Interaction of glycylglycine and Na+ at the mucosal border of Guinea-pig small intestine: A non-mutual stimulation of transport

Sodium-dependence of glycylglycine (Gly-Gly) influx and stimulation of Na⁺ transport by Gly-Gly were studied in everted sacs, sheet preparations and brush-border membrane vesicles isolated from guinea-pig ileum. Gly-Gly influx was found to be independent of the presence of Na⁺, while Na⁺ transport was stimulated by Gly-Gly as evidenced by increases in transmural potential difference (PD_t), short-circuit current (I_sc) and Na⁺ influx. The change in PD_t (ΔPD_t) induced by Gly-Gly was a saturable function of Gly-Gly concentration, showing a Michaelis-Menten type relationship. The half-saturation concentration for Gly-Gly estimated from the electrical data was nearly identical with that estimated from influx data. At a constant Gly-Gly concentration the relationship between I_sc and Na⁺ concentration was sigmoid, and the Hill coefficient was 1.5. Kinetic analysis according to Garay Garrahan indicates that each Gly-Gly carrier has two equivalent non-interacting binding sites for Na⁺, and that translocation of Na⁺ occurs when the two Na⁺ sites on the carrier loaded with Gly-Gly are occupied by Na⁺. However, our results indicate that the resultant Na⁺ flow is not capable of stimulating Gly-Gly translocation.     

Rates of peptide bond hydrolysis by cobalt(III) complexes: observation by rate of amino acid release

The rates of hydrolysis of eleven dipeptides (Gly-Gly; Gly-l-Leu; Gly-d,l-Val; Gly-l-Phe: d,l-Leu-Gly; l-Leu-l-Tyr; l-Pro-l-Tyr; l-Ala-l-Phe; l-Val-Gly; l-Val-l-Ile; l-Ile-l-Val) by cis-β-Co(trien)(OH)(H₂O)²⁺ under pseudo first order conditions (excess Co(III)) are reported. The rates are determined by the rate of amino acid release by liquid chromatography. The range of rates observed for the eleven dipeptides is 10.1. Two of the dipeptides are hydrolyzed more rapidly than Gly-Gly, the remaining six more slowly. Phosphate does not have any significant effect on the rate of peptide hydrolysis. Aquohydroxy-1,8-diamino-3,6-dithiaoctanecobalt(III)²⁺ did not hydrolyze Gly-Gly at 25°, pH8, and aquohydroxy-1,6-bis-(α-pyridyl)-2,5-diazahexanecobalt(III)²⁺ did not hydrolyze Gly-Gly at 65°, pH 8, to any significant extent.     

Proteasome Assembly Influences Interaction with Ubiquitinated Proteins and Shuttle Factors

A major fraction of intracellular protein degradation is mediated by the proteasome. Successful degradation of these substrates requires ubiquitination and delivery to the proteasome followed by protein unfolding and disassembly of the multiubiquitin chain. Enzymes, such as Rpn11, dismantle multiubiquitin chains, and mutations can affect proteasome assembly and activity. We report that different rpn11 mutations can affect proteasome interaction with ubiquitinated proteins. Moreover, proteasomes are unstable in rpn11-1 and do not form productive interactions with multiubiquitinated proteins despite high levels in cell extracts. However, increased levels of ubiquitinated proteins were found associated with shuttle factors. In contrast to rpn11-1, proteasomes expressing a catalytically inactive mutant (rpn11^AXA) were more stable and bound very high amounts of ubiquitinated substrates. Expression of the carboxyl-terminal domain of Rpn11 partially suppressed the growth and proteasome stability defects of rpn11-1. These results indicate that ubiquitinated substrates are preferentially delivered to intact proteasome.     

New insights into the type A glycan modification of Clostridioides difficile flagellar protein flagellin C by phosphoproteomics analysis

The type A glycan modification found in human pathogen Clostridioides difficile consists of a monosaccharide (GlcNAc) that is linked to an N-methylated threonine through a phosphodiester bond. This structure has previously been described on the flagellar protein flagellin C of several C. difficile strains and is important for bacterial motility. The study of post-translational modifications often relies on some type of enrichment strategy; however, a procedure for enrichment of this modification has not yet been demonstrated. In this study, we show that an approach that is commonly used in phosphoproteomics, Fe³⁺-immobilized metal affinity chromatography, also enriches for peptides with this unique post-translational modification. Using LC–MS/MS analyses of immobilized metal affinity chromatography–captured tryptic peptides, we observed not only type A-modified C. difficile flagellin peptides but also a variety of truncated/modified type A structures on these peptides. Using an elaborate set of mass spectrometry analyses, we demonstrate that one of these modifications consists of a type A structure containing a phosphonate (2-aminoethylphosphonate), a modification that is rarely observed and has hitherto not been described in C. difficile. In conclusion, we show that a common enrichment strategy results in reliable identification of peptides carrying a type A glycan modification, and that the results obtained can be used to advance models about its biosynthesis.     

Hepatocyte growth factor regulated tyrosine kinase substrate in the peripheral development and function of B-cells

Hepatocyte growth factor (HGF)-regulated tyrosine kinase substrate (Hrs) is a vesicular sorting protein that functions as one of the endosomal-sorting proteins required for transport (ESCRT). Hrs, which binds to ubiquitinated proteins through its ubiquitin-interacting motif (UIM), contributes to the lysosomal transport and degradation of ubiquitinated membrane proteins. However, little is known about the relationship between B-cell functions and ESCRT proteins in vivo. Here we examined the immunological roles of Hrs in B-cell development and functions using B-cell-specific Hrs-deficient (Hrs^flox/flox;mb1^cre/+:Hrs-cKO) mice, which were generated using a cre-LoxP recombination system. Hrs deficiency in B-cells significantly reduced T-cell-dependent antibody production in vivo and impaired the proliferation of B-cells treated in vitro with an anti-IgM monoclonal antibody but not with LPS. Although early development of B-cells in the bone marrow was normal in Hrs-cKO mice, there was a significant decrease in the number of the peripheral transitional B-cells and marginal zone B-cells in the spleen of Hrs-cKO mice. These results indicate that Hrs plays important roles during peripheral development and physiological functions of B lymphocytes.     

RUBI: rapid proteomic-scale prediction of lysine ubiquitination and factors influencing predictor performance

Post-translational modification of protein lysines was recently shown to be a common feature of eukaryotic organisms. The ubiquitin modification is regarded as a versatile regulatory mechanism with many important cellular roles. Large-scale datasets are becoming available for H. sapiens ubiquitination. However, using current experimental techniques the vast majority of their sites remain unidentified and in silico tools may offer an alternative. Here, we introduce Rapid UBIquitination (RUBI) a sequence-based ubiquitination predictor designed for rapid application on a genome scale. RUBI was constructed using an iterative approach. At each iteration, important factors which influenced performance and its usability were investigated. The final RUBI model has an AUC of 0.868 on a large cross-validation set and is shown to outperform other available methods on independent sets. Predicted intrinsic disorder is shown to be weakly anti-correlated to ubiquitination for the H. sapiens dataset and improves performance slightly. RUBI predicts the number of ubiquitination sites correctly within three sites for ca. 80 % of the tested proteins. The average potentially ubiquitinated proteome fraction is predicted to be at least 25 % across a variety of model organisms, including several thousand possible H. sapiens proteins awaiting experimental characterization. RUBI can accurately predict ubiquitination on unseen examples and has a signal across different eukaryotic organisms. The factors which influenced the construction of RUBI could also be tested in other post-translational modification predictors. One of the more interesting factors is the influence of intrinsic protein disorder on ubiquitinated lysines where residues with low disorder probability are preferred.     

A comparative study of complex formation in the reactions of gold(III) with Gly-Gly, Gly-l-Ala and Gly-l-His dipeptides

Proton NMR spectroscopy was applied to study the reactions of the dipeptides glycyl-glycine (Gly-Gly) and glycyl-l-alanine (Gly-l-Ala) with hydrogen tetrachloridoaurate(III) (H[AuCl₄]). All reactions were performed at pH 2.0 and 3.0 and at 40 °C. The final products in these reactions were [Au(Gly-Gly-κ³N_G1,N_G2,O_G2)Cl] and [Au(Gly-l-Ala-κ³N_G,N_A,O_A)Cl] complexes. Tridentate coordination of the corresponding dipeptides and square-planar geometry of these Au(III) complexes was confirmed by NMR (¹H and ¹³C) spectroscopy. This study showed that at pH < 3.0 the Au(III) ion was able to deprotonate the amide nitrogen atom. However this displacement reaction was very slow and the total concentration of the corresponding Au(III)-peptide complex formed after 5 days was less than 60% for the Gly-l-Ala or 70% for the Gly-Gly dipeptide. The kinetic data of the reactions between the Gly-Gly and Gly-l-Ala dipeptides and [AuCl₄]⁻ were compared with those for the histidine-containing Gly-l-His dipeptide. The differences in the reactivity of these three dipeptides with the Au(III) ion are discussed.     

Purification and Some Properties of Cyclo(Gly-Gly) Hydrolase from a Strain of Bacillus sp. No. 106

Bacillus sp. No. 106, which was isolated from soil, secreted an enzyme that hydrolyzed cyclo(Gly-Gly). The enzyme was purified to the ultracentrifugally homogeneous state and an activity more than 450-fold that of culture broth. The enzyme was activated by Na⁺, Mg²⁺, Ca²⁺, and Sr²⁺, and strongly inhibited by Ni²⁺, Cu²⁺, p-chloromercuribenzoate, and monoiodoacetic acid. The Km value for cyclo(Gly-Gly) was estimated to be 11.1 mm. The enzyme hydrolyzed only cyclo(Gly-Gly) among various diketopiperazines tested. Aslo, the enzyme was inert toward Gly-Gly, milk casein, and hemoglobin.     

Peptide Level Immunoaffinity Enrichment Enhances Ubiquitination Site Identification on Individual Proteins

Ubiquitination is a process that involves the covalent attachment of the 76-residue ubiquitin protein through its C-terminal di-glycine (GG) to lysine (K) residues on substrate proteins. This post-translational modification elicits a wide range of functional consequences including targeting proteins for proteasomal degradation, altering subcellular trafficking events, and facilitating protein-protein interactions. A number of methods exist for identifying the sites of ubiquitination on proteins of interest, including site-directed mutagenesis and affinity-purification mass spectrometry (AP-MS). Recent publications have also highlighted the use of peptide-level immunoaffinity enrichment of K-GG modified peptides from whole cell lysates for global characterization of ubiquitination sites. Here we investigated the utility of this technique for focused mapping of ubiquitination sites on individual proteins. For a series of membrane-associated and cytoplasmic substrates including erbB-2 (HER2), Dishevelled-2 (DVL2), and T cell receptor α (TCRα), we observed that K-GG peptide immunoaffinity enrichment consistently yielded additional ubiquitination sites beyond those identified in protein level AP-MS experiments. To assess this quantitatively, SILAC-labeled lysates were prepared and used to compare the abundances of individual K-GG peptides from samples prepared in parallel. Consistently, K-GG peptide immunoaffinity enrichment yielded greater than fourfold higher levels of modified peptides than AP-MS approaches. Using this approach, we went on to characterize inducible ubiquitination on multiple members of the T-cell receptor complex that are functionally affected by endoplasmic reticulum (ER) stress. Together, these data demonstrate the utility of immunoaffinity peptide enrichment for single protein ubiquitination site analysis and provide insights into the ubiquitination of HER2, DVL2, and proteins in the T-cell receptor complex.Ubiquitin is a highly conserved, 8 kDa protein that can be covalently attached to substrate proteins, leading to changes in protein stability, subcellular localization, and pathway activation. Ubiquitination occurs primarily on lysine residues via a multistep process that requires the concerted action of three enzymes. First an E1 ubiquitin-activating enzyme uses ATP to form a high-energy thioester bond with ubiquitin. This charged E1 can subsequently interact with and transfer ubiquitin to an E2 ubiquitin-conjugating enzyme. E3 ubiquitin-ligases ultimately provide specificity to the reaction by facilitating the transfer of ubiquitin from a charged E2 to the substrate protein (1). As ubiquitination dictates the fate of modified proteins, characterizing the residues within specific proteins that can be modified by ubiquitin provides mechanistic insight into many biological processes.Dysregulated ubiquitination of critical substrates has been associated with many human diseases including cancer and neurodegeneration (2–8). Currently, efforts are underway to gain a better understanding of factors modulating ubiquitination on a substrate by substrate basis. Because E3 ligases confer much of this specificity, many have become attractive as potential therapeutic targets (9). Understanding the precise targets of ubiquitination, and the stimuli that elicit this modification, will play a central role in validating these enzymes and their modulators as targets (3, 9–12).A series of biochemical methods are available for detecting ubiquitination on both endogenous and overexpressed proteins. For endogenous proteins, a common diagnostic for ubiquitination involves protein-level immunoprecipitation followed by Western blot analysis using an antibody recognizing ubiquitin. Many ubiquitinated proteins have shorter half-lives and are present at lower levels than their unmodified counterparts. To overcome this challenge, cells overexpressing substrates of interest are often treated with proteasomal or lysosomal inhibitors to stabilize ubiquitinated proteins. Although this method is diagnostic for the presence of ubiquitination, it does not reveal the exact site(s) of ubiquitination. For this, site-directed mutagenesis is commonly employed to identify residues that may be ubiquitinated. Lysine residues are substituted with arginines (individually or in combination) and the mutant protein is examined by immunoprecipitation-Western blot analysis. In some cases, because of the number of lysine residues and the size of the protein, this task can be challenging. Functional redundancy can result in the ubiquitination of alternative lysines when preferred sites are mutated. Conversely, mutagenesis can inhibit ubiquitination by blocking the ligase-substrate interaction even when the substituted lysine was not the primary target of the modification.Mass-spectrometry-based methods provide a means of generating direct evidence to demonstrate ubiquitination on a particular lysine. This can be achieved by immunoprecipitating the protein of interest, separating the captured proteins by SDS-PAGE, excising the high molecular weight modified protein, and performing in-gel tryptic digestion (referred to as the gel-based method). Tryptic digestion results in the generation of a di-glycine remnant that remains attached to ubiquitinated lysine residue. This remnant is derived from the C terminus of ubiquitin, and results in a mass shift of +114.0429 Da that can be detected by MS/MS. The use of multiple-reaction monitoring (MRM)-initiated detection has been reported to aid in identification of low abundance ubiquitinated peptides (13). Although gel-based methods have led to the successful identification of many ubiquitination sites (14–16), there are instances in which the sensitivity has not been sufficient to systematically define ubiquitination sites of interest.Recent publications have demonstrated that peptide level immunoaffinity enrichment, which takes advantage of antibodies raised against the di-glycine remnant (K-GG)¹ motif, is a powerful approach for cataloging ubiquitinated substrates from cell lysate (17–19). K-GG peptide immunoaffinity enrichment has been used to identify >5,000 sites from as little as 1 mg of input material in global profiling efforts (19) and to successfully map ubiquitination sites on the protein APOBEC3F (20). Considering the emerging role for this method in investigating ubiquitination on a global scale, we were interested in assessing its utility in the context of focused ubiquitination site mapping on individual proteins.To investigate this, ubiquitination on three different proteins was characterized: the receptor tyrosine-protein kinase (RTK) erbB-2 (HER2), Dishevelled-2 (DVL2), and T cell receptor α (TCRα). For these three likely ubiquitinated substrates, efforts to map ubiquitination sites using protein level immunoprecipitation methods were each met with limited success. In contrast, K-GG peptide immunoaffinity enrichment using comparable amounts of starting material yielded multiple high confidence ubiquitination site identifications for each substrate. Our results demonstrate the effectiveness of K-GG peptide immunoaffinity enrichment at identifying ubiquitination sites not seen by other methods, while in parallel elucidating valuable information regarding the ubiquitination status in a global manner.     

Molecular Characterization of the SUMO-1 Modification of RanGAP1 and Its Role in Nuclear Envelope Association

The mammalian guanosine triphosphate (GTP)ase-activating protein RanGAP1 is the first example of a protein covalently linked to the ubiquitin-related protein SUMO-1. Here we used peptide mapping, mass spectroscopy analysis, and mutagenesis to identify the nature of the link between RanGAP1 and SUMO-1. SUMO-1 is linked to RanGAP1 via glycine 97, indicating that the last 4 amino acids of this 101– amino acid protein are proteolytically removed before its attachment to RanGAP1. Recombinant SUMO-1 lacking the last four amino acids is efficiently used for modification of RanGAP1 in vitro and of multiple unknown proteins in vivo. In contrast to most ubiquitinated proteins, only a single lysine residue (K526) in RanGAP1 can serve as the acceptor site for modification by SUMO-1. Modification of RanGAP1 with SUMO-1 leads to association of RanGAP1 with the nuclear envelope (NE), where it was previously shown to be required for nuclear protein import. Sufficient information for modification and targeting resides in a 25-kD domain of RanGAP1. RanGAP1–SUMO-1 remains stably associated with the NE during many cycles of in vitro import. This indicates that removal of RanGAP1 from the NE is not a required element of nuclear protein import and suggests that the reversible modification of RanGAP1 may have a regulatory role.     

Protein Degradation and Quality Control in Cells from Laforin and Malin Knockout Mice

Lafora disease is a progressive myoclonus epilepsy caused by mutations in the EPM2A or EPM2B genes that encode a glycogen phosphatase, laforin, and an E3 ubiquitin ligase, malin, respectively. Lafora disease is characterized by accumulation of insoluble, poorly branched, hyperphosphorylated glycogen in brain, muscle, heart, and liver. The laforin-malin complex has been proposed to play a role in the regulation of glycogen metabolism and protein quality control. We evaluated three arms of the protein degradation/quality control process (the autophago-lysosomal pathway, the ubiquitin-proteasomal pathway, and the endoplasmic reticulum (ER) stress response) in mouse embryonic fibroblasts from Epm2a^−/−, Epm2b^−/−, and Epm2a^−/− Epm2b^−/− mice. The levels of LC3-II, a marker of autophagy, were decreased in all knock-out cells as compared with wild type even though they still showed a slight response to starvation and rapamycin. Furthermore, ribosomal protein S6 kinase and S6 phosphorylation were increased. Under basal conditions there was no effect on the levels of ubiquitinated proteins in the knock-out cells, but ubiquitinated protein degradation was decreased during starvation or stress. Lack of malin (Epm2b^−/− and Epm2a^−/− Epm2b^−/− cells) but not laforin (Epm2a^−/− cells) decreased LAMP1, a lysosomal marker. CHOP expression was similar in wild type and knock-out cells under basal conditions or with ER stress-inducing agents. In conclusion, both laforin and malin knock-out cells display mTOR-dependent autophagy defects and reduced proteasomal activity but no defects in the ER stress response. We speculate that these defects may be secondary to glycogen overaccumulation. This study also suggests a malin function independent of laforin, possibly in lysosomal biogenesis and/or lysosomal glycogen disposal.     

Depletion of Ubiquilin induces an augmentation in soluble ubiquitinated Drosophila TDP-43 to drive neurotoxicity in the fly

The proteostasis machinery has critical functions in metabolically active cells such as neurons. Ubiquilins (UBQLNs) may decide the fate of proteins, with its ability to bind and deliver ubiquitinated misfolded or no longer functionally required proteins to the ubiquitin-proteasome system (UPS) and/or autophagy. Missense mutations in UBQLN2 have been linked to X-linked dominant amyotrophic lateral sclerosis with frontotemporal dementia (ALS-FTD). Although aggregation-prone TAR DNA-binding protein 43 (TDP-43) has been recognized as a major component of the ubiquitin pathology, the mechanisms by which UBQLN involves in TDP-43 proteinopathy have not yet been elucidated in detail. We previously characterized a new Drosophila Ubiquilin (dUbqn) knockdown model that produces learning/memory and locomotive deficits during the proteostasis impairment. In the present study, we demonstrated that the depletion of dUbqn markedly affected the expression and sub-cellular localization of Drosophila TDP-43 (TBPH), resulting in a cytoplasmic ubiquitin-positive (Ub⁺) TBPH pathology. Although we found that the knockdown of dUbqn widely altered and affected the turnover of a large number of proteins, we herein showed that an augmented soluble cytoplasmic Ub⁺-TBPH is as a crucial source of neurotoxicity following the depletion of dUbqn. We demonstrated that dUbqn knockdown-related neurotoxicity may be rescued by either restoring the proteostasis machinery or reducing the expression of TBPH. These novel results extend our knowledge on the UBQLN loss-of-function pathomechanism and may contribute to the identification of new therapeutics for ALS-FTD and aging-related diseases.     

AMSH is required to degrade ubiquitinated proteins in the central nervous system

Deubiquitination is a biochemical process that mediates the removal of ubiquitin moieties from ubiquitin-conjugated substrates. AMSH (associated molecule with the SH3 domain of STAM) is a deubiquitination enzyme that participates in the endosomal sorting of several cell-surface molecules. AMSH impairment results in missorted ubiquitinated cargoes in vitro and severe neurodegeneration in vivo, but it is not known how AMSH deficiency causes neuronal damage in the brain. Here, we demonstrate that AMSH^−/− mice developed ubiquitinated protein accumulations as early as embryonic day 10 (E10), and that severe deposits were present in the brain at postnatal day 8 (P8) and P18. Interestingly, TDP-43 was found to accumulate and colocalize with glial marker-positive cells in the brain. Glutamate receptor and p62 accumulations were also found; these molecules colocalized with ubiquitinated aggregates in the brain. These data suggest that AMSH plays an important role in degrading ubiquitinated proteins and glutamate receptors in vivo. AMSH^−/− mice provide an animal model for neurodegenerative diseases, which are commonly characterized by the generation of proteinaceous aggregates.     

UBE2E Ubiquitin-conjugating Enzymes and Ubiquitin Isopeptidase Y Regulate TDP-43 Protein Ubiquitination

Trans-activation element DNA-binding protein of 43 kDa (TDP-43) characterizes insoluble protein aggregates in distinct subtypes of frontotemporal lobar degeneration and amyotrophic lateral sclerosis. TDP-43 mediates many RNA processing steps within distinct protein complexes. Here we identify novel TDP-43 protein interactors found in a yeast two-hybrid screen using an adult human brain cDNA library. We confirmed the TDP-43 interaction of seven hits by co-immunoprecipitation and assessed their co-localization in HEK293E cells. As pathological TDP-43 is ubiquitinated, we focused on the ubiquitin-conjugating enzyme UBE2E3 and the ubiquitin isopeptidase Y (UBPY). When cells were treated with proteasome inhibitor, ubiquitinated and insoluble TDP-43 species accumulated. All three UBE2E family members could enhance the ubiquitination of TDP-43, whereas catalytically inactive UBE2E3^C145S was much less efficient. Conversely, silencing of UBE2E3 reduced TDP-43 ubiquitination. We examined 15 of the 48 known disease-associated TDP-43 mutants and found that one was excessively ubiquitinated. This strong TDP-43^K263E ubiquitination was further enhanced by proteasomal inhibition as well as UBE2E3 expression. Conversely, UBE2E3 silencing and expression of UBPY reduced TDP-43^K263E ubiquitination. Moreover, wild-type but not active site mutant UBPY reduced ubiquitination of TDP-43 C-terminal fragments and of a nuclear import-impaired mutant. In Drosophila melanogaster, UBPY silencing enhanced neurodegenerative TDP-43 phenotypes and the accumulation of insoluble high molecular weight TDP-43 and ubiquitin species. Thus, UBE2E3 and UBPY participate in the regulation of TDP-43 ubiquitination, solubility, and neurodegeneration.     

Posttranslational Regulation of Human DNA Polymerase ι

Human DNA polymerases (pols) η and ι are Y-family DNA polymerase paralogs that facilitate translesion synthesis past damaged DNA. Both polη and polι can be monoubiquitinated in vivo. Polη has been shown to be ubiquitinated at one primary site. When this site is unavailable, three nearby lysines may become ubiquitinated. In contrast, mass spectrometry analysis of monoubiquitinated polι revealed that it is ubiquitinated at over 27 unique sites. Many of these sites are localized in different functional domains of the protein, including the catalytic polymerase domain, the proliferating cell nuclear antigen-interacting region, the Rev1-interacting region, and its ubiquitin binding motifs UBM1 and UBM2. Polι monoubiquitination remains unchanged after cells are exposed to DNA-damaging agents such as UV light (generating UV photoproducts), ethyl methanesulfonate (generating alkylation damage), mitomycin C (generating interstrand cross-links), or potassium bromate (generating direct oxidative DNA damage). However, when exposed to naphthoquinones, such as menadione and plumbagin, which cause indirect oxidative damage through mitochondrial dysfunction, polι becomes transiently polyubiquitinated via Lys¹¹- and Lys⁴⁸-linked chains of ubiquitin and subsequently targeted for degradation. Polyubiquitination does not occur as a direct result of the perturbation of the redox cycle as no polyubiquitination was observed after treatment with rotenone or antimycin A, which both inhibit mitochondrial electron transport. Interestingly, polyubiquitination was observed after the inhibition of the lysine acetyltransferase KATB3/p300. We hypothesize that the formation of polyubiquitination chains attached to polι occurs via the interplay between lysine acetylation and ubiquitination of ubiquitin itself at Lys¹¹ and Lys⁴⁸ rather than oxidative damage per se.     

An immunoaffinity purification method for the proteomic analysis of ubiquitinated protein complexes

Protein ubiquitination plays an important role in the regulation of many cellular processes, including protein degradation, cell cycle regulation, apoptosis, and DNA repair. To study the ubiquitin proteome we have established an immunoaffinity purification method for the proteomic analysis of endogenously ubiquitinated protein complexes. A strong, specific enrichment of ubiquitinated factors was achieved using the FK2 antibody bound to protein G-beaded agarose, which recognizes monoubiquitinated and polyubiquitinated conjugates. Mass spectrometric analysis of two FK2 immunoprecipitations (IPs) resulted in the identification of 296 FK2-specific proteins in both experiments. The isolation of ubiquitinated and ubiquitination-related proteins was confirmed by pathway analyses (using Ingenuity Pathway Analysis and Gene Ontology-annotation enrichment). Additionally, comparing the proteins that specifically came down in the FK2 IP with databases of ubiquitinated proteins showed that a high percentage of proteins in our enriched fraction was indeed ubiquitinated. Finally, assessment of protein–protein interactions revealed that significantly more FK2-specific proteins were residing in protein complexes than in random protein sets. This method, which is capable of isolating both endogenously ubiquitinated proteins and their interacting proteins, can be widely used for unraveling ubiquitin-mediated protein regulation in various cell systems and tissues when comparing different cellular states.     

Spatiotemporal Regulation of the Ubiquitinated Cargo-binding Activity of Rabex-5 in the Endocytic Pathway

Ubiquitin (Ub)-dependent endocytosis of membrane proteins requires precise molecular recognition of ubiquitinated cargo by Ub-binding proteins (UBPs). Many UBPs are often themselves monoubiquitinated, a mechanism referred to as coupled monoubiquitination, which prevents them from binding in trans to the ubiquitinated cargo. However, the spatiotemporal regulatory mechanism underlying the interaction of UBPs with the ubiquitinated cargo, via their Ub-binding domains (UBDs) remains unclear. Previously, we reported the interaction of Rabex-5, a UBP and guanine nucleotide exchange factor (GEF) for Rab5, with ubiquitinated neural cell adhesion molecule L1, via its motif interacting with Ub (MIU) domain. This interaction is critical for the internalization and sorting of the ubiquitinated L1 into endosomal/lysosomal compartments. The present study demonstrated that the interaction of Rabex-5 with Rab5 depends specifically on interaction of the MIU domain with the ubiquitinated L1 to drive its internalization. Notably, impaired GEF mutants and the Rabex-5^E213A mutant increased the flexibility of the hinge region in the HB-VPS9 tandem domain, which significantly affected their interactions with the ubiquitinated L1. In addition, GEF mutants increased the catalytic efficiency, which resulted in a reduced interaction with the ubiquitinated L1. Furthermore, the coupled monoubiquitination status of Rabex-5 was found to be significantly associated with interaction of Rabex-5 and the ubiquitinated L1. Collectively, our study reveals a novel mechanism, wherein the GEF activity of Rabex-5 acts as an intramolecular switch orchestrating ubiquitinated cargo-binding activity and coupled monoubiquitination to permit the spatiotemporal dynamic exchange of the ubiquitinated cargos.