Ubiquitin Family Research Articles

Genomic expression profiling of TNF-α-treated BDC2.5 diabetogenic CD4 + T cells

TNF-alpha plays an important role in immune regulation, inflammation, and autoimmunity. Chronic TNF exposure has been shown to down-modulate T cell responses. In a mouse T cell hybridoma model, TNF attenuated T cell receptor (TCR) signaling. We have confirmed that chronic TNF and anti-TNF exposure suppressed and increased T cell responses, respectively. In adult TCR (BDC2.5) transgenic nonobese diabetic mice, DNA microarray analysis of global gene expression in BDC2.5 CD4(+) T cells in response to chronic TNF or anti-TNF exposure showed that genes involved in functional categories including T cell signaling, cell cycle, proliferation, ubiquitination, cytokine synthesis, calcium signaling, and apoptosis were modulated. Genes such as ubiquitin family genes, cytokine inducible Src homology 2-containing genes, cyclin-dependent kinase inhibitors p21, p57, calmodulin family genes (calmodulin-1, -2, and -3) and calcium channel voltage-dependent, N type alpha1B subunit (CaV2.2) were induced by TNF, whereas Vav2, Rho GTPase-activating protein, calcium channel voltage-dependent, L type alpha1C subunit (CaV1.2), IL-1 receptor-associated kinase-1 and -2, and IL enhancer binding factor 3 were reduced by TNF. Genes such as CaV1.2 and proliferating cell nuclear antigen, repressed by TNF, were induced by anti-TNF treatment. Further, we showed that chronic TNF exposure impaired NF-kappaB and adaptor protein 1 transactivation activity, leading to T cell unresponsiveness. Thus, our results present a detailed picture of transcriptional programs affected by chronic TNF exposure and provide candidate target genes that may function to mediate TNF-induced T cell unresponsiveness.

Proteomic Analysis of SUMO Mediated Stress Responses in Yeast

Cells are constantly exposed to a wide variety of environmental stresses and their ability to maintain the integrity of their genome is essential to their survival. One cellular pathway that plays a key role in this cellular response is the SUMO modification pathway. SUMO is a member of the ubiquitin family of small protein modifiers and its covalent attachment to cellular targets is a critical regulatory step for a diverse range of cellular processes that includes transcriptional regulation, cell cycle progression, and cellular DNA damage response. In this study we are investigating how patterns of targets modified by SUMO changes in response to various stresses such as hydrogen peroxide, ethanol, nocodazole, and doxorubicin. This approach involves the use of a quantitative proteomics platform established in our laboratory and is built around a recently developed linear ion trap–orbitrap hybrid mass spectrometer. Ultimately, this work will enable us to understand how cells use SUMO pathways to protect themselves from genotoxic damage and offer insight into how the deregulation of this pathway, a common feature in cancers, may lead to genetic instability. These experiments are currently underway and the results from this work will be presented. Importantly, the SUMO pathway is highly conserved between yeast and humans so that important findings generated by our analysis will likely be directly applicable to humans.

Structural and electrostatic properties of ubiquitination and related pathways

Post-translational modification by ubiquitin and ubiquitin-like (UBL) proteins is a key mechanism for cellular control. The specificity of the enzymes of ubiquitination and their close paralogs is dependent on their molecular electrostatic potentials. For example, analysis of molecular electrostatic potentials and electrostatically key residues can account for the selectivity of different E1s (activating enzymes) and of different SUMO proteases. The molecular interactions of the ubiquitin conjugating enzymes, the ubiquitin family proteins (UFP) and UBL domains are discussed in detail. An interesting observation is that the Non Canonical Ubiquitin Conjugating Enzymes (NCUBEs) have electrostatic potentials that are more similar to the UBC9 orthologs, the SUMO conjugating enzymes, than they are to other ubiquitin conjugating enzymes. It had previously been suggested that UBC9 may select for SUMO based on its difference in electrostatic potential as compared to other E2s but the NCUBE exception suggests that this may not be the case. The web site http://www.ubiquitin-resource.org/ allows users to find the E2s most electrostatically similar to a query E2. Where possible, models have been made for all E2 domains in the SMART database (http://smart.embl-heidelberg.de/). A brief overview of molecular electrostatic potentials and their application to understanding protein function is also given.

Study of proteins associated with the Eimeria tenella refractile body by a proteomic approach

Refractile bodies (RB), whose function is still unknown, are specific structures of Eimeriidae parasites. In order to study their proteome, RB were purified from Eimeria tenella sporozoites by a new procedure using a reversible fixation followed by centrifugation. RB proteins were resolved by two-dimensional electrophoresis. Around 76 and 89 spots were detected on RB two-dimensional gels using gradients in the 3–10 and 4–7 range, respectively. RB proteins were located mainly between pH 5 and 7. RB gels were then compared with previously established maps of the entire sporozoite proteome. Proteins appearing in new spots were identified by mass spectrometry. Thirty protein isoforms were located in RB. Added to the already known RB proteins such as Eimepsin and SO7′, the new RB proteins were defined as haloacid dehalogenase, hydrolase, subtilase, lactacte dehydrogenase or ubiquitin family proteins. The RB proteome analysis confirmed the hypothesis that this structure is a reservoir for proteins necessary to invasion but also suggests that RB have energetic and metabolic functions.

The prokaryotic antecedents of the ubiquitin-signaling system and the early evolution of ubiquitin-like β-grasp domains

A systematic analysis of prokaryotic ubiquitin-related beta-grasp fold proteins provides new insights into the Ubiquitin family functional history.

UBL/UBA Ubiquitin Receptor Proteins Bind a Common Tetraubiquitin Chain

The ubiquitin-proteasome pathway is essential throughout the life cycle of a cell. This system employs an astounding number of proteins to ubiquitylate and to deliver protein substrates to the proteasome for their degradation. At the heart of this process is the large and growing family of ubiquitin receptor proteins. Within this family is an intensely studied group that contains both ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains: Rad23, Ddi1 and Dsk2. Although UBL/UBA family members are reported to regulate the degradation of other proteins, their individual roles in ubiquitin-mediated protein degradation has proven difficult to resolve due to their overlapping functional roles and interaction with each other and other ubiquitin family members. Here, we use a combination of NMR spectroscopy and molecular biology to reveal that Rad23 and Ddi1 interact with each other by using UBL/UBA domain interactions in a manner that does not preclude their interaction with ubiquitin. We demonstrate that UBL/UBA proteins can bind a common tetraubiquitin molecule and thereby provide strong evidence for a model in which chains adopt an opened structure to bind multiple receptor proteins. Altogether our results suggest a mechanism through which UBL/UBA proteins could protect chains from premature de-ubiquitylation and unnecessary elongation during their transit to the proteasome.

Detection of modification by ubiquitin-like proteins

Ubiquitin and ubiquitin-like proteins (Ubls) are conjugated to many target proteins either as monomeric units or as polymeric chains. There are at least 12 members of the ubiquitin family in the human genome and their conjugation dramatically alters the properties of the modified protein. The presence of highly active proteases that specifically deconjugate Ubls often means that, in the cell, the steady state level of modified protein is low. Detection of protein species modified by Ubls can therefore represent a significant challenge. Here, we describe methods that have been developed to allow detection of Ubl modified proteins both in vivo and in vitro.

Insights into E3 ligase activity revealed by a SUMO–RanGAP1–Ubc9–Nup358 complex

SUMO-1 (for small ubiquitin-related modifier) belongs to the ubiquitin (Ub) and ubiquitin-like (Ubl) protein family. SUMO conjugation occurs on specific lysine residues within protein targets, regulating pathways involved in differentiation, apoptosis, the cell cycle and responses to stress by altering protein function through changes in activity or cellular localization or by protecting substrates from ubiquitination. Ub/Ubl conjugation occurs in sequential steps and requires the concerted action of E2 conjugating proteins and E3 ligases. In addition to being a SUMO E3, the nucleoporin Nup358/RanBP2 localizes SUMO-conjugated RanGAP1 to the cytoplasmic face of the nuclear pore complex by means of interactions in a complex that also includes Ubc9, the SUMO E2 conjugating protein. Here we describe the 3.0-A crystal structure of a four-protein complex of Ubc9, a Nup358/RanBP2 E3 ligase domain (IR1-M) and SUMO-1 conjugated to the carboxy-terminal domain of RanGAP1. Structural insights, combined with biochemical and kinetic data obtained with additional substrates, support a model in which Nup358/RanBP2 acts as an E3 by binding both SUMO and Ubc9 to position the SUMO-E2-thioester in an optimal orientation to enhance conjugation.

SUMOylation of the Polyglutamine Repeat Protein, Ataxin-1, Is Dependent on a Functional Nuclear Localization Signal

SUMO (small ubiquitin-like modifier) is a member of the ubiquitin family of proteins. SUMO targets include proteins involved in numerous roles including nuclear transport and transcriptional regulation. The previous finding that mutant ataxin-1[82Q] disrupted promyelocytic leukemia (PML) oncogenic domains prompted us to determine whether ataxin-1 disrupts another component of PML oncogenic domains, Sp100 (100-kDa Speckled protein). Similar to the PML protein, mutant ataxin-1[82Q] redistributed Sp100 to mutant ataxin-1[82Q] nuclear inclusions. Based on the ability of PML and Sp100 to be covalently modified by SUMO, we investigated the ability of ataxin-1 to be SUMOylated. SUMO-1 was found to covalently modify the polyglutamine repeat protein ataxin-1. There was a decrease in ataxin-1 SUMOylation in the presence of the expanded polyglutamine tract, ataxin-1[82Q]. The phospho-mutant, ataxin-1[82Q]-S776A, restored SUMO levels to those of wild-type ataxin-1[30Q]. SUMOylation of ataxin-1 was dependent on a functional nuclear localization signal. Ataxin-1 SUMOylation was mapped to at least five lysine residues. Lys(16), Lys(194) preceding the polyglutamine tract, Lys(610)/Lys(697) in the C-terminal ataxin high mobility group domain, and Lys(746) all contribute to ataxin-1 SUMOylation.

Elucidation of IL-1/TGF-β interactions in mouse chondrocyte cell line by genome-wide gene expression

To elucidate the antagonism between interleukin-1 (IL-1) and transforming growth factor-beta (TGF-beta) at the gene expression level, as IL-1 and TGF-beta are postulated to be critical mediators of cartilage degeneration/protection in rheumatic diseases. The H4 chondrocyte cell line was validated by comparing metalloproteinase expression profile with intact murine cartilage by reverse transcription polymerase chain reaction. Genome-wide gene expression in the H4 cells in response to IL-1 and TGF-beta, alone and in combination, was analyzed by using oligonucleotide arrays negotiating approximately 12,000 genes. The response of cartilage and the H4 cell line to IL-1 and TGF-beta was comparable. Oligonucleotide array analysis demonstrated a mutual but asymmetrical antagonism as the dominant mode of interaction between IL-1 and TGF-beta. Cluster analysis revealed a remarkable selectivity in the mode of action exerted by TGF-beta on IL-1 regulated genes: antagonistic on pro-inflammatory genes whereas additive on growth regulators such as vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF). While the former cluster underlined the protective effect of TGF-beta, the latter underscored the adverse effect of TGF-beta. We further identified potentially novel classes of target genes under control of TGF-beta such as ras family, histones, proteasome components, and ubiquitin family, highlighting the importance of such genes in TGF signaling besides the well-characterized SMAD pathway. We identified a cluster of genes as potential targets mediating the adverse effect of TGF-beta such as fibrosis. Transcriptional regulation of ras GTPase and ubiquitin/proteasome pathways is likely to be a novel mechanism mediating the effect of TGF-beta and its interaction with IL-1. These down-stream genes and pathways can be targets in future therapy.

Native-state dynamics of the ubiquitin family: implications for function and evolution

Protein dynamics are integral to protein function. In recent years, the use of computer simulation to understand the molecular motions of proteins has become widespread. However, there are few such studies which compare the dynamics of proteins that are structurally and functionally related. In this study, we present native-state molecular dynamic simulations of four proteins which possess a ubiquitin-like fold. Three of these proteins are thought to have evolved from a common ancestral ubiquitin-like protein and have similarities in their function. A fourth protein, which is structurally homologous but which appears to have a different function, is also studied. Local fluctuations in the native state simulations are analysed, and conserved motions of the C-alpha backbone atoms are identified in residues which are important for function. In addition, the global dynamics of the proteins are analysed using the essential-dynamics method. This analysis reveals a slightly higher degree of conservation in dynamics for the three proteins which are functionally related. Both the global and local analyses illustrate how nature has optimized and conserved protein motions for specific biological activity within the ubiquitin family.

The ubiquitin family gathers: The Joint 59th Harden/EMBO Conference: The Ubiquitin–Proteasome System in Health and Disease

The ubiquitin family gathers: The Joint 59th Harden/EMBO Conference: The Ubiquitin–Proteasome System in Health and Disease

A Universal Strategy for Proteomic Studies of SUMO and Other Ubiquitin-like Modifiers

Post-translational modification by the conjugation of small ubiquitin-like modifiers is an essential mechanism to affect protein function. Currently, only a limited number of substrates are known for most of these modifiers, thus limiting our knowledge of their role and relevance for cellular physiology. Here, we report the development of a universal strategy for proteomic studies of ubiquitin-like modifiers. This strategy involves the development of stable transfected cell lines expressing a double-tagged modifier under the control of a tightly negatively regulated promoter, the induction of the expression and conjugation of the tagged modifier to cellular proteins, the tandem affinity purification of the pool of proteins covalently modified by the tagged modifier, and the identification of the modified proteins by LC and MS. By applying this methodology to the proteomic analysis of SUMO-1 and SUMO-3, we determined that SUMO-1 and SUMO-3 are stable proteins exhibiting half-lives of over 20 h, demonstrated that sumoylation with both SUMO-1 and SUMO-3 is greatly stimulated by MG-132 and heat shock treatment, demonstrated the preferential usage of either SUMO-1 or SUMO-3 for some known SUMO substrates, and identified 122 putative SUMO substrates of which only 27 appeared to be modified by both SUMO-1 and SUMO-3. This limited overlapping in the subset of proteins modified by SUMO-1 and SUMO-3 supports that the SUMO paralogues are likely to be functionally distinct. Three of the novel putative SUMO substrates identified, namely the polypyrimidine tract-binding protein-associated splicing factor PSF, the structural microtubular component alpha-tubulin, and the GTP-binding nuclear protein Ran, were confirmed as authentic SUMO substrates. The application of this universal strategy to the identification of the pool of cellular substrates modified by other ubiquitin-like modifiers will dramatically increase our knowledge of the biological role of the different ubiquitin-like conjugations systems in the cell.

Ubiquitin-mediated fluorescence complementation reveals that Jun ubiquitinated by Itch/AIP4 is localized to lysosomes.

Ubiquitin family peptide modifications regulate the functions and stabilities of many proteins. We have developed an approach for the visualization of ubiquitinated proteins in living cells designated ubiquitin-mediated fluorescence complementation (UbFC). This approach is based on complementation among fragments of fluorescent proteins when they are brought together by the covalent conjugation of ubiquitin fused to one fragment to a substrate protein fused to a complementary fragment. The UbFC strategy enables simultaneous visualization of proteins modified by different ubiquitin family peptides and comparison of their effects on protein localization. Visualization of ubiquitinated Jun revealed that it was localized predominantly to cytoplasmic structures. In contrast, Jun conjugated to small ubiquitin-related modifier 1 (SUMO1) was localized to subnuclear foci. Comparison of the distribution of ubiquitinated Jun with markers for various cytoplasmic compartments revealed that ubiquitinated Jun was localized to lysosomal vesicles. Fractionation of cell lysates confirmed that the majority of ubiquitinated Jun partitioned to the cytoplasmic fraction, and density gradient centrifugation analysis demonstrated that it cosedimented with lysosomal beta-hexosaminidase activity. Mutation of a recognition sequence for the E3 ligase Itch/AIP4 prevented Jun ubiquitination and stabilized it in cells. Inhibition of lysosomal protein degradation by bafilomycin or chloroquine stabilized Jun but had no effect on the stability of mutated Jun that was not ubiquitinated by Itch/AIP4. The visualization of ubiquitinated Jun in living cells has uncovered a lysosomal pathway for Jun degradation that involves ubiquitination by Itch/AIP4.

Raf RBD and ubiquitin proteins share similar folds, folding rates and mechanisms despite having unrelated amino acid sequences.

Recent experimental and theoretical studies in protein folding suggest that the rates and underlying mechanisms by which proteins attain the native state are largely determined by the topological complexity of a specific fold rather than by the fine details of the amino acid sequences. However, such arguments are based upon the examination of a limited number of protein folds. To test this view, we sought to investigate whether proteins belonging to the ubiquitin superfamily display similar folding behavior. To do so, we compared the folding-unfolding transitions of mammalian ubiquitin (mUbi) with those of its close yeast homologue (yUbi), and to those of the structurally related Ras binding domain (RBD) of the serine/threonine kinase raf that displays no apparent sequence homology with the ubiquitin family members. As demonstrated for mUbi [Krantz, B. A., and Sosnick, T. R. (2000) Biochemistry 39, 11696-11701], we show that a two-state transition model with no burst phase intermediate can describe folding of both yUbi and raf RBD. We further demonstrate that (1) all three proteins refold at rates that are within 1 order of magnitude (1800, 1100, and 370 s(-1) for mUbi, raf RBD, and yUbi, respectively), (2) both mUbi and raf RBD display similar refolding heterogeneity, and (3) the folding free energy barriers of both mUbi and raf RBD display a similar temperature dependence and sensitivity to a stabilizing agent or to mutations of a structurally equivalent central core residue. These findings are consistent with the view that rates and mechanisms for protein folding depend mostly on the complexity of the native structure topology rather than on the fine details of the amino acid sequence.

Identification of a ubiquitin family protein as a novel neutrophil chemotactic factor

Neutrophil chemotaxis is a process that is essential for the recruitment of neutrophils to an inflamed site. In the present study, we found a remarkable increase in neutrophil chemotactic activity in the lysate of red blood cells (RBC) of mice infected with murine malaria, Plasmodium yoelii . A neutrophil chemotactic factor with an apparent molecular weight of 17 kDa (IP17) was isolated from RBC by a combination of anion-exchange chromatography on DE52 and cation-exchange chromatography on Mono S. A comprehensive GenBank database search of N-terminal amino acid sequences and MALDI-TOF mass analysis of IP17 revealed that IP17 is identical to a murine homologue of ISG15/UCRP, a member of the ubiquitin family of proteins that are inducible by interferon-β. Recombinant mouse ISG15 showed neutrophil chemotactic activity comparable to that of natural IP17. IP17 showed specific chemotactic activity forward neutrophils and activated neutrophils to induce the release of eosinophil chemotactic factors. These results suggest that the ubiquitin family protein ISG15/UCRP has novel functions in neutrophil-mediated immune mechanisms.

Identification of a ubiquitin family protein as a novel neutrophil chemotactic factor

Neutrophil chemotaxis is a process that is essential for the recruitment of neutrophils to an inflamed site. In the present study, we found a remarkable increase in neutrophil chemotactic activity in the lysate of red blood cells (RBC) of mice infected with murine malaria, Plasmodium yoelii. A neutrophil chemotactic factor with an apparent molecular weight of 17 kDa (IP17) was isolated from RBC by a combination of anion-exchange chromatography on DE52 and cation-exchange chromatography on Mono S. A comprehensive GenBank database search of N-terminal amino acid sequences and MALDI-TOF mass analysis of IP17 revealed that IP17 is identical to a murine homologue of ISG15/UCRP, a member of the ubiquitin family of proteins that are inducible by interferon-β. Recombinant mouse ISG15 showed neutrophil chemotactic activity comparable to that of natural IP17. IP17 showed specific chemotactic activity forward neutrophils and activated neutrophils to induce the release of eosinophil chemotactic factors. These results suggest that the ubiquitin family protein ISG15/UCRP has novel functions in neutrophil-mediated immune mechanisms.

The Schizosaccharomyces pombe rad60 gene is essential for repairing double-strand DNA breaks spontaneously occurring during replication and induced by DNA-damaging agents.

To identify novel genes involved in DNA double-strand break (DSB) repair, we previously isolated Schizosaccharomyces pombe mutants which are hypersensitive to methyl methanesulfonate (MMS) and synthetic lethals with rad2. This study characterizes one of these mutants, rad60-1. The gene that complements the MMS sensitivity of this mutant was cloned and designated rad60. rad60 encodes a protein with 406 amino acids which has the conserved ubiquitin-2 motif found in ubiquitin family proteins. rad60-1 is hypersensitive to UV and gamma rays, epistatic to rhp51, and defective in the repair of DSBs caused by gamma-irradiation. The rad60-1 mutant is also temperature sensitive for growth. At the restrictive temperature (37 degrees C), rad60-1 cells grow for several divisions and then arrest with 2C DNA content; the arrested cells accumulate DSBs and have a diffuse and often aberrantly shaped nuclear chromosomal domain. The rad60-1 mutant is a synthetic lethal with rad18-X, and expression of wild-type rad60 from a multicopy plasmid partially suppresses the MMS sensitivity of rad18-X cells. rad18 encodes a conserved protein of the structural maintenance of chromosomes (SMC) family (A. R. Lehmann, M. Walicka, D. J. Griffiths, J. M. Murray, F. Z. Watts, S. McCready, and A. M. Carr, Mol. Cell. Biol. 15:7067-7080, 1995). These results suggest that S. pombe Rad60 is required to repair DSBs, which accumulate during replication, by recombination between sister chromatids. Rad60 may perform this function in concert with the SMC protein Rad18.

Structural studies of the interaction between ubiquitin family proteins and proteasome subunit S5a.

The 26S proteasome is essential for the proteolysis of proteins that have been covalently modified by the attachment of polyubiquitinated chains. Although the 20S core particle performs the degradation, the 19S regulatory cap complex is responsible for recognition of polyubiquitinated substrates. We have focused on how the S5a component of the 19S complex interacts with different ubiquitin-like (ubl) modules, to advance our understanding of how polyubiquitinated proteins are targeted to the proteasome. To achieve this, we have determined the solution structure of the ubl domain of hPLIC-2 and obtained a structural model of hHR23a by using NMR spectroscopy and homology modeling. We have also compared the S5a binding properties of ubiquitin, SUMO-1, and the ubl domains of hPLIC-2 and hHR23a and have identified the residues on their respective S5a contact surfaces. We provide evidence that the S5a-binding surface on the ubl domain of hPLIC-2 is required for its interaction with the proteasome. This study provides structural insights into protein recognition by the proteasome, and illustrates how the protein surface of a commonly utilized fold has highly evolved for various biological roles.

Post-translational modifications of proteins: acetylcholinesterase as a model system.

Analysis of the expressed protein complement of cells requires knowledge of the diversity of post-translational modifications that can occur and which can be transient or permanent. The modifications range from amino acid changes through to the addition of macromolecules: lipid, carbohydrate or protein. Many variants of the common amino acids can occur, which can affect the structure or function of the protein. The major class of modification, however, is represented by glycosylation, N-linked, O-linked, or glycosylphosphatidylinositol(GPI)-linked. Such modifications have roles in protein stability and folding, targeting and recognition. Glycosylated proteins can be found in all cellular compartments and, intracellularly, O-GlcNAc modification is commonplace. Lipid modification of proteins (acylation, prenylation, GPI-anchoring) is also common, resulting in membrane association, and can play an important role in cell signalling. Targeting and turnover of proteins can also be mediated via covalent protein addition, for example by members of the ubiquitin family. Limited proteolysis as a post-translational modification will be discussed, focusing on the family of membrane protein secretases, in particular in relation to the Alzheimer's amyloid precursor protein. Finally, acetylcholinesterase will be used as a model example to illustrate the diversity of modifications occurring on a single protein.