nucleosomes, we have developed an efficient strategy to chemically ligate intact ubiquitin and histone proteins at specific sites to generate near-native ubiquitin-histone conjugates. Because these chemically-ligated ubiquitin conjugates are hydrolysable, they enabled us to characterize in vitro the specificities of several histone deubiquitinases. To gain insight into the mechanisms that contribute to the specificities of these deubiquitinases, we used a free Ub sensor-based real-time assay to determine their Michaelis-Menten kinetics. Our results confirmed previously reported specificities of BAP1 and USP22, but also revealed specificities of other histone deubiquitinases that have been less well defined in the literature.

Quantitative Imaging of Proteins in Tissue by Stable Isotope Labeled Mimetic Liquid Extraction Surface Analysis Mass Spectrometry.

Absolute quantification of proteins in tissue is important for numerous fields of study. Liquid chromatography–mass spectrometry (LC–MS) methods are the norm but typically involve lengthy sample preparation including tissue homogenization, which results in the loss of information relating to spatial distribution. Here, we propose liquid extraction surface analysis (LESA) mass spectrometry (MS) of stable isotope labeled mimetic tissue models for the spatially resolved quantification of intact ubiquitin in rat and mouse brain tissue. Measured ubiquitin concentrations are in agreement with values found in the literature. Images of rat and mouse brain tissue demonstrate spatial variation in the concentration of ubiquitin and demonstrate the utility of spatially resolved quantitative measurement of proteins in tissue. Although we have focused on ubiquitin, the method has the potential for broader application to the absolute quantitation of any endogenous protein or protein-based drug in tissue.

Stepwise multipolyubiquitination of p53 by the E6AP-E6 ubiquitin ligase complex

The human papillomavirus (HPV) oncoprotein E6 specifically binds to E6AP (E6-associated protein), a HECT (homologous to the E6AP C terminus)-type ubiquitin ligase, and directs its ligase activity toward the tumor suppressor p53. To examine the biochemical reaction in vitro, we established an efficient reconstitution system for the polyubiquitination of p53 by the E6AP-E6 complex. We demonstrate that E6AP-E6 formed a stable ternary complex with p53, which underwent extensive polyubiquitination when the isolated ternary complex was incubated with E1, E2, and ubiquitin. Mass spectrometry and biochemical analysis of the reaction products identified lysine residues as p53 ubiquitination sites. A p53 mutant with arginine substitutions of its 18 lysine residues was not ubiquitinated. Analysis of additional p53 mutants retaining only one or two intact ubiquitination sites revealed that chain elongation at each of these sites was limited to 5-6-mers. We also determined the size distribution of ubiquitin chains released by en bloc cleavage from polyubiquitinated p53 to be 2-6-mers. Taken together, these results strongly suggest that p53 is multipolyubiquitinated with short chains by E6AP-E6. In addition, analysis of growing chains provided strong evidence for step-by-step chain elongation. Thus, we hypothesize that p53 is polyubiquitinated in a stepwise manner through the back-and-forth movement of the C-lobe, and the permissive distance for the movement of the C-lobe restricts the length of the chains in the E6AP-E6-p53 ternary complex. Finally, we show that multipolyubiquitination at different sites provides a signal for proteasomal degradation.

Global analysis of ubiquitome in PRRSV-infected pulmonary alveolar macrophages

Protein lysine ubiquitination is a dynamic reversible post-translational modification that plays key roles in modulating different cellular processes. Porcine reproductive and respiratory syndrome virus (PRRSV) is a notorious pathogen, causing tremendous economic losses for the global swine industry. The possible involvement of ubiquitination in PRRSV infection is unclear. So anti-ubiquitination-based enrichment and LC-MS were performed to investigate the global ubiquitination events triggered by PRRSV infection in pulmonary alveolar macrophages. We totally identified 4044 lysine ubiquitination sites on 1580 cellular proteins, of which 983 sites on 717 proteins were significantly altered at 36 h postinfection. A systematic, intensive bioinformatic analysis of the ubiquitome data suggested that PRRSV suppresses the host immune responses by manipulating the ubiquitination of important adaptors and effectors, including TRAF6, JAK1, STAT1, and ISGs. Ubiquitination was also observed on 15 PRRSV proteins, including important virus proteases and structural proteins that function in virus infectivity and neutralizing antibody elicitation. The efficient replication of PRRSV requires an intact ubiquitin–proteasome system. Our study is the first to analyze the global ubiquitination events in pulmonary alveolar macrophages during PRRSV infection. It provides insight into the molecular mechanisms of PRRSV pathogenesis, promoting the development of antiviral drugs. Biological significancePRRSV is a notorious pathogen which has been resulting in huge economic losses in the swine industry since the first outbreak. Therefore, more in-depth knowledge of the PRRSV immunoregulatory mechanisms and valid control methods to combat the virus are urgently needed. Ubiquitination is an important post-translational modification regulating various cellular processes. However, information about the possible involvement of ubiquitination responses to PRRSV infection is limited. In this study, a quantitative proteomic approach was first used to analyze ubiquitination level alteration in PRRSV-infected PAMs. We demonstrate that PRRSV can suppresses the host immune responses by manipulating the ubiquitination of important effectors that include TRAF6, JAK1, STAT1, and ISGs. Furthermore, 15 PRRSV proteins undergo ubiquitination and efficient replication of PRRSV requires an intact ubiquitin–proteasome system. Our study will significantly expand our knowledge about the molecular mechanisms of PRRSV pathogenesis and provides novel insights into the development of antiviral drugs.

Mdm2 selectively suppresses DNA damage arising from inhibition of topoisomerase II independent of p53.

Mdm2 is often overexpressed in tumors that retain wild-type TP53 but may affect therapeutic response independently of p53. Herein is shown that tumor cells with MDM2 amplification are selectively resistant to treatment with topoisomerase II poisons but not other DNA damaging agents. Tumor cells that overexpress Mdm2 have reduced DNA double-strand breaks in response to doxorubicin or etoposide. This latter result is not due to altered drug uptake. The selective attenuation of DNA damage in response to these agents is dependent on both Mdm2 levels and an intact ubiquitin ligase function. These findings reveal a novel, p53-independent activity of Mdm2 and have important implications for the choice of chemotherapeutic agents in the treatment of Mdm2-overexpressing tumors.

Abstract 37: Centrosome Destabilization Through the Ubiquitin-Proteasome Pathway Regulates Proplatelet Production

Background: Proteasome inhibitors such as bortezomib, a chemotherapeutic used to treat multiple myeloma, induce thrombocytopenia within days of initiation. The mechanism for this thrombocytopenia has been tied to data revealing that proteasome activity is essential for platelet formation. The major pathway of selective protein degradation uses ubiquitin as a marker that targets proteins for proteolysis by the proteasome. This pathway is previously unexplored in megakaryocytes (MKs). Objectives: We aim to define the mechanism by which the ubiquitin-proteasome pathway affects MK maturation and platelet production. Results: Pharmacologic inhibition of proteasome activity blocks proplatelet formation in megakaryocytes. To further characterize how this degradation was occurring, we probed distinct ubiquitin pathways. Inhibition of the ubiquitin-activating enzyme E1 significantly inhibited proplatelet formation up to 73%. In addition, inhibition of the deubiquitinase proteins UCHL5 and USP14 significantly inhibited proplatelet formation up to 83%. These data suggest that an intact ubiquitin pathway is necessary for proplatelet formation. Proteomic and polysome analyses of MKs undergoing proplatelet formation revealed a subset of proteins decreased in proplatelet-producing megakaryocytes, consistent with data showing that protein degradation is necessary for proplatelet formation. Specifically, the centrosome stabilizing proteins Aurora kinase (Aurk) A/B, Tpx2, Cdk1, and Plk1 were decreased in proplatelet-producing MKs. Furthermore, inhibition of AurkA and Plk1, but not Cdk1, significantly inhibited proplatelet formation in vitro over 83%. Conclusions: We hypothesize that proplatelet formation is triggered by centrosome destabilization and disassembly, and that the ubiquitin-proteasome pathway plays a crucial role in this transformation. Specifically, regulation of the AurkA/Plk1/Tpx2 pathway may be key in centrosome integrity and initiation of proplatelet formation. Determination of the mechanism by which the ubiquitin-proteasome pathway regulates the centrosome and facilitates proplatelet formation will allow us to design better strategies to target and reverse thrombocytopenia.

Combined Infrared Multiphoton Dissociation with Ultraviolet Photodissociation for Ubiquitin Characterization.

Herein we report the successful implementation of the consecutive and simultaneous photodissociation with high (213 nm) and low (10.6 μm) energy photons (HiLoPD, high-low photodissociation) on ubiquitin in a quadrupole-Orbitrap mass spectrometer. Absorption of high-energy UV photon is dispersed over the whole protein and stimulates extensive C–Cα backbone fragmentation, whereas low-energy IR photon gradually increases the internal energy and thus preferentially dissociates the most labile amide (C–N) bonds. We noticed that simultaneous irradiation of UV and IR lasers on intact ubiquitin in a single MS/MS experiment provides a rich and well-balanced fragmentation array of a/x, b/y, and z ions. Moreover, secondary fragmentation from a/x and z ions leads to the formation of satellite side-chain ions (d, v, and w) and can help to distinguish isomeric residues in a protein. Implementation of high-low photodissociation in a high-resolution mass spectrometer may offer considerable benefits to promote a comprehensive portrait of protein characterization.Graphical ᅟElectronic supplementary materialThe online version of this article (doi:10.1007/s13361-016-1419-8) contains supplementary material, which is available to authorized users.

Newborn Mouse Lens Proteome and Its Alteration by Lysine 6 Mutant Ubiquitin

Ubiquitin is a tag that often initiates degradation of proteins by the proteasome in the ubiquitin proteasome system. Targeted expression of K6W mutant ubiquitin (K6W-Ub) in the lens results in defects in lens development and cataract formation, suggesting critical functions for ubiquitin in lens. To study the developmental processes that require intact ubiquitin, we executed the most extensive characterization of the lens proteome to date. We quantified lens protein expression changes in multiple replicate pools of P1 wild-type and K6W-Ub-expressing mouse lenses. Lens proteins were digested with trypsin, peptides were separated using strong cation exchange and reversed-phase liquid chromatography, and tandem mass (MS/MS) spectra were collected with a linear ion trap. Transgenic mice that expressed low levels of K6W-Ub (low expressers) had normal, clear lenses at birth, whereas the lenses that expressed high levels of K6W-Ub (higher expressers) had abnormal lenses and cataracts at birth. A total of 2052 proteins were identified, of which 996 were reliably quantified and compared between wild-type and K6W-Ub transgenic mice. Consistent with a delayed developmental program, fiber-cell-specific proteins, such as γ-crystallins (γA, γB, γC, and γE), were down-regulated in K6W-Ub higher expressers. Up-regulated proteins were involved in energy metabolism, signal transduction, and proteolysis. The K6W-Ub low expressers exhibited delayed onset and milder cataract consistent with smaller changes in protein expression. Because lens protein expression changes occurred prior to lens morphological abnormalities and cataract formation in K6W-Ub low expressers, it appears that expression of K6W-Ub sets in motion a process of altered protein expression that results in developmental defects and cataract.

The Cancer-associated K351N Mutation Affects the Ubiquitination and the Translocation to Mitochondria of p53 Protein

Stress-induced monoubiquitination of p53 is a crucial event for the nuclear-cytoplasm-mitochondria trafficking and transcription-independent pro-apoptotic functions of p53. Although an intact ubiquitination pathway and a functional nuclear export sequence are required for p53 nuclear export, the role of specific residues within this region in regulating both processes remains largely unknown. Here we characterize the mechanisms accounting for the nuclear accumulation of a new point mutation (Lys-351 to Asn) in the nuclear export sequence of p53 identified in a cisplatin-resistant ovarian carcinoma cell line (A2780 CIS). We found that K351N substitution abrogates the monoubiquitination of p53 induced by both Mdm2 and MSL2 E3-ligases. As a consequence, cells expressing p53 K351N mutant showed defects in cisplatin-induced translocation of p53 to mitochondria, Bax oligomerization, and mitochondrial membrane depolarization. These data identify K351N as a critical mutation of p53 that contributes to the development and maintenance of resistance to cisplatin.

ABIN1 Protein Cooperates with TAX1BP1 and A20 Proteins to Inhibit Antiviral Signaling

Upon virus infection, the innate immune response provides the first line of protection and rapidly induces type I interferons (IFNα/β), which mediate potent antiviral effects. To maintain homeostasis and prevent autoimmunity, IFN production is tightly regulated; however, the mechanisms of negative regulation are poorly understood. Herein, we demonstrate that the A20 binding inhibitor of NF-κB 1 (ABIN1) is a novel negative regulator of antiviral signaling. Overexpression of ABIN1 inhibited IFN-β promoter activation in response to virus infection or poly(I:C) transfection, whereas siRNA-mediated knockdown of ABIN1 enhanced IFN-β production upon virus infection. ABIN1 interacted with the A20 regulatory molecule TAX1BP1 and was essential for the recruitment of TAX1BP1 and A20 to the noncanonical IκB kinases TBK1 and IKKi in response to poly(I:C) transfection. ABIN1 and TAX1BP1 together disrupted the interactions between the E3 ubiquitin ligase TRAF3 and TBK1/IKKi to attenuate lysine 63-linked polyubiquitination of TBK1/IKKi. Finally, an intact ubiquitin binding domain of ABIN1 was essential for ABIN1 to interact with TBK1/IKKi and inhibit IFN-β production upon poly(I:C) transfection or virus infection. Together, these results suggest that ABIN1 requires its ubiquitin binding domain and cooperates with TAX1BP1 and A20 to restrict antiviral signaling.

Epstein-Barr latent membrane protein 1 transformation site 2 activates NF-κB in the absence of NF-κB essential modifier residues 133–224 or 373–419

Epstein Barr virus latent membrane protein 1 (LMP1) induces NF-κB activation through transformation effector sites (TES) 1 and 2, both of which are critical for B-lymphocyte transformation. TES2 principally activates canonical NF-κB, which we confirm is NF-κB essential modifier (NEMO)-dependent and requires an intact ubiquitin binding in A20 binding inhibitor of NF-κB and NEMO (UBAN) domain. LMP1 TES2 activated NF-κB in Jurkat cell lines harboring NEMO truncated at 372 (A45) or NEMO with an in-frame deletion of 133-224 (2C), whereas TNFα, 12-O-Tetradecanoylphorbol-13-acetate, human T-cell leukemia virus 1 Tax, and CD40 did not. In both A45 and 2C Jurkat cell lines, LMP1 TES2-mediated NF-κB activation was blocked by siRNAs to TNFα receptor-associated factor 6 and NEMO, by IκB kinase inhibitors, and by the IκBα superrepressor, indicating that the NEMO mutants function to support canonical NF-κB activation. Expression of A45 or 2C mutants in NEMO-deficient murine embryonic fibroblasts reproduced the Jurkat phenotypes: LMP1 TES2 activated NF-κB in fibroblasts lacking NEMO amino acids 133-224 or 373-419, but TNFα and Tax did not. Further analysis indicated that TES2 did not activate NF-κB in cells expressing the double deletion mutant Δ133-224/Δ372-419. These data provide further evidence of the essential role for NEMO in LMP1 TES2 NF-κB activation and highlight the importance of unique domains within NEMO for sensing distinct NF-κB stimuli.

Cytomegalovirus Promoter Up-regulation Is the Major Cause of Increased Protein Levels of Unstable Reporter Proteins after Treatment of Living Cells with Proteasome Inhibitors

Fluorescent unstable proteins obtained by the fusion of a fluorescent protein coding sequence with specific amino acid sequences that promote its fast degradation have become popular to gauge the activity of the ubiquitin/proteasome system in living cells. The steady-state levels of expression of these unstable proteins is low in agreement with their short half-lives, and they accumulate in the cell upon treatment with proteasome inhibitors. We show here that this accumulation is mainly due to transcriptional up-regulation of the cytomegalovirus promoter by proteasome inhibitors and mediated, at least in part, by AP1 transactivation. These simple facts put under quarantine conclusions reached about the activity of the ubiquitin/proteasome pathway in animal cells in culture or in transgenic mice, where popular cytomegalovirus-driven constructs are used, as transcriptional regulation of the expression of the reporter protein construct and not degradation of the unstable protein by the ubiquitin/proteasome system may contribute significantly to the interpretation of the results observed.

Data‐dependent electron transfer dissociation of large peptides and medium size proteins in a QTOF instrument on a liquid chromatography timescale

Liquid chromatography (LC) electron transfer dissociation (ETD) tandem mass spectrometry (MS/MS) of protein digests is demonstrated in a hybrid quadrupole-hexapole orthogonal time-of-flight (OTOF) mass spectrometer. Analyte ions are selected in a mass-analyzing quadrupole, accumulated in the hexapole linear ETD reaction cell and mutually stored with ETD reagent anions. Product ions are collected in an ion cooler and then analyzed by an OTOF mass analyzer. The hexapole structure of the ETD reaction cell allows for a broad fragment ion mass range distribution and a high ion storage capacity. Analytically useful ETD OTOF-MS/MS spectra could be obtained at a rate of faster than 2 Hz. When used in conjunction with LC this high speed allows for several MS and MS/MS spectra to be obtained across each LC peak. An MS scan is used to select the precursor ions. With a 1 m flight tube and single reflection, resolutions of about 10 k and a mass accuracy of 5 ppm were achieved. When analyzing a 100 fmol solution of a tryptic digest of bovine serum albumin (BSA) by LC/ETD MS/MS, 27 unique peptides were identified with a summed Mascot score of 1316 using the Swiss Prot database. In addition, we explored the capability for analyzing small proteins with the present hybrid instrument. ETD MS/MS of intact ubiquitin ([M+12H](12+)) leads to the identification of the protein with a Mascot score of 264.

Nonuniform Isotope Patterns Produced by Collision-Induced Dissociation of Homogeneously Labeled Ubiquitin: Implications for Spatially Resolved Hydrogen/Deuterium Exchange ESI-MS Studies

There is an ongoing debate whether collision-induced dissociation (CID) of electrosprayed proteins after solution-phase hydrogen/deuterium exchange (HDX) is a viable approach for determining spatially resolved deuteration patterns. This work explores the use of two methods, source-CID and hexapole tandem mass spectrometry (MS/MS) on a quadrupole time-of-flight (Q-TOF) mass spectrometer, for measuring the fragment deuteration levels of regioselectively labeled ubiquitin. Both methods reveal that b-ions exhibit HDX levels significantly below that of the intact protein, whereas several y'' fragments are labeled to a much greater extent. These results are consistent with earlier source-CID data (Akashi, S.; Naito, Y.; Takio, K. Anal. Chem. 1999, 71, 4974-4980). However, the measured b-ion deuteration levels are in disagreement with the known solution-phase behavior of ubiquitin. Partial agreement is observed for y''-ions. Control experiments on homogeneously labeled ubiquitin (having the same average deuteration level at every exchangeable site) result in highly nonuniform fragment HDX levels. In particular, b-ions exhibit deuteration levels significantly below that of intact ubiquitin, thereby mimicking the behavior seen for the regioselectively labeled protein. This effect is likely caused by isotope fractionation during collisional activation, facilitated by the high mobility of charge carriers (scrambling) in the gas phase. The observation that the b-ion labeling behavior is largely independent of the spatial isotope distribution within solution-phase ubiquitin invalidates these ions as reporters of the protein deuteration pattern. This work questions the common practice of interpreting any nonuniformities in fragment deuteration as being indicative of regioselective solution-phase labeling. Artifactual deuterium enrichment or depletion during collisional activation may have contributed to the current lack of consensus as to whether HDX/CID represents a potentially viable tool for measuring solution-phase deuteration patterns.

PKR and PKR-like Endoplasmic Reticulum Kinase Induce the Proteasome-dependent Degradation of Cyclin D1 via a Mechanism Requiring Eukaryotic Initiation Factor 2α Phosphorylation

PKR and PKR-like Endoplasmic Reticulum Kinase Induce the Proteasome-dependent Degradation of Cyclin D1 via a Mechanism Requiring Eukaryotic Initiation Factor 2α Phosphorylation

Regulation of p53 Nuclear Export through Sequential Changes in Conformation and Ubiquitination

Wild-type p53 is a conformationally labile protein that undergoes nuclear-cytoplasmic shuttling. MDM2-mediated ubiquitination promotes p53 nuclear export by exposing or activating a nuclear export signal (NES) in the C terminus of p53. We observed that cancer-derived p53s with a mutant (primary antibody 1620-/pAb240+) conformation localized in the cytoplasm to a greater extent and displayed increased susceptibility to ubiquitination than p53s with a more wild-type (primary antibody 1620+/pAb240-) conformation. The cytoplasmic localization of mutant p53s required the C-terminal NES and an intact ubiquitination pathway. Mutant p53 ubiquitination occurred at lysines in both the DNA-binding domain (DBD) and C terminus. Interestingly, Lys to Arg mutations that inhibited ubiquitination restored nuclear localization to mutant p53 but had no apparent effect on p53 conformation. Further studies revealed that wild-type p53, like mutant p53, is ubiquitinated by MDM2 in both the DBD and C terminus and that ubiquitination in both regions contributes to its nuclear export. MDM2 binding can induce a conformational change in wild-type p53, but this conformational change is insufficient to promote p53 nuclear export in the absence of MDM2 ubiquitination activity. Taken together, these results support a stepwise model for mutant and wild-type p53 nuclear export. In this model, the conformational change induced by either the cancer-derived mutation or MDM2 binding precedes p53 ubiquitination. The addition of ubiquitin to DBD and C-terminal lysines then promotes nuclear export via the C-terminal NES.

AMSH is an endosome-associated ubiquitin isopeptidase.

The JAMM (JAB1/MPN/Mov34 metalloenzyme) motif has been proposed to provide the active site for isopeptidase activity associated with the Rpn11/POH1 subunit of the 19S-proteasome and the Csn5-subunit of the signalosome. We have looked for similar activity in associated molecule with the SH3 domain of STAM (AMSH), a JAMM domain–containing protein that associates with the SH3-domain of STAM, a protein, which regulates receptor sorting at the endosome. We demonstrate isopeptidase activity against K48-linked tetraubiquitin and K63-linked polyubiquitin chains to generate di-ubiquitin and free ubiquitin, respectively. An inactivating mutation (D348A) in AMSH leads to accumulation of ubiquitin on endosomes and the concomitant stabilization of a ubiquitinated form of STAM, which requires an intact ubiquitin interaction motif (UIM) within STAM. Short interfering RNA knockdown of AMSH enhances the degradation rate of EGF receptor (EGFR) following acute stimulation and ubiquitinated EGFR provides a substrate for AMSH in vitro. We propose that AMSH is a deubiquitinating enzyme with functions at the endosome, which oppose the ubiquitin-dependent sorting of receptors to lysosomes.

Cbl-mediated Ubiquitinylation Is Required for Lysosomal Sorting of Epidermal Growth Factor Receptor but Is Dispensable for Endocytosis

Ligand-induced down-regulation controls the signaling potency of the epidermal growth factor receptor (EGFR/ErbB1). Overexpression studies have identified Cbl-mediated ubiquitinylation of EGFR as a mechanism of ligand-induced EGFR down-regulation. However, the role of endogenous Cbl in EGFR down-regulation and the precise step in the endocytic pathway regulated by Cbl remain unclear. Using Cbl-/- mouse embryonic fibroblast cell lines, we demonstrate that endogenous Cbl is essential for ligand-induced ubiquitinylation and efficient degradation of EGFR. Further analyses using Chinese hamster ovary cells with a temperature-sensitive defect in ubiquitinylation confirm a crucial role of the ubiquitin machinery in Cbl-mediated EGFR degradation. However, internalization into early endosomes did not require Cbl function or an intact ubiquitin pathway. Confocal immunolocalization studies indicated that Cbl-dependent ubiquitinylation plays a critical role at the early endosome to late endosome/lysosome sorting step of EGFR down-regulation. These findings establish Cbl as the major endogenous ubiquitin ligase responsible for EGFR degradation, and show that the critical role of Cbl-mediated ubiquitinylation is at the level of endosomal sorting, rather than at the level of internalization.

Structure and Properties of a Dimeric N-Terminal Fragment of Human Ubiquitin.

Previous peptide dissection and kinetic experiments have indicated that in vitro folding of ubiquitin may proceed via transient species in which native-like structure has been acquired in the first 45 residues. A peptide fragment, UQ(1-51), encompassing residues 1 to 51 of ubiquitin was produced in order to test whether this portion has propensity for independent self-assembly. Surprisingly, the construct formed a folded symmetrical dimer that was stabilised by 0.8 M sodium sulphate at 298 K (the S state). The solution structure of the UQ(1-51) dimer was determined by multinuclear NMR spectroscopy. Each subunit of UQ(1-51) consists of an N-terminal β-hairpin followed by an α-helix and a final β-strand, with orientations similar to intact ubiquitin. The dimer is formed by the third β-strand of one subunit interleaving between the hairpin and third strand of the other to give a six-stranded β-sheet, with the two α-helices sitting on top. The helix-helix and strand portions of the dimer interface also mimic related features in the structure of ubiquitin. The structural specificity of the UQ(1-51) peptide is tuneable: as the concentration of sodium sulphate is decreased, near-native alternative conformations are populated in slow chemical exchange. Magnetization transfer experiments were performed to characterize the various species present in 0.35 M sodium sulphate, namely the S state and two minor forms. Chemical shift differences suggest that one minor form is very similar to the S state, while the other experiences a significant conformational change in the third strand. A segmental rearrangement of the third strand in one subunit of the S state would render the dimer asymmetric, accounting for most of our results. Similar small-scale transitions in proteins are often invoked to explain solvent exchange at backbone amide proton sites that have an intermediate level of protection.