Namrata D. Udeshi

Lenalidomide Causes Selective Degradation of IKZF1 and IKZF3 in Multiple Myeloma Cells

Drug With a (Re)Purpose Thalidomide, once infamous for its deleterious effects on fetal development, has re-emerged as a drug of great interest because of its beneficial immunomodulatory effects. A derivative drug called lenalidomide significantly extends the survival of patients with multiple myeloma, but the molecular mechanisms underlying its efficacy remain unclear (see the Perspective by Stewart). Building on a previous observation that thalidomide binds to cereblon, a ubiquitin ligase, Lu et al. (p. 305, published online 28 November) and Krönke et al. (p. 301, published online 28 November) show that in the presence of lenalidomide, cereblon selectively targets two B cell transcription factors (Ikaros family members, IKZF1 and IKZF3) for degradation. In myeloma cell lines and patient cells, down-regulation of IKZF1 and IKZF3 was necessary and sufficient for the drugs anticancer activity. Thus, lenalidomide may act, at least in part, by “grepurposing” a ubiquitin ligase. A drug with potent activity in multiple myeloma patients acts by inducing degradation of two specific transcription factors. [Also see Perspective by Stewart] Lenalidomide is a drug with clinical efficacy in multiple myeloma and other B cell neoplasms, but its mechanism of action is unknown. Using quantitative proteomics, we found that lenalidomide causes selective ubiquitination and degradation of two lymphoid transcription factors, IKZF1 and IKZF3, by the CRBN-CRL4 ubiquitin ligase. IKZF1 and IKZF3 are essential transcription factors in multiple myeloma. A single amino acid substitution of IKZF3 conferred resistance to lenalidomide-induced degradation and rescued lenalidomide-induced inhibition of cell growth. Similarly, we found that lenalidomide-induced interleukin-2 production in T cells is due to depletion of IKZF1 and IKZF3. These findings reveal a previously unknown mechanism of action for a therapeutic agent: alteration of the activity of an E3 ubiquitin ligase, leading to selective degradation of specific targets.

Integrated proteomic analysis of post-translational modifications by serial enrichment

We report a mass spectrometry–based method for the integrated analysis of protein expression, phosphorylation, ubiquitination and acetylation by serial enrichments of different post-translational modifications (SEPTM) from the same biological sample. This technology enabled quantitative analysis of nearly 8,000 proteins and more than 20,000 phosphorylation, 15,000 ubiquitination and 3,000 acetylation sites per experiment, generating a holistic view of cellular signal transduction pathways as exemplified by analysis of bortezomib-treated human leukemia cells.

EMRE is an Essential Component of the Mitochondrial Calcium Uniporter Complex

EMRE Emerges Concentrations of calcium within mitochondria are tightly regulated and modulate physiological mitochondrial functions, including control of metabolism and cell death. Sancak et al. (p. 1379, published online 14 November) complete the molecular characterization of the mitochondrial calcium uniporter (MCU), the multicomponent channel that allows concentration of calcium within the organelle. They identified a small protein termed “essential MCU regulator”—or EMRE—which was required for calcium transport activity of the fully assembled uniporter. A final but essential protein component involved in maintaining mitochondrial calcium levels is discovered. The mitochondrial uniporter is a highly selective calcium channel in the organelle’s inner membrane. Its molecular components include the EF-hand–containing calcium-binding proteins mitochondrial calcium uptake 1 (MICU1) and MICU2 and the pore-forming subunit mitochondrial calcium uniporter (MCU). We sought to achieve a full molecular characterization of the uniporter holocomplex (uniplex). Quantitative mass spectrometry of affinity-purified uniplex recovered MICU1 and MICU2, MCU and its paralog MCUb, and essential MCU regulator (EMRE), a previously uncharacterized protein. EMRE is a 10-kilodalton, metazoan-specific protein with a single transmembrane domain. In its absence, uniporter channel activity was lost despite intact MCU expression and oligomerization. EMRE was required for the interaction of MCU with MICU1 and MICU2. Hence, EMRE is essential for in vivo uniporter current and additionally bridges the calcium-sensing role of MICU1 and MICU2 with the calcium-conducting role of MCU.

O-GlcNAc Regulates FoxO Activation in Response to Glucose

FoxO proteins are key transcriptional regulators of nutrient homeostasis and stress response. The transcription factor FoxO1 activates expression of gluconeogenic, including phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, and also activates the expression of the oxidative stress response enzymes catalase and manganese superoxide dismutase. Hormonal and stress-dependent regulation of FoxO1 via acetylation, ubiquitination, and phosphorylation, are well established, but FoxOs have not been studied in the context of the glucose-derived O-linked β-N-acetylglucosamine (O-GlcNAc) modification. Here we show that O-GlcNAc on hepatic FoxO1 is increased in diabetes. Furthermore, O-GlcNAc regulates FoxO1 activation in response to glucose, resulting in the paradoxically increased expression of gluconeogenic genes while concomitantly inducing expression of genes encoding enzymes that detoxify reactive oxygen species. GlcNAcylation of FoxO provides a new mechanism for direct nutrient control of transcription to regulate metabolism and stress response through control of FoxO1 activity.

Enrichment and Site Mapping of O-Linked N-Acetylglucosamine by a Combination of Chemical/Enzymatic Tagging, Photochemical Cleavage, and Electron Transfer Dissociation Mass Spectrometry

Numerous cellular processes are regulated by the reversible addition of either phosphate or O-linked β-N-acetylglucosamine (O-GlcNAc) to nuclear and cytoplasmic proteins. Although sensitive methods exist for the enrichment and identification of protein phosphorylation sites, those for the enrichment of O-GlcNAc-containing peptides are lacking. Reported here is highly efficient methodology for the enrichment and characterization of O-GlcNAc sites from complex samples. In this method, O-GlcNAc-modified peptides are tagged with a novel biotinylation reagent, enriched by affinity chromatography, released from the solid support by photochemical cleavage, and analyzed by electron transfer dissociation mass spectrometry. Using this strategy, eight O-GlcNAc sites were mapped from a tau-enriched sample from rat brain. Sites of GlcNAcylation were characterized on important neuronal proteins such as tau, synucleins, and methyl CpG-binding protein 2.

Lenalidomide induces ubiquitination and degradation of CK1α in del(5q) MDS.

Lenalidomide is a highly effective treatment for myelodysplastic syndrome (MDS) with deletion of chromosome 5q (del(5q)). Here, we demonstrate that lenalidomide induces the ubiquitination of casein kinase 1A1 (CK1α) by the E3 ubiquitin ligase CUL4–RBX1–DDB1–CRBN (known as CRL4CRBN), resulting in CK1α degradation. CK1α is encoded by a gene within the common deleted region for del(5q) MDS and haploinsufficient expression sensitizes cells to lenalidomide therapy, providing a mechanistic basis for the therapeutic window of lenalidomide in del(5q) MDS. We found that mouse cells are resistant to lenalidomide but that changing a single amino acid in mouse Crbn to the corresponding human residue enables lenalidomide-dependent degradation of CK1α. We further demonstrate that minor side chain modifications in thalidomide and a novel analogue, CC-122, can modulate the spectrum of substrates targeted by CRL4CRBN. These findings have implications for the clinical activity of lenalidomide and related compounds, and demonstrate the therapeutic potential of novel modulators of E3 ubiquitin ligases.

A PGC-1α-O-GlcNAc Transferase Complex Regulates FoxO Transcription Factor Activity in Response to Glucose

Metabolic and stress response gene regulation is crucial for the survival of an organism to a changing environment. Three key molecules that sense nutrients and broadly affect gene expression are the FoxO transcription factors, the transcriptional co-activator PGC-1α, and the dynamic post-translational modification, O-linked β-N-acetylglucosamine (O-GlcNAc). Here we identify novel post-translational modifications of PGC-1α, including O-GlcNAc, and describe a novel mechanism for how PGC-1α co-activates transcription by FoxOs. In liver, in cultured cells, and in vitro with recombinant proteins, PGC-1α binds to O-GlcNAc transferase and targets the enzyme to FoxOs, resulting in their increased GlcNAcylation and increased transcriptional activity. Furthermore, glucose-enhanced activation of FoxO1 occurs via this PGC-1α-O-GlcNAc transferase-mediated GlcNAcylation. Therefore, one mechanism by which PGC-1α can serve as a co-activator of transcription is by targeting the O-GlcNAc transferase to increase GlcNAcylation of specific transcription factors important to nutrient/stress sensing and energy metabolism.

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

Detection of endogenous ubiquitination sites by mass spectrometry has dramatically improved with the commercialization of anti-di-glycine remnant (K-ε-GG) antibodies. Here, we describe a number of improvements to the K-ε-GG enrichment workflow, including optimized antibody and peptide input requirements, antibody cross-linking, and improved off-line fractionation prior to enrichment. This refined and practical workflow enables routine identification and quantification of ∼20,000 distinct endogenous ubiquitination sites in a single SILAC experiment using moderate amounts of protein input.

iTRAQ Labeling is Superior to mTRAQ for Quantitative Global Proteomics and Phosphoproteomics

Labeling of primary amines on peptides with reagents containing stable isotopes is a commonly used technique in quantitative mass spectrometry. Isobaric labeling techniques such as iTRAQ™ or TMT™ allow for relative quantification of peptides based on ratios of reporter ions in the low m/z region of spectra produced by precursor ion fragmentation. In contrast, nonisobaric labeling with mTRAQ™ yields precursors with different masses that can be directly quantified in MS1 spectra. In this study, we compare iTRAQ- and mTRAQ-based quantification of peptides and phosphopeptides derived from EGF-stimulated HeLa cells. Both labels have identical chemical structures, therefore precursor ion- and fragment ion-based quantification can be directly compared. Our results indicate that iTRAQ labeling has an additive effect on precursor intensities, whereas mTRAQ labeling leads to more redundant MS2 scanning events caused by triggering on the same peptide with different mTRAQ labels. We found that iTRAQ labeling quantified nearly threefold more phosphopeptides (12,129 versus 4,448) and nearly twofold more proteins (2,699 versus 1,597) than mTRAQ labeling. Although most key proteins in the EGFR signaling network were quantified with both techniques, iTRAQ labeling allowed quantification of twice as many kinases. Accuracy of reporter ion quantification by iTRAQ is adversely affected by peptides that are cofragmented in the same precursor isolation window, dampening observed ratios toward unity. However, because of tighter overall iTRAQ ratio distributions, the percentage of statistically significantly regulated phosphopeptides and proteins detected by iTRAQ and mTRAQ was similar. We observed a linear correlation of logarithmic iTRAQ to mTRAQ ratios over two orders of magnitude, indicating a possibility to correct iTRAQ ratios by an average compression factor. Spike-in experiments using peptides of defined ratios in a background of nonregulated peptides show that iTRAQ quantification is less accurate but not as variable as mTRAQ quantification.

Methods for Quantification of in vivo Changes in Protein Ubiquitination following Proteasome and Deubiquitinase Inhibition

Ubiquitination plays a key role in protein degradation and signal transduction. Ubiquitin is a small protein modifier that is adducted to lysine residues by the combined function of E1, E2, and E3 enzymes and is removed by deubiquitinating enzymes. Characterization of ubiquitination sites is important for understanding the role of this modification in cellular processes and disease. However, until recently, large-scale characterization of endogenous ubiquitination sites has been hampered by the lack of efficient enrichment techniques. The introduction of antibodies that specifically recognize peptides with lysine residues that harbor a di-glycine remnant (K-ε-GG) following tryptic digestion has dramatically improved the ability to enrich and identify ubiquitination sites from cellular lysates. We used this enrichment technique to study the effects of proteasome inhibition by MG-132 and deubiquitinase inhibition by PR-619 on ubiquitination sites in human Jurkat cells by quantitative high performance mass spectrometry. Minimal fractionation of digested lysates prior to immunoaffinity enrichment increased the yield of K-ε-GG peptides three- to fourfold resulting in detection of up to ∼3300 distinct K-GG peptides in SILAC triple encoded experiments starting from 5 mg of protein per label state. In total, we identify 5533 distinct K-ε-GG peptides of which 4907 were quantified in this study, demonstrating that the strategy presented is a practical approach to perturbational studies in cell systems. We found that proteasome inhibition by MG-132 and deubiquitinase inhibition by PR-619 induces significant changes to the ubiquitin landscape, but that not all ubiquitination sites regulated by MG-132 and PR-619 are likely substrates for the ubiquitin-proteasome system. Additionally, we find that the proteasome and deubiquitinase inhibitors studied induced only minor changes in protein expression levels regardless of the extent of regulation induced at the ubiquitin site level. We attribute this finding to the low stoichiometry of the majority ubiquitination sites identified in this study.